EP4565275A1 - Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions - Google Patents

Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions

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Publication number
EP4565275A1
EP4565275A1 EP23753840.0A EP23753840A EP4565275A1 EP 4565275 A1 EP4565275 A1 EP 4565275A1 EP 23753840 A EP23753840 A EP 23753840A EP 4565275 A1 EP4565275 A1 EP 4565275A1
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EP
European Patent Office
Prior art keywords
alkylene
alkyl
group
composition
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP23753840.0A
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German (de)
French (fr)
Inventor
Zhengxin Dong
Jonathan Mcgee
Griffin GILDERSLEEVE
Daniel KALLIN
Meike GANGLUFF
Hossam HEFESHA
Ehsan MEHRAVAR
Olaf OTHERSEN
Azzurra APRICENO
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Biontech SE
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Biontech SE
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Publication of EP4565275A1 publication Critical patent/EP4565275A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/40—Polyamides containing oxygen in the form of ether groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • NUCLEIC ACID COMPOSITIONS COMPRISING AMPHIPHILIC OLIGO ETHYLENE GLYCOL (OEG)-CONJUGATED COMPOUNDS AND METHODS OF USING SUCH COMPOUNDS AND COMPOSITIONS
  • the present disclosure relates generally to the field of nucleic acid (such as DNA or RNA, in particular mRNA) compositions comprising an amphiphilic oligo ethylene glycol (OEG)-conjugated compound (as alternative to PEG lipids), to the use of such compositions, in particular for delivering nucleic acids to cells of a subject or in therapy, to such amphiphilic OEG-conjugated compounds, and to conjugates of such amphiphilic OEG-conjugated compounds.
  • nucleic acid such as DNA or RNA, in particular mRNA
  • OEG amphiphilic oligo ethylene glycol
  • a recombinant nucleic acid such as DNA or RNA
  • a recombinant nucleic acid may be administered in naked form to a subject in need thereof; however, usually a recombinant nucleic acid is administered using a composition.
  • nucleic acid such as RNA
  • the nanoparticles are intended to protect the nucleic acid, such as RNA, from degradation, enable delivery of the nucleic acid, such as RNA, to the target site and facilitate cellular uptake and processing by the target cells.
  • the efficiency of the nucleic acid delivery depends, in part, on the molecular composition of the nanoparticle and can be influenced by numerous parameters, including particle size, formulation, and charge or grafting with molecular moieties, such as polyethylene glycol (PEG) or other ligands.
  • PEG polyethylene glycol
  • lipid nanoparticles are manufactured by mixing an aqueous phase of the nucleic acid, such as RNA, with an organic phase of the lipids a certain fraction of PEG- conjugated lipid (such PEG-conjugated lipids have at least 30 consecutive ethylene glycol repeating units) in the lipid mixture is required, otherwise the particles aggregate during or after the mixing step.
  • the size of the particles can be adjusted.
  • the particle size may be adjusted by variation of the molar mass of the PEG moiety of the PEGylated lipids. Typical sizes which are accessible are in the range between 30 and 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). So-formed particles have additionally the advantage, that, due to the PEG fraction, they interact less with serum components, and have a longer circulation half-life, which is desirable in many drug delivery approaches. Without PEG-lipids, no particles with discrete size can be formed; the particles form large aggregates and precipitate.
  • PEG-lipids facilitate particle self-assembly by providing a steric barrier at the surface of nascent particles formed when nucleic acids are rapidly mixed in ethanol solutions containing lipids to bind the nucleic acid, such as RNA.
  • PEG steric hindrance prevents inter-particle fusion and promotes the formation of a homogeneous population of LNPs where diameters ⁇ 100 nm can be achieved.
  • PEGylation of nanoparticles may lead as well to several effects which are detrimental to the intended use for drug delivery.
  • PEGylation of liposomes and LNPs is known to reduce the cellular uptake and endosomal escape, thus reducing at the end the overall transfection efficiency.
  • the PEG shell provides a steric barrier to efficient binding of particles to the cell and also hinders endosomal release by preventing membrane fusion between the liposome and the endosomal membrane. This is why the type of PEG-lipid and the amount of PEG-lipid used must be always carefully adjusted. It should provide sufficient stealth effect for in vivo and stabilization aspects on the one hand, while not hindering transfection on the other. This phenomenon is known as the "PEG Dilemma".
  • anti-PEG antibodies Due to broad use of PEGs as ingredient of food, cosmetic, hygienic products, and medicines, a certain percentage of the general population have "pre-existing" anti-PEG antibodies.
  • the anti-PEG antibodies are associated with decreased efficacy of pegylated drugs and hypersensitivity reactions that can lead to severe allergic symptoms.
  • RNA nucleic acid
  • mRNA nucleic acid
  • protein knock-down therapies using inhibitory RNA such as siRNA
  • antisense oligonucleotides or DNA based therapies are examples of nucleic acid, such as RNA, into cells which avoid the disadvantages accompanied by use of PEG.
  • the present disclosure addresses this and other needs.
  • compositions, methods, polymer-conjugated compounds (which are also referred to as amphiphilic OEG-conjugated compounds herein), and conjugates described herein fulfill the above-mentioned requirements.
  • polymer-conjugated compounds described herein are not bound by antibodies raised against PEG structures and are stable under physiological conditions.
  • the polymer component of the polymer- conjugated compounds as well as the polymer-conjugated compounds and their conjugates can be synthesized by well-known procedures, such as solid-phase peptide synthesis (SPPS).
  • SPPS solid-phase peptide synthesis
  • the polymer- conjugated compounds and conjugates can be end-group functionalized with different moieties to modulate charge or to introduce specific molecular moieties like ligands.
  • the present disclosure provides a composition
  • a composition comprising (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein
  • X 2 and X 1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester, preferably optionally substituted amide, optionally substituted thioamide, or ester;
  • Y is -CH 2 -, -(CH 2 ) 2 -, or -(CH 2 ) 3 -; z is 2 to 24; and n is 1 to 100.
  • This composition is also referred to as a nucleic acid composition herein.
  • anti-PEG antibodies polyclonal as well as IgG and IgM anti-PEG antibodies
  • PEG i.e., having at least 30 consecutive ethylene glycol repeating units
  • the present application shows that a polymer comprising the structure of formula (I) is stable under physiological conditions.
  • X 2 and X 1 taken together are an optionally substituted amide.
  • X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Cus alkyl.
  • X 1 is -NR 1 - and X 2 is -C(O)-, wherein R 1 is hydrogen or Cus alkyl.
  • X 2 and X 1 taken together are an optionally substituted thioamide.
  • X 1 is -C(S)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Cus alkyl.
  • X 1 is -NR 1 - and X 2 is -C(S)-, wherein R 1 is hydrogen or C1-8 alkyl.
  • X 2 and X 1 taken together are an ester.
  • X 1 is -C(O)- and X 2 is -O.
  • X 1 is -O- then X 2 is -C(O).
  • X 2 and X 1 taken together are a thioester.
  • X 1 is -C(S)- and X 2 is -O-.
  • X 1 is -O- then X 2 is -C(S)-.
  • X 1 is -C(O)- then X 2 is -S-.
  • X 1 is -S- then X 2 is -C(O)-.
  • X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Cus alkyl.
  • R 1 may be hydrogen or methyl.
  • R 1 is hydrogen.
  • Y is -CH2- or -(CFFh-. In some embodiments, Y is -CH2-.
  • the polymer comprises the following general formula (II): wherein R 1 is hydrogen or Cus alkyl.
  • R 1 is hydrogen or methyl.
  • R 1 may be hydrogen.
  • R 1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R 1 may be H or methyl in each repeating unit).
  • R 1 in at least one repeating unit differs from R 1 in another repeating unit (e.g., for at least one repeating unit R 1 is one specific alkyl (such as H), and for at least one different repeating unit R 1 is a different specific alkyl (such as methyl)).
  • z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
  • the polymer comprises the following general formula (III): wherein R 1 is hydrogen or Cus alkyl.
  • R 1 is hydrogen or methyl.
  • R 1 may be hydrogen.
  • R 1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R 1 may be H or methyl in each repeating unit).
  • R 1 in at least one repeating unit differs from R 1 in another repeating unit (e.g., for at least one repeating unit R 1 is one specific alkyl (such as H), and for at least one different repeating unit R 1 is a different specific alkyl (such as methyl)).
  • the polymer comprises the following general formula (IV):
  • the polymer comprises the following general formula (IVa):
  • n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
  • the one or more hydrophobic chains are located at either the X 1 end or the X 2 end of the polymer.
  • the one or more hydrophobic chains are independently selected from non-cyclic, preferably straight, hydrocarbyl groups, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid.
  • the hydrocarbyl groups have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.
  • the hydrocarbyl groups may be saturated or unsaturated. If the polymer-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different.
  • the polymer-conjugated compound comprises two hydrophobic chains
  • said two hydrophobic chains are the same.
  • said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated.
  • the polymer-conjugated compound comprises the following general formula (V) or (V’): wherein
  • X 2 and X 1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
  • Y is -CH 2 -, -(CH 2 ) 2 -, or -(CH 2 ) 3 -;
  • R 2 is a moiety comprising the one or more hydrophobic chains
  • R 3 is selected from the group consisting of H, C1-6 alkyl, C 2 -6 alkynyl, -OR 20 , -SR 20 , halogen, -CN, -N 3 , -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N 3 , C 2 -6 alkynyl, -COOH, -COOCH 3 , -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide
  • X 2 and X 1 taken together are an optionally substituted amide.
  • X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Cus alkyl.
  • X 1 is -NR 1 - and X 2 is -C(O)-, wherein R 1 is hydrogen or Cus alkyl.
  • X 2 and X 1 taken together are an optionally substituted thioamide.
  • X 1 is -C(S)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Cus alkyl.
  • X 1 is -NR 1 - and X 2 is -C(S)-, wherein R 1 is hydrogen or Cus alkyl.
  • X 2 and X 1 taken together are an ester.
  • X 1 is -C(O)- and X 2 is -O.
  • X 1 is -O- then X 2 is -C(O).
  • X 2 and X 1 taken together are a thioester.
  • X 1 is -C(S)- and X 2 is -O-.
  • X 1 is -O- then X 2 is -C(S).
  • X 1 is -C(O)- then X 2 is -S-.
  • X 1 is -S- then X 2 is -C(O)-.
  • X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Cus alkyl.
  • R 1 may be hydrogen or methyl.
  • R 1 is hydrogen.
  • Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-. In some embodiments of formula (V) or (V’), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
  • R 1 is hydrogen or methyl.
  • R 1 may be hydrogen.
  • R 1 at each occurrence i.e., in each repeating unit
  • R 1 is the same (e.g., R 1 may be H or methyl in each repeating unit).
  • R 1 in at least one repeating unit differs from R 1 in another repeating unit (e.g., for at least one repeating unit R 1 is one specific alkyl (such as H), and for at least one different repeating unit R 1 is a different specific alkyl (such as methyl)).
  • n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
  • the polymer-conjugated compound comprises the following general formula (VI) or (VI’): wherein z, n, R 2 , and R 3 are as defined for Formula (V) and (V’); and R 1 is hydrogen or Cus alkyl.
  • R 1 is hydrogen or methyl.
  • R 1 may be hydrogen.
  • R 1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R 1 may be H or methyl in each repeating unit).
  • R 1 in at least one repeating unit differs from R 1 in another repeating unit (e.g., for at least one repeating unit R 1 is one specific alkyl (such as H), and for at least one different repeating unit R 1 is a different specific alkyl (such as methyl)).
  • z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
  • n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
  • the polymer-conjugated compound comprises the following general formula (VII) or (VII’): wherein n, R 2 , and R 3 are as defined for formula (V) and (V’); and R 1 is hydrogen or Cus alkyl.
  • R 1 is hydrogen or methyl.
  • R 1 may be hydrogen.
  • R 1 at each occurrence i.e., in each repeating unit
  • R 1 is the same (e.g., R 1 may be H or methyl in each repeating unit).
  • R 1 in at least one repeating unit differs from R 1 in another repeating unit (e.g., for at least one repeating unit R 1 is one specific alkyl (such as H), and for at least one different repeating unit R 1 is a different specific alkyl (such as methyl)).
  • n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
  • the polymer-conjugated compound comprises the following general formula (VIII), (Villa), (VIII’), or (Villa’): wherein n, R 2 , and R 3 are as defined for formula (V) and (V’).
  • n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
  • R 2 is R 4 or -L ⁇ R ⁇ p, wherein each R 4 is independently a hydrophobic chain, such as a hydrocarbyl group; L 1 is a linker; and p is 1 or 2.
  • L 1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R 4 , to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate,
  • L 1 further comprises at least one additional difunctional moiety via which R 2 is attached to either X 1 in formula (V) (or to the carbonyl group of any one of formulas (VI), (VII), (VIII), and (Villa)) or X 2 in formula (V’) (or the N atom of any one of formulas (VI’), (VII’), (VIII’), and (Villa’)).
  • the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydr
  • L 1 is selected from the group consisting of [*-C(O)O] p (C1-6- alkylene)OP(O)(OR 27 )O(C1-6-alkylene)-, [*-C(O)O] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-alkylene)- NR 26 -, [*-C(O)O] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-alkylene)C(O)-, [*-OC(O)] p (C1-6-alkylene)- OP(O)(OR 27 )O(C1-6-alkylene)-, [*-OC(O)] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-alkylene)NR 26 -, [*- OC(O)] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-
  • L 1 is selected from the group consisting of [*-C(O)O] p (C 1-6 -alky lene)- OP(O)(OR 27 )O(C1-6-alkylene)-, [*-C(O)O] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-alkylene)NH-, [*- C(O)O] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-alkylene)C(O)-, [*-OC(O)] p (C w -alkylene)-OP(O)(OR 27 )- O(C1-6-alkylene)-, [*-OC(O)] p (C1-6-alkylene)-OP(O)(OR 27 )O(C1-6-alkylene)NH-, [*-OC(O)] p (Ci- 6 - alkylene)OP(O)(OR 27 )O(C
  • R 2 is selected from the group consisting of [R 4 C(O)O] p (C2-3-alkylene)-OP(O)(OR 27 )O(Ci. 3 -alkylene)-, [R 4 C(O)O] p (C2-3-alkylene)- OP(O)(OR 27 )O(Ci.
  • R 2 is (R 4 )(R 26 )N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R 26 is selected from the group consisting of H and C1-6 alkyl; R 27 is selected from the group consisting of H
  • R 2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R 2 is a monoalkylamine moiety.
  • each of the one or more hydrophobic chains is independently a non-cyclic, preferably straight, hydrocarbyl group, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid.
  • the one or more hydrocarbyl groups independently have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.
  • the one or more hydrocarbyl groups may be saturated or unsaturated.
  • the polymer-conjugated compound comprises two or more hydrophobic chains
  • these chains can be the same or different.
  • said two hydrophobic chains are the same.
  • said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated and/or said two hydrophobic chains differ in their length.
  • the one or more hydrophobic chains include the hydrocarbyl chains of fatty acids, in particular the hydrocarbyl chains of naturally occurring fatty acids, such as the hydrocarbyl chains of naturally occurring fatty acids and having at least 8 carbon atoms.
  • the one or more hydrophobic chains include the hydrocarbyl chains of caprylic alcohol, capric alcohol, lauric alcohol, myristic alcohol, palmitic alcohol, stearic alcohol, arachidic alcohol, behenic alcohol, lignoceric alcohol, cerotic alcohol, oleic alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.
  • R 2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R 2 is a monomyristylamine moiety.
  • R 3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R 21 , -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)
  • R 3 is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R 21 , -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and a member of a targeting pair;
  • R 3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(0)NH2, -C(0)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair.
  • the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody (including fragments or derivatives thereof) specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine -gly
  • the polymer-conjugated compound has one of the following wherein n is 5 to 25; R 3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH,
  • R 27 is H or a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); and in each case -C(O)Ci7H 3 5 refers to the moiety -C(O)(CH 2 )I 6 CH 3 (stearoyl), in each case -C(O)Ci5H 3i refers to the moiety -C(
  • n is 7 to 16, e.g., 7 to 14 (preferably 8, 10, 12, 14, or 16); and/or R 3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N 3 , C 2 -6 alkynyl, an antigen, and an antibody.
  • substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N 3 , C 2 -6 alkynyl, an antigen, and an antibody.
  • polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains" .
  • the polymer-conjugated compound has any one of formulas (V-l), (V-5), (V-17), and (V-25).
  • the composition is substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units.
  • PEG polyethylene glycol
  • the composition is also substantially free of another polymer- conjugated lipid.
  • the another polymer-conjugated lipid is a polysarcosine- conjugated lipid and/or a conjugate comprising hydrophobic chains and a polyoxazoline (POX) and/or polyoxazine (POZ) polymer.
  • water is the main component in the composition and/or the total amount of solvent(s) other than water contained in the composition is less than about 1.0% (v/v), such as less than about 0.5% (v/v).
  • the amount of water contained in the composition may be at least 50% (w/w), such as at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), at least 90% (w/w), or at least 95% (w/w).
  • the amount of water contained in the composition may be at least 50% (w/w), such as at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), or at least 90% (w/w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition may be at least 95% (w/w).
  • the total amount of solvent(s) other than water contained in the composition may be less than about 0.5% (v/v), such as less than about 0.4% (v/v), less than about 0.3% (v/v), less than about 0.2% (v/v), less than about 0.1% (v/v), less than about 0.05% (v/v), less than about 0.01% (v/v), or less than about 0.005% (v/v).
  • a cryoprotectant which is liquid under normal conditions will not be considered as a solvent other than water but as cryoprotectant.
  • the total amount of solvent(s) other than water contained in the composition may be less than about 0.5% (v/v), such as less than about 0.4% (v/v), does not apply to cryoprotectants which are liquids under normal conditions.
  • the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg/1 to about 500 mg/1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg/1 to about 100 mg/1.
  • the concentration of the nucleic acid (in particular RNA) in the composition is about 5 mg/1 to about 500 mg/1, such as about 10 mg/1 to about 400 mg/1, about 10 mg/1 to about 300 mg/1, about 10 mg/1 to about 200 mg/1, about 10 mg/1 to about 150 mg/1, or about 10 mg/1 to about 100 mg/1, preferably about 10 mg/1 to about 140 mg/1, more preferably about 20 mg/1 to about 130 mg/1, more preferably about 30 mg/1 to about 120 mg/1.
  • the concentration of the nucleic acid (in particular RNA) in the composition is about 5 mg/1 to about 150 mg/1, such as about 10 mg/1 to about 140 mg/1, about 20 mg/1 to about 130 mg/1, about 25 mg/1 to about 125 mg/1, about 30 mg/1 to about 120 mg/1, about 35 mg/1 to about 115 mg/1, about 40 mg/1 to about 110 mg/1, about 45 mg/1 to about 105 mg/1, or about 50 mg/1 to about 100 mg/1.
  • the concentration of the nucleic acid (in particular RNA) in the composition is 1 mg/1 to about 50 mg/1 or about 10 mg/1 to about 100 mg/1.
  • the composition comprises a cryoprotectant. In some embodiments of the first aspect, the composition is substantially free of a cryoprotectant.
  • the cationically ionizable lipid comprises a head group which includes at least one tertiary amine moiety.
  • the cationically ionizable lipid has the structure of Formula (X) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 10 , L 20 , G 1 , G 2 , G 3 , R 35 , R 36 , and R 37 are as defined herein.
  • the cationically ionizable lipid is selected from the following: the structures X-l to X-36 (shown herein); or the structures A to G (shown herein).
  • the cationically ionizable lipid is the lipid having the structure X-3.
  • the cationically ionizable lipid is DPL-14 (i.e., the lipid having the structure G).
  • the cationically ionizable lipid has the structure of Formula (XI): wherein Ri, R2 R3, R4, L2, G2, and m are as defined herein.
  • the cationically ionizable lipid is selected from the structures (XIV-1), (XIV-2), and (XIV-3) (shown herein).
  • the cationically ionizable lipid is the lipid having the structure XIV-1
  • the cationically ionizable lipid is the lipid having the structure XIV-2.
  • the cationically ionizable lipid is the lipid having the structure XIV-3.
  • the cationic or cationically ionizable lipid comprises 2,3- dioleyloxy-l-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)- N,N,N -trimethylammonium chloride (DOTAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-
  • the cationically ionizable lipid is completely or partially replaced by a cationic lipid.
  • the cationic lipid is selected from the structures XV- 1 to XV -6 (shown herein).
  • the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol of the total lipid present in the composition.
  • the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of a phospholipid and a steroid.
  • the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmito
  • the phospholipid comprises from about 5 mol % to about 30 mol % of the total lipid present in the composition.
  • the steroid comprises a sterol. In some preferred embodiments, the steroid comprises or is cholesterol.
  • the steroid comprises from about 10 mol % to about 60 mol % of the total lipid present in the composition.
  • the cationic or cationically ionizable lipid comprises from about 20 mol % to about 70 mol % of the total lipid present in the composition;
  • the polymer-conjugated compound (amphiphilic OEG-conjugated compound) comprises from about 0.5 mol % to about 15 mol % (such as from about 2 mol % to about 6 mol % or from about 2 mol % to about 5 mol %) of the total lipid present in the composition;
  • the phospholipid comprises from about 5 mol % to about 25 mol % of the total lipid present in the composition;
  • the steroid comprises from about 20 mol % to about 55 mol % of the total lipid present in the composition.
  • the composition further comprises one or more additional lipids.
  • the one or more additional lipids may comprise a cationic lipid.
  • a cationic lipid is present, the sum of (1) the amount the cationically ionizable lipid and (2) the amount of cationic lipid is used for calculations.
  • the amount of cationically ionizable lipid in a composition should be from about 20 mol % to about 70 mol % and the composition should also contain a cationic lipid, then the sum of (1) the amount the cationically ionizable lipid and (2) the amount of cationic lipid is to be from about 20 mol % to about 70 mol %.
  • the only lipids contained in the composition are the cationic or cationically ionizable lipid, the steroid, the neutral lipid, and the polymer-conjugated compound comprising the polymer of formula (I) as defined herein (i.e., the amphiphilic OEG-conjugated compound), in particular the cationic or cationically ionizable lipid, the steroid, the phospholipid, and the polymer-conjugated compound comprising the polymer of formula (I) as defined herein.
  • the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the cationic or cationically ionizable lipid, and at least a portion of the polymer-conjugated compound comprising the polymer of formula (I) as defined herein.
  • the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof.
  • the particles comprise or are LNPs.
  • the particles comprise or are liposomes.
  • the particles comprise or are LPXs.
  • the particles comprise or are mixtures of LNPs and liposomes. In some embodiments, the particles comprise or are mixtures of LNPs and LPXs. In some embodiments, the particles comprise or are mixtures of liposomes and LPXs. In some embodiments, the particles comprise or are mixtures of LNPs, liposomes, and LPXs.
  • the particles comprise essentially all of lipids, (in particular all of the cationic or cationically ionizable lipid, the one or more additional lipids, if present, and the and the polymer- conjugated compound comprising the polymer of formula (I) as defined herein) present in the composition.
  • the particles comprise at least 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acid (in particular RNA) present in the composition.
  • the particles comprise at least 75%, preferably at least 85% of the nucleic acid (in particular RNA) present in the composition.
  • the aqueous phase is substantially free of the nucleic acid.
  • the composition comprises particles dispersed in an aqueous phase
  • the nucleic acid such as RNA
  • the particles have a size of from about 30 nm to about 500 nm. In some embodiments, the particles have a size from about 50 nm to about 150 nm.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA, preferably mRNA or inhibitory RNA, (e.g., siRNA).
  • RNA preferably mRNA or inhibitory RNA, (e.g., siRNA).
  • the nucleic acid is RNA (such as mRNA) and (i) comprises a modified nucleoside in place of uridine; (ii) has a coding sequence which is codon-optimized; and/or (iii) has a coding sequence whose G/C content is increased compared to the wild-type coding sequence.
  • the modified nucleoside is selected from pseudouridine (y), N1 -methyl - pseudouridine (ml ⁇
  • the nucleic acid is RNA (such as mRNA) and comprises at least one or more of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly-A sequence.
  • the RNA (such as mRNA) comprises all of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly-A sequence.
  • the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence preferably is an interrupted sequence of A nucleotides.
  • the 5’ cap is a capl or cap2 structure.
  • the nucleic acid is RNA (such as mRNA) and encodes one or more polypeptides.
  • the one or more polypeptides are pharmaceutically active polypeptides and/or comprise an epitope for inducing an immune response against an antigen in a subject.
  • the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof.
  • the pathogen is a pathogen causing an infectious disease.
  • the nucleic acid is inhibitory RNA (such as siRNA) and selectively hybridizes to and/or is specific for a target mRNA.
  • the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, in particular a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease.
  • the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.
  • the present disclosure relates to a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect. It is understood that any embodiment described herein in the context of the first aspect may also apply to any embodiment of the second aspect.
  • the present disclosure relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described herein in the context of the first or second aspect may also apply to any embodiment of the third aspect.
  • the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.
  • the present disclosure relates to a nucleic acid composition of the first aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, or third aspect may also apply to any embodiment of the fourth aspect.
  • the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
  • the present disclosure relates to a nucleic acid composition of the first aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, or fourth aspect may also apply to any embodiment of the fifth aspect.
  • the subject is a mammal, such as a human.
  • a polymer-conjugated compound also referred to herein as amphiphilic OEG-conjugated compound
  • a polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains.
  • Preferred embodiments of the polymer- conjugated compound of the sixth aspect are specified in the first aspect and are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains".
  • the present disclosure provides conjugate of (a) the polymer-conjugated compound of the sixth aspect containing a member of a targeting pair; and (b) a compound comprising the other member of the targeting pair.
  • the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (such as an antigen or epitope), or an antibody.
  • the conjugate has one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen.
  • the conjugate has one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, r 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH 2 )i6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen.
  • the polymer-conjugated compound containing a member of a targeting pair has the following formula: wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety - C(O)(CH 2 )I 6 CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen; the compound comprising the other member of the targeting pair is a compound comprising (i) an antibody specific for said antigen if Pept is said antigen; or (ii) an antigen if Pept is an antibody specific for said antigen; and the polymer-conjugated compound containing a member of a targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) said antibody specific for said antigen and (2) said antigen.
  • the present disclosure provides a composition
  • a composition comprising (i) nucleic acid (such as DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of the seventh aspect.
  • the present disclosure relates to a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a composition of the eighth aspect. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect may also apply to any embodiment of the ninth aspect.
  • the present disclosure relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth aspect may also apply to any embodiment of the tenth aspect.
  • the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.
  • the present disclosure relates to a composition of the eighth aspect for use in a method for treating or preventing a disease or disorder in a subj ect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect may also apply to any embodiment of the eleventh aspect.
  • the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
  • the present disclosure relates to a composition of the eighth aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspect may also apply to any embodiment of the twelfth aspect.
  • the present disclosure provides a method of transfecting cells, comprising adding a composition of the first or eighth aspect to cells; and incubating the mixture of the composition and cells for a sufficient amount of time.
  • the nucleic acid is DNA or RNA (such as mRNA) and encodes a pharmaceutically active protein
  • the mixture of the composition and cells is incubated for a time sufficient to allow the expression of the pharmaceutically active protein.
  • the nucleic acid is inhibitory RNA (such as siRNA) directed against a target mRNA
  • the mixture of the composition and cells is incubated for a time sufficient to allow the inhibition of the transcription and/or translation of the target mRNA.
  • the sufficient amount of time is at least one hour (such at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and/or up to about 48 hours (such as up to about 36 or up to about 24 hours).
  • the method is conducted in vivo (i.e., the cells form part of an organ, a tissue and/or an organism of a subject). In some embodiments of the thirteenth aspect, the method is conducted in vitro (i.e., the cells do not form part of an organ, a tissue and/or an organism of a subject, e.g., the cells are an ex vivo cell culture). It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth aspect may also apply to any embodiment of the thirteenth aspect.
  • the present disclosure provides a pharmaceutical composition comprising a conjugate of the seventh aspect or a composition of the first or eighth aspect.
  • the pharmaceutical composition further comprises one or more of pharmaceutically acceptable carriers, diluents and excipients. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect may also apply to any embodiment of the fourteenth aspect.
  • the present disclosure provides a kit comprising a composition of the first or eighth aspect, a polymer-conjugated compound (amphiphilic OEG-conjugated compound) of the sixth aspect, a conjugate of the seventh aspect, or a pharmaceutical composition as described herein (such as a pharmaceutical composition of the fourteenth aspect).
  • the kit is for use in therapy, such as for inducing an immune response.
  • the kit is for use in inducing an immune response against a pathogen, such as for treating or preventing an infectious disease.
  • the present disclosure provides a method for preparing an amphiphilic OEG- conjugated compound comprising the following steps: (a) providing an intermediate compound having the formula (VII) or (VIE) as disclosed herein, wherein, for formula (VII), R 2 is OH, and R 3 is H, acetyl, or Fmoc; and, for formula (VIE), R 2 is H, acetyl, or Fmoc, and R 3 is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, in particular a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, thereby obtaining the amphiphilic OEG-conjugated compound.
  • an organic molecule in particular a compound comprising a phosphatidylethanolamine moiety, a tocophe
  • Figure 1 Scheme of the anti-PEG ELISA assay used for determining the binding of anti -PEG polyclonal antibodies to different antigens (PEG, AEEA, pSAR).
  • Biotin-labeled antigens biotin- PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NEE-pSAR
  • IxPBS 0.1% CHAPS
  • Figure 2 Results of the anti-PEG ELISA assay of Figure 1 for different biotin-labeled antigens (biotin- PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NH2-pSAR).
  • Figure 3 Results of a further anti-PEG ELISA assay using monoclonal anti PEG IgG (A) or monoclonal anti PEG IgM (B) and different antigens (PEG36, capped-AEEA14, NH2-AEEA14).
  • the PEG36 had a molecular weight of about 1.6 kDa.
  • the capped AEEA14 was Ac-AEEA14.
  • Figure 4 Stability of pAEEA under physiological conditions.
  • Ac-AEEA14 (A) and NH2-AEEAI4 (B) were incubated in mouse or human plasma for 0 h to 72 h.
  • Figure 5 Particle size and PDI of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Particle size and PDI of BM LNPs formulation are also included.
  • FIG. 6 Zeta potential of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Zeta potential of BM formulations is also included.
  • Figure 7 Accessible RNA of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • FIG. 8 Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items.
  • the horizontal dashed line shows the level of SC5b-9 formation for PBS.
  • Figure 9 Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid.
  • Figure 11 Particle size and PDI of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Particle size and PDI of BM LNPs formulation are also included.
  • FIG. 12 Zeta potential of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Zeta potential of BM formulations is also included.
  • Figure 13 Accessible RNA of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • Figure 14 Quantification of SI protein expression after transfection with VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid.
  • A Variation in VE-AEEA14-AC composition vs. MFI (Mean Fluorescence Intensity) of the overall cell population. Data was fitted using a quadratic polynomial function.
  • Figure 15 Yield and purity of peptide intermediates.
  • Peptide intermediates Ac-(AEEA)8-OH (A-D) and Ac-(AEEA)i4-OH (E-H) were synthesized and their yields and purities were determined using UPLC (A, B, E, F) and mass spectrometry (C, D, G, H).
  • Samples for UPLC and mass spectrometry were taken either directly after cleavage from the resin, i.e., prior to the QC method (crude purities; Figures 15 A, C, E, G), of after the QC method (final purities; Figures 15 B, D, F, H).
  • Figure 16 Monitoring of the synthesis of Ac-(AEEA)8-a-tocopherol. Samples of the reaction were taken and analyzed using the methods described herein. UPLC chromatograms are shown for samples taken (A) after 5 minutes and (B) at the end of the reaction time (4 h).
  • Figure 17 Purity of the amphiphilic compounds Ac-(AEEA)8-a-tocopherol (A, B), Ac-(AEEA)i4-a- tocopherol (C, D), AC-(AEEA)I 4 -DMA (E, F), Ac-(AEEA) 8 -DMG (G, H), and AC-(AEEA)I 4 -DSPE (I, J) as shown by exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J), respectively.
  • Figure 18 Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). Particle size and PDI of BM LNPs formulation are also included.
  • FIG. 19 Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). Zeta potential of BM formulations is also included.
  • Figure 20 Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • FIG. 21 Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items.
  • the horizontal dashed line shows the level of SC5b-9 formation for PBS.
  • Figure 22 Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 23 Quantification of SI protein expression after transfection with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • A Variation in composition vs. MFI (Mean Fluorescence Intensity) of the overall cell population. Data was fitted using a quadratic polynomial function.
  • Figure 24 Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 25 Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 26 Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • Figure 28 T-cell targeting using LNPs with different stealth lipids and Alfa lipids.
  • LNPs were formulated with different lipid compositions (Cargo: Thy 1.1 RNA/Luc RNA/Np proxy Venus 1: 1:2 w/w; N/P ratio: 6; Lipid mix: HY501/Cholesterol/DSPC/stealth lipid/Alfa lipid.
  • the lipid ratio has been selected as [47.5/40.5/10/1.8/0.2] for the following combinations of stealth lipid and Alfa lipid: C16 PEG2k Ceramide/DSPE PEG2k Alfa, DSPE PEG2k/DSPE PEG2k Alfa, DSPE-AEEA14/DSPE- AEEA14-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa.
  • the lipid ratio has been selected as [47.5/38.5/10/3.8/0.2] for the following combinations of stealth lipid and Alfa lipid: VE-PEGlk/DSPE- PEG2k-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa.
  • Figure 29 Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 30 Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 31 Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • Figure 32 Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
  • Figure 33 Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 35 Particle size and PDI of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 36 Zeta potential of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 37 Accessible RNA of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • FIG. 38 Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items.
  • the horizontal dashed line shows the level of SC5b-9 formation for PBS.
  • Figure 39 Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 41 Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 42 Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • FIG. 43 Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items.
  • the horizontal dashed line shows the level of SC5b-9 formation for PBS.
  • Figure 44 Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
  • Figure 46 Particle size and PDI of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
  • Figure 47 Zeta potential of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
  • Figure 48 Accessible RNA of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
  • DODMA amphiphilic OEG-conjugated compound
  • DODAB various cationical or cationically ionizable lipids
  • A RiboGreen Assay
  • B agarose gel electrophoresis
  • Figure 49 Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
  • Figure 50 Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
  • DODMA amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
  • Figure 52 Particle size and PDI of functionalized LNPs prepared with 5 different OEG-conjugated compounds as stealth lipids.
  • Figure 53 Agarose gel electrophoresis of controls, untreated functionalized LNPs (upper row) and functionalized LNPs treated with release solution (lower row).
  • Figure 54 Particle size and PDI of functionalized LNPs prepared respectively with OEG-conjugated compounds ((A) C14-pAEEA14-Ac, (B) DSPE-pAEEA14-Ac, (C) VitE-pAEEA14-Ac, (D) DMA- pAEEA14-Ac and (E) VitE-pAEEA8-Ac) subjected to three freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.
  • OEG-conjugated compounds ((A) C14-pAEEA14-Ac, (B) DSPE-pAEEA14-Ac, (C) VitE-pAEEA14-Ac, (D) DMA- pAEEA14-Ac and (E) VitE-pAEEA8-Ac) subjected to three freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.
  • Figure 55 In vitro transfection with LNPs.
  • A) The percentages of transfected cells (CD 14+ Monocytes, CD 19+ B cells, CD4+ T cells or CD8+ T cells) within all transfected PBMCs (Transfection, y-axes) per tested formulation.
  • B) Total cell counts acquired by flow cytometry within 20 seconds. Numbers are corrected relative to counting beads. BD FACS Lyric was used for sample acquisition. FlowJo was used for data analysis.
  • Figure 56 Ligand-mediated transfection of T cells in vivo. Intramuscular injection of naked or LNP- formulated luciferase- and Thy 1.1 -encoding RNA mixtures (1 : 1 weight-to-weight mix) in B6- hCD3EDG transgeneic mice (1 pg RNA dose per injection side; 2 pg total RNA dose per mouse). Analysis performed 18h after injection.
  • A Drainage analyzed via ex vivo bioluminescence imaging of popliteal, inguinal, axially and brachial lymph nodes, and spleens.
  • B Cell-type specific transfection analyzed by flow-cytometry via detection of delivered Thy 1.
  • C T-cell activation status analyzed via mean fluorescent intensity of CD69 surface expression within depicted organ (following staining with anti-CD69-APC antibody).
  • LN lymph nodes
  • LNP lipid nanoparticle
  • NK natural killer
  • PMN polymorphonuclear cells.
  • the term typically indicates deviation from the indicated numerical value by ⁇ 5%, such as ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.9%, ⁇ 0.8%, ⁇ 0.7%, ⁇ 0.6%, ⁇ 0.5%, ⁇ 0.4%, ⁇ 0.3%, ⁇ 0.2%, ⁇ 0.1%, ⁇ 0.05%, and for example ⁇ 0.01%.
  • ⁇ 5% such as ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.9%, ⁇ 0.8%, ⁇ 0.7%, ⁇ 0.6%, ⁇ 0.5%, ⁇ 0.4%, ⁇ 0.3%, ⁇ 0.2%, ⁇ 0.1%, ⁇ 0.05%, and for example ⁇ 0.01%.
  • the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
  • Terms such as “enhance” and “increase” as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level. In some embodiments, these terms relate to an increase or enhancement by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.
  • physiological pH refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.
  • physiological conditions refer to the conditions (in particular pH and temperature) in a living subject, in particular a human.
  • physiological conditions mean a physiological pH and/or a temperature of about 37°C.
  • % (w/v) refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (ml).
  • % by volume or “% (v/v)” (or “% v/v”) refers to volume percent, which is a unit of concentration measuring the amount of a liquid substance in milliliters (ml) expressed as a percent of the total volume of solution in milliliters (ml).
  • % by weight or “% (w/w)” (or “% w/w”) refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).
  • mol % is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.
  • mol % of the total lipid is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100.
  • total lipid includes lipids and lipid-like material.
  • ionic strength refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges.
  • ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge.
  • E is taken over all the different kinds of ions (i) in solution.
  • the term "ionic strength" in some embodiments relates to the presence of monovalent ions.
  • divalent inorganic ions in particular divalent inorganic cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is in one embodiment sufficiently low so as to prevent degradation of the RNA.
  • the concentration or effective concentration of divalent inorganic ions is below the catalytic level for hydrolysis of the phosphodiester bonds between RNA nucleotides.
  • the concentration of free divalent inorganic ions is 20 pM or less. In one embodiment, there are no or essentially no free divalent inorganic ions.
  • a “monovalent” compound relates to a compound having only one functional group of interest.
  • a monovalent anion relates to a compound having only one negatively charged group, preferably under physiological conditions.
  • a “divalent” or “dibasic” compound relates to a compound having two functional groups of interest.
  • a dibasic organic acid has two acid groups.
  • An example of a divalent cation is Ca 2+ .
  • a "polyvalent” or “polybasic” compound relates to a compound having three or more functional groups of interest.
  • a polybasic organic acid has three or more acid groups.
  • a "monovalent moiety” relates to a monoradical, i.e., a moiety having a valence of 1.
  • Typical monovalent moieties include alkyl, alkenyl, aryl, etc.
  • a “divalent moiety” or “bivalent moiety” relates to a diradical, i.e., a moiety having a valence of 2.
  • Typical divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, etc.
  • a further example of a divalent moiety is the C1-6-alkylene moiety in the group [* -S] p (C1-6 -alkylene)-, if p is 1 (resulting in the group *-S(C1-6-alkylene)-, such as *-S-(CH2)g- or *-S-CH2-, wherein * represents the attachment point to R 4 ).
  • a “polyvalent moiety” relates to a polyradical, i.e., a moiety having a valence of at least 3.
  • a "trivalent moiety” relates to a triradical, i. e., a moiety having a valence of 3. For example, by removing a further H atom of an alkylene group the resulting alkylene is trivalent.
  • a further example of a trivalent moiety is the C1-6-alkylene moiety in the group [*-S] p (C1-6-alkylene)-, if p is 2 (resulting in the group [*-S]2(C1-6-alkylene)-, such as *-S-CH(S-*)(CH2)s- or *-S-CH(S-*)(CH2)-, wherein * represents the attachment point to R 4 ).
  • C1-6 -alkyltriyl moiety in the group [*-C(O)NH](C1-6-alkyltriyl)- (here the [*-C(O)NH] moiety as well as a further hydrophobic chain are bound to the C1-6 -alkyltriyl moiety which, in turn, is attached to X 1 (for formula (V)) or to X 2 (for formula (V’)), either directly or through at least one additional difunctional moiety).
  • a [*-C(O)NH](C1-6- alkyltriyl) moiety as an example for L 1 , wherein C1-6 -alkyltriyl is directly attached to another hydrophobic chain R 4 may comprise at least the following structures: wherein one of ww represents the bond by which the C1-6 -alkyltriyl is attached to [*-C(O)NH] and the other ww represents the bond by which the C1-6-alkyltriyl is attached (either directly or via an additional difunctional moiety) to the remainder of the compound (e.g., the compound of formula (V)).
  • Molar ratio refers to the ratio between the amounts in moles of any two substances. For example, if a first substance is present in a composition in an amount of 1 millimole (mmol) and a second substance is present in the composition in an amount of 2 millimole (mmol), the molar ratio of the first substance to the second substance is 1:2 or 0.5.
  • Oleality refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.
  • lyophilizing refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase.
  • surrounding pressure e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less
  • spray-drying refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.
  • the term "reconstitute” relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.
  • freezing relates to the solidification of a liquid, usually with the removal of heat. In some embodiments, freezing is reverse action to thawing.
  • thawing relates to the liquification of a solid, usually with the addition of heat. In some embodiments, thawing is reverse action to freezing.
  • aqueous phase as used herein in relation to a composition/formulation comprising particles, in particular LNPs, liposomes, and/or lipoplexes, means the mobile or liquid phase, i.e., the continuous water phase including all components dissolved therein but (formally) excluding the particles.
  • particles such as LNPs
  • the aqueous phase is free of X is such manner as it is practically and realistically feasible, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight.
  • the particles dispersed in the aqueous phase may comprise compound X in an amount of more than 1% by weight.
  • the term "recombinant” in the context of the present disclosure means “made through genetic engineering”. In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • found in nature means "present in nature” and includes known objects as well as objects that have not yet been discovered and/or isolated from nature, but that may be discovered and/or isolated in the future from a natural source.
  • room temperature and “ambient temperature” are used interchangeably herein and refer to temperatures from at least about 15°C, preferably from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.
  • alkyl refers to a monoradical of a saturated straight or branched hydrocarbon.
  • the alkyl group comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, abbreviated as C1-12 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as C1-10 alkyl), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms.
  • Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1- methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl- propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n- decyl, n-undecyl, n-dodecyl, and the like.
  • alkylene refers to a diradical of a saturated straight or branched hydrocarbon.
  • the alkylene comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms.
  • alkylene groups include methylene, ethylene (i.e., 1,1 -ethylene, 1,2-ethylene), propylene (i.e., 1,1 -propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2- propylene (-C(CH3)2-), and 1,3-propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2- butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1 -pentylene, 1,2-pentylene, 1,3- pentylene, 1,4-pentylene, 1,5 -pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1-
  • the straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene).
  • 1,4-butylene can also be called tetramethylene
  • tetramethylene a polymer of methylene
  • a “substituted alkylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • alkenyl refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • the alkenyl group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenyl group comprises from 2 to 12, abbreviated as C2-12 alkenyl, (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • alkenyl groups include vinyl, 1- propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5 -hexenyl, 1 -heptenyl, 2-heptenyl, 3 -heptenyl, 4- heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7- octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-
  • a "substituted alkenyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • alkynyl refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen.
  • Alkynyl groups can optionally have one or more carbon carbon double bonds.
  • the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carboncarbon triple bonds is 4.
  • the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.
  • a "substituted alkynyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • alkenylene refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2,
  • carbon-carbon double bonds such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • alkenylene groups include ethen- 1,2-diyl, vinylidene (also called ethenylidene), 1 -propen- 1,2-diyl, 1 -propen- 1,3 -diyl, 1 -propen-2,3 -diyl, allylidene, 1-buten- 1,2-diyl, l-buten-l,3-diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, 1- buten-3,4-diyl, 2-buten- 1,2-diyl, 2-buten-l,3-diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl, and the like.
  • a "substituted alkenylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • cycloalkyl represents cyclic non-aromatic versions of “alkyl” and "alkenyl” with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms.
  • Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl.
  • the cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).
  • a “substituted cycloalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to
  • hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • cycloalkylene represents cyclic non-aromatic versions of "alkylene” and is a geminal, vicinal or isolated diradical.
  • the cycloalkylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and/or (ii) is 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered).
  • the cycloalkylene is a mono-, bi- or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkylene.
  • the ending "ylene” for cycloalkylene moieties as specified above, one can also use the ending "diyl” (e.g., 1,2-cyclopropylene can also be called cyclopropan-l,2-diyl).
  • Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g., tricyclo[3.3. 1. l 3 ’ 7 ]decan-2,2- diyl).
  • a “substituted cycloalkylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • cycloalkenylene represents cyclic non-aromatic versions of “alkenylene” and is a geminal, vicinal or isolated diradical.
  • the maximal number of carbon-carbon double bonds in the cycloalkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the cycloalkenylene group by 2 and, if the number of carbon atoms in the cycloalkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an cycloalkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4.
  • the cycloalkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • the cycloalkenylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and/or (ii) is 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered).
  • the cycloalkenylene is a mono-, bi- or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkenylene.
  • exemplary cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene.
  • a “substituted cycloalkenylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • aryl refers to a monoradical of an aromatic cyclic hydrocarbon.
  • the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl).
  • exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl.
  • aryl refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes.
  • substituted aryl means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (z.e., 2-, 3-, or 4-methoxyphenyl).
  • heteroaryl or “heteroaromatic ring” means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N.
  • heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S.
  • it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S.
  • heteroaryl groups include fiiranyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazoly
  • Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl.
  • a “substituted heteroaryl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • heterocyclyl or “heterocyclic ring” means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, or N.
  • a heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms.
  • the maximum number of O atoms is 1
  • the maximum number of S atoms is 1
  • the maximum total number of O and S atoms is 2.
  • heterocyclyl is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups.
  • exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides.
  • a “substituted heterocyclyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • heterocycloalkylene as used herein means a heterocyclyl group as defined above which contains at least one ring heteroatom (such as those selected from the group consisting of O, S, N, B, Si, and P) and in which one hydrogen atom has been removed resulting in a geminal, vicinal or isolated diradical.
  • the heteroatoms of the heterocycloalkylene group are selected from the group consisting of O, S, and N.
  • the heterocycloalkylene may be O/S-heterocycloalkylene, such as O-heterocycloalkylene.
  • the maximum number of O atoms is 1
  • the maximum number of S atoms is 1
  • the maximum total number of O and S atoms is 2.
  • the heterocycloalkylene may be monocyclic or polycyclic (such as bi- or tricyclic).
  • the heterocycloalkylene is a mono-, bi- or tricyclic 4- to 14-membered (i.e., 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 4- to 12-membered or 4- to 10-membered) heterocycloalkylene.
  • heterocycloalkylene is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups (preferably partially or completely hydrogenated forms of the above-mentioned mono-, bi-, or tricyclic heteroaryl groups) in which one hydrogen atom has been removed from the same carbon atom resulting in a geminal diradical.
  • a heterocycloalkylene is saturated or unsaturated (i.e., it contains one or more double bonds within the ring) but cannot be aromatic.
  • a “substituted heterocycloalkylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the substituent other than hydrogen is a 1 st level substituent, a 2 nd level substituent, or a 3 rd level substituent as specified herein.
  • partially hydrogenated form of an unsaturated compound or group as used herein means that part of the unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group without removing all unsaturated moieties.
  • completely hydrogenated form of an unsaturated compound or group is used herein interchangeably with the term “perhydro” and means that all unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group.
  • partially hydrogenated forms of a 5-membered heteroaryl group include dihydro forms of said 5- membered heteroaryl group (such as 2,3 -dihydrofuran or 2,5 -dihydrofuran), whereas the tetrahydro form of said 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a completely hydrogenated (or perhydro) form of said 5 -membered heteroaryl group.
  • dihydro forms of said 5- membered heteroaryl group such as 2,3 -dihydrofuran or 2,5 -dihydrofuran
  • the tetrahydro form of said 5-membered heteroaryl group e.g., tetrahydrofuran, i.e., THF
  • partially hydrogenated forms include di- and tetrahydro forms (such as di- and tetrahydropyridyl), whereas the hexahydro form (such as piperidinyl in case of the heteroaryl pyridyl) is the completely hydrogenated (or perhydro) derivative of said 6-membered heteroaryl group. Consequently, a hexahydro form of an aryl or heteroaryl can only be considered a partially hydrogenated form according to the present disclosure if the aryl or heteroaryl contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms.
  • aromatic as used in the context of hydrocarbons means that the whole molecule has to be aromatic.
  • a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure.
  • a bi- or polycyclic aryl such as naphthyl
  • the resulting hydrogenated bi- or polycyclic structure is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic).
  • heteroaryl i.e., a dihydro variant of indolyl
  • heterocyclyl i.e., a dihydro variant of indolyl
  • hydrocarbyl as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule.
  • hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched.
  • Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.).
  • hydrocarbyl groups are C1-30 alkyl (such as Cg-30 alkyl, Cx-24 alkyl, or C 10-20 alkyl), C2-30 alkenyl (such as Cg-30 alkenyl, Cx-24 alkenyl, or C 10-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(C1-6 alkyl).
  • the hydrocarbyl group is optionally substituted (e.g., with one or more 1 st level substituents, one or more 2 nd level substituents, or one or more 3 rd level substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively nonpolar.
  • the hydrocarbyl group is the hydrocarbyl chain of naturally occurring fatty acids and may have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably linear) C8-20 alkyl chain, e.g., a (preferably linear) Cio-is alkyl chain).
  • hydrogen atom(s) may be replaced with a group (i.e., a 1 st level substituent) different from hydrogen such as alkyl (preferably, C1-6 alkyl), alkenyl (preferably, C2-6 alkenyl), alkynyl (preferably, C2-6 alkynyl), aryl (preferably, 6- to 14-membered aryl), heteroaryl (preferably, 3- to 14- membered heteroaryl), cycloalkyl (preferably, 3- to 14-membered cycloalkyl), heterocyclyl (preferably, 3- to 14-membered heterocyclyl), halogen, -CN, azido, -NO2, -OR 71 , -N(R 72 )(R
  • Typical 1 st level substituents are preferably selected from the group consisting of CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14-membered (such as 5- or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR 71 , -N(R 72 )(R 73 ), -S(0)o-2R 71 , -S(O)I.
  • 1 st level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3.
  • Particularly preferred 3 rd level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3.
  • halogen such as F, Cl, or Br
  • -CF3 such as halogen (e.g., F, Cl, or Br)
  • tertiary amine moiety as used herein relates to a moiety containing a nitrogen atom which is substituted with three organic substituents (wherein the substituents may be the same or different from each other).
  • the organic substituents are selected from hydrocarbyl groups (such as alkyl groups, in particular C1-6 alkyl groups) which are optionally substituted (e.g., with one or more 1 st level substituents, one or more 2 nd level substituents, or one or more 3 rd level substituents as defined herein).
  • hydrocarbyl groups such as alkyl groups, in particular C1-6 alkyl groups
  • optionally substituted e.g., with one or more 1 st level substituents, one or more 2 nd level substituents, or one or more 3 rd level substituents as defined herein).
  • filament relates to any process that involves removal or separation of at least one component (such as permeable molecules like salts, small proteins, solvents etc.,) of a liquid composition based on the molecular size of the components contained in the composition.
  • This separation may use micro-molecule permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., for dialysis).
  • filtrating comprise dialyzing, tangential flow filtrating and diafiltrating.
  • substantially free of X means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of X in such manner as it is practically and realistically feasible.
  • the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture.
  • substantially free of a lipid comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of a lipid comprising at least 30 consecutive ethylene glycol repeating units in such manner as it is practically and realistically feasible.
  • PEG polyethylene glycol
  • the amount of a lipid comprising at least 30 consecutive ethylene glycol repeating units in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture.
  • 1% by weight e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.0
  • nucleic acid integrity means the percentage of the full-length (i.e., non-fragmented) nucleic acid to the total amount of nucleic acid (i.e., non-fragmented plus fragmented nucleic acid) contained in a sample.
  • the nucleic acid integrity may be determined by chromatographically separating the nucleic acid (e.g., using capillary electrophoresis), determining the peak area of the main nucleic acid peak (i. e. , the peak area of the full-length (z. e., non-fragmented) nucleic acid), determining the peak area of the total nucleic acid, and dividing the peak area of the main nucleic acid peak by the peak area of the total nucleic acid.
  • RNA integrity means the percentage of the full- length (i.e., non-fragmented) RNA to the total amount of RNA (i.e., non-fragmented plus fragmented RNA) contained in a sample.
  • the RNA integrity may be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of the main RNA peak (i.e., the peak area of the full-length (i. e . , non-fragmented) RNA), determining the peak area of the total RNA, and dividing the peak area of the main RNA peak by the peak area of the total RNA.
  • cryoprotectant relates to a substance that is added to a preparation (e.g., formulation or composition) in order to protect the active ingredients of the preparation during the freezing stages.
  • lyoprotectant relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.
  • peptide comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds.
  • protein or “polypeptide” refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide", “polypeptide", and “protein” are used herein usually as synonyms.
  • a "therapeutic peptide or protein” has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount.
  • a therapeutic peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder.
  • a therapeutic peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition.
  • the term "therapeutic peptide or protein” includes entire peptides or proteins, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a peptide or protein.
  • therapeutically active peptides or proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.
  • therapeutically active peptides or proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.
  • therapeutically active peptide or protein and “pharmaceutically active peptide or protein” are used interchangeably herein.
  • portion refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said structure.
  • part and fragment are used interchangeably herein and refer to a continuous element.
  • a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure.
  • the term “part” means a portion of the composition.
  • a part of a composition may any portion from 0. 1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.
  • “Fragment” with reference to an amino acid sequence (peptide, polypeptide or protein), relates to apart of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus.
  • a fragment shortened at the C-terminus is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame.
  • a fragment shortened at the N-terminus is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation.
  • a fragment of an amino acid sequence comprises, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from an amino acid sequence.
  • a fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.
  • a fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the amino acid sequence.
  • a part or fragment of a peptide, polypeptide or protein preferably has at least one functional property of the peptide, polypeptide or protein from which it has been derived.
  • Such functional properties comprise a pharmacological activity, the interaction with other peptides, polypeptides or proteins, an enzymatic activity, the interaction with antibodies, and the selective binding of nucleic acids.
  • a pharmacological active fragment of a peptide, polypeptide or protein has at least one of the pharmacological activities of the peptide, polypeptide or protein from which the fragment has been derived.
  • a part or fragment of a peptide, polypeptide or protein preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids of the peptide or protein.
  • a part or fragment of a peptide or protein preferably comprises a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the peptide or protein.
  • Variant as used herein and with reference to an amino acid sequence (peptide, polypeptide, or protein), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid).
  • the parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence.
  • the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, and preferably from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.
  • wild type or “WT” or “native” with respect to an amino acid sequence is meant an amino acid sequence that is found in nature, including allelic variations.
  • a wild type amino acid sequence, peptide, polypeptide or protein has an amino acid sequence that has not been intentionally modified.
  • wild type or “WT” or “native” with respect to a nucleic acid sequence is meant a nucleic acid sequence that is found in nature, including allelic variations.
  • a wild type coding sequence is meant to be a coding sequence that is found in nature and that has not been intentionally modified.
  • a "coding sequence”, as sued herein means the portion of a nucleic acid (e.g., a gene's DNA or RNA) that codes for protein.
  • G/C content or “G/C content” means the percentage of bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C).
  • the G/C content may be given for a specific portion of DNA or RNA or for an entire genome.
  • the G/C content refers to a portion, it may denote the G/C content of an individual gene or portion of a gene (domain), a group of genes or gene clusters, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.
  • variants of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • variant includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring.
  • variant includes, in particular, fragments of an amino acid sequence.
  • Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.
  • Amino acid addition variants comprise amino- and/or carboxy -terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein.
  • Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C- terminal truncation variants.
  • Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties.
  • amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • conservative amino acid substitutions include substitutions within the following groups:
  • the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence.
  • the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids.
  • the degree of similarity or identity is given for the entire length of the reference amino acid sequence.
  • the alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: meedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
  • Sequnce identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
  • % identical and % identity are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J.
  • NCBI National Center for Biotechnology Information
  • the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used.
  • the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides.
  • the degree of similarity or identity is given for the entire length of the reference sequence.
  • Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.
  • amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation.
  • the manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example.
  • the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
  • a fragment or variant of an amino acid sequence is preferably a "functional fragment” or “functional variant”.
  • the term "functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent.
  • one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived.
  • the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence.
  • the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
  • amino acid sequence "derived from” a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence.
  • amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof.
  • Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
  • the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
  • isolated means altered or removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”.
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • the RNA (such as mRNA) used in the present disclosure is in substantially purified form.
  • a solution (preferably an aqueous solution) of RNA (such as mRNA) in substantially purified form contains a first buffer system.
  • genetic modification or simply "modification” includes the transfection of cells with nucleic acid.
  • transfection relates to the introduction of nucleic acids, in particular RNA, into a cell.
  • the term “transfection” also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient.
  • a cell for transfection of a nucleic acid described herein can be present in vitro (e.g., in cell culture) or in vivo, e.g., the cell can form part of an organ, a tissue and/or an organism of a patient.
  • transfection can be transient or stable.
  • RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.
  • an analog of a peptide, polypeptide or protein is a modified form of said peptide, polypeptide or protein from which it has been derived and has at least one functional property of said peptide, polypeptide or protein.
  • a pharmacological active analog of a peptide, polypeptide or protein has at least one of the pharmacological activities of the peptide, polypeptide or protein from which the analog has been derived.
  • modifications include any chemical modification and comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the protein, polypeptide or peptide, such as carbohydrates, lipids and/or proteins or peptides.
  • analogs of proteins, polypeptides or peptides include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective/blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand.
  • the term “analog” also extends to all functional chemical equivalents of said proteins, polypeptides and peptides.
  • a nucleic acid such as RNA (e.g., mRNA) encoding a peptide, polypeptide or protein is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide, polypeptide or protein.
  • the cell may express the encoded peptide, polypeptide or protein intracellularly (e.g. in the cytoplasm and/or in the nucleus), may secrete the encoded peptide, polypeptide or protein, and/or may express it on the surface.
  • nucleic acid expressing and “nucleic acid encoding” or similar terms are used interchangeably herein and with respect to a particular peptide, polypeptide or protein mean that the nucleic acid, if present in the appropriate environment, preferably within a cell, can be expressed to produce said peptide, polypeptide or protein.
  • Activation refers to the state of a cell (e.g., an immune effector cell such as T cell) that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions.
  • activated immune effector cells refers to, among other things, immune effector cells that are undergoing cell division.
  • the term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.
  • clonal expansion refers to a process wherein a specific entity is multiplied.
  • the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified.
  • expansion leads to differentiation of the immune effector cells.
  • an “antigen” covers any substance that will elicit an immune response and/or any substance against which an immune response or an immune mechanism such as a cellular response and/or humoral response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules.
  • an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T-cells).
  • the term "antigen" may comprise any molecule which comprises at least one epitope, such as a T cell epitope.
  • an antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, which may be specific for the antigen (including cells expressing the antigen).
  • an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.
  • any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may be a humoral or cellular immune response or both.
  • the antigen is presented by a cell, preferably by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen.
  • An antigen may be a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen.
  • an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.
  • disease-associated antigen is used in its broadest sense to refer to any antigen associated with a disease.
  • a disease-associated antigen is a molecule which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and/or a humoral antibody response against the disease.
  • Disease-associated antigens include pathogen-associated antigens, i.e., antigens which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.
  • the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells.
  • the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein.
  • the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, preferably CD4+ and CD8+ lymphocytes, in particular via the modulation of the activity of a T-cell receptor.
  • tumor antigen refers to a constituent of cancer cells which may be derived from the cytoplasm, the cell surface or the cell nucleus. In particular, it refers to those antigens which are produced intracellularly or as surface antigens on tumor cells.
  • tumor antigens include the carcinoembryonal antigen, al -fetoprotein, isoferritin, and fetal sulphoglycoprotein, a2-H-ferroprotein and y-fetoprotein, as well as various virus tumor antigens.
  • a tumor antigen comprises any antigen which is characteristic for tumors or cancers as well as for tumor or cancer cells with respect to type and/or expression level.
  • viral antigen refers to any viral component having antigenic properties, i.e., being able to provoke an immune response in an individual.
  • the viral antigen may be a viral ribonucleoprotein or an envelope protein.
  • bacterial antigen refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the bacterial antigen may be derived from the cell wall or cytoplasm membrane of the bacterium.
  • epitope refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies T cells or B cells, in particular when presented in the context of MHC molecules.
  • An epitope of a protein may comprise a continuous or discontinuous portion of said protein and, e.g., may be between about 5 and about 100, between about 5 and about 50, between about 8 and about 0, between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the epitope in the context of the present disclosure is a T cell epitope.
  • Terms such as “epitope”, “fragment of an antigen”, “immunogenic peptide” and “antigen peptide” are used interchangeably herein and, e.g., may relate to an incomplete representation of an antigen which is, e.g., capable of eliciting an immune response against the antigen or a cell expressing or comprising and presenting the antigen.
  • the terms relate to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules.
  • epitope refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system.
  • the epitope may be recognized by T cells, B cells or antibodies.
  • An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is between about 10 and about 25 amino acids in length.
  • epitope includes T cell epitopes.
  • T cell epitope refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules.
  • major histocompatibility complex and the abbreviation "MHC” includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells.
  • the proteins encoded by the MHC are expressed on the surface of cells, and display both selfantigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell.
  • the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective.
  • the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.
  • the peptide and protein antigen can be 2 to 100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.
  • the peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein.
  • vaccine antigen i.e., an antigen whose inoculation into a subject induces an immune response
  • the vaccine antigen is recognized by an immune effector cell.
  • the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen.
  • the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell.
  • an antigen is expressed in a diseased cell (such as tumor cell or an infected cell).
  • an antigen is presented by a diseased cell (such as tumor cell or an infected cell).
  • an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC.
  • binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells.
  • binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
  • an antigen is expressed on the surface of a diseased cell (such as tumor cell or an infected cell).
  • an antigen receptor is a CAR which binds to an extracellular domain or to an epitope in an extracellular domain of an antigen.
  • a CAR binds to native epitopes of an antigen present on the surface of living cells.
  • binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on cells results in stimulation, priming and/or expansion of said T cells.
  • binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
  • an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In some embodiments, an antibody or B cell receptor binds to native epitopes of an antigen.
  • the term "expressed on the cell surface” or "associated with the cell surface” means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell.
  • a part may be, e.g., at least 4, at least 8, at least 12, or at least 20 amino acids.
  • the association may be direct or indirect.
  • the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell.
  • a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.
  • Cell surface or “surface of a cell” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules.
  • An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by, e.g., antigen-specific antibodies added to the cells.
  • an antigen expressed on the surface of cells is an integral membrane protein having an extracellular portion which may be recognized by a CAR.
  • extracellular portion or “exodomain” in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell.
  • the term refers to one or more extracellular loops or domains or a fragment thereof.
  • T cell and "T lymphocyte” are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells.
  • T helper cells CD4+ T cells
  • CTLs cytotoxic T cells
  • antigen-specific T cell or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted, in particular when presented on the surface of antigen presenting cells or diseased cells such as cancer cells in the context of MHC molecules and preferably exerts effector functions of T cells.
  • T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen.
  • T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be measured.
  • the RNA in particular mRNA
  • target shall mean an agent such as a cell or tissue which is a target for an immune response such as a cellular immune response.
  • Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen.
  • the target cell is a cell expressing an antigen and preferably presenting said antigen with class I MHC.
  • Antigen processing refers to the degradation of an antigen into processing products which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells to specific T-cells.
  • Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells.
  • the term "professional antigen presenting cells” relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell.
  • Professional antigen presenting cells comprise dendritic cells and macrophages.
  • non-professional antigen presenting cells relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma.
  • exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.
  • dendritic cell refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells.
  • dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node.
  • Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In some embodiments, the dendritic cells are splenic dendritic cells.
  • macrophage refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are splenic macrophages.
  • antigen-responsive CTL is meant a CD8 + T-cell that is responsive to an antigen or a peptide derived from said antigen, which is presented with class I MHC on the surface of antigen presenting cells.
  • CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-y and TNF-a, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells.
  • CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.
  • immune response and “immune reaction” are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and may refer to a cellular immune response, a humoral immune response, or both.
  • the term “immune response to” or “immune response against” with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent.
  • An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, such as CD4 + and CD8 + T-lymphocytes, e.g., CD8 + T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.
  • T-lymphocytes such as CD4 + and CD8 + T-lymphocytes, e.g., CD8 + T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.
  • the terms "inducing an immune response” and “eliciting an immune response” and similar terms in the context of the present disclosure refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both.
  • the immune response may be protective/preventive/prophylactic and/or therapeutic.
  • the immune response may be directed against any immunogen or antigen or antigen peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV or RSV)).
  • inducing in this context may mean that there was no immune response against a particular antigen or pathogen before induction, but it may also mean that there was a certain level of immune response against a particular antigen or pathogen before induction and after induction said immune response is enhanced.
  • inducing the immune response in this context also includes “enhancing the immune response”.
  • after inducing an immune response in an individual said individual is protected from developing a disease such as an infectious disease or a cancerous disease or the disease condition is ameliorated by inducing an immune response.
  • cellular immune response means to include a cellular response directed to cells characterized by expression of an antigen and/or presentation of an antigen with class I or class II MHC.
  • the cellular response relates to cells called T cells or T lymphocytes which act as either "helpers” or “killers".
  • the helper T cells also termed CD4 + T cells
  • the killer cells also termed cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs kill cells such as diseased cells.
  • the term "humoral immune response” refers to a process in living organisms wherein antibodies are produced in response to agents and organisms, which they ultimately neutralize and/or eliminate.
  • the specificity of the antibody response is mediated by T and/or B cells through membrane-associated receptors that bind antigen of a single specificity.
  • B lymphocytes divide, which produces memory B cells as well as antibody secreting plasma cell clones, each producing antibodies that recognize the identical antigenic epitope as was recognized by its antigen receptor.
  • Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the resulting escalation in antibody response when re-exposed to a specific antigen.
  • antibody refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which is able to specifically bind to an epitope on an antigen under typical physiological conditions, preferably with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity).
  • the term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • the term “antibody” includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing.
  • Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • the variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the CDRs of a VH are termed HCDR1, HCDR2 and HCDR3, the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein.
  • immunoglobulin relates to proteins of the immunoglobulin superfamily, such as to antigen receptors such as antibodies or the B cell receptor (BCR).
  • the immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold.
  • the term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins.
  • Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies.
  • Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds.
  • immunoglobulin domains such as the VL (variable light chain) domain, CL (constant light chain) domain, VH (variable heavy chain) domain, and the CH (constant heavy chain) domains CHI, CH2, CH3, and CH4.
  • immunoglobulin heavy chains There are five types of mammalian immunoglobulin heavy chains, i.e., a, 8, a, y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM.
  • the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus.
  • the "polydispersity index” is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.
  • one procedure to determine the hydrodynamic radius of a particle or a population of particles is to measure the DLS signal of said particle or population of particles (such as DLS signal of particles such as LNPs contained in a formulation or composition as disclosed herein or the DLS signal of a particle peak obtained from subjecting such a formulation or composition to field-flow fractionation).
  • aggregate as used herein relates to a cluster of particles, wherein the particles are identical or very similar and adhere to each other in a non-covalently manner (e.g., via ionic interactions, H bridge interactions, dipole interactions, and/or van der Waals interactions).
  • light scattering refers to the physical process where light is forced to deviate from a straight trajectory by one or more paths due to localized non-uniformities in the medium through which the light passes.
  • UV means ultraviolet and designates a band of the electromagnetic spectrum with a wavelength from 10 nm to 400 nm, i.e., shorter than that of visible light but longer than X-rays.
  • multi-angle light scattering or “MALS” as used herein relates to a technique for measuring the light scattered by a sample into a plurality of angles.
  • Multi -angle means in this respect that scattered light can be detected at different discrete angles as measured, for example, by a single detector moved over a range including the specific angles selected or an array of detectors fixed at specific angular locations.
  • the light source used in MALS is a laser source (MALLS: multi -angle laser light scattering).
  • the Zimm plot is a graphical presentation using the following equation (or the reciprocal thereof): wherein c is the mass concentration of the particles in the solvent (g/mL); A ?
  • K* is an optical constant that is equal to 4TI 2 T
  • the Berry plot is calculated the following term or the reciprocal thereof: wherein c, Re and K* are as defined above.
  • the Debye plot is calculated the following term or the reciprocal thereof: wherein c, Re and K* are as defined above.
  • SLS static light scattering
  • a high-intensity monochromatic light usually a laser, is launched in a solution containing the particles.
  • One or many detectors are used to measure the scattering intensity at one or many angles. The angular dependence is needed to obtain accurate measurements of both molar mass and size for all macromolecules of radius.
  • simultaneous measurements at several angles relative to the direction of incident light known as multi-angle light scattering (MALS) or multiangle laser light scattering (MALLS) is generally regarded as the standard implementation of static light scattering.
  • MALS multi-angle light scattering
  • MALLS multiangle laser light scattering
  • Immunogenicity is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal.
  • the innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term "repeating unit” relates to an elementary unit which periodically repeats itself along the polymeric chain of a polymer and which is derived from one monomer. Although the structures of the repeating unit and its corresponding monomer are often coincident, they may differ from each other.
  • the term "functional moiety” as used herein relates to a group of atoms in a molecule with distinctive chemical properties, wherein the atoms of the functional moiety are linked to each other and to the rest of the molecule by covalent bonds.
  • the atoms of the functional moiety comprise at least one atom selected from the group consisting of O, N, and S.
  • Functional moieties may be monovalent (such as hydroxy, cyano, nitro, or amide (e.g., -C(0)NHCH3)) or divalent (such as amide (e.g., -C(O)NH-), carbonyl (-C(O)-), or ester (e.g., -OC(O)-).
  • the functional moiety provides hydrophilicity to the group to which the functional moiety is bound, e.g., by providing at least one hydrogen bond acceptor/donor and/or at least one charge (positive or negative) to the group to which the functional moiety is bound.
  • the functional moiety comprises a hydrogen bond acceptor (such as a carbonyl moiety), a hydrogen bond donor (such as a hydroxyl moiety, -NH- (of, e.g., an amide moiety), or thiol moiety) or both (e.g., an amide moiety), and/or is charged (e.g., phosphate, amino, or ammonium moiety).
  • a hydrogen bond acceptor such as a carbonyl moiety
  • a hydrogen bond donor such as a hydroxyl moiety, -NH- (of, e.g., an amide moiety), or thiol moiety) or both (e.g., an amide moiety)
  • is charged e.g., phosphate, amino, or ammonium moiety.
  • Examples of monovalent functional moieties include hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbono
  • divalent functional moieties include ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine,
  • hydroxyl or "hydroxy" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -OH.
  • halogen as used herein with respect to a functional moiety, in particular as component of a linker, means fluoro, choloro, bromo, or iodo.
  • cyano as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -CN.
  • zido as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group N3.
  • nitro as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -NO2.
  • each of R 72 and R 73 is independently H or a hydrocarbyl group, such as selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl), wherein each of the hydrocarbyl groups (such as each of the C1-6 alkyl, aryl, and aryl(C1-6 alkyl) groups) is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the hydrocarbyl group (such as C1-6 alkyl, aryl, or aryl(C1-6 alkyl) group)), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 .
  • a hydrocarbyl group such as selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl)
  • each of the hydrocarbyl groups such as each of the C1-6 alkyl, ary
  • ammonium as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -N + (R 72 )2(R 73 ), wherein R 72 and R 73 are as defined for the term "amino".
  • thiol or "sulfanyl” as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -SH.
  • diisulfanyl as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -SSH.
  • amide or "amido” as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)NH- (including its isomerically arranged structure -NHC(O)-, unless it is specified to the contrary).
  • each of both ends of the amide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (if both ends are linked to the same organic group the amide moiety is also referred to as lactam).
  • An amide group may be monovalent (e.g., -C(O)NRR or -NRC(O)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)NR- or -NRC(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • ester as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)O- (including its isomerically arranged structure -OC(O)-, unless it is specified to the contrary).
  • each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (if both ends are linked to the same organic group the ester moiety is also referred to as lactone).
  • An ester group may be monovalent (e.g., -C(O)OR or -OC(O)R, wherein R is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino”) or divalent (e.g., -C(O)O- or -OC(O)-)).
  • R is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino”
  • divalent e.g., -C(O)O- or -OC(O)-
  • ether as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -O-, wherein each of both ends of the ether structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • An ether group may be monovalent (e.g., -OR, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -O-).
  • sulfide or "thioether” as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S-, wherein each of both ends of the sulfide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • a sulfide group may be monovalent (e.g., -SR, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -S-).
  • disulfide as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SS-, wherein each of both ends of the disulfide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • a disulfide group may be monovalent (e.g., -SSR, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -SS-).
  • diselenide as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SeSe-, wherein each of both ends of the diselenide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • a diselenide group may be monovalent (e.g., -SeSeR, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -SeSe-).
  • sulfoxide as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the sulfinyl structure -S(O)-, wherein each of both ends of the sulfoxide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • a sulfoxide group may be monovalent (e.g., -S(O)R, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (as, e.g., -S(O)-).
  • sulfone as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the sulfonyl structure -S(O)2-, wherein each of both ends of the sulfone structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • a sulfone group may be monovalent (e.g., -S(O)2R, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (as, e.g., -S(O)2-).
  • a sulfamate group may be monovalent (e.g., -OS(O)2N(R)2 or -N(R)S(0)20R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -0S(0)2N(R)- or -N(R)S(0)20-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • An urea group may be monovalent (e.g., -N(R)C(0)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(O)N(R)-, wherein each R is H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • thiourea as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(S)N(R)-, wherein one of both ends of the thiourea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • an organic group e.g., an alkylene group as further component of the linker
  • a thiourea group may be monovalent (e.g., -N(R)C(S)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(S)N(R)-, wherein each R is H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • carbonyl as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)-, wherein one of both ends of the carbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (if both ends are linked to C atoms of organic groups the carbonyl moiety is also referred to as "keto" moiety).
  • an organic group e.g., an alkylene group as further component of the linker
  • a carbonyl group may be monovalent (e.g., -C(O)R, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)-).
  • thiocarbonyl as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(S)-, wherein one of both ends of the thiocarbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group.
  • an organic group e.g., an alkylene group as further component of the linker
  • a thiocarbonyl group may be monovalent (e.g., -C(S)R, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -C(S)-).
  • orthoester as used herein with respect to a functional moiety, in particular as component of a linker, relates to a moiety comprising a C atom to which three alkoxy groups (i.e., -OR, wherein R is an organic group (e.g., an alkylene group as further component of the linker), such one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino" are attached.
  • An exemplary formula of an orthoester comprises the structure (-0) r C(0R)3- r -, wherein each R is independently an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; r is 1 or 2; and each of both ends of the orthoester structure is covalently linked to a C atom of a further organic group or of two further separate organic groups.
  • an orthoester comprises the structure (-O) r C(OR 25 )3 r-, wherein each R 25 is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl) which is optionally substituted (e.g., with one or more 1 st level substituents, 2 nd level substituents, or 3 rd level substituents as defined herein); r is 1 or 2; and each of both ends of the orthoester structure is covalently linked to a C atom of a further organic group or of two further separate organic groups.
  • An orthoester group may be monovalent (e.g., -C(0R)3 or -0C(0R)2R, wherein each R is an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., (-0)2C(0R)(R) or -OC(OR)2-, wherein each R is an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • thioate or "thioester” as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(O)- (including its isomerically arranged structures -C(O)S-, _-OC(S), and -C(S)O-, unless it is specified to the contrary), wherein each of both ends of the thioate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • a thioate group may be monovalent (e.g., -SC(O)R or -C(O)SR or -OC(S)R or -C(S)OR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)- or -C(O)S- or -OC(S)- or -C(S)O-).
  • a dithioate group may be monovalent (e.g., -SC(S)R or -C(S)SR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)- or -C(S)S-).
  • an organic group e.g., an alkylene group as further component of the linker
  • an organic group e.g., an alkylene group as further component of the linker
  • each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"
  • thionylamino as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(S)NR- (including its isomerically arranged structure -N(R)C(S)-, unless it is specified to the contrary), wherein one of both ends of the thionylamino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • an organic group e.g., an alkylene group as further component of the linker
  • each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amin
  • a thionylamino group may be monovalent (e.g., -C(S)NRR or -N(R)C(S)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -C(S)NR- or -N(R)C(S)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • carbonothioate as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(S)O- or -OC(O)S- (including its isomerically arranged structure -SC(O)O-, unless it is specified to the contrary), wherein each of both ends of the carbonothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker).
  • an organic group e.g., an alkylene group as further component of the linker
  • a carbonothioate group may be monovalent (e.g., -OC(S)OR’ or -OC(O)SR’ or -SC(O)OR’, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(S)O- or -OC(O)S- or -SC(O)O-).
  • a carbonodithioate group may be monovalent (e.g., -SC(O)SR’ -OC(S)SR’ or -SC(S)OR’, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)S- or -OC(S)S- or -SC(S)O-).
  • carbonotrithioate as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)S-, wherein each of both ends of the carbonotrithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker).
  • a carbonotrithioate group may be monovalent (e.g., -SC(S)SR, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)S-).
  • an organic group e.g., an alkylene group as further component of the linker
  • an organic group e.g., an alkylene group as further component of the linker
  • a carbamate group may be monovalent (e.g., -OC(O)NRR or -N(R)C(O)OR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(O)NR- or -N(R)C(O)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • carbamodithioate as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)NR- (including its isomerically arranged structure -N(R)C(S)S-, unless it is specified to the contrary), wherein the S end of the carbamodithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • an organic group e.g., an alkylene group as further component of the linker
  • N end is covalently linked to H or to a C atom of the same or another organic group
  • each R is independently H or
  • an organic group e.g., an alkylene group as further component of the linker
  • N end is covalently linked to H or to a C atom of the same or another organic group
  • each R is independently
  • carbamothioate as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including their isomerically arranged structures -SC(O)NR- or -OC(S)NR-, unless it is specified to the contrary), wherein the O/S end of the carbamothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • an organic group e.g., an alkylene group as further component of the linker
  • N end is covalently linked to a C
  • a carbamothioate group may be monovalent (e.g., -N(R)C(O)SR’ or -N(R)C(S)OR’ or -SC(O)NRR or -OC(S)NRR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(O)S- or -N(R)C(S)O- or -SC(O)NR- or -OC(S)NR-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • each 'ww represents the bond by which the acylhydrazone is covalently linked to the further organic group(s) (e.g., an alkylene group as further component of the linker).
  • hydrazine as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and each of both ends of the hydrazine structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker).
  • a hydrazine comprises the structure -N(R 26 )N(R 26 )-, wherein each R 26 is independently H or a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl), which is optionally substituted (e.g., with one or more 1 st level substituents, 2 nd level substituents, or 3 rd level substituents as defined herein); and each of both ends of the hydrazine structure is covalently linked to a C atom of a further organic group or of two further separate organic groups.
  • a hydrazine group may be monovalent (e.g., -N(R)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)N(R)-, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • An exemplary chemical formula of an oxime is shown below: wherein each AAAAV represents the bond by which the oxime is covalently linked to the further organic group(s).
  • acetal as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCH(R’)O-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and each of both O atoms of the acetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker).
  • an acetal comprises the structure -OCH(R 25 )O-, wherein R 25 is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1 st level substituents, 2 nd level substituents, or 3 rd level substituents as defined herein); and each of both O atoms of the acetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups.
  • R 25 is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)
  • An acetal group may be monovalent (e.g., -OCH(R’)OR’, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -OCH(R’)O-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • hemiacetal as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCH(OH)-, wherein each of both ends of the hemiacetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker).
  • a hemiacetal group may be monovalent (e.g., -OCH(OH)OR’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino”) or divalent (e.g., -OCH(OH)-).
  • ketal as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(R’)(R’)O-, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and each of both O atoms of the ketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker).
  • a ketal comprises the structure -OC(R 25 )(R 25 )O -, wherein each R 25 is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1 st level substituents, 2 nd level substituents, or 3 rd level substituents as defined herein); and each of both O atoms of the ketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups.
  • An ketal group may be monovalent (e.g., -OC(R’)(R’)OR’, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(R’)(R’)O-, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • hemiketal as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCR’ (OH)-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and each of both ends of the hemiketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker).
  • a hemiketal comprises the structure -OCR 25 (OH)-, wherein R 25 is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1 st level substituents, 2 nd level substituents, or 3 rd level substituents as defined herein); and each of both ends of the hemiketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups.
  • a hemiketal group may be monovalent (e.g., -0C(R’)2(0H), wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -OCR’(OH)-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • imide as used herein with respect to a functional moiety, in particular within a as component of, relates to a group comprising the structure -C(O)N(R)C(O)-, wherein R is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and each of both ends of the imide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • An imide group may be monovalent (e.g., -C(O)N(R)C(O)R’, wherein R is independently H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)N(R)C(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R 72 indicated above in the definition of the term "amino").
  • non-cyclic as used herein in the context of organic groups relates to open-chain organic groups which contain no rings.
  • Open-chain or acyclic organic groups may be straight (i.e., they contain only one unbranched chain without any sidechain) or branched (i.e., the main chain comprises one or more sidechains).
  • An organic group which is "substituted with one or more substituents” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the organic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the organic group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the one or more substituents may be selected from the 1 st level substituents, 2 nd level substituents, or 3 rd level substituents described herein.
  • hydrogen bond or "H-bond” as used herein means a non-covalent bond (in some embodiments a primarily electrostatic force of attraction) between (i) a hydrogen atom which is covalently bound to a more electronegative atom or group, and (ii) a lone pair of electrons of another electronegative atom.
  • the more electronegative atom or group includes nitrogen atoms and oxygen atoms; thus, examples of groups in which a hydrogen atom is covalently bound to a more electronegative atom or group include amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (as such (as in respective thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) groups))).
  • groups in which a hydrogen atom is covalently bound to a more electronegative atom or group include amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional
  • the lone pair of electrons of another electronegative atom is a lone pair of an oxygen atom present in a carbonyl group or a lone pair of a nitrogen atom present in a primary, secondary or tertiary amino group.
  • hydrogen bond donor means an atom, ion, or a molecule component of a hydrogen bond which supplies the bridging (shared) hydrogen atom.
  • a hydrogen bond donor includes amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (as such (as in respective thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) groups))).
  • hydrogen bond acceptor means an atom, ion, or a molecule component of a hydrogen bond which does not supply the bridging (shared) hydrogen atom.
  • a hydrogen bond acceptor comprises at least one lone pair of electrons.
  • hydrogen bond acceptors include carbonyl moieties and primary, secondary and tertiary amino groups.
  • stealth is used herein to describe the ability of the particles described herein not to be detected and then sequestered and/or degraded, or to be hardly detected and then sequestered and/or degraded, and/or to be detected and then sequestered and/or degraded late, by the immune system of the host to which they are administered.
  • phosphatidylethanolamine means diacylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight).
  • each acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms.
  • the acyl moiety may be saturated or unsaturated (such as monounsaturated).
  • both acyl moieties may be saturated or unsaturated (such as monounsaturated).
  • one acyl moiety is saturated and the other is unsaturated (such as monounsaturated).
  • phosphatidylethanolamine moiety means a monovalent radical of phosphatidylethanolamine, preferably that in which a hydrogen atom of the amino group has been removed.
  • DSPE distearoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(O)Ci7H 3 5 refers to the moiety -C(O)(CH 2 )igCH 3 (stearoyl).
  • disearoylphosphatidylethanolamine moiety means a monovalent radical of DSPE, preferably that in which a hydrogen atom of the amino group has been removed.
  • DPPE dipalmitoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(0)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl).
  • dimitoylphosphatidylethanolamine moiety means a monovalent radical of DPPE, preferably that in which a hydrogen atom of the amino group has been removed.
  • dioleoylphosphatidylethanolamine moiety means a monovalent radical of DOPE, preferably that in which a hydrogen atom of the amino group has been removed.
  • the term "palmitoyloleoylphosphatidylethanolamine moiety” means a monovalent radical of POPE, preferably that in which a hydrogen atom of the amino group has been removed.
  • tocopherol means a group of four compounds (i.e., a-tocopherol, P-tocopherol, y-tocopherol, and 5-tocopherol) having the following formula: wherein each of Ru and R2 is independently H or methyl. In a-tocopherol, Ru and Rt2 are both methyl; in P-tocopherol, Ri is methyl, and Rt2 is H; in y-tocopherol, Ru is H, and Rt2 is methyl; and in 5- tocopherol, Rti and Rt2 are both H.
  • the term "tocopherol moiety” or "tocopheryl moiety” means a monovalent radical of tocopherol, preferably that in which the hydrogen atom of the hydroxy group has been removed.
  • each acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms.
  • the acyl moiety may be saturated or unsaturated (such as monounsaturated).
  • both acyl moieties may be saturated or unsaturated (such as monounsaturated).
  • one acyl moiety is saturated and the other is unsaturated (such as monounsaturated).
  • DMG means 1,2-dimyristoylglycerol, i.e., a diacylglyceride of the above formula, wherein both acyl groups are -C(O)(CH2)i2CH 3 (myristoyl).
  • diacylglyceride moiety means a monovalent radical of diacylglyceride, preferably that in which a hydrogen atom of the hydroxy group has been removed.
  • each alkyl moiety preferably is straight. In some embodiments, each alkyl moiety has at least 8 carbon atoms. Preferably, each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl moieties include -(CFDnCFE (stearyl), -(CFDisCFE (palmityl), and -(CH2)I 3 CH 3 (myristyl).
  • DMA means 1,2-dimyristylamine, i.e., a dialkylamine of the above formula, wherein both alkyl groups are -(CH2)I 3 CH 3 (myristyl).
  • dialkylamine moiety means a monovalent radical of dialkylamine, preferably that in which the hydrogen atom of the amino group has been removed.
  • ceramide means acyl sphingosine having the following formula: or a salt thereof, wherein -C13H27 refers to the moiety -(CFDnCFE; and acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight).
  • the acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms.
  • the acyl moiety may be saturated or unsaturated (such as monounsaturated).
  • palmitoyl ceramide means a ceramide of the above formula, wherein acyl is -C(O)(CH2)i4CH3 (palmitoyl).
  • ceramide moiety means a monovalent radical of ceramide, preferably that in which the hydrogen atom of a hydroxy group (preferably the hydrogen of the terminal (primary) hydroxy group) has been removed.
  • MAA means monoalkylamine having the formula H2N(alkyl) or a salt thereof, wherein the alkyl moiety preferably is straight.
  • the alkyl moiety has at least 8 carbon atoms.
  • the alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably the alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms.
  • alkyl moieties include -(CIDnCFE (stearyl), -(CIDisCFE (palmityl), and -(CTDBCTE (myristyl).
  • MMA means myristylamine, i.e., a monoalkylamine of the above formula, wherein the alkyl group is - (CH 2 )i3CH 3 (myristyl).
  • monoalkylamine moiety means a monovalent radical of monoalkylamine, preferably that in which one of the hydrogen atoms of the amino group has been removed.
  • nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof.
  • the term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
  • a nucleic acid can be isolated.
  • isolated nucleic acid means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • RNA polymerase RNA polymerase
  • nucleoside (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.
  • the five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine.
  • the five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively.
  • thymidine is more commonly written as “dT” ("d” represents “deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA).
  • uridine is found in RNA and not DNA.
  • the remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.
  • the nucleic acid is DNA.
  • DNA relates to a nucleic acid molecule which includes deoxyribonucleotide residues.
  • the DNA contains all or a majority of deoxyribonucleotide residues.
  • deoxyribonucleotide refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group.
  • DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA.
  • a molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the nucleic acid is RNA.
  • RNA means a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group.
  • RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides.
  • altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered/modified nucleotides or nucleosides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs.
  • a molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule .
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • RNA includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA).
  • RNA refers to mRNA.
  • the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein.
  • Said RNA may express the encoded peptide, polypeptide, or protein.
  • said RNA may be RNA encoding and expressing a pharmaceutically active peptide or protein.
  • RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein.
  • a cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface.
  • the RNA can be non-coding RNA such as antisense -RNA, micro RNA (miRNA) or siRNA.
  • the nucleic acid is mRNA.
  • mRNA means "messenger-RNA” and includes a “transcript” which may be generated by using a DNA template.
  • mRNA encodes a peptide, polypeptide or protein.
  • an mRNA comprises a 5'-UTR, a peptide/protein coding region, and a 3'-UTR.
  • mRNA is preferably generated by in vitro transcription (IVT) from a DNA template.
  • IVTT in vitro transcription
  • the in vitro transcription methodology is known to the skilled person, and a variety of in vitro transcription kits is commercially available.
  • mRNA is single -stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.
  • dsRNA means double-stranded RNA and is RNA with two partially or completely complementary strands.
  • the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.
  • the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.
  • nucleotides such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000
  • the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide/polypeptide/protein coding region and a 3' untranslated region (3'-UTR).
  • the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis.
  • the RNA (such as mRNA) is produced by in vitro transcription using a DNA template.
  • the in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.
  • in vitro transcription kits are commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribeTM T7 kit, HiScribeTM T7 ARCA mRNA kit), Promega (such as RiboMAXTM, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribeTM).
  • Thermo Fisher Scientific such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc.
  • HiScribeTM T7 kit such as HiScribeTM T7 kit, HiScribeTM T7 ARCA mRNA kit
  • Promega such as RiboMAXTM, HeLaScribe®, Riboprobe® systems
  • Jena Bioscience such as SP6 or T
  • modified RNA such as mRNA
  • correspondingly modified nucleotides such as modified naturally occurring nucleotides, non- naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and/or added to the mRNA after transcription.
  • RNA such as mRNA
  • IVT-RNA in vitro transcribed RNA
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • the in vitro transcription is controlled by a T7 or SP6 promoter.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the RNA (such as mRNA) is “replicon RNA” (such as “replicon mRNA”) or simply a “replicon”, in particular “self-replicating RNA” (such as “self-replicating mRNA”) or “self-amplifying RNA” (or “self-amplifying mRNA”).
  • the replicon or self-replicating RNA (such as self-replicating mRNA) is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856).
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5 ’-cap, and a 3’ poly(A) tail.
  • the genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non-structural proteins are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome.
  • first ORF is larger than the second ORF, the ratio being roughly 2: 1.
  • RNA eukaryotic messenger RNA
  • (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non- structural poly-protein (nsP1234).
  • Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • the RNA (such as mRNA) described herein (e.g., contained in the compositions of the present disclosure and/or used in the methods of the present disclosure) contains one or more modifications, e.g., in order to increase its stability and/or increase translation efficiency and/or decrease immunogenicity and/or decrease cytotoxicity.
  • the RNA in order to increase expression of the RNA (such as mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein.
  • modifications are described, for example, in WO 2007/036366 and PCT/EP2019/056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and/or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the G/C content of the RNA).
  • the term "modification" in the context of modified mRNA according to the present disclosure preferably relates to any modification of an mRNA which is not naturally present in said RNA (such as mRNA).
  • the RNA (such as mRNA) described herein comprises a 5'-cap structure. In some embodiments, the mRNA does not have uncapped 5 '-triphosphates. In some embodiments, the RNA (such as mRNA) described herein may comprise a conventional 5'-cap and/or a 5'-cap analog.
  • inventional 5'-cap refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine 5 '-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'- end of the next nucleotide of the mRNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the mRNA).
  • Gppp guanosine 5 '-triphosphate
  • the guanosine may be methylated at position N 7 (resulting in the cap structure m 7 Gppp).
  • 5'-cap analog refers to a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3 '-position of the m 7 guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called antireverse cap analogs (ARCAs)).
  • ARCAs antireverse cap analogs
  • Particularly preferred 5 '-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the P-phosphate (such as m2 7 ’ 2 O G(5')ppSp(5')G (referred to as beta-S-ARCA or P-S-ARCA)), as described in PCT/EP2019/056502.
  • RNA such as mRNA
  • a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5 '-cap compound, wherein said 5 '-cap structure is co-transcriptionally incorporated into the generated RNA (such as mRNA) strand, or the RNA (such as mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the mRNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
  • capping enzymes for example, capping enzymes of vaccinia virus.
  • the RNA (such as mRNA) comprises a 5 '-cap structure selected from the group consisting of m2 7 ’ 2 O G(5’)ppSp(5')G (in particular its DI diastereomer), m2 7 ’ 3 O G(5')ppp(5')G, and m2 7 ’ 3 O Gppp(mi 2 O )ApG.
  • RNA encoding a peptide, polypeptide or protein comprising an antigen or epitope comprises m2 7 ’ 2 O G(5’)ppSp(5')G (in particular its DI diastereomer) as 5'-cap structure.
  • the RNA (such as mRNA) comprises a capO, capl, or cap2, preferably capl or cap2.
  • capO means the structure "m 7 GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'.
  • capl means the structure "m 7 GpppNm”, wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'.
  • cap2 means the structure "m 7 GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.
  • the 5'-cap analog beta-S-ARCA (P-S-ARCA) has the following structure:
  • the "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)” is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S- ARCA(D2)) and thus exhibits a shorter retention time.
  • the HPLC preferably is an analytical HPLC.
  • a Supelcosil LC-18-T RP column preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml/min can be applied.
  • VWD UV-detection
  • FLD fluorescence detection
  • the 5 '-cap analog m2 7 ’ 3 O Gppp(mi 2 O )ApG (also referred to as m2 7 ’ 3 O G(5')ppp(5')m 2 O ApG) which is a building block of a capl has the following structure:
  • An exemplary capO mRNA comprising P-S-ARCA and mRNA has the following structure:
  • An exemplary capO mRNA comprising m2 7 ’ 3 °G(5')ppp(5')G and mRNA has the following structure:
  • An exemplary capl mRNA comprising m2 7 ’ 3 O Gppp(mi 2 O )ApG and mRNA has the following structure:
  • the RNA (such as mRNA) comprises a 3’-poly(A) sequence.
  • poly-A tail or "poly-A sequence” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA (such as mRNA) molecule.
  • Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs (such as mRNAs) described herein.
  • An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical.
  • RNAs such as mRNAs
  • RNAs can have a poly-A tail attached to the free 3 '-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template -dependent RNA polymerase.
  • poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of mRNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).
  • the poly-A tail may be of any length.
  • a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly-A tail typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • consists of means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides.
  • a nucleotide or “A” refers to adenylate.
  • a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly-A tail (coding strand) is referred to as poly (A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed.
  • the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • no nucleotides other than A nucleotides flank a poly-A tail at its 3 '-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.
  • a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly- A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides.
  • the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides. In some embodiments, the poly-A tail comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the poly-A sequence has a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3.
  • the term "untranslated region" or “UTR” relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5 '-UTR) and/or 3' (downstream) of an open reading frame (3 -UTR).
  • a 5 '-UTR if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap.
  • a 3'-UTR if present, is located at the 3 '-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does preferably not include the poly-A sequence.
  • the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence.
  • Incorporation of a 3 '-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency.
  • a synergistic effect may be achieved by incorporating two or more of such 3'- UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)).
  • the 3'-UTRs may be autologous or heterologous to the RNA (preferably mRNA) into which they are introduced.
  • the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2 -globin, alpha 1 -globin, or beta-globin, preferably betaglobin, more preferably human beta-globin.
  • the RNA (preferably mRNA) may be modified by the replacement of the existing 3 '-UTR with or the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2 -globin, alpha 1 -globin, beta-globin, preferably beta-globin, more preferably human beta-globin.
  • a globin gene such as alpha2 -globin, alpha 1 -globin, beta-globin, preferably beta-globin, more preferably human beta-globin.
  • the RNA (such as mRNA) used in present disclosure comprises a 5 ’-UTR comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.
  • the RNA (such as mRNA) used in present disclosure comprises a 3 ’-UTR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.
  • RNA such as mRNA
  • the RNA (such as mRNA) described herein may have modified ribonucleotides in order to increase its stability and/or decrease immunogenicity and/or decrease cytotoxicity.
  • uridine in the RNA (such as mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside.
  • the modified nucleoside is a modified uridine.
  • the modified uridine replacing uridine is selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml ⁇
  • the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5 -methoxy-uridine (mo5U), 5 -aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5 -bromo-uridine),
  • 4-thio-l- methyl-pseudouridine 3 -methyl -pseudouridine (m3i
  • 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl- uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl- uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5- (2 -carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.
  • RNA preferably mRNA which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "T-modificd”.
  • ml'P-modified means that the RNA (preferably mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine).
  • m5U-modified means that the RNA (preferably mRNA) contains 5 -methyluridine (replacing partially or completely, preferably completely, uridine).
  • RNA preferably mRNA
  • N(l)-methylpseudouridine replacing completely uridine
  • the codons of the RNA (preferably mRNA) described in the present disclosure may further be optimized, e.g., to increase the G/C content of the RNA and/or to replace codons which are rare in the cell (or subject) in which the peptide or protein of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject).
  • the amino acid sequence encoded by the RNA described in the present disclosure is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence.
  • This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence.
  • the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.
  • coding regions are preferably codon-optimized for optimal expression in a subject to be treated using the RNA (preferably mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (preferably mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".
  • the guanosine/cytosine (G/C) content of the coding region of the RNA (preferably mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA (preferably mRNA) is preferably not modified compared to the amino acid sequence encoded by the wild type RNA.
  • This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA (preferably mRNA). Sequences having an increased G (guanosine )/C (cytosine) content are more stable than sequences having an increased A (adenosine )/U (uracil) content.
  • RNA preferably mRNA
  • codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.
  • the G/C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.
  • a combination of the above described modifications i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and/or 3'- UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5 -methylcytidine for cytidine and/or pseudouridine (T) or N(l)-methylpseudouridine (m I T) or 5 -methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency.
  • synthetic nucleotides e.g., 5 -methylcytidine for cytidine and/or pseudouridine (T) or N(l)-methylpseudouridine (m I T) or 5 -methyluridine (
  • the RNA (preferably mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure; (ii) incorporation of a poly- A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5 -methylcytidine for cytidine and/or pseudouridine (T) or N(l)- methylpseudouridine (m I T) or 5 -methyluridine (m5U) for uridine); and (v) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5 -methylcytidine for cytidine
  • the RNA (preferably mRNA) described in the present disclosure comprises a capl or cap2, preferably a capl structure.
  • the poly-A sequence comprises at least 100 nucleotides.
  • the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 3.
  • the a 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.
  • the 3’-UTR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.
  • the disclosure involves the targeted delivery of the RNA (preferably mRNA) disclosed herein to certain cells or tissues.
  • the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen.
  • Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is in particular preferred if the RNA (preferably mRNA) administered is RNA (preferably mRNA) encoding an antigen or epitope for inducing an immune response.
  • the target cell is a spleen cell.
  • the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen.
  • the target cell is a dendritic cell in the spleen.
  • the "lymphatic system” is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph.
  • the lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph.
  • the primary or central lymphoid organs generate lymphocytes from immature progenitor cells.
  • the thymus and the bone marrow constitute the primary lymphoid organs.
  • Secondary or peripheral lymphoid organs which include lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.
  • the target cell is a T cell.
  • Lipid-based RNA (such as mRNA) delivery systems have an inherent preference to the liver. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates).
  • the target organ is liver and the target tissue is liver tissue.
  • the delivery to such target tissue is preferred, in particular, if presence of mRNA or of the encoded peptide or protein in this organ or tissue is desired and/or if it is desired to express large amounts of the encoded peptide or protein and/or if systemic presence of the encoded peptide or protein, in particular in significant amounts, is desired or required.
  • the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell.
  • the RNA is RNA (preferably mRNA) encoding a peptide or protein and the RNA is translated by the target cell to produce the peptide or protein.
  • the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell in the tumor microenvironment.
  • the target cell is a blood cell. In some embodiments, the target cell is a cell in the lymph nodes. In some embodiments, the target cell is a cell in the lung In some embodiments, the target cell is a cell in the skin. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is aNK cell. In some embodiments, the target cell is a monocyte.
  • nucleic acid (such as RNA) particle (such as RNA LNP) compositions described herein may be used for delivering nucleic acid (such as RNA, preferably mRNA) to such target cell.
  • the present disclosure also relates to a method for delivering nucleic acid (such as RNA, preferably mRNA) to a target cell in a subject comprising the administration of the nucleic acid (such as RNA, preferably mRNA) compositions described herein to the subject.
  • the RNA is delivered to the cytosol of the target cell.
  • the RNA is RNA (preferably mRNA) encoding a peptide or protein and the RNA is translated by the target cell to produce the peptide or protein.
  • the nucleic acid is an inhibitory RNA.
  • inhibitory RNA means RNA which selectively hybridizes to and/or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and/or translation thereof.
  • Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA.
  • antisense RNA refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and/or translation of said mRNA.
  • An antisense transcript of a nucleic acid or of a part thereof may form a duplex with naturally occurring mRNA and thus prevent accumulation of or translation of the mRNA.
  • Another possibility is the use of ribozymes for inactivating a nucleic acid.
  • the antisense RNA may hybridize with an N-terminal or 5' upstream site such as a translation initiation site, transcription initiation site or promoter site. In some embodiments, the antisense RNA may hybridize with a 3 '-untranslated region or mRNA splicing site.
  • the size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.
  • the antisense RNA has a length of at least 2,700 nucleotides (such as at least 2,800, at least 2,900, at least 3,000, at least 3,100, at least 3,200, at least 3,300, at least 3,400, at least 3,500, at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900, at least 5,000 nucleotides).
  • 2,700 nucleotides such as at least 2,800, at least 2,900, at least 3,000, at least 3,100, at least 3,200, at least 3,300, at least 3,400, at least 3,500, at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700,
  • antisense RNA may be modified as required.
  • antisense RNA may be stabilized by one or more modifications having a stabilizing effect.
  • modifications include modified phosphodiester linkages (such as methylphosphonate, phosphorothioate, phosphorodithioate or phosphoramidate linkages instead of naturally occurring phosphodiester linkages) and 2'-substitutions (e.g., 2'-fluoro, 2'-O-alkyl (such as 2'-O-methyl, 2'-O-propyl, or 2'-O-pentyl) and 2'-O-allyl).
  • modified phosphodiester linkages such as methylphosphonate, phosphorothioate, phosphorodithioate or phosphoramidate linkages instead of naturally occurring phosphodiester linkages
  • 2'-substitutions e.g., 2'-fluoro, 2'-O-alkyl (such as 2'-O-methyl, 2'-O-propyl, or
  • phosphorothioate linkages are substituted partially for phosphodiester linkages.
  • the ribose moiety is substituted partially at the 2'-position with O-alkyl (such as 2'-O- methyl).
  • An antisense RNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence").
  • a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3'-direction) from the start codon.
  • the target sequence can, however, be located in the 5'- or 3 '-untranslated regions, or in the region nearby the start codon.
  • Antisense RNA can be obtained using a number of techniques known to those of skill in the art. For example, antisense RNA can be chemically synthesized or recombinantly produced using methods known in the art. Preferably, antisense RNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • small interfering RNA or "siRNA” as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs.
  • the sense and antisense strands of siRNAs can comprise two complementary, single-stranded RNA molecules
  • the siRNAs according to the present disclosure, comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. That is, the sense region and antisense region can be covalently connected via a linker molecule.
  • the linker molecule can be a polynucleotide or non-nucleotide linker, but is preferably a polynucleotide linker.
  • the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.
  • the siRNA can also comprise a 3'-overhang.
  • a "3'-overhang” refers to at least one unpaired nucleotide extending from the 3'-end of an RNA strand.
  • the siRNA comprises at least one 3 '-overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
  • both strands of the siRNA molecule i.e., after the siRNA molecule is cleaved intracellularly by the "Dicer" protein
  • the length of the overhangs can be the same or different for each strand.
  • the 3'- overhang is present on both strands of the siRNA, and is 2 nucleotides in length.
  • each strand of the siRNA can comprise 3 '-overhangs of dideoxythymidylic acid ("TT") or diuridylic acid ("uu").
  • the 3'-overhangs can be also stabilized against degradation.
  • the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotides in the 3 '-overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation.
  • the absence of a 2'- hydroxyl in the 2'-deoxythymidine significantly enhances the nuclease resistance of the 3'-overhang in tissue culture medium.
  • target mRNA refers to an RNA molecule that is a target for downregulation.
  • the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein.
  • the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease.
  • the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.
  • siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence”).
  • Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide”, revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. "The siRNA User Guide” is available on the world wide web at a website maintained by Dr. Thomas Tuschl, Uaboratory of RNA Molecular Biology, Rockefeller University, New York, USA, and can be found by accessing the website of the Rockefeller University and searching with the keyword "siRNA”.
  • the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.
  • a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3'- direction) from the start codon.
  • siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. application no. 2002/0086356 of Tuschl et al., the entire disclosure of which is herein incorporated by reference. siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
  • siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for transcribing siRNA used in the present disclosure from a plasmid include, for example, the U6 or Hl RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • plasmids suitable for transcribing siRNA are within the skill in the art.
  • miRNA refers to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradation and/or prevent translation of target mRNAs.
  • miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively.
  • miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can be obtained using a number of techniques known to those of skill in the art.
  • miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.).
  • miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (preferably mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a nucleic acid sequence e.g., an ORF
  • polypeptides e.g., a peptide or protein, preferably a pharmaceutically active peptide or protein.
  • RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence (e.g., an ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein, and is capable of expressing said peptide or protein, in particular if transferred into a cell or subject.
  • the RNA (preferably mRNA) described in the present disclosure contains a coding region (ORF) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein.
  • ORF coding region
  • an "open reading frame” or "ORF” is a continuous stretch of codons beginning with a start codon and ending with a stop codon.
  • RNA preferably mRNA
  • RNA encoding a pharmaceutically active peptide or protein
  • pharmaceutically active RNA or “pharmaceutically active mRNA”
  • RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide, e.g., two, three, four or more peptides or polypeptides.
  • RNA which is pharmaceutically active in its own; thus, pharmaceutically active RNA may alternatively be non-coding RNA such as antisense- RNA, micro RNA (miRNA), siRNA, one or more strands of RNA interference (RNAi), short hairpin RNAs (shRNAs), or precursor of a siRNA or microRNA-like RNA, targeted to a target transcript, e.g., a transcript of an endogenous disease-related transcript of a subject.
  • RNAi micro RNA
  • siRNA one or more strands of RNA interference
  • shRNAs short hairpin RNAs
  • precursor of a siRNA or microRNA-like RNA targeted to a target transcript, e.g., a transcript of an endogenous disease-related transcript of a subject.
  • the term "pharmaceutically active peptide or protein” means a peptide or protein that can be used in the treatment of an individual where the expression of the peptide or protein would be of benefit, e.g., in ameliorating the symptoms of a disease or disorder.
  • a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder.
  • a pharmaceutically active peptide or protein has a positive or advantageous effect on the condition or disease state of an individual when administered to the individual in a therapeutically effective amount.
  • pharmaceutically active peptides and proteins include, but are not limited to, immunostimulants, e.g., cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins.
  • immunostimulants e.g., cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins.
  • the pharmaceutically active peptide and polypeptide includes a replacement protein.
  • an “immunostimulant” is any substance that stimulates the immune system by inducing activation or increasing activity of any of the immune system's components, in particular immune effector cells.
  • the immunostimulant may be pro-inflammatory (e.g., when treating infections or cancer), or antiinflammatory (e.g., when treating autoimmune diseases).
  • the immunostimulant is a cytokine or a variant thereof.
  • cytokines include interferons, such as interferon-alpha (IFN-a) or interferon-gamma (IFN-y), interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor.
  • the immunostimulant includes an adjuvant-type immunostimulatory agent such as APC Toll-like Receptor agonists or costimulatory/cell adhesion membrane proteins.
  • Toll -like Receptor agonists include costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.
  • cytokines relates to proteins which have a molecular weight of about 5 to 60 kDa (such as about 5 to 20 kDa) and which participate in cell signaling (e.g., paracrine, endocrine, and/or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors.
  • Cytokines differ from hormones in that (i) they usually act at much more variable concentrations than hormones and (ii) generally are made by a broad range of cells (nearly all nucleated cells can produce cytokines).
  • Interferons are usually characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface, thereby preventing viral replication within the cells. The interferons can be grouped into two types. IFN- gamma is the sole type II interferon; all others are type I interferons.
  • cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte -macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFNa), interferon beta (IFNP), interferon gamma (INFy), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL- 12), and interleukin 21 (IL-21).
  • EPO erythropoietin
  • CSF colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte -macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • BMP bone morphogenetic protein
  • IFNa interferon alfa
  • a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof.
  • a cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal.
  • One particularly preferred cytokine is interferon-a.
  • Immunostimulants may be provided to a subject by administering to the subject nucleic acid (such as DNA or RNA) encoding an immunostimulant in a formulation for preferential delivery of nucleic acid to liver or liver tissue.
  • nucleic acid such as DNA or RNA
  • the delivery of nucleic acid (such as DNA or RNA) to such target organ or tissue is preferred, in particular, if it is desired to express large amounts of the immunostimulant and/or if systemic presence of the immunostimulant, in particular in significant amounts, is desired or required.
  • Nucleic acid (such as DNA or RNA) delivery systems have an inherent preference to the liver. This pertains to lipid-based particles, cationic and neutral nanoparticles, in particular lipid nanoparticles.
  • cytokines involved in T cell proliferation and/or maintenance.
  • suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.
  • nucleic acid (such as DNA or RNA) encoding an immunostimulant may be administered in a formulation for preferential delivery of nucleic acid (such as DNA or RNA) to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen.
  • the delivery of an immunostimulant to such target tissue is preferred, in particular, if presence of the immunostimulant in this organ or tissue is desired (e.g., for inducing an immune response, in particular in case immunostimulants such as cytokines are required during T-cell priming or for activation of resident immune cells), while it is not desired that the immunostimulant is present systemically, in particular in significant amounts (e.g., because the immunostimulant has systemic toxicity).
  • suitable immunostimulants are cytokines involved in T cell priming.
  • suitable cytokines include IL12, IL15, IFN-a, or IFN-P, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.
  • Interferons are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.
  • interferons are typically divided among three classes: type I interferon, type II interferon, and type III interferon.
  • IFNAR IFN-a/p receptor
  • type I interferons present in humans are IFNa, IFNp, IFNs, IFNK and IFNco.
  • type I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.
  • the IFNa proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection.
  • the genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA 14, IFNA 16, IFNA 17, IFNA21. These genes are found together in a cluster on chromosome 9.
  • the IFNp proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFNp have been described, IFNpi and IFNP3. The natural and recombinant forms of IFNpi have antiviral, antibacterial, and anticancer properties.
  • Type II interferon IFNy in humans
  • IL12 Type II interferon
  • type II interferons are released by cytotoxic T cells and T helper cells.
  • Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of virus or fungal infections. In general, type I and II interferons are responsible for regulating and activating the immune response.
  • a type I interferon is preferably IFNa or IFNp, more preferably IFNa.
  • an interferon may be a naturally occurring interferon or a functional fragment or variant thereof.
  • An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.
  • Interleukins are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins.
  • an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof.
  • An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.
  • Immunostimulant polypeptides described herein can be prepared as fusion or chimeric polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulant).
  • the immunostimulant may be fused to an extended-PK group, which increases circulation half-life.
  • extended-PK groups are described infra. It should be understood that other PK groups that increase the circulation half-life of immunostimulants such as cytokines, or variants thereof, are also applicable to the present disclosure.
  • the extended-PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).
  • PK is an acronym for "pharmacokinetic” and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject.
  • an "extended-PK group” refers to a protein, peptide, or moiety that increases the circulation half-life of a biologically active molecule when fused to or administered together with the biologically active molecule.
  • examples of an extended-PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549).
  • an extended-PK immunostimulant refers to an immunostimulant moiety in combination with an extended-PK group.
  • the extended-PK immunostimulant is a fusion protein in which an immunostimulant moiety is linked or fused to an extended-PK group.
  • the serum half-life of an extended-PK immunostimulant is increased relative to the immunostimulant alone (i.e., the immunostimulant not fused to an extended-PK group).
  • the serum half-life of the extended-PK immunostimulant is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer relative to the serum half-life of the immunostimulant alone.
  • the serum half-life of the extended-PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7- fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35- fold, 40-fold, or 50-fold greater than the serum half-life of the immunostimulant alone.
  • half-life refers to the time taken for the serum or plasma concentration of a compound such as a peptide or polypeptide to reduce by 50%, in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms.
  • An extended-PK immunostimulant suitable for use herein is stabilized in vivo and its half-life increased by, e.g., fusion to serum albumin (e.g., HSA or MSA), which resist degradation and/or clearance or sequestration.
  • the half-life can be determined in any manner known per se, such as by pharmacokinetic analysis.
  • Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).
  • the albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding an albumin.
  • the therapeutic protein and albumin, once part of the albumin fusion protein may each be referred to as a "portion", "region” or “moiety” of the albumin fusion protein (e.g., a "therapeutic protein portion” or an "albumin protein portion”).
  • an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin).
  • an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, e.g. a liver cell, and secreted into the circulation.
  • Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and/or assembly into multimeric proteins.
  • albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin.
  • Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.
  • albumin refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin.
  • albumin refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules.
  • the albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon.
  • the albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.
  • the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.
  • HSA human serum albumin
  • HA human albumin
  • albumin and serum albumin are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
  • a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fusion state.
  • the albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability.
  • Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin.
  • one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used.
  • the HSA fragment is the mature form of HSA.
  • an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.
  • albumin may be naturally occurring albumin or a fragment or variant thereof.
  • Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.
  • the albumin fusion protein comprises albumin as the N-terminal portion, and a therapeutic protein as the C-terminal portion.
  • an albumin fusion protein comprising albumin as the C-terminal portion, and a therapeutic protein as the N-terminal portion may also be used.
  • the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin.
  • the therapeutic proteins fused at the N- and C-termini are the same therapeutic proteins.
  • the therapeutic proteins fused at the N- and C-termini are different therapeutic proteins.
  • the different therapeutic proteins are both cytokines.
  • Fc region refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains.
  • Fc domain refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain.
  • an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.
  • an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
  • An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain.
  • the Fc domain may be derived from an immunoglobulin of any species and/or any subtype, including, but not limited to, a human IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody.
  • the Fc domain encompasses native Fc and Fc variant molecules.
  • any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule.
  • the Fc domain has reduced effector function (e.g., FcyR binding).
  • the Fc domains of a polypeptide described herein may be derived from different immunoglobulin molecules.
  • an Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule.
  • an Fc domain can comprise a chimeric hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
  • an extended-PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "Fc domain").
  • the Fc domain does not contain a variable region that binds to antigen.
  • Fc domains suitable for use in the present disclosure may be obtained from a number of different sources.
  • an Fc domain is derived from a human immunoglobulin.
  • the Fc domain is from a human IgGl constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.
  • rodent e.g. a mouse, rat, rabbit, guinea pig
  • non-human primate e.g. chimpanzee, mac
  • the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgGl, IgG2, IgG3, and IgG4.
  • Fc domain gene sequences e.g., mouse and human constant region gene sequences
  • Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and/or with a particular modification to reduce immunogenicity.
  • Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art recognized techniques.
  • the extended-PK group is a serum albumin binding protein such as those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422,
  • the extended-PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are herein incorporated by reference in their entirety.
  • the extended-PK group is a serum immunoglobulin binding protein such as those disclosed in US2007/0178082, US2014/0220017, and US2017/0145062, which are herein incorporated by reference in their entirety.
  • the extended-PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012/0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012/0094909.
  • Fn3(HSA) a Fn3 protein that binds to human serum albumin.
  • the extended-PK immunostimulant can employ one or more peptide linkers.
  • peptide linker refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended-PK moiety and an immunostimulant moiety) in a linear amino acid sequence of a polypeptide chain.
  • peptide linkers may be used to connect an immunostimulant moiety to an HSA domain.
  • Linkers suitable for fusing the extended-PK group to, e.g., an immunostimulant are well known in the art.
  • Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers.
  • the linker is a glycine-serine- polypeptide linker, i.e., a peptide that consists of glycine and serine residues.
  • a pharmaceutically active peptide or protein comprises a replacement protein.
  • the present disclosure provides a method for treatment of a subject having a disorder requiring protein replacement (e.g., protein deficiency disorders) comprising administering to the subject RNA as described herein encoding a replacement protein.
  • protein replacement refers to the introduction of a protein (including functional variants thereof) into a subject having a deficiency in such protein.
  • the term also refers to the introduction of a protein into a subject otherwise requiring or benefiting from providing a protein, e.g., suffering from protein insufficiency.
  • disorder characterized by a protein deficiency refers to any disorder that presents with a pathology caused by absent or insufficient amounts of a protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in a biologically inactive protein product. Protein insufficiency can be involved in infectious diseases, immunosuppression, organ failure, glandular problems, radiation illness, nutritional deficiency, poisoning, or other environmental or external insults.
  • hormones relates to a class of signaling molecules produced by glands, wherein signaling usually includes the following steps: (i) synthesis of a hormone in a particular tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) relay and amplification of the signal; and (vi) breakdown of the hormone.
  • Hormones differ from cytokines in that (1) hormones usually act in less variable concentrations and (2) generally are made by specific kinds of cells.
  • a "hormone” is a peptide or protein hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormones (such as human grown hormone or bovine somatotropin), oxytocin, atrial -natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptins.
  • Adhesion molecules relates to proteins which are located on the surface of a cell and which are involved in binding of the cell with other cells or with the extracellular matrix (ECM).
  • Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Particular examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.
  • Integrins are also involved in signal transduction.
  • integrins upon ligand binding, integrins modulate cell signaling pathways, e.g., pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTK).
  • RTK receptor tyrosine kinases
  • Such regulation can lead to cellular growth, division, survival, or differentiation or to apoptosis.
  • integrins include:
  • immunoglobulins or “immunoglobulin superfamily” refers to molecules which are involved in the recognition, binding, and/or adhesion processes of cells. Molecules belonging to this superfamily share the feature that they contain a region known as immunoglobulin domain or fold.
  • immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD 19), antigen receptor accessory molecules (e.g., CD-3y, CD3-5, CD-3a, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and other.
  • antibodies e.g., IgG
  • TCRs T cell receptors
  • MHC major histocompatibility complex
  • co-receptors e.g., CD4, CD8, CD 19
  • antigen receptor accessory molecules e.g., CD-3y, CD3-5, CD-3a, CD79a, CD79b
  • co-stimulatory or inhibitory molecules e.g., CD28, CD80, CD86
  • immunologically active compound relates to any compound altering an immune response, preferably by inducing and/or suppressing maturation of immune cells, inducing and/or suppressing cytokine biosynthesis, and/or altering humoral immunity by stimulating antibody production by B cells.
  • Immunologically active compounds possess potent immunostimulating activity including, but not limited to, antiviral and antitumor activity, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2 mediated diseases.
  • Immunologically active compounds can be useful as vaccine adjuvants.
  • immunologically active compounds include interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T cell receptors, chimeric antigen receptors (CARs), immunoglobulins, and antigens, in particular tumor-associated antigens, pathogen- associated antigens (such as bacterial, parasitic, or viral antigens), allergens, autoantigens, hormones (insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin
  • nucleic acid such as DNA or RNA, in particular, mRNA
  • nucleic acid described in the present disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject.
  • the "peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject” is also designated herein as "vaccine antigen", “peptide and protein antigen” or simply "antigen”.
  • the RNA (in particular, mRNA) encoding vaccine antigen is a single -stranded, 5' capped mRNA that is translated into the respective protein upon entering cells of a subject being administered the RNA, e.g., antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • the RNA (i) contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence); (ii) is modified for optimized efficacy of the RNA (e.g., increased translation efficacy, decreased immunogenicity, and/or decreased cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (in particular cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml ⁇
  • nucleosides e.g., synthetic nucleosides selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml ⁇
  • beta-S-ARCA(Dl) is utilized as specific capping structure at the 5'-end of the RNA.
  • the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.
  • the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.
  • the poly(A) sequence is 110 nucleotides in length and consists of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence was designed to enhance RNA stability and translational efficiency in dendritic cells.
  • the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3.
  • the RNA comprises a modified nucleoside in place of uridine.
  • the modified nucleoside replacing (partially or completely, preferably completely) uridine is selected from the group consisting of pseudouridine (y), N1 -methyl -pseudouridine (ml ⁇
  • the RNA encoding the vaccine antigen has a coding sequence (a) which is codon-optimized, (b) the G/C content of which is increased compared to the wild type coding sequence, or (c) both (a) and (b).
  • the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is expressed in cells of the subject to provide the vaccine antigen. In some embodiments, expression of the vaccine antigen is at the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is transiently expressed in cells of the subject. In some embodiments, the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is administered systemically. In some embodiments, after systemic administration of the nucleic acid (such as DNA or RNA) encoding the vaccine antigen, expression of the nucleic acid encoding the vaccine antigen in spleen occurs.
  • nucleic acid encoding the vaccine antigen after systemic administration of the nucleic acid encoding the vaccine antigen, expression of the nucleic acid encoding the vaccine antigen in antigen presenting cells, preferably professional antigen presenting cells occurs.
  • the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells.
  • no or essentially no expression of the nucleic acid encoding the vaccine antigen in lung and/or liver occurs.
  • expression of the nucleic acid encoding the vaccine antigen in spleen is at least 5-fold the amount of expression in lung.
  • the vaccine antigen comprises an epitope for inducing an immune response against an antigen in a subject.
  • the vaccine antigen comprises an antigenic sequence for inducing an immune response against an antigen in a subject.
  • Such antigenic sequence may correspond to a target antigen or disease-associated antigen, e.g., a protein of an infectious agent (e.g., viral or bacterial antigen) or tumor antigen, or may correspond to an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or the immunogenic variant thereof.
  • the antigenic sequence may comprise at least an epitope of a target antigen or disease-associated antigen or an immunogenic variant thereof.
  • the antigenic sequences e.g., epitopes, suitable for use according to the disclosure typically may be derived from a target antigen, i.e. the antigen against which an immune response is to be elicited.
  • a target antigen i.e. the antigen against which an immune response is to be elicited.
  • the antigenic sequences contained within the vaccine antigen may be a target antigen or a fragment or variant of a target antigen.
  • the antigenic sequence or a procession product thereof may bind to the antigen receptor such as TCR or CAR carried by immune effector cells.
  • the antigenic sequence is selected from the group consisting of the antigen expressed by a target cell to which the immune effector cells are targeted or a fragment thereof, or a variant of the antigenic sequence or the fragment.
  • a vaccine antigen which is provided to a subject according to the present disclosure by administering nucleic acid (such as DNA or RNA) encoding the vaccine antigen preferably results in the induction of an immune response, e.g., in the stimulation, priming and/or expansion of immune effector cells, in the subject being provided the vaccine antigen.
  • Said immune response e.g., stimulated, primed and/or expanded immune effector cells, is preferably directed against a target antigen, in particular a target antigen expressed by diseased cells, tissues and/or organs, i.e., a disease-associated antigen.
  • a vaccine antigen may comprise the disease-associated antigen, or a fragment or variant thereof. In some embodiments, such fragment or variant is immunologically equivalent to the disease-associated antigen.
  • fragment of an antigen or “variant of an antigen” means an agent which results in the induction of an immune response, e.g., in the stimulation, priming and/or expansion of immune effector cells, which immune response, e.g., stimulated, primed and/or expanded immune effector cells, targets the antigen, i.e. a disease-associated antigen, in particular when presented by diseased cells, tissues and/or organs.
  • the vaccine antigen may correspond to or may comprise the disease-associated antigen, may correspond to or may comprise a fragment of the disease- associated antigen or may correspond to or may comprise an antigen which is homologous to the disease- associated antigen or a fragment thereof.
  • the vaccine antigen comprises a fragment of the disease- associated antigen or an amino acid sequence which is homologous to a fragment of the disease- associated antigen
  • said fragment or amino acid sequence may comprise an epitope of the disease- associated antigen to which the antigen receptor of the immune effector cells is targeted or a sequence which is homologous to an epitope of the disease-associated antigen.
  • a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence being homologous to an immunogenic fragment of a disease-associated antigen.
  • an "immunogenic fragment of an antigen” preferably relates to a fragment of an antigen which is capable of inducing an immune response against, e.g., stimulating, priming and/or expanding immune effector cells carrying an antigen receptor binding to, the antigen or cells expressing the antigen.
  • the vaccine antigen (similar to the disease-associated antigen) provides the relevant epitope for binding by the antigen receptor present on the immune effector cells.
  • the vaccine antigen or a fragment thereof is expressed on the surface of a cell such as an antigen-presenting cell (optionally in the context of MHC) so as to provide the relevant epitope for binding by immune effector cells.
  • the vaccine antigen may be a recombinant antigen.
  • the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is expressed in cells of a subject to provide the antigen or a procession product thereof for binding by the antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and/or expansion of the immune effector cells.
  • autoantigen or "self-antigen” refers to an antigen which originates from within the body of a subject (i.e., the autoantigen can also be called “autologous antigen") and which produces an abnormally vigorous immune response against this normal part of the body. Such vigorous immune reactions against autoantigens may be the cause of "autoimmune diseases”.
  • an antigen is expressed on the surface of a diseased cell (such as tumor cell or an infected cell).
  • an antigen receptor is a CAR which binds to an extracellular domain or to an epitope in an extracellular domain of an antigen.
  • a CAR binds to native epitopes of an antigen present on the surface of living cells.
  • binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on cells results in stimulation, priming and/or expansion of said T cells.
  • binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
  • an amino acid sequence enhancing antigen processing and/or presentation is fused, either directly or through a linker, to an antigenic peptide or polypeptide (antigenic sequence).
  • the nucleic acid such as DNA or RNA
  • the nucleic acid described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence enhancing antigen processing and/or presentation.
  • an antigen for vaccination which may be administered in the form of nucleic acid (such as DNA or RNA) coding therefor comprises a naturally occurring antigen or a fragment such as an epitope thereof.
  • amino acid sequences enhancing antigen processing and/or presentation are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the C-terminus of an amino acid sequence which breaks immunological tolerance), without being limited thereto.
  • Amino acid sequences enhancing antigen processing and/or presentation as defined herein preferably improve antigen processing and presentation.
  • the amino acid sequence enhancing antigen processing and/or presentation as defined herein includes, without being limited thereto, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof.
  • a secretory sequence e.g., a sequence comprising the amino acid sequence of SEQ ID NO: 4, may be fused to the N-terminus of the antigenic peptide or polypeptide.
  • an amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or a functional fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.
  • an amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 5.
  • a secretory sequence e.g., a sequence comprising the amino acid sequence of SEQ ID NO: 4, may be fused to the N-terminus of the antigenic peptide or polypeptide.
  • Amino acid sequences derived from tetanus toxoid of Clostridium tetani may be employed to overcome self-tolerance mechanisms in order to efficiently mount an immune response to self-antigens by providing T-cell help during priming.
  • tetanus toxoid heavy chain includes epitopes that can bind promiscuously to MHC class II alleles and induce CD4 + memory T cells in almost all tetanus vaccinated individuals.
  • TT tetanus toxoid
  • p2 QYIKANSKFIGITEL; TT830-844; SEQ ID NO: 9
  • pl6 MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT578-609; SEQ ID NO: 10.
  • the p2 epitope was already used for peptide vaccination in clinical trials to boost anti-melanoma activity.
  • Non-clinical data showed that RNA vaccines encoding both a tumor antigen plus promiscuously binding tetanus toxoid sequences lead to enhanced CD8 + T-cell responses directed against the tumor antigen and improved break of tolerance.
  • Immunomonitoring data from patients vaccinated with vaccines including those sequences fused in frame with the tumor antigen-specific sequences reveal that the tetanus sequences chosen are able to induce tetanus-specific T-cell responses in almost all patients.
  • an amino acid sequence which breaks immunological tolerance is fused, either directly or through a linker, e.g., a linker having the amino acid sequence according to SEQ ID NO: 7, to the antigenic peptide or polypeptide.
  • a linker e.g., a linker having the amino acid sequence according to SEQ ID NO: 7, to the antigenic peptide or polypeptide.
  • amino acid sequences which break immunological tolerance are preferably located at the C- terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence enhancing antigen processing and/or presentation, wherein the amino acid sequence which breaks immunological tolerance and the amino acid sequence enhancing antigen processing and/or presentation may be fused either directly or through a linker, e.g., a linker having the amino acid sequence according to SEQ ID NO: 8), without being limited thereto.
  • Amino acid sequences which break immunological tolerance as defined herein preferably improve T cell responses.
  • the amino acid sequence which breaks immunological tolerance as defined herein includes, without being limited thereto, sequences derived from tetanus toxoid-derived helper sequences p2 and pl6 (P2P16), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 6 or a functional variant thereof.
  • an amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6, or a functional fragment of the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6.
  • an amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 6.
  • cap 5'-cap structure selected from the group consisting of m2 7 ’ 2 O G(5’)ppSp(5')G (in particular its DI diastereomer), m2 7 ’ 3 °G(5')ppp(5')G, and m2 7 ’ 3 O Gppp(mi 2 O )ApG.
  • hAg-Kozak 5'-UTR sequence of the human alpha-globin mRNA with an optimized ‘Kozak sequence’ to increase translational efficiency.
  • sec/MITD Fusion-protein tags derived from the sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), which have been shown to improve antigen processing and presentation.
  • Sec corresponds to the 78 bp fragment coding for the secretory signal peptide, which guides translocation of the nascent polypeptide chain into the endoplasmatic reticulum.
  • MITD corresponds to the transmembrane and cytoplasmic domain of the MHC class I molecule, also called MHC class I trafficking domain.
  • Antigen Sequences encoding the respective vaccine antigen/epitope.
  • Glycine-serine linker (GS): Sequences coding for short peptide linkers predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.
  • P2P16 Sequence coding for tetanus toxoid-derived helper epitopes to break immunological tolerance.
  • the 3'-UTR is a combination of two sequence elements derived from the “amino terminal enhancer of split” (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.
  • AES amino terminal enhancer of split
  • A30L70 A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells.
  • vaccine RNA described herein has one of the following structures: cap-hAg-Kozak-sec-GS(l)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 beta-S-ARCA(Dl)-hAg-Kozak-sec-GS(l)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70
  • vaccine antigen described herein has the structure: sec-GS( l)-Antigen-GS(2)-P2P 16-GS(3)-MITD
  • hAg-Kozak comprises the nucleotide sequence of SEQ ID NO: 1.
  • sec comprises the amino acid sequence of SEQ ID NO: 4.
  • P2P16 comprises the amino acid sequence of SEQ ID NO: 6.
  • MITD comprises the amino acid sequence of SEQ ID NO: 5.
  • GS(1) comprises the amino acid sequence of SEQ ID NO: 7.
  • GS(2) comprises the amino acid sequence of SEQ ID NO: 8.
  • GS(3) comprises the amino acid sequence of SEQ ID NO: 8.
  • FI comprises the nucleotide sequence of SEQ ID NO: 2.
  • A30L70 comprises the nucleotide sequence of SEQ ID NO: 3.
  • the sequence encoding the vaccine antigen/epitope comprises a modified nucleoside replacing (partially or completely, preferably completely) uridine, wherein the modified nucleoside is selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (m I ⁇
  • the sequence encoding the vaccine antigen/epitope is codon-optimized.
  • the G/C content of the sequence encoding the vaccine antigen/epitope is increased compared to the wild type coding sequence.
  • non-professional antigen presenting cells relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma.
  • exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.
  • dendritic cell refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells.
  • dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node.
  • Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In some embodiments, the dendritic cells are splenic dendritic cells.
  • macrophage refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are splenic macrophages.
  • allergen refers to a kind of antigen which originates from outside the body of a subject (i.e., the allergen can also be called “heterologous antigen”) and which produces an abnormally vigorous immune response in which the immune system of the subject fights off a perceived threat that would otherwise be harmless to the subject.
  • allergen usually is an antigen which is able to stimulate a type-I hypersensitivity reaction in atopic individuals through immunoglobulin E (IgE) responses.
  • IgE immunoglobulin E
  • allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phi p 5), and proteins of dust mites (e.g., Der p 2).
  • peanut proteins e.g., Ara h 2.02
  • ovalbumin e.g., ovalbumin
  • grass pollen proteins e.g., Phi p 5
  • proteins of dust mites e.g., Der p 2
  • growth factors refers to molecules which are able to stimulate cellular growth, proliferation, healing, and/or cellular differentiation. Typically, growth factors act as signaling molecules between cells.
  • growth factors include particular cytokines and hormones which bind to specific receptors on the surface of their target cells.
  • growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), such as VEGFA, epidermal growth factor (EGF), insulin-like growth factor, ephrins, macrophage colonystimulating factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, neuregulins, neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti- apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P)), and tumor necrosis factor-alpha (TNF-a).
  • BMPs bone morphogenetic proteins
  • protease inhibitors refers to molecules, in particular peptides or proteins, which inhibit the function of proteases.
  • Protease inhibitors can be classified by the protease which is inhibited (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serpins).
  • protease inhibitors include serpins, such as alpha 1 -antitrypsin, aprotinin, and be statin.
  • enzymes refers to macromolecular biological catalysts which accelerate chemical reactions. Like any catalyst, enzymes are not consumed in the reaction they catalyze and do not alter the equilibrium of said reaction. Unlike many other catalysts, enzymes are much more specific.
  • an enzyme is essential for homeostasis of a subject, e.g., any malfunction (in particular, decreased activity which may be caused by any of mutation, deletion or decreased production) of the enzyme results in a disease.
  • examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.
  • receptors refers to protein molecules which receive signals (in particular chemical signals called ligands) from outside a cell.
  • signals in particular chemical signals called ligands
  • the binding of a signal (e.g., ligand) to a receptor causes some kind of response of the cell, e.g., the intracellular activation of a kinase.
  • Receptors include transmembrane receptors (such as ion channel-linked (ionotropic) receptors, G protein-linked (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (such as cytoplasmic receptors and nuclear receptors).
  • receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand- 1 (PSGL-1).
  • PSGL-1 P-selectin glycoprotein ligand- 1
  • growth factor receptors refers to receptors which bind to growth factors.
  • apoptosis regulators refers to molecules, in particular peptides or proteins, which modulate apoptosis, i.e., which either activate or inhibit apoptosis.
  • Apoptosis regulators can be grouped into two broad classes: those which modulate mitochondrial function and those which regulate caspases.
  • the first class includes proteins (e.g., BCL-2, BCL-xL) which act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol.
  • proapoptotic proteins e.g., BAX, BAK, BIM
  • the second class includes proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP which block the activation of caspases.
  • transcription factors relates to proteins which regulate the rate of transcription of genetic information from DNA to messenger RNA, in particular by binding to a specific DNA sequence. Transcription factors may regulate cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and/or in response to signals from outside the cell, such as a hormone. Transcription factors contain at least one DNA-binding domain which binds to a specific DNA sequence, usually adjacent to the genes which are regulated by the transcription factors. Particular examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.
  • tumor suppressor proteins relates to molecules, in particular peptides or proteins, which protect a cell from one step on the path to cancer.
  • Tumor-suppressor proteins (usually encoded by corresponding tumor-suppressor genes) exhibit a weakening or repressive effect on the regulation of the cell cycle and/or promote apoptosis.
  • Their functions may be one or more of the following: repression of genes essential for the continuing of the cell cycle; coupling the cell cycle to DNA damage (as long as damaged DNA is present in a cell, no cell division should take place); initiation of apoptosis, if the damaged DNA cannot be repaired; metastasis suppression (e.g., preventing tumor cells from dispersing, blocking loss of contact inhibition, and inhibiting metastasis); and DNA repair.
  • tumor-suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI/SNF (SWItch/Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3).
  • the term "structural proteins” refers to proteins which confer stiffness and rigidity to otherwise-fluid biological components.
  • reprogramming factors or "reprogramming transcription factors” relates to molecules, in particular peptides or proteins, which, when expressed in somatic cells optionally together with further agents such as further reprogramming factors, lead to reprogramming or de-differentiation of said somatic cells to cells having stem cell characteristics, in particular pluripotency.
  • reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.
  • genomic engineering proteins relates to proteins which are able to insert, delete or replace DNA in the genome of a subject.
  • genomic engineering proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9).
  • blood proteins relates to peptides or proteins which are present in blood plasma of a subject, in particular blood plasma of a healthy subject.
  • Blood proteins have diverse functions such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), protease inhibitors (e.g., alpha 1 -antitrypsin), etc.
  • blood proteins include thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony stimulating factor (G-CSF), modified Factor VIII, and anticoagulants.
  • the pharmaceutically active peptide or protein is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2), and interleukin 10 (IL- 11), more preferably EPO; (ii) an adhesion molecule, in particular an integrin; (iii) an immunoglobulin, in particular an antibody; (iv) an immunologically active compound, in particular an antigen, such as a viral or bacterial antigen, e.g., an antigen of SARS-CoV-2; (v) a hormone, in particular vasopressin, insulin or growth hormone; (vi) a growth factor, in particular VEGFA; (vii) a protease inhibitor, in particular alpha 1 -antitrypsin; (viii) an enzyme, preferably selected from the group consisting of herpes simplex virus type 1 thymidine kinase (HSV1-TK), he
  • a pharmaceutically active peptide or protein comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or protein to a subject elicits an immune response against the one or more antigens or one or more epitopes in a subject which may be therapeutic or partially or fully protective.
  • the nucleic acid (such as DNA or RNA, preferably mRNA) encodes at least one epitope, e.g. , at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.
  • the target antigen is a tumor antigen and the antigenic sequence (e.g., an epitope) is derived from the tumor antigen.
  • the tumor antigen may be a "standard” antigen, which is generally known to be expressed in various cancers.
  • the tumor antigen may also be a "neo-antigen", which is specific to an individual’s tumor and has not been previously recognized by the immune system.
  • a neo-antigen or neo-epitope may result from one or more cancer-specific mutations in the genome of cancer cells resulting in amino acid changes.
  • the vaccine antigen preferably comprises an epitope or a fragment of said neo-antigen comprising one or more amino acid changes.
  • tumor antigens include, without limitation, p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1 , CASP-8, CDC27/m, CDK4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6- AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A 10, MAGE-A11, or MAGE-
  • Cancer mutations vary with each individual. Thus, cancer mutations that encode novel epitopes (neoepitopes) represent attractive targets in the development of vaccine compositions and immunotherapies.
  • the efficacy of tumor immunotherapy relies on the selection of cancer-specific antigens and epitopes capable of inducing a potent immune response within a host.
  • Nucleic acid such as DNA or RNA
  • DCs Dendritic cells residing in the spleen represent antigen-presenting cells of particular interest for RNA expression of immunogenic epitopes or antigens such as tumor epitopes.
  • the use of multiple epitopes has been shown to promote therapeutic efficacy in tumor vaccine compositions.
  • Rapid sequencing of the tumor mutanome may provide multiple epitopes for individualized vaccines which can be encoded by nucleic acid (such as DNA or RNA, in particular mRNA) described herein, e.g., as a single polypeptide wherein the epitopes are optionally separated by linkers.
  • the nucleic acid (such as DNA or RNA, in particular mRNA) encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.
  • Exemplary embodiments include nucleic acid (such as DNA or RNA, in particular mRNA) that encodes at least five epitopes (termed a "pentatope”), nucleic acid (such as DNA or RNA, in particular mRNA) that encodes at least ten epitopes (termed a "decatope”), and nucleic acid (such as DNA or RNA, in particular mRNA) that encodes at least twenty epitopes (termed an "eicosatope”).
  • the epitope is derived from a pathogen-associated antigen.
  • the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof.
  • the pathogen is selected from viruses, bacteria, fungi, parasites, and other microorganisms.
  • viruses include, but are not limited to, are severe acute respiratory syndrome coronavirus (SARS-CoV), such as SARS-CoV2, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) (e.g., CMV5), human herpesviruses (HHV) (e.g., HHV6, 7 or 8), herpes simplex viruses (HSV), bovine herpes virus (BHV) (e.g., BHV4), equine herpes virus (EHV) (e.g., EHV2), human T-Cell leukemia viruses (HTLV)5, Varicella-Zoster virus (VZV), measles virus, papovaviruses (JC and BK), hepatitis viruses (e.g., HBV or HCV), myxoma virus, adenoviruses, rhinoviruses, enteroviruses, parvoviruses, polyoma
  • Exemplary diseases caused by viral infection include, but are not limited to, SARS, acquired immune deficiency syndrome (AIDS), measles, chicken pox, cytomegalovirus infections, genital herpes, hepatitis (such as hepatitis B or C), influenza (flu, such as human flu, swine flu, dog flu, horse flu, and avian flu), HPV infection, shingles, rabies, common cold, gastroenteritis, rubella, and mumps.
  • SARS acquired immune deficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • measles such as hepatitis B or C
  • influenza flu, such as human flu, swine flu, dog flu, horse flu, and avian flu
  • HPV infection shingles, rabies, common cold, gastroenteritis, rubella, and mumps.
  • Exemplary bacteria include, but are not limited to, Campylobacter (such as Campylobacter jejuni), Enterobacter species, Enterococcus faecium, Enterococcus faecalis, Escherichia coli (e.g., F. coli O157:H7), Group A streptococci, Haemophilus influenzae, Helicobacter pylori, listeria, Mycobacterium tuberculosis, Pseudomonas aeruginosa, S.
  • Campylobacter such as Campylobacter jejuni
  • Enterobacter species Enterococcus faecium
  • Enterococcus faecalis Enterococcus faecalis
  • Escherichia coli e.g., F. coli O157:H7
  • Group A streptococci Haemophilus influenzae
  • Helicobacter pylori listeria
  • pneumoniae Salmonella, Shigella, Staphylococcus aureus, Staphylococcus epidermidis, Borrelia and Rickettsia, Chlamydiaceae, Neisseria gonorrhoeae, Bordetella pertussis, Clostridium tetani, Neisseria meningitidis, Streptococcus (such as Streptococcus pneumoniae or Streptococcus pyogenes), and Treponema pallidum.
  • Exemplary diseases caused by bacterial infection include, but are not limited to, anthrax, cholera, diphtheria, foodbome illnesses, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, gonorrhea, pertussis, tetanus, meningitis, scarlet fever, and syphilis.
  • Exemplary parasites include, but are not limited to, Plasmodium, Trypanosoma, Leishmania, Trichomonas, Dientamoeba, Giardia, Entamoeba histolytica, Naegleria, Isospora, Toxoplasma, Sarcocystis, Rhinosporidium seeberi, and Balantidium.
  • Exemplary diseases caused by parasite infection include, but are not limited to, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, proamoebiasis, giardiasis, amebic dysentery, coccidiosis, toxoplasmosis, sarcocystosis, rhinosporidiosis, and balantidiasis.
  • the pathogen is an infectious pathogen, in particular a pathogen causing an infectious disease, such as a viral disease, a bacterial disease, or a parasitic disease.
  • the pathogen is a virus, bacterium, or parasite.
  • the nucleic acid such as DNA or RNA, in particular mRNA
  • compositions described herein can be used to prevent and/or treat an infectious disease caused by said pathogen.
  • the epitope is derived from a viral antigen.
  • the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof.
  • the nucleic acid such as DNA or RNA, in particular mRNA
  • the present disclosure encodes an amino acid sequence comprising a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof.
  • the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or the immunogenic variant thereof.
  • the nucleic acid such as DNA or RNA, in particular, mRNA
  • the coronavirus is MERS-CoV.
  • the coronavirus is SARS-CoV.
  • the coronavirus is SARS-CoV-2.
  • the nucleic acid e.g., DNA or RNA, in particular, mRNA
  • the RNA is a modified RNA, in particular a stabilized mRNA.
  • the RNA comprises a modified nucleoside in place of at least one uridine.
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is independently selected from pseudouridine (y), Nl-methyl-pseudouridine (ml ⁇
  • the RNA (in particular, mRNA) described herein comprises a modified nucleoside in place of uridine.
  • the modified nucleoside is selected from pseudouridine (y), Nl-methyl- pseudouridine (ml ⁇
  • the RNA in particular, mRNA
  • the RNA comprises a 5’ cap.
  • rm 73 °Gppp(mi 2 °) ApG is utilized as specific capping structure at the 5'-end of the mRNA.
  • the RNA (in particular, mRNA) encoding an antigen (in particular the vaccine RNA) comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.
  • the RNA (in particular, mRNA) encoding an antigen (in particular the vaccine RNA) comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.
  • the RNA (in particular, mRNA) encoding an antigen (in particular the vaccine RNA) comprises a poly-A sequence.
  • the poly-A sequence comprises at least 100 nucleotides.
  • the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 3.
  • the antigen (such as a tumor antigen or vaccine antigen) is preferably administered as single -stranded, 5' capped RNA (preferably mRNA) that is translated into the respective protein upon entering cells of a subject being administered the RNA.
  • the RNA contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence).
  • beta-S-ARCA(Dl) is utilized as specific capping structure at the 5'-end of the RNA.
  • mj 7,3 °Gppp(mi 2 °)ApG is utilized as specific capping structure at the 5'- end of the RNA.
  • the 5'-UTR sequence is derived from the human alpha-globin mRNA and optionally has an optimized 'Kozak sequence' to increase translational efficiency.
  • a combination of two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • F amino terminal enhancer of split
  • I mitochondrial encoded 12S ribosomal RNA
  • two re-iterated 3'-UTRs derived from the human beta-globin mRNA are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • a poly (A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues is used.
  • This poly(A) sequence was designed to enhance RNA stability and translational efficiency.
  • vaccine RNA described herein may comprise, from 5' to 3', one of the following structures: Cap-5'-UTR-Vaccine Antigen-Encoding Sequence-3 '-UTR-Poly(A) or beta-S-ARCA(D l)-hAg-Kozak-Vaccine Antigen-Encoding Sequence-FI-A30L70.
  • RNA or RNA encoding the above described vaccine antigen may be non-modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA) or self-amplifying RNA (saRNA).
  • uRNA uridine containing mRNA
  • modRNA nucleoside modified mRNA
  • saRNA self-amplifying RNA
  • the above described RNA or RNA encoding the above described vaccine antigen is nucleoside modified mRNA (modRNA).
  • Non-modified uridine messenger RNA The active principle of the non-modified messenger RNA (uRNA) is a single-stranded mRNA that is translated upon entering a cell.
  • each uRNA preferably contains common structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5 '-cap, 5'-UTR, 3'-UTR, poly(A)-tail).
  • the preferred 5’ cap structure is beta-S-ARCA(Dl) (m2 7 ’ 2 O GppSpG).
  • the preferred 5'- UTR and 3'-UTR comprise the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively.
  • the preferred poly(A)-tail comprises the sequence of SEQ ID NO: 3.
  • hAg -Kozak mean the 5'-UTR sequence of the human alpha-globin mRNA with an optimized ‘Kozak sequence’ to increase translational efficiency;
  • FI element means that the 3'-UTR is a combination of two sequence elements derived from the "amino terminal enhancer of split” (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I).
  • AES amino terminal enhancer of split
  • A30L70 means a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells
  • GS means a glycineserine linker, i.e., sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.
  • each modRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5 '-cap, 5'-UTR, 3'-UTR, poly(A)-tail). Compared to the uRNA, modRNA contains 1-methyl-pseudouridine instead of uridine.
  • the preferred 5’ cap structure is m2 7 ’ 3 °Gppp(mi 2 °)ApG.
  • the preferred 5'-UTR and 3 '-UTR comprise the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively.
  • the preferred poly(A)-tail comprises the sequence of SEQ ID NO: 3.
  • RNA Self-amplifying RNA
  • the active principle of the self-amplifying mRNA (saRNA) drug substance is a single-stranded RNA, which self-amplifies upon entering a cell, and the vaccine antigen is translated thereafter.
  • the coding region of saRNA contains two open reading frames (ORFs).
  • the 5’-ORF encodes the RNA-dependent RNA polymerase such as Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase).
  • VEEV Venezuelan equine encephalitis virus
  • replicase RNA-dependent RNA polymerase
  • the replicase ORF is followed 3’ by a subgenomic promoter and a second ORF encoding the antigen.
  • saRNA UTRs contain 5’ and 3’ conserved sequence elements (CSEs) required for selfamplification.
  • CSEs conserved sequence elements
  • the saRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5 '-cap, 5'-UTR, 3'-UTR, poly(A)-tail).
  • the saRNA preferably contains uridine.
  • the preferred 5’ cap structure is beta-S-ARCA(Dl) (m2 7 ’ 2 O GppSpG).
  • Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle.
  • the saRNA does not encode for alphaviral structural proteins that are required for genome packaging or cell entry, therefore generation of replication competent viral particles is very unlikely to not possible.
  • Replication does not involve any intermediate steps that generate DNA.
  • the use/uptake of saRNA therefore poses no risk of genomic integration or other permanent genetic modification within the target cell.
  • the saRNA itself prevents its persistent replication by effectively activating innate immune response via recognition of dsRNA intermediates.
  • a secretory signal peptide may be fused to the antigen-encoding regions preferably in a way that the sec is translated as N terminal tag.
  • sec corresponds to the secretory signal peptide of the S protein.
  • Sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins may be used as GS/Linkers.
  • nucleic acid such as DNA or RNA, preferably mRNA
  • an antigen such as a tumor antigen or a vaccine antigen
  • the nucleic acid is transiently expressed in cells of the subject.
  • the RNA is in vitro transcribed.
  • expression of the antigen is at the cell surface.
  • the antigen is expressed and presented in the context of MHC.
  • expression of the antigen is into the extracellular space, i.e., the antigen is secreted.
  • the antigen molecule or a procession product thereof may bind to an antigen receptor such as a BCR or TCR carried by immune effector cells, or to antibodies.
  • Said immune response is preferably directed against a target antigen.
  • a vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In one embodiment, such fragment or variant is immunologically equivalent to the target antigen.
  • fragment of an antigen or “variant of an antigen” means an agent which results in the induction of an immune response which immune response targets the antigen, i.e. a target antigen.
  • the vaccine antigen may correspond to or may comprise the target antigen, may correspond to or may comprise a fragment of the target antigen or may correspond to or may comprise an antigen which is homologous to the target antigen or a fragment thereof.
  • a vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence being homologous to an immunogenic fragment of a target antigen.
  • An "immunogenic fragment of an antigen” according to the disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against the target antigen.
  • the vaccine antigen may be a recombinant antigen.
  • immunologically equivalent means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect.
  • immunologically equivalent is preferably used with respect to the immunological effects or properties of antigens or antigen variants used for immunization.
  • an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.
  • a molecule which is immunologically equivalent to an antigen exhibits the same or essentially the same properties and/or exerts the same or essentially the same effects regarding the stimulation, priming and/or expansion of T cells as the antigen to which the T cells are targeted.
  • the RNA (preferably mRNA) used in the present disclosure is non-immunogenic.
  • RNA encoding an immunostimulant may be administered according to the present disclosure to provide an adjuvant effect.
  • the RNA encoding an immunostimulant may be standard RNA or non-immunogenic RNA.
  • non-immunogenic RNA refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non- immunogenic, i.e., than would have been induced by standard RNA (stdRNA).
  • stdRNA standard RNA
  • non-immunogenic RNA which is also termed modified RNA (modRNA) herein, is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or limiting the amount of double -stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA), e.g., during in vitro transcription, and/or by removing double -stranded RNA (dsRNA), e.g., following in vitro transcription.
  • dsRNA double -stranded RNA
  • non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.
  • dsRNA double-stranded RNA
  • any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA.
  • modified nucleosides that suppress RNA- mediated activation of innate immune receptors.
  • the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase.
  • the modified nucleobase is a modified uracil.
  • the nucleoside comprising a modified nucleobase is selected from the group consisting of 3 -methyl -uridine (m 3 U), 5- methoxy-uridine (mo 5 U), 5 -aza-uridine, 6-aza-uridine, 2-thio-5 -aza-uridine, 2-thio-uridine (s 2 U), 4- thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy-uridine (ho 5 U), 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5 -iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5 -carboxymethyl -uridine (cm 5 U), 1 -carboxymethylpseudouridine, 5 -carboxyhydroxymethyl
  • 2-thio-2'-O-methyl -uridine (s 2 Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3, 2'-O-dimethyl -uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-0H-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E- propenylamino)uridine.
  • the nucleoside comprising a modified nucleobase is pseudouridine (y), N1 -methyl -pseudouridine (m I ⁇
  • the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.
  • RNA preferably mRNA
  • IVT in vitro transcription
  • dsRNA double -stranded RNA
  • formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis.
  • UTP may be added once or several times during synthesis of mRNA.
  • dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix.
  • PS-DVB polystyrene-divinylbenzene
  • an enzymatic based method using E. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used.
  • dsRNA can be separated from ssRNA by using a cellulose material.
  • an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.
  • Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524.
  • remove or “removal” refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance.
  • a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the nonseparated mixture of first and second substances.
  • the removal of dsRNA (especially dsmRNA) from non-immunogenic RNA comprises a removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA.
  • the non-immunogenic RNA is free or essentially free of dsRNA.
  • the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA.
  • the purified preparation of single-stranded nucleoside modified RNA (especially mRNA) is substantially free of double stranded RNA (dsRNA).
  • the purified preparation is at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).
  • RNA may be taken as a measure for the amount of dsRNA in the sample.
  • a sample containing a known amount of dsRNA may be used as a reference.
  • RNA may be spotted onto a membrane, e.g., nylon blotting membrane.
  • the membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v/v) TWEEN-20) containing 5% (w/v) skim milk powder.
  • the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary).
  • the membrane After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
  • a secondary antibody e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
  • the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence.
  • translation is enhanced by a factor of 2-fold relative to its unmodified counterpart.
  • translation is enhanced by a 3 -fold factor.
  • translation is enhanced by a 4-fold factor.
  • translation is enhanced by a 5 -fold factor.
  • translation is enhanced by a 6-fold factor.
  • translation is enhanced by a 7-fold factor.
  • translation is enhanced by an 8-fold factor.
  • translation is enhanced by a 9-fold factor.
  • translation is enhanced by a 10-fold factor.
  • translation is enhanced by a 15 -fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In one embodiment, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold.
  • the non-immunogenic RNA exhibits significantly less innate immunogenicity than standard RNA with the same sequence.
  • the non- immunogenic RNA exhibits an innate immune response that is 2-fold less than its unmodified counterpart.
  • innate immunogenicity is reduced by a 3 -fold factor.
  • innate immunogenicity is reduced by a 4-fold factor.
  • innate immunogenicity is reduced by a 5 -fold factor.
  • innate immunogenicity is reduced by a 6-fold factor.
  • innate immunogenicity is reduced by a 7-fold factor.
  • innate immunogenicity is reduced by a 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15 -fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.
  • the term “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity.
  • the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response.
  • the term refers to a decrease such that the non- immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non- immunogenic RNA.
  • the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.
  • the nucleic acid e.g., DNA or RNA, in particular, mRNA
  • the nucleic acid described herein is formulated or is to be formulated as a liquid, a solid, or a combination thereof.
  • the nucleic acid e.g., DNA or RNA, in particular, mRNA
  • the nucleic acid described herein is formulated or is to be formulated for injection.
  • the nucleic acid e.g., DNA or RNA, in particular, mRNA
  • the nucleic acid described herein is formulated or is to be formulated for intramuscular administration.
  • the nucleic acid e.g., DNA or RNA, in particular, mRNA
  • a composition e.g., a nucleic acid composition, in particular a pharmaceutical composition.
  • the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc),
  • the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid selected from the group consisting of DODMA, DOTMA, DPL14, 3D-P- DMA, formula (X), and formula (XI); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (VJ’), (VI), (VI’), (VII), (VII’),
  • the one or more additional lipids are selected from neutral lipids and combinations thereof.
  • the neutral lipids include phospholipids, steroid lipids, and combinations thereof.
  • the one or more additional lipids are a combination of a phospholipid and a steroid lipid.
  • the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any one of formulas A to G; (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (I), (IX
  • the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (X); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (VJ’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (
  • the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (XI) (e.g., any one of formulas (Xlla), (Xllb), (Xllla), (Xlllb), (XIV-1), (XIV-2), and (XIV-3); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’
  • the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any one of formulas (XV- 1) to (XV-6); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’),
  • V-23 (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35),
  • V-49 (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61),
  • the nucleic acid is DNA.
  • the nucleic acid is RNA, in particular mRNA.
  • the composition in particular the pharmaceutical composition, is a vaccine.
  • the composition in particular the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the nucleic acid e.g., DNA or RNA
  • the composition, in particular the pharmaceutical composition is/are a component of a kit.
  • the kit further comprises instructions for use of the nucleic acid (e.g., DNA or RNA) for therapeutically or prophylactically treating an infection, e.g., a viral infection, such as a coronavirus infection, in a subject.
  • the coronavirus is a betacoronavirus.
  • the coronavirus is a sarbecovirus.
  • the coronavirus is SARS-CoV- 2.
  • the subject is a human.
  • the nucleic acid (e.g., DNA or RNA) in the nucleic acid (e.g., DNA or RNA) compositions (e.g., in the nucleic acid (e.g., DNA or RNA) particles) described herein is at a concentration from about 0.002 mg/mL to about 5 mg/mL, from about 0.002 mg/mL to about 2 mg/mL, from about 0.005 mg/mL to about 2 mg/mL, from about 0.01 mg/mL to about 1 mg/mL, from about 0.05 mg/mL to about 0.5 mg/mL or from about 0. 1 mg/mL to about 0.5 mg/mL.
  • the nucleic acid e.g., DNA or RNA
  • the nucleic acid is at a concentration from about 0.005 mg/mL to about 0.1 mg/mL, from about 0.005 mg/mL to about 0.09 mg/mL, from about 0.005 mg/mL to about 0.08 mg/mL, from about 0.005 mg/mL to about 0.07 mg/mL, from about 0.005 mg/mL to about 0.06 mg/mL, or from about 0.005 mg/mL to about 0.05 mg/mL.
  • Nucleic acids such as DNA or RNA, in particular mRNA, described herein may be present in particles comprising (i) the nucleic acid, (ii) at least one cationic or cationically ionizable compound; and (iii) a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII),
  • the particles comprise (i) the nucleic acid (such as RNA, in particular mRNA); (ii) at least one cationic or cationically ionizable compound as disclosed herein; (iii) a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I); and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VF), (VII), (VII’), (VV), (V
  • V-32 (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44),
  • V-45 (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57),
  • V-58 (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-
  • nucleic acid e.g., DNA or RNA
  • particles have been described previously to be suitable for delivery of nucleic acid (e.g., DNA or RNA) in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60).
  • nucleic acid e.g., DNA or RNA
  • nanoparticle encapsulation of nucleic acid e.g., DNA or RNA
  • Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles.
  • nucleic acid such as RNA
  • organic e.g., ethanolic
  • a lipid mixture containing a cationically ionizable lipid at pH of, e.g., 5
  • electrostatic interaction leads to particle formation coincident with efficient encapsulation of the nucleic acid (e.g., DNA or RNA) drug substance.
  • RNA-LNP nucleic acid particle
  • pH 7-8 pH 7-8
  • charge -neutral particles display longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are cleared rapidly by the reticuloendothelial system.
  • the low pH of the endosome renders the particles (such as LNPs) fusogenic and allows for release of the nucleic acid (such as RNA) into the cytosol of the target cell.
  • the term "particle” relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds.
  • the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids, amphiphilic polymers, and/or amphiphilic proteins/polypeptides).
  • amphiphilic substance means that the substance possesses both hydrophilic and lipophilic properties.
  • the envelope may also comprise additional substances (e.g., additional lipids and/or additional polymers) which do not have to be amphiphilic.
  • the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids, amphiphilic polymers, and/or amphiphilic proteins/polypeptides) optionally in combination with additional substances (e.g., additional lipids and/or additional polymers) which do not have to be amphiphilic.
  • the term "particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure.
  • micro-sized means that all three external dimensions of the particle are in the microscale, i.e., between 1 and 5 pm.
  • the term "particle” includes lipoplex particles (LPXs), lipid nanoparticles (LNPs), polyplex particles, lipopolyplex particles, virus-like particles (VLPs), and mixtures thereof (e.g., a mixture of two or more of particle types, such as a mixture of LPXs and VLPs or a mixture of LNPs and VLPs).
  • LPXs lipoplex particles
  • LNPs lipid nanoparticles
  • VLPs virus-like particles
  • mixtures thereof e.g., a mixture of two or more of particle types, such as a mixture of LPXs and VLPs or a mixture of LNPs and VLPs.
  • a “nucleic acid particle” can be used to deliver nucleic acid (such as RNA, in particular mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid, a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (
  • Nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, liposome-based, polyplex-based, lipopolyplex-based, virus-like (VLP)-based formulations and mixtures thereof.
  • nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, and/or liposome-based formulations.
  • the cationic or cationically ionizable lipid and the polymer-conjugated compound as disclosed herein and, if present, additional lipids combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.
  • particles comprise an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and nucleic acid (such as RNA, especially mRNA) as described herein.
  • particles comprise or consist of a cationic/cationically ionizable lipid (in particular, a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein); helper lipids such as a neutral lipid (such as a phospholipid), and a steroid (such as cholesterol), and combinations thereof; and an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vb’), (Vb
  • V-23 (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35),
  • V-36 (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48),
  • V-49 (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61),
  • V-62 and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25).
  • the nucleic acid (such as RNA) particles disclosed herein comprise (i) nucleic acid (such as RNA, especially mRNA) as described herein; (ii) an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, (such as a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; a cationically ionizable lipid of formula (XI) disclosed herein; or a cationic lipid as disclosed herein); (iii) a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (
  • a steroid such as cholesterol
  • a neutral lipid such as a phospholipid
  • the steroid is cholesterol
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE.
  • the amphiphilic OEG- conjugated compound is a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V
  • V-20 (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32),
  • V-33 (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45),
  • V-46 (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58),
  • the steroid is cholesterol;
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and
  • the amphiphilic OEG-conjugated compound is a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (I
  • V-35 (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47),
  • V-48 (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60),
  • the nucleic acid such as RNA, in particular, mRNA
  • the nucleic acid is bound by cationically ionizable lipid (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein) that, in the case of LNPs, occupies the central core of the LNPs.
  • the polymer- conjugated compound forms the surface of the particles (such as LNPs), along with phospholipids.
  • the particles are substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units. In some embodiments, the particles are substantially free of PEG lipids having at least 30 consecutive ethylene glycol repeating units and substantially free of polysarcosine-conjugated lipids.
  • PEG polyethylene glycol
  • the particles are substantially free of polymer-conjugated lipids other than the polymer- conjugated compounds comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe
  • V-23 (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35),
  • V-36 (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48),
  • V-49 (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61),
  • the surface comprises a bilayer.
  • cholesterol and cationically ionizable lipid in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein
  • in charged and uncharged forms can be distributed throughout the particles such as LNPs.
  • a lipoplex is obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid (in particular RNA) and a phase comprising a dispersion of lipids.
  • the lipid phase comprises liposomes.
  • liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase.
  • a prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.
  • liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups.
  • cationic lipids employed in formulating liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.
  • lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with nucleic acids (such as DNAs or RNAs).
  • nucleic acids such as DNAs or RNAs.
  • formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid-lipoplexes.
  • these formulations are characterized by their poor encapsulation of the nucleic acid (such as DNA or RNA) and incomplete entrapment of the nucleic acid.
  • an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and nucleic acid (such as RNA, especially mRNA) as described herein.
  • electrostatic interactions between positively charged liposomes made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids
  • negatively charged nucleic acid such as RNA, especially mRNA
  • Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as a cationically ionizable lipid of formula (I), DOTMA and/or DODMA, and additional lipids, such as DSPC.
  • a nucleic acid (such as RNA, especially mRNA) lipoplex particle is a nanoparticle.
  • a lipid nanoparticle is obtainable from direct mixing of nucleic acid (such as RNA) in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol.
  • nucleic acid such as RNA
  • lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.
  • the lipids comprise a cationically ionizable lipid (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), a steroid as disclosed herein (such as cholesterol), a neutral lipid as disclosed herein (such as a phospholipid), and an amphiphilic OEG-conjugated compound as specified herein (e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (
  • V-12 (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24),
  • V-25 (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37),
  • V-38 (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)).
  • particles described herein comprise a cationically ionizable lipid as disclosed herein (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), a steroid as disclosed herein (such as cholesterol), a neutral lipid as disclosed herein (such as a phospholipid), and a polymer-conjugated compound as disclosed herein (z. e.
  • an amphiphilic OEG-conjugated compound as specified herein e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IX), (
  • nucleic acid particles (especially RNA particles such as RNA LNPs (e.g., mRNA particles such as mRNA LNPs)) comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features,
  • nucleic acid such as RNA, e.g., mRNA
  • nucleic acid may be noncovalently associated with a particle as described herein.
  • the nucleic acid such as RNA, especially mRNA
  • the nucleic acid may be adhered to the outer surface of the particle (surface nuclei acid (such as surface RNA, especially surface mRNA)) and/or may be contained in the particle (encapsulated nucleic acid (such as surface RNA, especially encapsulated mRNA)).
  • nanoparticle refers to a particle comprising nucleic acid (especially RNA such as mRNA) as described herein and at least one cationic lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm (preferably, between 10 and 990 nm, such as between 15 and 900 nm, between 20 and 800 nm, between 30 and 700 nm, between 40 and 600 nm, or between 50 and 500 nm).
  • the longest and shortest axes do not differ significantly.
  • the size of a particle is its diameter.
  • the particle has an average diameter suitable for intravenous administration.
  • Nucleic acid particles described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05.
  • PDI polydispersity index
  • the nucleic acid particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0. 1 to about 0.3.
  • lipoplex particle relates to a particle that contains an amphiphilic lipid, in particular cationic amphiphilic lipid, and nucleic acid (especially RNA such as mRNA) as described herein. Electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic amphiphilic lipids) and negatively charged nucleic acid (especially RNA such as mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic amphiphilic lipid, such as DOTMA, and additional lipids, such as DOPE or DSPC.
  • a nucleic acid (especially RNA such as mRNA) lipoplex particle is a nanoparticle.
  • lipid nanoparticle relates to a nano-sized lipid containing particle.
  • polyplex particle relates to a particle that contains an amphiphilic polymer, in particular a cationic amphiphilic polymer, and nucleic acid (especially RNA such as mRNA) as described herein. Electrostatic interactions between positively charged cationic amphiphilic polymers and negatively charged nucleic acid (especially RNA such as mRNA) results in complexation and spontaneous formation of nucleic acid polyplex particles. Positively charged amphiphilic polymers suitable for the preparation of polyplex particle include protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine and histone.
  • a nucleic acid (especially RNA such as mRNA) polyplex particle is a nanoparticle.
  • lipopolyplex particle relates to particle that contains amphiphilic lipid (in particular cationic amphiphilic lipid) as described herein, amphiphilic polymer (in particular cationic amphiphilic polymer) as described herein, and nucleic acid (especially RNA such as mRNA) as described herein.
  • a nucleic acid (especially RNA such as mRNA) lipopolyplex particle is a nanoparticle.
  • virus-like particle refers to a molecule that closely resembles a virus, but which does not contain any genetic material of said virus and, thus, is non-infectious.
  • VLPs contain nucleic acid (preferably RNA) as described herein, said nucleic acid (preferably RNA) being heterologous to the virus(es) from which the VLPs are derived.
  • VLPs can be synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. In one embodiment, combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs.
  • VLPs can be produced from components of a wide variety of virus families including Hepatitis B virus (HBV) (small HBV derived surface antigen (HBsAg)), Parvoviridae (e.g., adeno-associated virus), Papillomaviridae (e.g., HPV), Retroviridae (e.g., HIV), Flaviviridae (e.g., Hepatitis C virus) and bacteriophages (e.g. QP, AP205).
  • HBV Hepatitis B virus
  • HBsAg small HBV derived surface antigen
  • Parvoviridae e.g., adeno-associated virus
  • Papillomaviridae e.g., HPV
  • Retroviridae e.g., HIV
  • Flaviviridae e.g., Hepatitis C virus
  • bacteriophages e.g. QP, AP205

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Abstract

The present disclosure relates generally to the field of nucleic acid (such as DNA or RNA, in particular mRNA) compositions comprising an amphiphilic oligo ethylene glycol (OEG)-conjugated compound (as alternative to PEG lipids), to the use of such compositions, in particular for delivering nucleic acids to cells of a subject or in therapy, to such amphiphilic OEG-conjugated compounds, and to conjugates of such amphiphilic OEG-conjugated compounds.

Description

NUCLEIC ACID COMPOSITIONS COMPRISING AMPHIPHILIC OLIGO ETHYLENE GLYCOL (OEG)-CONJUGATED COMPOUNDS AND METHODS OF USING SUCH COMPOUNDS AND COMPOSITIONS
Technical Field
The present disclosure relates generally to the field of nucleic acid (such as DNA or RNA, in particular mRNA) compositions comprising an amphiphilic oligo ethylene glycol (OEG)-conjugated compound (as alternative to PEG lipids), to the use of such compositions, in particular for delivering nucleic acids to cells of a subject or in therapy, to such amphiphilic OEG-conjugated compounds, and to conjugates of such amphiphilic OEG-conjugated compounds.
Background
The use of a recombinant nucleic acid (such as DNA or RNA) for delivery of foreign genetic information into target cells is well known. A recombinant nucleic acid may be administered in naked form to a subject in need thereof; however, usually a recombinant nucleic acid is administered using a composition. For example, nucleic acid, such as RNA, may be delivered to a subject using different delivery vehicles, based mostly on cationic polymers or lipids which together with the nucleic acid form nanoparticles. The nanoparticles are intended to protect the nucleic acid, such as RNA, from degradation, enable delivery of the nucleic acid, such as RNA, to the target site and facilitate cellular uptake and processing by the target cells. The efficiency of the nucleic acid delivery depends, in part, on the molecular composition of the nanoparticle and can be influenced by numerous parameters, including particle size, formulation, and charge or grafting with molecular moieties, such as polyethylene glycol (PEG) or other ligands.
Grafting with PEG is considered to reduce serum interactions, to increase serum stability and to increase circulation time, which can be helpful for certain targeting approaches. Ligands which bind to receptors at the target site can help to improve targeting efficacy. Furthermore, PEGylation can be used for particle engineering. For example, if lipid nanoparticles (LNPs) are manufactured by mixing an aqueous phase of the nucleic acid, such as RNA, with an organic phase of the lipids a certain fraction of PEG- conjugated lipid (such PEG-conjugated lipids have at least 30 consecutive ethylene glycol repeating units) in the lipid mixture is required, otherwise the particles aggregate during or after the mixing step. It has been shown that by variation of the molar fraction of PEG-lipids comprising PEG at different molar masses the size of the particles can be adjusted. As well, the particle size may be adjusted by variation of the molar mass of the PEG moiety of the PEGylated lipids. Typical sizes which are accessible are in the range between 30 and 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). So-formed particles have additionally the advantage, that, due to the PEG fraction, they interact less with serum components, and have a longer circulation half-life, which is desirable in many drug delivery approaches. Without PEG-lipids, no particles with discrete size can be formed; the particles form large aggregates and precipitate. Thus, one of the primary roles of PEG-lipids is to facilitate particle self-assembly by providing a steric barrier at the surface of nascent particles formed when nucleic acids are rapidly mixed in ethanol solutions containing lipids to bind the nucleic acid, such as RNA. PEG steric hindrance prevents inter-particle fusion and promotes the formation of a homogeneous population of LNPs where diameters <100 nm can be achieved.
Despite these advantages, PEGylation of nanoparticles may lead as well to several effects which are detrimental to the intended use for drug delivery. PEGylation of liposomes and LNPs is known to reduce the cellular uptake and endosomal escape, thus reducing at the end the overall transfection efficiency. Indeed, the PEG shell provides a steric barrier to efficient binding of particles to the cell and also hinders endosomal release by preventing membrane fusion between the liposome and the endosomal membrane. This is why the type of PEG-lipid and the amount of PEG-lipid used must be always carefully adjusted. It should provide sufficient stealth effect for in vivo and stabilization aspects on the one hand, while not hindering transfection on the other. This phenomenon is known as the "PEG Dilemma".
Besides lowering transfection efficiency, PEGylation has been associated with accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and/or complement activation as well as storage diseases (Bendele A et al., 1998, Toxicolocical Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; S.M. Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection in rats of PEG-grafted liposomes may significantly alter the pharmacokinetic behavior of a second dose when this second dose is administered after an interval of several days (Laverman P et al., 2001, J. Pharmacol. Exp. Ther. 298(2), 607-12; Ishida et al., 2006, J. Control Release 115(3), 251-8). The phenomenon of "accelerated blood clearance" (ABC) appears to be related to the PEG content of liposomes. The presence of anti- PEG antibodies in the plasma induces a higher clearance of the particles by the Monophagocyte System (MPS) which at the end reduces the efficacy of the drug.
Due to broad use of PEGs as ingredient of food, cosmetic, hygienic products, and medicines, a certain percentage of the general population have "pre-existing" anti-PEG antibodies. The anti-PEG antibodies are associated with decreased efficacy of pegylated drugs and hypersensitivity reactions that can lead to severe allergic symptoms.
As PEG may induce immune responses there is a need to avoid it for certain applications where multiple injections are needed. Examples are therapies using nucleic acid (such as RNA, in particular mRNA), for example for protein replacement therapy. Here, the risk can be particularly high due to the potential intrinsic immunogenicity of nucleic acid (in particular RNA). Other examples are protein knock-down therapies using inhibitory RNA (such as siRNA), antisense oligonucleotides or DNA based therapies. Thus, there remains a need in the art for efficient compositions and methods for introducing nucleic acid, such as RNA, into cells which avoid the disadvantages accompanied by use of PEG. The present disclosure addresses this and other needs.
The inventors surprisingly found that the compositions, methods, polymer-conjugated compounds (which are also referred to as amphiphilic OEG-conjugated compounds herein), and conjugates described herein fulfill the above-mentioned requirements. In particular, it is demonstrated that the polymer-conjugated compounds described herein are not bound by antibodies raised against PEG structures and are stable under physiological conditions. The polymer component of the polymer- conjugated compounds as well as the polymer-conjugated compounds and their conjugates can be synthesized by well-known procedures, such as solid-phase peptide synthesis (SPPS). The polymer- conjugated compounds and conjugates can be end-group functionalized with different moieties to modulate charge or to introduce specific molecular moieties like ligands.
Summary
The present invention is defined by the appended claims.
In a first aspect, the present disclosure provides a composition comprising (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester, preferably optionally substituted amide, optionally substituted thioamide, or ester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100. This composition is also referred to as a nucleic acid composition herein.
As demonstrated in the present application, anti-PEG antibodies (polyclonal as well as IgG and IgM anti-PEG antibodies) which have been raised against PEG (i.e., having at least 30 consecutive ethylene glycol repeating units) and which bind to such PEG do not bind to a polymer comprising the structure of formula (I). Furthermore, the present application shows that a polymer comprising the structure of formula (I) is stable under physiological conditions. In some embodiments of the first aspect (in particular with respect to formula (I)), X2 and X1 taken together are an optionally substituted amide. Thus, in some embodiments, X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. In some embodiments, X1 is -NR1- and X2 is -C(O)-, wherein R1 is hydrogen or Cus alkyl.
In some embodiments of the first aspect (in particular with respect to formula (I)), X2 and X1 taken together are an optionally substituted thioamide. Thus, in some embodiments, X1 is -C(S)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. In some embodiments, X1 is -NR1- and X2 is -C(S)-, wherein R1 is hydrogen or C1-8 alkyl.
In some embodiments of the first aspect (in particular with respect to formula (I)), X2 and X1 taken together are an ester. Thus, in some embodiments, X1 is -C(O)- and X2 is -O. In some embodiments, X1 is -O- then X2 is -C(O).
In some embodiments of the first aspect (in particular with respect to formula (I)), X2 and X1 taken together are a thioester. Thus, in some embodiments, X1 is -C(S)- and X2 is -O-. In some embodiments, X1 is -O- then X2 is -C(S)-. In some embodiments, X1 is -C(O)- then X2 is -S-. In some embodiments, X1 is -S- then X2 is -C(O)-.
In some embodiments of the first aspect (in particular with respect to formula (I)), X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. For example, R1 may be hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments of the first aspect (in particular with respect to formula (I)), Y is -CH2- or -(CFFh-. In some embodiments, Y is -CH2-.
In some embodiments of the first aspect, the polymer comprises the following general formula (II): wherein R1 is hydrogen or Cus alkyl. In some embodiments of formula (II), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of the first aspect (in particular with respect to any one of formulas (I) and (II)), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of the first aspect, the polymer comprises the following general formula (III): wherein R1 is hydrogen or Cus alkyl. In some embodiments of formula (III), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of the first aspect, the polymer comprises the following general formula (IV):
In some embodiments of the first aspect, the polymer comprises the following general formula (IVa):
(IVa).
In some embodiments of the first aspect (in particular with respect to any one of formulas (I), (II), (III), (IV), and (IVa)), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the first aspect (in particular with respect to any one of formulas (I), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are located at either the X1 end or the X2 end of the polymer.
In some embodiments of the first aspect (in particular with respect to any one of Formulas (I), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are independently selected from non-cyclic, preferably straight, hydrocarbyl groups, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the hydrocarbyl groups have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl groups may be saturated or unsaturated. If the polymer-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different. For example, if the polymer-conjugated compound comprises two hydrophobic chains, in some embodiments said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated.
In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (V) or (V’): wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-;
R2 is a moiety comprising the one or more hydrophobic chains;
R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.
In some embodiments of formula (V) or (V’), X2 and X1 taken together are an optionally substituted amide. Thus, in some embodiments, X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. In some embodiments, X1 is -NR1- and X2 is -C(O)-, wherein R1 is hydrogen or Cus alkyl.
In some embodiments of formula (V) or (V’), X2 and X1 taken together are an optionally substituted thioamide. Thus, in some embodiments, X1 is -C(S)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. In some embodiments, X1 is -NR1- and X2 is -C(S)-, wherein R1 is hydrogen or Cus alkyl.
In some embodiments of formula (V) or (V’), X2 and X1 taken together are an ester. Thus, in some embodiments, X1 is -C(O)- and X2 is -O. In some embodiments, X1 is -O- then X2 is -C(O).
In some embodiments of formula (V) or (V’), X2 and X1 taken together are a thioester. Thus, in some embodiments, X1 is -C(S)- and X2 is -O-. In some embodiments, X1 is -O- then X2 is -C(S). In some embodiments, X1 is -C(O)- then X2 is -S-. In some embodiments, X1 is -S- then X2 is -C(O)-.
In some embodiments of formula (V) or (V’), X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. For example, R1 may be hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments of formula (V) or (V’), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-. In some embodiments of formula (V) or (V’), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of formula (V) or (V’), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of formula (V) or (V’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VI) or (VI’): wherein z, n, R2, and R3 are as defined for Formula (V) and (V’); and R1 is hydrogen or Cus alkyl.
In some embodiments of formula (VI) or (VF), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of formula (VI) or (VF), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of formula (VI) or (VF), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16. In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VII) or (VII’): wherein n, R2, and R3 are as defined for formula (V) and (V’); and R1 is hydrogen or Cus alkyl.
In some embodiments of formula (VII) or (VII’), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of formula (VII) or (VII’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VIII), (Villa), (VIII’), or (Villa’): wherein n, R2, and R3 are as defined for formula (V) and (V’).
In some embodiments of formula (VIII), (Villa), (VIII’), or (Villa’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R2 is R4 or -L^R^p, wherein each R4 is independently a hydrophobic chain, such as a hydrocarbyl group; L1 is a linker; and p is 1 or 2.
In some embodiments, L1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.
In some embodiments, L1 comprises a functional moiety selected from the group consisting of [*- C(O)O]p(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-, [*-NHC(O)]p(C1-6-alkylene)-, [*-C(O)NH]P(C1-6- alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6 -alkylene)-, [*-S(O)2]p(C1-6-alkylene)-, [(*- O)rC(OR25)3.r](C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*-C(R25)(=N-N(R26)C(O)-)]P(CI_6- alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p(C1-6-alkylene)-, [*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-, [*=C(=N(OH))]p(Cw-alkylene)-, [*-OC(R25)(R26)O]P(C1-6- alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-)pN(R26)2.p, and [*-C(O)NH](CI-6 -alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6 -alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25 is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments, L1 further comprises at least one additional difunctional moiety via which R2 is attached to either X1 in formula (V) (or to the carbonyl group of any one of formulas (VI), (VII), (VIII), and (Villa)) or X2 in formula (V’) (or the N atom of any one of formulas (VI’), (VII’), (VIII’), and (Villa’)). In some embodiments, the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1 further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6 -alkylene group from each other.
In some embodiments, L1 is selected from the group consisting of [*-C(O)O]p(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)- NR26-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*- OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)O-
P(O)(OR27)O(C1-6-alkylene)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*- NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)O-
P(O)(OR27)O(C1-6-alkylene)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-NR26-, [*- C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6- alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*- C(O)O]p(C1-6-alkylene), [*-OC(O)]p(C 1-6 -alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*- C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27 is selected from the group consisting of H, Cu 6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments, L1 is selected from the group consisting of [*-C(O)O]p(C 1-6 -alky lene)- OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*- C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(Cw-alkylene)-OP(O)(OR27)- O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(Ci-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(0)]P(CI-6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)-, [*-C(O)NH]p(C 1-6 -alky lene)OP(O)(OR27)O(C 1-6 -alkylene)NH-, [*-C(O)NH]p(C1-6- alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6- alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6-alkyltriyl)O- or L1 is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*- C(O)O]p(C1-6-alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*-C(O)NH]p(Ci- 6-alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen- 6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R2 is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(Ci.3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(Ci.3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(Ci.3-alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)- O(Ci.3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(Ci.3-alkylene)C(O)-,
[R4NHC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4NHC(O)]p(C2-3-alkylene)-
OP(O)(OR27)O(C1-3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)NH]p(C2-3-alkylene)-
OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(C1-3-alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O-, or R2 is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VF), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2 is a monoalkylamine moiety.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VF), (VII), (VIE), (VIII), (Villa), (Vlir), and (Villa’)), each of the one or more hydrophobic chains (i.e., each of R4) is independently a non-cyclic, preferably straight, hydrocarbyl group, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the one or more hydrocarbyl groups independently have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The one or more hydrocarbyl groups may be saturated or unsaturated. If the polymer-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different. For example, if the polymer-conjugated compound comprises two hydrophobic chains, in some embodiments said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated and/or said two hydrophobic chains differ in their length. Examples of the one or more hydrophobic chains include the hydrocarbyl chains of fatty acids, in particular the hydrocarbyl chains of naturally occurring fatty acids, such as the hydrocarbyl chains of naturally occurring fatty acids and having at least 8 carbon atoms. Specific examples the one or more hydrophobic chains include the hydrocarbyl chains of caprylic alcohol, capric alcohol, lauric alcohol, myristic alcohol, palmitic alcohol, stearic alcohol, arachidic alcohol, behenic alcohol, lignoceric alcohol, cerotic alcohol, oleic alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (VIII’), and (Villa’)), R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2 is a monomyristylamine moiety.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R3 is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair. In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(0)NH2, -C(0)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair.
In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody (including fragments or derivatives thereof) specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine -glycine -aspartic acid (RGD) peptide - avf>3 integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor.
In some embodiments of the first aspect, the polymer-conjugated compound has one of the following wherein n is 5 to 25; R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH,
-COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair; R27 is H or a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); and in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)I6CH3 (stearoyl), in each case -C(O)Ci5H3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)CI3H22 refers to the moiety -C(O)(CH2)I2CH3 (myristoyl), in each case -Ci4H29 refers to the moiety -(CH2)I3CH3 (myristyl), in each case -CI3H22 refers to the moiety -(CH2)I2CH3, and in each case -C(O)CI?H33 refers to the moiety -czs-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). In some embodiments, n is 7 to 16, e.g., 7 to 14 (preferably 8, 10, 12, 14, or 16); and/or R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, C2-6 alkynyl, an antigen, and an antibody.
Further preferred embodiments of the polymer-conjugated compound (in particular with respect to any one of formulas (I), (II), (III), (IV), (IVa), (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (VIII’), and (Villa’)) are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains" . In some preferred embodiments of the first aspect, the polymer-conjugated compound has any one of formulas (V-l), (V-5), (V-17), and (V-25).
In some embodiments of the first aspect, the composition is substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units.
In some embodiments of the first aspect, the composition is also substantially free of another polymer- conjugated lipid. In some embodiments, the another polymer-conjugated lipid is a polysarcosine- conjugated lipid and/or a conjugate comprising hydrophobic chains and a polyoxazoline (POX) and/or polyoxazine (POZ) polymer.
In some embodiments of the first aspect, water is the main component in the composition and/or the total amount of solvent(s) other than water contained in the composition is less than about 1.0% (v/v), such as less than about 0.5% (v/v). For example, the amount of water contained in the composition may be at least 50% (w/w), such as at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), at least 90% (w/w), or at least 95% (w/w). In particular, if the composition comprises a cryoprotectant, the amount of water contained in the composition may be at least 50% (w/w), such as at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), or at least 90% (w/w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition may be at least 95% (w/w). Additionally, or alternatively, the total amount of solvent(s) other than water contained in the composition may be less than about 0.5% (v/v), such as less than about 0.4% (v/v), less than about 0.3% (v/v), less than about 0.2% (v/v), less than about 0.1% (v/v), less than about 0.05% (v/v), less than about 0.01% (v/v), or less than about 0.005% (v/v). In this respect, a cryoprotectant which is liquid under normal conditions will not be considered as a solvent other than water but as cryoprotectant. In other words, the above optional limitation that the total amount of solvent(s) other than water contained in the composition may be less than about 0.5% (v/v), such as less than about 0.4% (v/v), does not apply to cryoprotectants which are liquids under normal conditions.
In some embodiments of the first aspect, the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg/1 to about 500 mg/1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg/1 to about 100 mg/1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 5 mg/1 to about 500 mg/1, such as about 10 mg/1 to about 400 mg/1, about 10 mg/1 to about 300 mg/1, about 10 mg/1 to about 200 mg/1, about 10 mg/1 to about 150 mg/1, or about 10 mg/1 to about 100 mg/1, preferably about 10 mg/1 to about 140 mg/1, more preferably about 20 mg/1 to about 130 mg/1, more preferably about 30 mg/1 to about 120 mg/1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 5 mg/1 to about 150 mg/1, such as about 10 mg/1 to about 140 mg/1, about 20 mg/1 to about 130 mg/1, about 25 mg/1 to about 125 mg/1, about 30 mg/1 to about 120 mg/1, about 35 mg/1 to about 115 mg/1, about 40 mg/1 to about 110 mg/1, about 45 mg/1 to about 105 mg/1, or about 50 mg/1 to about 100 mg/1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is 1 mg/1 to about 50 mg/1 or about 10 mg/1 to about 100 mg/1.
In some embodiments of the first aspect, the composition comprises a cryoprotectant. In some embodiments of the first aspect, the composition is substantially free of a cryoprotectant.
In some embodiments of the first aspect, the cationically ionizable lipid comprises a head group which includes at least one tertiary amine moiety.
In some embodiments of the first aspect, the cationically ionizable lipid has the structure of Formula (X) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L10, L20, G1, G2, G3, R35, R36, and R37 are as defined herein. In some embodiments, the cationically ionizable lipid is selected from the following: the structures X-l to X-36 (shown herein); or the structures A to G (shown herein). In some embodiments, the cationically ionizable lipid is the lipid having the structure X-3. In some embodiments, the cationically ionizable lipid is DPL-14 (i.e., the lipid having the structure G). In some embodiments of the first aspect, the cationically ionizable lipid has the structure of Formula (XI): wherein Ri, R2 R3, R4, L2, G2, and m are as defined herein. In some embodiments, the cationically ionizable lipid is selected from the structures (XIV-1), (XIV-2), and (XIV-3) (shown herein). In some embodiments, the cationically ionizable lipid is the lipid having the structure XIV-1 In some embodiments, the cationically ionizable lipid is the lipid having the structure XIV-2. In some embodiments, the cationically ionizable lipid is the lipid having the structure XIV-3.
In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises 2,3- dioleyloxy-l-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)- N,N,N -trimethylammonium chloride (DOTAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.
In some embodiments of the first aspect, the cationically ionizable lipid is completely or partially replaced by a cationic lipid. In some embodiments, the cationic lipid is selected from the structures XV- 1 to XV -6 (shown herein).
In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol of the total lipid present in the composition.
In some embodiments of the first aspect, the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of a phospholipid and a steroid.
In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1- oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl- phosphatidylethanolamine (DPyPE).
In some embodiments, the phospholipid comprises from about 5 mol % to about 30 mol % of the total lipid present in the composition.
In some embodiments, the steroid comprises a sterol. In some preferred embodiments, the steroid comprises or is cholesterol.
In some embodiments, the steroid comprises from about 10 mol % to about 60 mol % of the total lipid present in the composition.
In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 70 mol % of the total lipid present in the composition; the polymer-conjugated compound (amphiphilic OEG-conjugated compound) comprises from about 0.5 mol % to about 15 mol % (such as from about 2 mol % to about 6 mol % or from about 2 mol % to about 5 mol %) of the total lipid present in the composition; the phospholipid comprises from about 5 mol % to about 25 mol % of the total lipid present in the composition; and the steroid comprises from about 20 mol % to about 55 mol % of the total lipid present in the composition.
In some embodiments of the first aspect, the composition further comprises one or more additional lipids. For example, the one or more additional lipids may comprise a cationic lipid. In these embodiments, where a cationic lipid is present, the sum of (1) the amount the cationically ionizable lipid and (2) the amount of cationic lipid is used for calculations. E.g., if the amount of cationically ionizable lipid in a composition should be from about 20 mol % to about 70 mol % and the composition should also contain a cationic lipid, then the sum of (1) the amount the cationically ionizable lipid and (2) the amount of cationic lipid is to be from about 20 mol % to about 70 mol %.
In some embodiments of the first aspect, the only lipids contained in the composition are the cationic or cationically ionizable lipid, the steroid, the neutral lipid, and the polymer-conjugated compound comprising the polymer of formula (I) as defined herein (i.e., the amphiphilic OEG-conjugated compound), in particular the cationic or cationically ionizable lipid, the steroid, the phospholipid, and the polymer-conjugated compound comprising the polymer of formula (I) as defined herein.
In some embodiments of the first aspect, the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the cationic or cationically ionizable lipid, and at least a portion of the polymer-conjugated compound comprising the polymer of formula (I) as defined herein. In some embodiments, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. In some embodiments, the particles comprise or are LNPs. In some embodiments, the particles comprise or are liposomes. In some embodiments, the particles comprise or are LPXs. In some embodiments, the particles comprise or are mixtures of LNPs and liposomes. In some embodiments, the particles comprise or are mixtures of LNPs and LPXs. In some embodiments, the particles comprise or are mixtures of liposomes and LPXs. In some embodiments, the particles comprise or are mixtures of LNPs, liposomes, and LPXs.
In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the particles comprise essentially all of lipids, (in particular all of the cationic or cationically ionizable lipid, the one or more additional lipids, if present, and the and the polymer- conjugated compound comprising the polymer of formula (I) as defined herein) present in the composition.
In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the particles comprise at least 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acid (in particular RNA) present in the composition. In some embodiments, the particles comprise at least 75%, preferably at least 85% of the nucleic acid (in particular RNA) present in the composition.
In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of the nucleic acid.
In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the nucleic acid (such as RNA) is encapsulated within or associated with the particles. In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the particles have a size of from about 30 nm to about 500 nm. In some embodiments, the particles have a size from about 50 nm to about 150 nm.
In some embodiments of the second aspect, the nucleic acid is DNA.
In some embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA, (e.g., siRNA).
In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and (i) comprises a modified nucleoside in place of uridine; (ii) has a coding sequence which is codon-optimized; and/or (iii) has a coding sequence whose G/C content is increased compared to the wild-type coding sequence. In some embodiments, the modified nucleoside is selected from pseudouridine (y), N1 -methyl - pseudouridine (ml\|/), and 5 -methyl -uridine (m5U).
In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and comprises at least one or more of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly-A sequence. In some embodiments, the RNA (such as mRNA) comprises all of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence preferably is an interrupted sequence of A nucleotides. In some embodiments, the 5’ cap is a capl or cap2 structure.
In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and encodes one or more polypeptides. In some embodiments, the one or more polypeptides are pharmaceutically active polypeptides and/or comprise an epitope for inducing an immune response against an antigen in a subject.
In some embodiments of the first aspect, the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof. In some embodiments, the pathogen is a pathogen causing an infectious disease.
In some embodiments of the first aspect, the nucleic acid is inhibitory RNA (such as siRNA) and selectively hybridizes to and/or is specific for a target mRNA. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, in particular a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.
In a second aspect, the present disclosure relates to a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect. It is understood that any embodiment described herein in the context of the first aspect may also apply to any embodiment of the second aspect.
In a third aspect, the present disclosure relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described herein in the context of the first or second aspect may also apply to any embodiment of the third aspect.
In a fourth aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. In a related aspect, the present disclosure relates to a nucleic acid composition of the first aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, or third aspect may also apply to any embodiment of the fourth aspect.
In a fifth aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. In a related aspect, the present disclosure relates to a nucleic acid composition of the first aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, or fourth aspect may also apply to any embodiment of the fifth aspect.
In some embodiments of the second to fifth aspect, the subject is a mammal, such as a human. In a sixth aspect, the present disclosure provides a polymer-conjugated compound (also referred to herein as amphiphilic OEG-conjugated compound) comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains. Preferred embodiments of the polymer- conjugated compound of the sixth aspect are specified in the first aspect and are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains".
In a seventh aspect, the present disclosure provides conjugate of (a) the polymer-conjugated compound of the sixth aspect containing a member of a targeting pair; and (b) a compound comprising the other member of the targeting pair. In some embodiments, the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (such as an antigen or epitope), or an antibody. In some embodiments of the seventh aspect, the conjugate has one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen. In some embodiments of the seventh aspect, the conjugate has one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, r 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)i6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen. In some embodiments of the seventh aspect, the polymer-conjugated compound containing a member of a targeting pair has the following formula: wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety - C(O)(CH2)I6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen; the compound comprising the other member of the targeting pair is a compound comprising (i) an antibody specific for said antigen if Pept is said antigen; or (ii) an antigen if Pept is an antibody specific for said antigen; and the polymer-conjugated compound containing a member of a targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) said antibody specific for said antigen and (2) said antigen.
It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth or sixth aspect may also apply to any embodiment of the seventh aspect.
In an eighth aspect, the present disclosure provides a composition comprising (i) nucleic acid (such as DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of the seventh aspect.
It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, or seventh aspect may also apply to any embodiment of the eighth aspect.
In a ninth aspect, the present disclosure relates to a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a composition of the eighth aspect. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect may also apply to any embodiment of the ninth aspect.
In a tenth aspect, the present disclosure relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth aspect may also apply to any embodiment of the tenth aspect. In an eleventh aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. In a related aspect, the present disclosure relates to a composition of the eighth aspect for use in a method for treating or preventing a disease or disorder in a subj ect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect may also apply to any embodiment of the eleventh aspect.
In a twelfth aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. In a related aspect, the present disclosure relates to a composition of the eighth aspect for use in a method for treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspect may also apply to any embodiment of the twelfth aspect.
In a thirteenth aspect, the present disclosure provides a method of transfecting cells, comprising adding a composition of the first or eighth aspect to cells; and incubating the mixture of the composition and cells for a sufficient amount of time. In some embodiments, in particular those, where the nucleic acid is DNA or RNA (such as mRNA) and encodes a pharmaceutically active protein, the mixture of the composition and cells is incubated for a time sufficient to allow the expression of the pharmaceutically active protein. In some embodiments, in particular those, where the nucleic acid is inhibitory RNA (such as siRNA) directed against a target mRNA, the mixture of the composition and cells is incubated for a time sufficient to allow the inhibition of the transcription and/or translation of the target mRNA. In some embodiments, the sufficient amount of time is at least one hour (such at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and/or up to about 48 hours (such as up to about 36 or up to about 24 hours).
In some embodiments of the thirteenth aspect, the method is conducted in vivo (i.e., the cells form part of an organ, a tissue and/or an organism of a subject). In some embodiments of the thirteenth aspect, the method is conducted in vitro (i.e., the cells do not form part of an organ, a tissue and/or an organism of a subject, e.g., the cells are an ex vivo cell culture). It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth aspect may also apply to any embodiment of the thirteenth aspect.
In a fourteenth aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate of the seventh aspect or a composition of the first or eighth aspect. In some embodiments, the pharmaceutical composition further comprises one or more of pharmaceutically acceptable carriers, diluents and excipients. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect may also apply to any embodiment of the fourteenth aspect.
In a further aspect, the present disclosure provides a kit comprising a composition of the first or eighth aspect, a polymer-conjugated compound (amphiphilic OEG-conjugated compound) of the sixth aspect, a conjugate of the seventh aspect, or a pharmaceutical composition as described herein (such as a pharmaceutical composition of the fourteenth aspect). In some embodiments, the kit is for use in therapy, such as for inducing an immune response. In some embodiments, the kit is for use in inducing an immune response against a pathogen, such as for treating or preventing an infectious disease.
In a further aspect, the present disclosure provides a method for preparing an amphiphilic OEG- conjugated compound comprising the following steps: (a) providing an intermediate compound having the formula (VII) or (VIE) as disclosed herein, wherein, for formula (VII), R2 is OH, and R3 is H, acetyl, or Fmoc; and, for formula (VIE), R2 is H, acetyl, or Fmoc, and R3 is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, in particular a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, thereby obtaining the amphiphilic OEG-conjugated compound. Preferred embodiments of this aspect are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains". It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect may also apply to any embodiment of this aspect of a method for preparing an amphiphilic OEG-conjugated compound. Brief description of the Figures
Figure 1: Scheme of the anti-PEG ELISA assay used for determining the binding of anti -PEG polyclonal antibodies to different antigens (PEG, AEEA, pSAR). Biotin-labeled antigens (biotin- PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NEE-pSAR) were synthesized and captured on Neutravidin-coated plates in wash buffer (IxPBS, 0.1% CHAPS) for 2 h at RT and under slow shaking. After washing with wash buffer (4x), the plates were incubated with different amounts of rabbit anti-PEG polyclonal sera (7 ng/ml, 3 ng/ml, 167 ng/ml, or 830 ng/ml) in assay buffer (IxPBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT and under slow shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody was added (diluted at 1:5000) and the plates were incubated for 1 h at RT and under slow shaking. After washing with wash buffer (5x), HRP substrate was added and the fluorescence signal was measured.
Figure 2: Results of the anti-PEG ELISA assay of Figure 1 for different biotin-labeled antigens (biotin- PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NH2-pSAR).
Figure 3: Results of a further anti-PEG ELISA assay using monoclonal anti PEG IgG (A) or monoclonal anti PEG IgM (B) and different antigens (PEG36, capped-AEEA14, NH2-AEEA14). The PEG36 had a molecular weight of about 1.6 kDa. The capped AEEA14 was Ac-AEEA14.
Figure 4: Stability of pAEEA under physiological conditions. Ac-AEEA14 (A) and NH2-AEEAI4 (B) were incubated in mouse or human plasma for 0 h to 72 h.
Figure 5: Particle size and PDI of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Particle size and PDI of BM LNPs formulation are also included.
Figure 6: Zeta potential of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Zeta potential of BM formulations is also included.
Figure 7: Accessible RNA of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 8: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
Figure 9: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Figure 10: Viability of all tested formulations after transfection with DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. The vability of BM_2 and other PEG containing formulations are also included. Plotted are mean values (n=3) +/- StDev.
Figure 11: Particle size and PDI of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Particle size and PDI of BM LNPs formulation are also included.
Figure 12: Zeta potential of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Zeta potential of BM formulations is also included.
Figure 13: Accessible RNA of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 14: Quantification of SI protein expression after transfection with VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. (A) Variation in VE-AEEA14-AC composition vs. MFI (Mean Fluorescence Intensity) of the overall cell population. Data was fitted using a quadratic polynomial function. (B) Viability of all tested formulations. The MFI and viability of BM_2 and other PEG containing formulations are also included. Plotted are mean values (n=3) +/- StDev.
Figure 15: Yield and purity of peptide intermediates. Peptide intermediates Ac-(AEEA)8-OH (A-D) and Ac-(AEEA)i4-OH (E-H) were synthesized and their yields and purities were determined using UPLC (A, B, E, F) and mass spectrometry (C, D, G, H). Samples for UPLC and mass spectrometry were taken either directly after cleavage from the resin, i.e., prior to the QC method (crude purities; Figures 15 A, C, E, G), of after the QC method (final purities; Figures 15 B, D, F, H).
Figure 16: Monitoring of the synthesis of Ac-(AEEA)8-a-tocopherol. Samples of the reaction were taken and analyzed using the methods described herein. UPLC chromatograms are shown for samples taken (A) after 5 minutes and (B) at the end of the reaction time (4 h).
Figure 17: Purity of the amphiphilic compounds Ac-(AEEA)8-a-tocopherol (A, B), Ac-(AEEA)i4-a- tocopherol (C, D), AC-(AEEA)I4-DMA (E, F), Ac-(AEEA)8-DMG (G, H), and AC-(AEEA)I4-DSPE (I, J) as shown by exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J), respectively. Figure 18: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). Particle size and PDI of BM LNPs formulation are also included.
Figure 19: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). Zeta potential of BM formulations is also included.
Figure 20: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 21: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
Figure 22: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 23: Quantification of SI protein expression after transfection with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). (A) Variation in composition vs. MFI (Mean Fluorescence Intensity) of the overall cell population. Data was fitted using a quadratic polynomial function. (B) Viability of all tested formulations. The MFI and viability of BM_1 and BM_2 and other control formulations are also included. Plotted are mean values (n=3) +/- StDev.
Figure 24: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 25: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 26: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 27: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 27A, B), a murine macrophage cell line (Raw) (Figures 27C, D), and a hepatocarcinoma cell line (HepG2) (Figures 27E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) +/- StDev.
Figure 28: T-cell targeting using LNPs with different stealth lipids and Alfa lipids. LNPs were formulated with different lipid compositions (Cargo: Thy 1.1 RNA/Luc RNA/Np proxy Venus 1: 1:2 w/w; N/P ratio: 6; Lipid mix: HY501/Cholesterol/DSPC/stealth lipid/Alfa lipid. The lipid ratio has been selected as [47.5/40.5/10/1.8/0.2] for the following combinations of stealth lipid and Alfa lipid: C16 PEG2k Ceramide/DSPE PEG2k Alfa, DSPE PEG2k/DSPE PEG2k Alfa, DSPE-AEEA14/DSPE- AEEA14-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa. The lipid ratio has been selected as [47.5/38.5/10/3.8/0.2] for the following combinations of stealth lipid and Alfa lipid: VE-PEGlk/DSPE- PEG2k-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa. The LNPs were equipped with aCD3 VHH X NbAlfa ligand via post functionalization [w/w* = ligand to cargo ratio 0.48]; RNA concentration: 0.1 pg/pl). Diameter of all LNPs is between 100 to 170 nm with a PDI below 0.4 as determined via DLS measurement.
For transfection studies, 10 pl (1000 ng dose) of respective formulations were pre-diluted in 50 pl X- Vivo 15 in an ultra-low adhesion 96 well plate. 106 thawed human PBMC were diluted in 50 pl of 100% clotted PHS and added to nanoparticle dilution. After 30 min of incubation (37°C, 5% CO2) 30 pl of each transfection reaction was transferred to second ultra-low adhesion 96 well plate and 170 pl of X- Vivo 15 medium + 100 U/ml IL2 was added per well. Cell dilutions were cultivated for additional 18 h (37°C, 5% CO2). In the following cell-type specific transfection (Thy 1.1) was analyzed via flowcytometry. Depicted are the percentages of transfected cell (CD2 negative cells, CD 19+ B cells, CD4+ T cells and CD8+ T cells) within all transfected PBMCs (Transfection, y-axes) per tested formulation condition.
Figure 29: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 30: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 31: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B). Figure 32: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
Figure 33: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 34: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 34A, B), a murine macrophage cell line (Raw) (Figures 34C, D), and a hepatocarcinoma cell line (HepG2) (Figures 34E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) +/- StDev.
Figure 35: Particle size and PDI of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 36: Zeta potential of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 37: Accessible RNA of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 38: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
Figure 39: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 40: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 40A, B), a murine macrophage cell line (Raw) (Figures 40C, D), and a hepatocarcinoma cell line (HepG2) (Figures 40E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA- loaded LNPs. Plotted are mean values (n=3) +/- StDev. Figure 41: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 42: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 43: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.
Figure 44: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).
Figure 45: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 45A, B), a murine macrophage cell line (Raw) (Figures 45C, D), and a hepatocarcinoma cell line (HepG2) (Figures 45E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) +/- StDev.
Figure 46: Particle size and PDI of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
Figure 47: Zeta potential of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
Figure 48: Accessible RNA of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).
Figure 49: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS. Figure 50: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).
Figure 51 : In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing an amphiphilic OEG- conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) in skeletal muscle cell line (C2C12) (Figures 51A, B), a murine macrophage cell line (Raw) (Figures 51C, D), and a hepatocarcinoma cell line (HepG2) (Figures 5 IE, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) +/- StDev.
Figure 52: Particle size and PDI of functionalized LNPs prepared with 5 different OEG-conjugated compounds as stealth lipids.
Figure 53: Agarose gel electrophoresis of controls, untreated functionalized LNPs (upper row) and functionalized LNPs treated with release solution (lower row).
Figure 54: Particle size and PDI of functionalized LNPs prepared respectively with OEG-conjugated compounds ((A) C14-pAEEA14-Ac, (B) DSPE-pAEEA14-Ac, (C) VitE-pAEEA14-Ac, (D) DMA- pAEEA14-Ac and (E) VitE-pAEEA8-Ac) subjected to three freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.
Figure 55: In vitro transfection with LNPs. A) The percentages of transfected cells (CD 14+ Monocytes, CD 19+ B cells, CD4+ T cells or CD8+ T cells) within all transfected PBMCs (Transfection, y-axes) per tested formulation. B) Total cell counts acquired by flow cytometry within 20 seconds. Numbers are corrected relative to counting beads. BD FACS Lyric was used for sample acquisition. FlowJo was used for data analysis.
Figure 56: Ligand-mediated transfection of T cells in vivo. Intramuscular injection of naked or LNP- formulated luciferase- and Thy 1.1 -encoding RNA mixtures (1 : 1 weight-to-weight mix) in B6- hCD3EDG transgeneic mice (1 pg RNA dose per injection side; 2 pg total RNA dose per mouse). Analysis performed 18h after injection. (A) Drainage analyzed via ex vivo bioluminescence imaging of popliteal, inguinal, axially and brachial lymph nodes, and spleens. (B) Cell-type specific transfection analyzed by flow-cytometry via detection of delivered Thy 1. 1 RNA expression in immune cell subtypes within the popliteal and inguinal lymph nodes, and spleens. (C) T-cell activation status analyzed via mean fluorescent intensity of CD69 surface expression within depicted organ (following staining with anti-CD69-APC antibody). LN, lymph nodes; LNP, lipid nanoparticle; NK, natural killer; PMN, polymorphonuclear cells.
Description of the sequences
The following table provides a listing of certain sequences referenced herein.
Table 1 : Description of the sequences
Detailed Description
Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser/Heathcook, "Organische Chemie", VCH, 1990; Beyer/Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey/Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rbmpp Chemie Lexikon, Falbe/Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Definitions
In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of' means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of'. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of' or "consisting of'. Likewise, at each occurrence in the present application, the term "consisting essentially of' may be replaced with the term "consisting of'.
The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.
Where used herein, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and/or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein. In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±5%, such as ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.
Terms such as "enhance" and "increase" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level. In some embodiments, these terms relate to an increase or enhancement by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.
"Physiological pH" as used herein refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.
"Physiological conditions" as used herein refer to the conditions (in particular pH and temperature) in a living subject, in particular a human. Preferably, physiological conditions mean a physiological pH and/or a temperature of about 37°C.
As used in the present disclosure, "% (w/v)" (or "% w/v") refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (ml).
As used in the present disclosure, "% by volume" or "% (v/v)" (or "% v/v") refers to volume percent, which is a unit of concentration measuring the amount of a liquid substance in milliliters (ml) expressed as a percent of the total volume of solution in milliliters (ml). As used in the present disclosure, "% by weight" or "% (w/w)" (or "% w/w") refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).
As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.
As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like material.
The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum E is taken over all the different kinds of ions (i) in solution.
According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions.
Regarding the presence of divalent inorganic ions, in particular divalent inorganic cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is in one embodiment sufficiently low so as to prevent degradation of the RNA. In one embodiment, the concentration or effective concentration of divalent inorganic ions is below the catalytic level for hydrolysis of the phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent inorganic ions is 20 pM or less. In one embodiment, there are no or essentially no free divalent inorganic ions.
A "monovalent" compound relates to a compound having only one functional group of interest. For example, a monovalent anion relates to a compound having only one negatively charged group, preferably under physiological conditions. A "divalent" or "dibasic" compound relates to a compound having two functional groups of interest. For example, a dibasic organic acid has two acid groups. An example of a divalent cation is Ca2+.
A "polyvalent" or "polybasic" compound relates to a compound having three or more functional groups of interest. For example, a polybasic organic acid has three or more acid groups.
A "monovalent moiety" relates to a monoradical, i.e., a moiety having a valence of 1. Typical monovalent moieties include alkyl, alkenyl, aryl, etc.
A "divalent moiety" or "bivalent moiety" relates to a diradical, i.e., a moiety having a valence of 2. Typical divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, etc. A further example of a divalent moiety is the C1-6-alkylene moiety in the group [* -S]p(C1-6 -alkylene)-, if p is 1 (resulting in the group *-S(C1-6-alkylene)-, such as *-S-(CH2)g- or *-S-CH2-, wherein * represents the attachment point to R4).
A "polyvalent moiety" relates to a polyradical, i.e., a moiety having a valence of at least 3. E.g., a "trivalent moiety" relates to a triradical, i. e., a moiety having a valence of 3. For example, by removing a further H atom of an alkylene group the resulting alkylene is trivalent. A further example of a trivalent moiety is the C1-6-alkylene moiety in the group [*-S]p(C1-6-alkylene)-, if p is 2 (resulting in the group [*-S]2(C1-6-alkylene)-, such as *-S-CH(S-*)(CH2)s- or *-S-CH(S-*)(CH2)-, wherein * represents the attachment point to R4). Another example of a trivalent moiety is the C1-6 -alkyltriyl moiety in the group [*-C(O)NH](C1-6-alkyltriyl)- (here the [*-C(O)NH] moiety as well as a further hydrophobic chain are bound to the C1-6 -alkyltriyl moiety which, in turn, is attached to X1 (for formula (V)) or to X2 (for formula (V’)), either directly or through at least one additional difunctional moiety). Thus, a [*-C(O)NH](C1-6- alkyltriyl) moiety as an example for L1, wherein C1-6 -alkyltriyl is directly attached to another hydrophobic chain R4, may comprise at least the following structures: wherein one of ww represents the bond by which the C1-6 -alkyltriyl is attached to [*-C(O)NH] and the other ww represents the bond by which the C1-6-alkyltriyl is attached (either directly or via an additional difunctional moiety) to the remainder of the compound (e.g., the compound of formula (V)). In case the C1-6 -alkyltriyl is also substituted with one -OH moiety, at least the following structures are encompassed: "Molar ratio", as used herein, refers to the ratio between the amounts in moles of any two substances. For example, if a first substance is present in a composition in an amount of 1 millimole (mmol) and a second substance is present in the composition in an amount of 2 millimole (mmol), the molar ratio of the first substance to the second substance is 1:2 or 0.5.
"Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.
The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used herein interchangeably.
The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.
The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.
The term "freezing" relates to the solidification of a liquid, usually with the removal of heat. In some embodiments, freezing is reverse action to thawing.
The term "thawing" relates to the liquification of a solid, usually with the addition of heat. In some embodiments, thawing is reverse action to freezing.
The term "aqueous phase" as used herein in relation to a composition/formulation comprising particles, in particular LNPs, liposomes, and/or lipoplexes, means the mobile or liquid phase, i.e., the continuous water phase including all components dissolved therein but (formally) excluding the particles. Thus, if particles, such as LNPs, are dispersed in an aqueous phase and the aqueous phase is to be substantially free of compound X, the aqueous phase is free of X is such manner as it is practically and realistically feasible, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight. However, it is possible that, at the same time, the particles dispersed in the aqueous phase may comprise compound X in an amount of more than 1% by weight. The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.
The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and/or isolated from nature, but that may be discovered and/or isolated in the future from a natural source.
As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, preferably from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.
The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, abbreviated as C1-12 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as C1-10 alkyl), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1- methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl- propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n- decyl, n-undecyl, n-dodecyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein. Examples of a substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.
The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1 -ethylene, 1,2-ethylene), propylene (i.e., 1,1 -propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2- propylene (-C(CH3)2-), and 1,3-propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2- butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1 -pentylene, 1,2-pentylene, 1,3- pentylene, 1,4-pentylene, 1,5 -pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6- hexylene, and 1,1 -isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3- heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6- octylene, 1,7-octylene, 1,8-octylene, and 1,1 -isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 12, abbreviated as C2-12 alkenyl, (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1- propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5 -hexenyl, 1 -heptenyl, 2-heptenyl, 3 -heptenyl, 4- heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7- octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1- decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1- undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8- undecenyl, 9-undecenyl, 10-undecenyl, 1 -dodecenyl, 2-dodecenyl, 3 -dodecenyl, 4-dodecenyl, 5- dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carboncarbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds. A "substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2,
3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen- 1,2-diyl, vinylidene (also called ethenylidene), 1 -propen- 1,2-diyl, 1 -propen- 1,3 -diyl, 1 -propen-2,3 -diyl, allylidene, 1-buten- 1,2-diyl, l-buten-l,3-diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, 1- buten-3,4-diyl, 2-buten- 1,2-diyl, 2-buten-l,3-diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to
4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "cycloalkylene" represents cyclic non-aromatic versions of "alkylene" and is a geminal, vicinal or isolated diradical. In certain embodiments, the cycloalkylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and/or (ii) is 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered). In one embodiment the cycloalkylene is a mono-, bi- or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkylene. Generally, instead of using the ending "ylene" for cycloalkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-cyclopropylene can also be called cyclopropan-l,2-diyl). Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g., tricyclo[3.3. 1. l3’7]decan-2,2- diyl). A "substituted cycloalkylene " means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "cycloalkenylene" represents cyclic non-aromatic versions of "alkenylene" and is a geminal, vicinal or isolated diradical. Generally, the maximal number of carbon-carbon double bonds in the cycloalkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the cycloalkenylene group by 2 and, if the number of carbon atoms in the cycloalkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an cycloalkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. In certain embodiments, the cycloalkenylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and/or (ii) is 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered). In one embodiment the cycloalkenylene is a mono-, bi- or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkenylene. Exemplary cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. A "substituted cycloalkenylene " means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. A "substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein. Examples of a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (z.e., 2-, 3-, or 4-methoxyphenyl).
The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include fiiranyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein.
The term "heterocycloalkylene" as used herein means a heterocyclyl group as defined above which contains at least one ring heteroatom (such as those selected from the group consisting of O, S, N, B, Si, and P) and in which one hydrogen atom has been removed resulting in a geminal, vicinal or isolated diradical. In some embodiments, the heteroatoms of the heterocycloalkylene group are selected from the group consisting of O, S, and N. For example, the heterocycloalkylene may be O/S-heterocycloalkylene, such as O-heterocycloalkylene. In some embodiments, in each ring of the heterocycloalkylene group the maximum number of O atoms is 1 , the maximum number of S atoms is 1 , and the maximum total number of O and S atoms is 2. The heterocycloalkylene may be monocyclic or polycyclic (such as bi- or tricyclic). In some embodiments, the heterocycloalkylene is a mono-, bi- or tricyclic 4- to 14-membered (i.e., 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 4- to 12-membered or 4- to 10-membered) heterocycloalkylene. The term "heterocycloalkylene" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups (preferably partially or completely hydrogenated forms of the above-mentioned mono-, bi-, or tricyclic heteroaryl groups) in which one hydrogen atom has been removed from the same carbon atom resulting in a geminal diradical. Thus, in some embodiments, a heterocycloalkylene is saturated or unsaturated (i.e., it contains one or more double bonds within the ring) but cannot be aromatic. A "substituted heterocycloalkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as specified herein. The expression "partially hydrogenated form" of an unsaturated compound or group as used herein means that part of the unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group without removing all unsaturated moieties. The phrase "completely hydrogenated form" of an unsaturated compound or group is used herein interchangeably with the term "perhydro" and means that all unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group. For example, partially hydrogenated forms of a 5-membered heteroaryl group (containing 2 double bonds in the ring, such as furan) include dihydro forms of said 5- membered heteroaryl group (such as 2,3 -dihydrofuran or 2,5 -dihydrofuran), whereas the tetrahydro form of said 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a completely hydrogenated (or perhydro) form of said 5 -membered heteroaryl group. Likewise, for a 6-membered heteroaryl group having 3 double bonds in the ring (such as pyridyl), partially hydrogenated forms include di- and tetrahydro forms (such as di- and tetrahydropyridyl), whereas the hexahydro form (such as piperidinyl in case of the heteroaryl pyridyl) is the completely hydrogenated (or perhydro) derivative of said 6-membered heteroaryl group. Consequently, a hexahydro form of an aryl or heteroaryl can only be considered a partially hydrogenated form according to the present disclosure if the aryl or heteroaryl contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms.
The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi- or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic). A similar distinction is made within the present application between heteroaryl and heterocyclyl. For example, indolinyl, i.e., a dihydro variant of indolyl, is classified as heterocyclyl for the purposes of the present disclosure, since only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.
The term "hydrocarbyl" as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Particular examples of hydrocarbyl groups are C1-30 alkyl (such as Cg-30 alkyl, Cx-24 alkyl, or C 10-20 alkyl), C2-30 alkenyl (such as Cg-30 alkenyl, Cx-24 alkenyl, or C 10-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(C1-6 alkyl). In some embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more 1st level substituents, one or more 2nd level substituents, or one or more 3rd level substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively nonpolar. In some embodiments, the hydrocarbyl group is the hydrocarbyl chain of naturally occurring fatty acids and may have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably linear) C8-20 alkyl chain, e.g., a (preferably linear) Cio-is alkyl chain).
The term "optionally substituted" indicates that one or more (such as 1 to the maximum number of hydrogen atoms bound to a group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atom(s) may be replaced with a group (i.e., a 1st level substituent) different from hydrogen such as alkyl (preferably, C1-6 alkyl), alkenyl (preferably, C2-6 alkenyl), alkynyl (preferably, C2-6 alkynyl), aryl (preferably, 6- to 14-membered aryl), heteroaryl (preferably, 3- to 14- membered heteroaryl), cycloalkyl (preferably, 3- to 14-membered cycloalkyl), heterocyclyl (preferably, 3- to 14-membered heterocyclyl), halogen, -CN, azido, -NO2, -OR71, -N(R72)(R73), -S(0)o-2R71, -S(O)I.2OR71, -OS(O)I.2R71, -OS(O)I.2OR71, -S(O)I.2N(R72)(R73), -OS(O)I.2N(R72)(R73),
-N(R71)S(O)I-2R71, -NR71S(O)I-2OR71, -NR71S(O)I-2N(R72)(R73), -OP(O)(OR71)2, -C(=X1)R71, -C(=XI)XIR71, -XiC(=Xi)R71, and -XiC(=Xi)X1R71, and/or any two 1st level substituents which are bound to the same carbon atom of a cycloalkyl or heterocyclyl group may join together to form =Xi, wherein each of the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups of the 1st level substituent may themselves be substituted by one or more (e.g., one, two or three) substituents (i.e., a 2nd level substituent) selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14- membered heterocyclyl, halogen, -CF3, -CN, azido, -NO2, -OR81, -N(R82)(R83), -S(0)o-2R81, -S(O)I-2OR81, -OS(O)I-2R81, -OS(O)I-2OR81, -S(O)I-2N(R82)(R83), -OS(O)I-2N(R82)(R83),
-N(R81)S(O)I-2R81, -NR81S(O)I-2OR81, -NR81S(O)I-2N(R82)(R83), -OP(O)(OR81)2, -C(=X2)R81, -C(=X2)X2R81, -X2C(=X2)R81, and -X2C(=X2)X2R81, and/or any two 2nd level substituents which are bound to the same carbon atom of a cycloalkyl or heterocyclyl group being a 1st level substituent may join together to form =X2, wherein each of the C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14-membered heterocyclyl groups of the 2nd level substituent is optionally substituted with one or more (e.g., one, two or three) substituents (i.e., a 3rd level substituent) independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -OCF3, -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(CI.3 alkyl)2, -NHS(O)2(CI_3 alkyl), -S(O)2NH2.Z(CI-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=O)(C1-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl) C(=NH)NH2-Z(C 1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl, and/or any two 3rd level substituents which are bound to the same carbon atom of a 3- to 14-membered cycloalkyl or heterocyclyl group being a 2nd level substituent may join together to form =0, =S, =NH, or =N(CI-3 alkyl); wherein each of R71, R72, and R73 is independently selected from the group consisting of H, CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl, wherein each of the CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 3- to 7- membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -OCF3, =0, -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(CI-3 alkyl)z, -C(=0)(CM alkyl), -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CI-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl; each of R81, R82, and R83 is independently selected from the group consisting of H, CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 3- to 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered heterocyclyl, wherein each of the CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 3- to 6- membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -OCF3, =0, -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(C1-3 alkyl)z, -C(=0)(CM alkyl), -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CI-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl; and each of Xi and X2 is independently selected from O, S, and N(R84), wherein R84 is H or C1-3 alkyl.
Typical 1st level substituents are preferably selected from the group consisting of CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14-membered (such as 5- or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR71, -N(R72)(R73), -S(0)o-2R71, -S(O)I.2OR71, -OS(O)I.2R71, -OS(O)I.2OR71, -S(O)I.2N(R72)(R73), -OS(O)I.2N(R72)(R73),
-N(R71)S(O)I.2R71, -NR71S(O)I.2OR71, -C(=XI)R71, -C(=XI)XIR71, -XIC(=XI)R71, and -XiC(=Xi)XiR71, such as CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 6-membered aryl, 5- or 6-membered heteroaryl, 3- to 7- membered cycloalkyl, 3- to 7-membered (such as 5- or 6-membered) heterocyclyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(C1-3 alkyl)z, -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CI-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl; wherein Xi is independently selected from O, S, NH and N(CHs); and each of R71, R72, and R73 is as defined above or, preferably, is independently selected from the group consisting of H, CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(CI-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=O)(C1-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. In some embodiments, 1st level substituents are selected from the group consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -0CH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and Ci. 3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, 1st level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3.
Typical 2nd level substituents are preferably selected from the group consisting of CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 6- or 10-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, =0, =S, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(C1-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CM alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. Particular examples of 2nd level substituents are independently selected from the group consisting of C1-3 alkyl, phenyl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, =0, =S, -CF3, -CN, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CM alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. Particularly preferred 2nd level substituents are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, phenyl, =0, and =S.
Typical 3rd level substituents are preferably selected from the group consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=0)0H, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. Particularly preferred 3rd level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3. The term "tertiary amine moiety" as used herein relates to a moiety containing a nitrogen atom which is substituted with three organic substituents (wherein the substituents may be the same or different from each other). In some embodiments, the organic substituents are selected from hydrocarbyl groups (such as alkyl groups, in particular C1-6 alkyl groups) which are optionally substituted (e.g., with one or more 1st level substituents, one or more 2nd level substituents, or one or more 3rd level substituents as defined herein).
The term "filtrating" as used herein relates to any process that involves removal or separation of at least one component (such as permeable molecules like salts, small proteins, solvents etc.,) of a liquid composition based on the molecular size of the components contained in the composition. This separation may use micro-molecule permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., for dialysis). Thus, examples of filtrating comprise dialyzing, tangential flow filtrating and diafiltrating.
The expression "substantially free of X", as used herein, means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of X in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture.
For example, "substantially free of a lipid comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units" as used herein, means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of a lipid comprising at least 30 consecutive ethylene glycol repeating units in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of a lipid comprising at least 30 consecutive ethylene glycol repeating units, the amount of a lipid comprising at least 30 consecutive ethylene glycol repeating units in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture. Similar considerations apply to expressions containing the phrase "substantially free of (such as "substantially free of a sarcosinylated lipid", "substantially free of a (POX) -conjugated and/or polyoxazine (POZ)-conjugated lipid ", and "substantially free of any polymer- conjugated lipid other than the amphiphilic OEG-conjugated compound").
The expression "nucleic acid integrity" means the percentage of the full-length (i.e., non-fragmented) nucleic acid to the total amount of nucleic acid (i.e., non-fragmented plus fragmented nucleic acid) contained in a sample. The nucleic acid integrity may be determined by chromatographically separating the nucleic acid (e.g., using capillary electrophoresis), determining the peak area of the main nucleic acid peak (i. e. , the peak area of the full-length (z. e., non-fragmented) nucleic acid), determining the peak area of the total nucleic acid, and dividing the peak area of the main nucleic acid peak by the peak area of the total nucleic acid. Likewise, the expression "RNA integrity" means the percentage of the full- length (i.e., non-fragmented) RNA to the total amount of RNA (i.e., non-fragmented plus fragmented RNA) contained in a sample. The RNA integrity may be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of the main RNA peak (i.e., the peak area of the full-length (i. e . , non-fragmented) RNA), determining the peak area of the total RNA, and dividing the peak area of the main RNA peak by the peak area of the total RNA.
The term "cryoprotectant" relates to a substance that is added to a preparation (e.g., formulation or composition) in order to protect the active ingredients of the preparation during the freezing stages.
The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.
According to the present disclosure, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" or "polypeptide " refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide", "polypeptide", and "protein" are used herein usually as synonyms.
A "therapeutic peptide or protein" has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In some embodiments, a therapeutic peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term "therapeutic peptide or protein" includes entire peptides or proteins, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a peptide or protein. Examples of therapeutically active peptides or proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines. The terms "therapeutic peptide or protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.
The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure.
The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term "part" means a portion of the composition. For example, a part of a composition may any portion from 0. 1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.
"Fragment", with reference to an amino acid sequence (peptide, polypeptide or protein), relates to apart of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the amino acid sequence.
According to the present disclosure, a part or fragment of a peptide, polypeptide or protein preferably has at least one functional property of the peptide, polypeptide or protein from which it has been derived. Such functional properties comprise a pharmacological activity, the interaction with other peptides, polypeptides or proteins, an enzymatic activity, the interaction with antibodies, and the selective binding of nucleic acids. E.g., a pharmacological active fragment of a peptide, polypeptide or protein has at least one of the pharmacological activities of the peptide, polypeptide or protein from which the fragment has been derived. A part or fragment of a peptide, polypeptide or protein preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids of the peptide or protein. A part or fragment of a peptide or protein preferably comprises a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the peptide or protein.
"Variant", as used herein and with reference to an amino acid sequence (peptide, polypeptide, or protein), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, and preferably from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.
By "wild type" or "WT" or "native" with respect to an amino acid sequence is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide, polypeptide or protein has an amino acid sequence that has not been intentionally modified. Likewise by "wild type" or "WT" or "native" with respect to a nucleic acid sequence is meant a nucleic acid sequence that is found in nature, including allelic variations. For example, a wild type coding sequence is meant to be a coding sequence that is found in nature and that has not been intentionally modified.
A "coding sequence", as sued herein means the portion of a nucleic acid (e.g., a gene's DNA or RNA) that codes for protein.
The expression "guanosine/cytosine (G/C) content" or "G/C content" means the percentage of bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). The G/C content may be given for a specific portion of DNA or RNA or for an entire genome. When the G/C content refers to a portion, it may denote the G/C content of an individual gene or portion of a gene (domain), a group of genes or gene clusters, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.
For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.
Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and/or carboxy -terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups:
- glycine, alanine;
- valine, isoleucine, leucine;
- aspartic acid, glutamic acid;
- asparagine, glutamine;
- serine, threonine;
- lysine, arginine; and
- phenylalanine, tyrosine.
In some embodiments, the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: meedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
"Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC =align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.
Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.
The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
In some embodiments, a fragment or variant of an amino acid sequence (peptide, polypeptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response (immunogenic fragment). In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
In some embodiments, "isolated" means altered or removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In some embodiments, the RNA (such as mRNA) used in the present disclosure is in substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of RNA (such as mRNA) in substantially purified form contains a first buffer system.
The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid.
The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro (e.g., in cell culture) or in vivo, e.g., the cell can form part of an organ, a tissue and/or an organism of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.
The disclosure includes analogs of a peptide, polypeptide or protein. According to the present disclosure, an analog of a peptide, polypeptide or protein is a modified form of said peptide, polypeptide or protein from which it has been derived and has at least one functional property of said peptide, polypeptide or protein. E.g., a pharmacological active analog of a peptide, polypeptide or protein has at least one of the pharmacological activities of the peptide, polypeptide or protein from which the analog has been derived. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the protein, polypeptide or peptide, such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, "analogs" of proteins, polypeptides or peptides include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective/blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand. The term "analog" also extends to all functional chemical equivalents of said proteins, polypeptides and peptides.
As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.
According to various embodiments of the present disclosure, a nucleic acid such as RNA (e.g., mRNA) encoding a peptide, polypeptide or protein is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide, polypeptide or protein. The cell may express the encoded peptide, polypeptide or protein intracellularly (e.g. in the cytoplasm and/or in the nucleus), may secrete the encoded peptide, polypeptide or protein, and/or may express it on the surface.
According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide, polypeptide or protein mean that the nucleic acid, if present in the appropriate environment, preferably within a cell, can be expressed to produce said peptide, polypeptide or protein.
"Activation" or "stimulation", as used herein, refers to the state of a cell (e.g., an immune effector cell such as T cell) that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers to, among other things, immune effector cells that are undergoing cell division.
The term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.
The term "clonal expansion" or "expansion" refers to a process wherein a specific entity is multiplied. In some embodiments, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. In some embodiments, expansion leads to differentiation of the immune effector cells.
An "antigen" according to the present disclosure covers any substance that will elicit an immune response and/or any substance against which an immune response or an immune mechanism such as a cellular response and/or humoral response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an "antigen" relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T-cells). According to the present disclosure, the term "antigen" may comprise any molecule which comprises at least one epitope, such as a T cell epitope. In some embodiments, an antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, which may be specific for the antigen (including cells expressing the antigen). In some embodiments, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.
According to the present disclosure, any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may be a humoral or cellular immune response or both. In the context of some embodiments of the present disclosure, the antigen is presented by a cell, preferably by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen. An antigen may be a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.
The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and/or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.
In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein. In particular, the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, preferably CD4+ and CD8+ lymphocytes, in particular via the modulation of the activity of a T-cell receptor.
The term "tumor antigen" or "tumor-associated antigen" refers to a constituent of cancer cells which may be derived from the cytoplasm, the cell surface or the cell nucleus. In particular, it refers to those antigens which are produced intracellularly or as surface antigens on tumor cells. For example, tumor antigens include the carcinoembryonal antigen, al -fetoprotein, isoferritin, and fetal sulphoglycoprotein, a2-H-ferroprotein and y-fetoprotein, as well as various virus tumor antigens. According to some embodiments of the present disclosure, a tumor antigen comprises any antigen which is characteristic for tumors or cancers as well as for tumor or cancer cells with respect to type and/or expression level.
The term "viral antigen" refers to any viral component having antigenic properties, i.e., being able to provoke an immune response in an individual. The viral antigen may be a viral ribonucleoprotein or an envelope protein.
The term "bacterial antigen" refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasm membrane of the bacterium.
The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies T cells or B cells, in particular when presented in the context of MHC molecules. An epitope of a protein may comprise a continuous or discontinuous portion of said protein and, e.g., may be between about 5 and about 100, between about 5 and about 50, between about 8 and about 0, between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, the epitope in the context of the present disclosure is a T cell epitope. Terms such as "epitope", "fragment of an antigen", "immunogenic peptide" and "antigen peptide" are used interchangeably herein and, e.g., may relate to an incomplete representation of an antigen which is, e.g., capable of eliciting an immune response against the antigen or a cell expressing or comprising and presenting the antigen. In some embodiments, the terms relate to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. The term "epitope" refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes T cell epitopes.
The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both selfantigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC/peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC/peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.
The peptide and protein antigen can be 2 to 100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.
The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein. In some embodiments, vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response, is recognized by an immune effector cell. In some embodiments, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell.
In some embodiments, an antigen is expressed in a diseased cell (such as tumor cell or an infected cell).
In some embodiments, an antigen is presented by a diseased cell (such as tumor cell or an infected cell). In some embodiments, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In some embodiments, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells. In some embodiments, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
In some embodiments, an antigen is expressed on the surface of a diseased cell (such as tumor cell or an infected cell). In some embodiments, an antigen receptor is a CAR which binds to an extracellular domain or to an epitope in an extracellular domain of an antigen. In some embodiments, a CAR binds to native epitopes of an antigen present on the surface of living cells. In some embodiments, binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells. In some embodiments, binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
In some embodiments, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In some embodiments, an antibody or B cell receptor binds to native epitopes of an antigen.
The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. In this context, a part may be, e.g., at least 4, at least 8, at least 12, or at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.
"Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by, e.g., antigen-specific antibodies added to the cells. In some embodiments, an antigen expressed on the surface of cells is an integral membrane protein having an extracellular portion which may be recognized by a CAR.
The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. In some embodiments, the term refers to one or more extracellular loops or domains or a fragment thereof.
The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells. The term "antigen-specific T cell" or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted, in particular when presented on the surface of antigen presenting cells or diseased cells such as cancer cells in the context of MHC molecules and preferably exerts effector functions of T cells. T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be measured. In certain embodiments of the present disclosure, the RNA (in particular mRNA) encodes at least one epitope.
The term "target" shall mean an agent such as a cell or tissue which is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell expressing an antigen and preferably presenting said antigen with class I MHC.
"Antigen processing" refers to the degradation of an antigen into processing products which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells to specific T-cells. Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells.
The term "professional antigen presenting cells" relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.
The term "non-professional antigen presenting cells" relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.
The term "dendritic cell" (DC) refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In some embodiments, the dendritic cells are splenic dendritic cells.
The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are splenic macrophages.
By "antigen-responsive CTL" is meant a CD8+ T-cell that is responsive to an antigen or a peptide derived from said antigen, which is presented with class I MHC on the surface of antigen presenting cells.
According to the disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-y and TNF-a, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.
The terms "immune response" and "immune reaction" are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the disclosure, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, such as CD4+ and CD8+ T-lymphocytes, e.g., CD8+ T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.
The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective/preventive/prophylactic and/or therapeutic. The immune response may be directed against any immunogen or antigen or antigen peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV or RSV)). "Inducing" in this context may mean that there was no immune response against a particular antigen or pathogen before induction, but it may also mean that there was a certain level of immune response against a particular antigen or pathogen before induction and after induction said immune response is enhanced. Thus, "inducing the immune response" in this context also includes "enhancing the immune response". In some embodiments, after inducing an immune response in an individual, said individual is protected from developing a disease such as an infectious disease or a cancerous disease or the disease condition is ameliorated by inducing an immune response. The terms "cellular immune response", "cellular response", "cell-mediated immunity" or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen and/or presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes which act as either "helpers" or "killers". The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill cells such as diseased cells.
The term "humoral immune response" refers to a process in living organisms wherein antibodies are produced in response to agents and organisms, which they ultimately neutralize and/or eliminate. The specificity of the antibody response is mediated by T and/or B cells through membrane-associated receptors that bind antigen of a single specificity. Following binding of an appropriate antigen and receipt of various other activating signals, B lymphocytes divide, which produces memory B cells as well as antibody secreting plasma cell clones, each producing antibodies that recognize the identical antigenic epitope as was recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the resulting escalation in antibody response when re-exposed to a specific antigen.
The term "antibody" as used herein, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which is able to specifically bind to an epitope on an antigen under typical physiological conditions, preferably with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3, the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy-terminus in the following order: CHI, CH2, CH3). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.
The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The terms "binding region" and "antigen-binding region" are used herein interchangeably and refer to the region which interacts with the antigen and comprises both a VH region and a VL region. An antibody as used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions.
As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in WO 2007/059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(l 1):484- 90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l): 111-24); and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al. , Science 242. 423-426 (1988) and Huston et al. , PNAS USA 85. 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.
The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, such as to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the VL (variable light chain) domain, CL (constant light chain) domain, VH (variable heavy chain) domain, and the CH (constant heavy chain) domains CHI, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, i.e., a, 8, a, y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.
The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s). By "cell characterized by presentation of an antigen" or "cell presenting an antigen" or "MHC molecules which present an antigen on the surface of an antigen presenting cell" or similar expressions is meant a cell such as a diseased cell, in particular a tumor cell or an infected cell, or an antigen presenting cell presenting the antigen or an antigen peptide, either directly or following processing, in the context of MHC molecules, preferably MHC class I and/or MHC class II molecules, most preferably MHC class I molecules.
In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA (especially mRNA) may be translated into peptide, polypeptide or protein.
The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence.
With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.
In the context of the present disclosure, the term "RNA encodes" means that the RNA, if present in the appropriate environment, such as within cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein it encodes during the process of translation.
The term "serum" as used herein means the fluid resulting from the removal of cells and clotting factors from whole blood, such as whole blood obtained from humans or mice. In some embodiments, serum is human serum or mouse serum.
A medical preparation, in particular kit, described herein may comprise instructional material or instructions. As used herein, "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions of the present disclosure or be shipped together with a container which contains the compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient. The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.
Prodrugs of a particular compound described herein are those compounds that upon administration to an individual undergo chemical conversion under physiological conditions to provide the particular compound. Additionally, prodrugs can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the particular compound when, for example, placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Exemplary prodrugs are esters (using an alcohol or a carboxy group contained in the particular compound) or amides (using an amino or a carboxy group contained in the particular compound) which are hydrolyzable in vivo. Specifically, any amino group which is contained in the particular compound and which bears at least one hydrogen atom can be converted into a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.
In the present specification, a structural formula of a compound may represent a certain isomer of said compound. It is to be understood, however, that the present disclosure includes all isomers such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers and the like which occur structurally and isomer mixtures and is not limited to the description of the formula. Furthermore, in the present specification, a structural formula of a compound may represent a specific salt and/or solvate of said compound. It is to be understood, however, that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates) and is not limited to the description of the specific salt and/or solvate.
"Isomers" are compounds having the same molecular formula but differ in structure ("structural isomers") or in the geometrical (spatial) positioning of the functional groups and/or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers which are non-superimposable mirrorimages of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is denoted by the prefix (±). "Diastereomers" are stereoisomers which are non- superimposable and which are not mirror-images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; i.e., the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto-enol- tautomerism. "Conformers" are stereoisomers that can be interconverted just by rotations about formally single bonds, and include - in particular - those leading to different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane. The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non-stoichiometric. A "hydrate" is a solvate wherein the solvent is water.
In isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced by a deuterium or tritium atom. Exemplary isotopes which can be used in the present disclosure include deuterium, tritium, nC, 13C, 14C, 15N, 18F, 32P, 32S, 35S, 36C1, and 125I.
The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the Zaverage-
In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.
The "radius of gyration" (abbreviated herein as Rg) of a particle about an axis of rotation is the radial distance of a point from the axis of rotation at which, if the whole mass of the particle is assumed to be concentrated, its moment of inertia about the given axis would be the same as with its actual distribution of mass. Mathematically, Rg is the root mean square distance of the particle's components from either its center of mass or a given axis. For example, for a macromolecule composed of n mass elements, of masses m, (i = 1, 2, 3, ... , n), located at fixed distances s, from the center of mass, Rg is the square-root of the mass average of Si2 over all mass elements and can be calculated as follows:
The radius of gyration can be determined or calculated experimentally, e.g., by using light scattering. In particular, for small scattering vectors q the structure function S is defined as follows: wherein N is the number of components (Guinier's law). The "DIO value", in particular regarding a quantitative size distribution of particles, is the diameter at which 10% of the particles have a diameter less than this value. The DIO value is a means to describe the proportion of the smallest particles within a population of particles (such as within a particle peak obtained from a field-flow fractionation).
"D50 value", in particular regarding a quantitative size distribution of particles, is the diameter at which 50% of the particles have a diameter less than this value. The D50 value is a means to describe the mean particle size of a population of particles (such as within a particle peak obtained from a field-flow fractionation).
The "D90 value", in particular regarding a quantitative size distribution of particles, is the diameter at which 90% of the particles have a diameter less than this value. The "D95", "D99", and "DI 00" values have corresponding meanings. The D90, D95, D99, and DI 00 values are means to describe the proportion of the larger particles within a population of particles (such as within a particle peak obtained from a field-flow fractionation).
The "hydrodynamic radius" (which is sometimes called "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as said particle. The hydrodynamic radius is related to the mobility of the particle, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a greater hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags a greater number of water molecules with it as it moves through the solution. Since the actual dimensions of the particle in a solvent are not directly measurable, the hydrodynamic radius may be defined by the Stokes-Einstein equation:
R — kB D _ - T h 6 ■ n ■ r/ ■ D wherein kB is the Boltzmann constant; T is the temperature; is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, e.g., by using dynamic light scattering (DLS). Thus, one procedure to determine the hydrodynamic radius of a particle or a population of particles (such as the hydrodynamic radius of particles such as LNPs contained in a formulation or composition as disclosed herein or the hydrodynamic radius of a particle peak obtained from subjecting such a formulation or composition to field-flow fractionation) is to measure the DLS signal of said particle or population of particles (such as DLS signal of particles such as LNPs contained in a formulation or composition as disclosed herein or the DLS signal of a particle peak obtained from subjecting such a formulation or composition to field-flow fractionation). The term "aggregate" as used herein relates to a cluster of particles, wherein the particles are identical or very similar and adhere to each other in a non-covalently manner (e.g., via ionic interactions, H bridge interactions, dipole interactions, and/or van der Waals interactions).
The expression "light scattering" as used herein refers to the physical process where light is forced to deviate from a straight trajectory by one or more paths due to localized non-uniformities in the medium through which the light passes.
The term "UV" means ultraviolet and designates a band of the electromagnetic spectrum with a wavelength from 10 nm to 400 nm, i.e., shorter than that of visible light but longer than X-rays.
The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring the light scattered by a sample into a plurality of angles. "Multi -angle" means in this respect that scattered light can be detected at different discrete angles as measured, for example, by a single detector moved over a range including the specific angles selected or an array of detectors fixed at specific angular locations. In one preferred embodiment, the light source used in MALS is a laser source (MALLS: multi -angle laser light scattering). Based on the MALS signal of a composition comprising particles and by using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), it is possible to determine the radius of gyration (Rg) and, thus, the size of said particles. Preferably, the Zimm plot is a graphical presentation using the following equation (or the reciprocal thereof): wherein c is the mass concentration of the particles in the solvent (g/mL); A ? is the second virial coefficient (mol-mL/g2); P(3) is a form factor relating to the dependence of scattered light intensity on angle; Re is the excess Rayleigh ratio (cm 1); and K* is an optical constant that is equal to 4TI2T|O (dw/dc)2Z(iU AA"1 , where r|0 is the refractive index of the solvent at the incident radiation (vacuum) wavelength, Xo is the incident radiation (vacuum) wavelength (nm), NA is Avogadro’s number (mol 1), and dn/dc is the differential refractive index increment (mL/g) (cf., e.g., Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); B.H. Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry plot is calculated the following term or the reciprocal thereof: wherein c, Re and K* are as defined above. Preferably, the Debye plot is calculated the following term or the reciprocal thereof: wherein c, Re and K* are as defined above. The expression "dynamic light scattering" or "DLS" as used herein refers to a technique to determine the size and size distribution profde of particles, in particular with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is shot through a polarizer and into a sample. The scattered light then goes through a second polarizer where it is detected and the resulting image is projected onto a screen. The particles in the solution are being hit with the light and diffract the light in all directions. The diffracted light from the particles can either interfere constructively (light regions) or destructively (dark regions). This process is repeated at short time intervals and the resulting set of speckle patterns are analyzed by an autocorrelator that compares the intensity of light at each spot over time.
The expression "static light scattering" or "SLS" as used herein refers to a technique to determine the size and size distribution profde of particles, in particular with respect to the radius of gyration of the particles, and/or the molar mass of particles. A high-intensity monochromatic light, usually a laser, is launched in a solution containing the particles. One or many detectors are used to measure the scattering intensity at one or many angles. The angular dependence is needed to obtain accurate measurements of both molar mass and size for all macromolecules of radius. Hence simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multiangle laser light scattering (MALLS), is generally regarded as the standard implementation of static light scattering.
"Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.
As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.
As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.
The term "repeating unit" relates to an elementary unit which periodically repeats itself along the polymeric chain of a polymer and which is derived from one monomer. Although the structures of the repeating unit and its corresponding monomer are often coincident, they may differ from each other. The term "functional moiety" as used herein relates to a group of atoms in a molecule with distinctive chemical properties, wherein the atoms of the functional moiety are linked to each other and to the rest of the molecule by covalent bonds. Preferably, the atoms of the functional moiety comprise at least one atom selected from the group consisting of O, N, and S. Functional moieties may be monovalent (such as hydroxy, cyano, nitro, or amide (e.g., -C(0)NHCH3)) or divalent (such as amide (e.g., -C(O)NH-), carbonyl (-C(O)-), or ester (e.g., -OC(O)-). In some embodiments, the functional moiety provides hydrophilicity to the group to which the functional moiety is bound, e.g., by providing at least one hydrogen bond acceptor/donor and/or at least one charge (positive or negative) to the group to which the functional moiety is bound. In certain embodiments, the functional moiety comprises a hydrogen bond acceptor (such as a carbonyl moiety), a hydrogen bond donor (such as a hydroxyl moiety, -NH- (of, e.g., an amide moiety), or thiol moiety) or both (e.g., an amide moiety), and/or is charged (e.g., phosphate, amino, or ammonium moiety). Examples of monovalent functional moieties include hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties. Examples of divalent functional moieties include ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.
The term "hydroxyl" or "hydroxy" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -OH.
The term "halogen" as used herein with respect to a functional moiety, in particular as component of a linker, means fluoro, choloro, bromo, or iodo.
The term "cyano" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -CN. The term "azido" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group N3.
The term "nitro" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -NO2.
The term "amino" as used herein with respect to a functional moiety, in particular as component of a linker, includes unsubstituted amino (i.e., the group -NH2) and substituted amino (i.e., mono- or disubstituted amino, wherein one or two of the hydrogen atoms have been replaced with a group other than hydrogen). An amino group may be monovalent (e.g., -NRR, wherein each R is independently H or an organic group, such as R72 or R73 as defined below) or divalent (e.g., -NR-, wherein R is H or an organic group, such as R72 as defined below). In some embodiments, the term "amino" means the group -N(R72)(R73), wherein R72 and R73 are, in each case, independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R72 and R73 may join together with the nitrogen atom to which they are attached to form the group -N=CR75R76, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; R75 and R76 are independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -NHyR802-y, or R75 and R76 may join together with the atom to which they are attached to form a ring which is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; y is an integer from 0 to 2; R80 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; and R70 is other than H, preferably a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as disclosed herein. In some embodiments, each of R72 and R73 is independently H or a hydrocarbyl group, such as selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl), wherein each of the hydrocarbyl groups (such as each of the C1-6 alkyl, aryl, and aryl(C1-6 alkyl) groups) is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the hydrocarbyl group (such as C1-6 alkyl, aryl, or aryl(C1-6 alkyl) group)), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70.
The term "ammonium" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -N+(R72)2(R73), wherein R72 and R73 are as defined for the term "amino".
The term "thiol" or "sulfanyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -SH.
The term "disulfanyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -SSH.
The term "carboxyl" or "carboxy" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -COOH.
The term "amide" or "amido" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)NH- (including its isomerically arranged structure -NHC(O)-, unless it is specified to the contrary). Preferably, each of both ends of the amide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (if both ends are linked to the same organic group the amide moiety is also referred to as lactam). An amide group may be monovalent (e.g., -C(O)NRR or -NRC(O)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)NR- or -NRC(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "ester" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)O- (including its isomerically arranged structure -OC(O)-, unless it is specified to the contrary). Preferably, each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (if both ends are linked to the same organic group the ester moiety is also referred to as lactone). An ester group may be monovalent (e.g., -C(O)OR or -OC(O)R, wherein R is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)O- or -OC(O)-)). The term "ether" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -O-, wherein each of both ends of the ether structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). An ether group may be monovalent (e.g., -OR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -O-).
The term "sulfide" or "thioether" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S-, wherein each of both ends of the sulfide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A sulfide group may be monovalent (e.g., -SR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -S-).
The term "disulfide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SS-, wherein each of both ends of the disulfide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A disulfide group may be monovalent (e.g., -SSR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SS-).
The term "diselenide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SeSe-, wherein each of both ends of the diselenide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A diselenide group may be monovalent (e.g., -SeSeR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SeSe-).
The term "sulfoxide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the sulfinyl structure -S(O)-, wherein each of both ends of the sulfoxide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A sulfoxide group may be monovalent (e.g., -S(O)R, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (as, e.g., -S(O)-).
The term "sulfone" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the sulfonyl structure -S(O)2-, wherein each of both ends of the sulfone structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A sulfone group may be monovalent (e.g., -S(O)2R, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (as, e.g., -S(O)2-).
The term "sulfite" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OS(O)O-, wherein one of the two ends of the sulfite structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or to a C atom of the same or another organic group (e.g., an alkylene group as further component of the linker). A sulfite group may be monovalent (e.g., -OS(O)OR, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OS(O)O-).
The term "sulfate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OS(O)2O-, wherein one of the two ends of the sulfate structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or to a C atom of the same or another organic group (e.g., an alkylene group as further component of the linker). A sulfate group may be monovalent (e.g., -OS(O)2OR, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OS(O)2O-).
The term "phosphate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OP(O)(OR)O-, wherein one of the two ends of the phosphate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A phosphate group may be monovalent (e.g., -OP(O)(OR)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OP(O)(OR)O-, wherein R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "sulfmamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S(O)N(R)-, wherein the S end of the sulfmamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the N end is covalently linked to H or to a C atom of the same or another organic group (R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A sulfmamide group may be monovalent (e.g., -S(O)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -S(O)N(R)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "sulfonamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S(O)2N(R)-, wherein the S end of the sulfonamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the N end is covalently linked to H or to a C atom of the same or another organic group (R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A sulfonamide group may be monovalent (e.g., -S(O)2N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -S(0)2N(R)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "sulfamate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -0S(0)2N(R)- (including its isomerically arranged structure -N(R)S(0)20-, unless it is specified to the contrary), wherein one of both ends of the sulfamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A sulfamate group may be monovalent (e.g., -OS(O)2N(R)2 or -N(R)S(0)20R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -0S(0)2N(R)- or -N(R)S(0)20-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "sulfiirous diamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)S(O)N(R)-, wherein one of both ends of the sulfiirous diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A sulfiirous diamide group may be monovalent (e.g., -N(R)S(O)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)S(O)N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "sulfuric diamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)S(0)2N(R)-, wherein one of both ends of the sulfuric diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A sulfuric diamide group may be monovalent (e.g., -N(R)S(O)2N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)S(0)2N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "urea" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(O)N(R)-, wherein one of both ends of the urea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). An urea group may be monovalent (e.g., -N(R)C(0)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(O)N(R)-, wherein each R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "thiourea" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(S)N(R)-, wherein one of both ends of the thiourea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A thiourea group may be monovalent (e.g., -N(R)C(S)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(S)N(R)-, wherein each R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbonyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)-, wherein one of both ends of the carbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (if both ends are linked to C atoms of organic groups the carbonyl moiety is also referred to as "keto" moiety). A carbonyl group may be monovalent (e.g., -C(O)R, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)-).
The term "thiocarbonyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(S)-, wherein one of both ends of the thiocarbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group. A thiocarbonyl group may be monovalent (e.g., -C(S)R, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(S)-).
The term "orthoester" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a moiety comprising a C atom to which three alkoxy groups (i.e., -OR, wherein R is an organic group (e.g., an alkylene group as further component of the linker), such one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") are attached. An exemplary formula of an orthoester comprises the structure (-0)rC(0R)3-r-, wherein each R is independently an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; r is 1 or 2; and each of both ends of the orthoester structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. In some embodiments, an orthoester comprises the structure (-O)rC(OR25)3 r-, wherein each R25 is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl) which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); r is 1 or 2; and each of both ends of the orthoester structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. An orthoester group may be monovalent (e.g., -C(0R)3 or -0C(0R)2R, wherein each R is an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., (-0)2C(0R)(R) or -OC(OR)2-, wherein each R is an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "thioate" or "thioester" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(O)- (including its isomerically arranged structures -C(O)S-, _-OC(S), and -C(S)O-, unless it is specified to the contrary), wherein each of both ends of the thioate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A thioate group may be monovalent (e.g., -SC(O)R or -C(O)SR or -OC(S)R or -C(S)OR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)- or -C(O)S- or -OC(S)- or -C(S)O-).
The term "dithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)- (including its isomerically arranged structure -C(S)S-, unless it is specified to the contrary), wherein each of both ends of the dithioate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A dithioate group may be monovalent (e.g., -SC(S)R or -C(S)SR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)- or -C(S)S-).
The term "imidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)- (including its isomerically arranged structure -C(=NR)O-, unless it is specified to the contrary), wherein each of both ends of the imidate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). An imidate group may be monovalent (e.g., -OC(=NR)R’ or -C(=NR)OR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)- or -C(=NR)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). The term "imino" or "imine" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(=NR)-, wherein one of both ends of the imino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). The moiety -C(=NR)H is also called "aldimine", and the moiety -C(=NR)R’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"), is also called "ketimine". An imino group may be monovalent (e.g., -C(=NR)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(=NR)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "imidothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(=NR)S- (including its isomerically arranged structure -SC(=NR)-, unless it is specified to the contrary), wherein one of both ends of the imidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). An imidothioate group may be monovalent (e.g., -C(=NR)SR or -SC(=NR)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(=NR)S- or -SC(=NR)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "thionylamino" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(S)NR- (including its isomerically arranged structure -N(R)C(S)-, unless it is specified to the contrary), wherein one of both ends of the thionylamino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A thionylamino group may be monovalent (e.g., -C(S)NRR or -N(R)C(S)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(S)NR- or -N(R)C(S)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbonate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(O)O-, wherein each of both ends of the carbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonate group may be monovalent (e.g., -OC(O)OR’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(O)O-).
The term "carbonothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(S)O- or -OC(O)S- (including its isomerically arranged structure -SC(O)O-, unless it is specified to the contrary), wherein each of both ends of the carbonothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonothioate group may be monovalent (e.g., -OC(S)OR’ or -OC(O)SR’ or -SC(O)OR’, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(S)O- or -OC(O)S- or -SC(O)O-).
The term "carbonodithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(O)S- or -OC(S)S- (including its isomerically arranged structure -SC(S)O-, unless it is specified to the contrary), wherein each of both ends of the carbonodithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonodithioate group may be monovalent (e.g., -SC(O)SR’ -OC(S)SR’ or -SC(S)OR’, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)S- or -OC(S)S- or -SC(S)O-).
The term "carbonotrithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)S-, wherein each of both ends of the carbonotrithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonotrithioate group may be monovalent (e.g., -SC(S)SR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)S-). The term "guanidino" or "imidamido" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(=NR)NR- (wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"), wherein one of both ends of the guanidino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group. A guanidino group may be monovalent (e.g., -N(R)C(=NR)NRR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(=NR)NR-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbamimidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)NR- (including its isomerically arranged structure -N(R)C(=NR)O-, unless it is specified to the contrary), wherein the O end of the carbamimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other (N) end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbamimidate group may be monovalent (e.g., -OC(=NR)NRR or -N(R)C(=NR)OR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)NR- or -N(R)C(=NR)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbonimidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)O-, wherein each of the ends of the carbonimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbonimidate group may be monovalent (e.g., -OC(=NR)OR’, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)O-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbamate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(O)NR- (including its isomerically arranged structure -N(R)C(O)O-, unless it is specified to the contrary), wherein the O end of the carbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbamate group may be monovalent (e.g., -OC(O)NRR or -N(R)C(O)OR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(O)NR- or -N(R)C(O)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbamodithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)NR- (including its isomerically arranged structure -N(R)C(S)S-, unless it is specified to the contrary), wherein the S end of the carbamodithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbamodithioate group may be monovalent (e.g., -SC(S)NRR or -N(R)C(S)SR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)NR- or -N(R)C(S)S-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbonodithioimidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(=NR)S-, wherein each of the ends of the carbonodithioimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbonodithioimidate group may be monovalent (e.g., -SC(=NR)SR’, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(=NR)S-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbamimidothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(=NR)NR- (including its isomerically arranged structure -N(R)C(=NR)S-, unless it is specified to the contrary), wherein the S end of the carbamimidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbamimidothioate group may be monovalent (e.g., -SC(=NR)NRR or -N(R)C(=NR)SR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -SC(=NR)NR- or -N(R)C(=NR)S-, wherein each R is independently H or an organic group, such one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbamothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including their isomerically arranged structures -SC(O)NR- or -OC(S)NR-, unless it is specified to the contrary), wherein the O/S end of the carbamothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbamothioate group may be monovalent (e.g., -N(R)C(O)SR’ or -N(R)C(S)OR’ or -SC(O)NRR or -OC(S)NRR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(O)S- or -N(R)C(S)O- or -SC(O)NR- or -OC(S)NR-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "carbonimidothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)S- (including its isomerically arranged structure -SC(=NR)O-, unless it is specified to the contrary), wherein the O/S end of the carbonimidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). A carbonimidothioate group may be monovalent (e.g., -OC(=NR)SR’ or -SC(=NR)OR’, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)S- or -SC(=NR)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "acylhydrazone" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(R’)(=N-N(R)C(O)-) (including its isomerically arranged structure (-C(O)(N(R)-N=)C(R’)-, unless it is specified to the contrary) and/or =C(=N- N(R)C(O)R’), wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; each R is H or an organic group, such as such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each of both ends of the acylhydrazone structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, an acylhydrazone comprises the structure -C(R25)(=N-N(R26)C(O)-) (including its isomerically arranged structure (-C(O)(N(R26)-N=)C(R25)-, unless it is specified to the contrary) and/or =C(=N-N(R26)C(O)R25), wherein each R25 is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl) which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); each R26 is independently H or a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl), which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); and each of both ends of the acylhydrazone structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. Exemplary chemical structures of an acylhydrazone are shown below: wherein each 'ww represents the bond by which the acylhydrazone is covalently linked to the further organic group(s) (e.g., an alkylene group as further component of the linker). A acylhydrazone group may be monovalent (e.g., -C(R’)(=N-N(R)C(O)R’) or -C(0)(N(R)-N=)C(R’)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(R’)(=N-N(R)C(O)-, -C(O)(N(R)-N=)C(R’)-, or =C(=N- N(R26)C(O)R25), wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each R is H an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino".
The term "hydrazine" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each of both ends of the hydrazine structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, a hydrazine comprises the structure -N(R26)N(R26)-, wherein each R26 is independently H or a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl), which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); and each of both ends of the hydrazine structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. A hydrazine group may be monovalent (e.g., -N(R)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -N(R)N(R)-, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "oxime" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure =C(=N(OH)), wherein each of both ends of the oxime structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). An exemplary chemical formula of an oxime is shown below: wherein each AAAAV represents the bond by which the oxime is covalently linked to the further organic group(s). An oxime group may be monovalent (e.g., -C(=N(OH))(R), wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., =C(=N(OH))).
The term "acetal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCH(R’)O-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each of both O atoms of the acetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, an acetal comprises the structure -OCH(R25)O-, wherein R25 is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); and each of both O atoms of the acetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. An acetal group may be monovalent (e.g., -OCH(R’)OR’, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OCH(R’)O-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "hemiacetal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCH(OH)-, wherein each of both ends of the hemiacetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). A hemiacetal group may be monovalent (e.g., -OCH(OH)OR’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OCH(OH)-).
The term "ketal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(R’)(R’)O-, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each of both O atoms of the ketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, a ketal comprises the structure -OC(R25)(R25)O -, wherein each R25 is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); and each of both O atoms of the ketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. An ketal group may be monovalent (e.g., -OC(R’)(R’)OR’, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OC(R’)(R’)O-, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "hemiketal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCR’ (OH)-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each of both ends of the hemiketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, a hemiketal comprises the structure -OCR25(OH)-, wherein R25 is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1st level substituents, 2nd level substituents, or 3rd level substituents as defined herein); and each of both ends of the hemiketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. A hemiketal group may be monovalent (e.g., -0C(R’)2(0H), wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -OCR’(OH)-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino").
The term "imide" as used herein with respect to a functional moiety, in particular within a as component of, relates to a group comprising the structure -C(O)N(R)C(O)-, wherein R is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and each of both ends of the imide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). An imide group may be monovalent (e.g., -C(O)N(R)C(O)R’, wherein R is independently H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., -C(O)N(R)C(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72 indicated above in the definition of the term "amino"). The term "non-cyclic" as used herein in the context of organic groups relates to open-chain organic groups which contain no rings. "Open-chain" or "acyclic" organic groups may be straight (i.e., they contain only one unbranched chain without any sidechain) or branched (i.e., the main chain comprises one or more sidechains).
An organic group which is "substituted with one or more substituents" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the organic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the organic group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the one or more substituents may be selected from the 1st level substituents, 2nd level substituents, or 3rd level substituents described herein.
The expression "hydrogen bond" or "H-bond" as used herein means a non-covalent bond (in some embodiments a primarily electrostatic force of attraction) between (i) a hydrogen atom which is covalently bound to a more electronegative atom or group, and (ii) a lone pair of electrons of another electronegative atom. In some embodiments, the more electronegative atom or group includes nitrogen atoms and oxygen atoms; thus, examples of groups in which a hydrogen atom is covalently bound to a more electronegative atom or group include amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (as such (as in respective thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) groups))). In some embodiments, the lone pair of electrons of another electronegative atom is a lone pair of an oxygen atom present in a carbonyl group or a lone pair of a nitrogen atom present in a primary, secondary or tertiary amino group.
The expression "hydrogen bond donor" as used herein means an atom, ion, or a molecule component of a hydrogen bond which supplies the bridging (shared) hydrogen atom. In some embodiments, a hydrogen bond donor includes amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (as such (as in respective thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) groups))).
The expression "hydrogen bond acceptor" as used herein means an atom, ion, or a molecule component of a hydrogen bond which does not supply the bridging (shared) hydrogen atom. In some embodiments, a hydrogen bond acceptor comprises at least one lone pair of electrons. Examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary and tertiary amino groups. The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and/or degraded, or to be hardly detected and then sequestered and/or degraded, and/or to be detected and then sequestered and/or degraded late, by the immune system of the host to which they are administered.
The term "phosphatidylethanolamine" means diacylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight). In some embodiments, each acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety may be saturated or unsaturated (such as monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (such as monounsaturated). In some embodiments, one acyl moiety is saturated and the other is unsaturated (such as monounsaturated). Examples of acyl moieties include -C(O)(CH2)I6CH3 (stearoyl), -C(O)(CH2)i4CH3 (palmitoyl), -C(O)(CH2)I2CH3 (myristoyl), and -CZ5-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The term "phosphatidylethanolamine moiety" means a monovalent radical of phosphatidylethanolamine, preferably that in which a hydrogen atom of the amino group has been removed.
The term "DSPE" means distearoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl). The term "distearoylphosphatidylethanolamine moiety" means a monovalent radical of DSPE, preferably that in which a hydrogen atom of the amino group has been removed.
The term "DPPE" means dipalmitoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(0)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl). The term "dipalmitoylphosphatidylethanolamine moiety" means a monovalent radical of DPPE, preferably that in which a hydrogen atom of the amino group has been removed.
The term "DOPE" means dioleoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(O)CI7H33 refers to the moiety -cz5-C(O)(CH2)7-CH=CH- (CH2)7CH3 (oleoyl). The term "dioleoylphosphatidylethanolamine moiety" means a monovalent radical of DOPE, preferably that in which a hydrogen atom of the amino group has been removed.
The term "POPE" means palmitoyloleoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl); and -C(O)CI7H33 refers to the moiety -cz5-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The term "palmitoyloleoylphosphatidylethanolamine moiety" means a monovalent radical of POPE, preferably that in which a hydrogen atom of the amino group has been removed.
The term "tocopherol" means a group of four compounds (i.e., a-tocopherol, P-tocopherol, y-tocopherol, and 5-tocopherol) having the following formula: wherein each of Ru and R2 is independently H or methyl. In a-tocopherol, Ru and Rt2 are both methyl; in P-tocopherol, Ri is methyl, and Rt2 is H; in y-tocopherol, Ru is H, and Rt2 is methyl; and in 5- tocopherol, Rti and Rt2 are both H. The term "tocopherol moiety" or "tocopheryl moiety" means a monovalent radical of tocopherol, preferably that in which the hydrogen atom of the hydroxy group has been removed.
The term "DAG" means diacylglyceride having the following formula: or a salt thereof, wherein in each case acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight). In some embodiments, each acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety may be saturated or unsaturated (such as monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (such as monounsaturated). In some embodiments, one acyl moiety is saturated and the other is unsaturated (such as monounsaturated). Examples of acyl moieties include -C(O)(CH2)i6CH3 (stearoyl), -C(O)(CH2)i4CH3 (palmitoyl), -C(O)(CH2)i2CH3 (myristoyl), and -CA-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). For example, DMG means 1,2-dimyristoylglycerol, i.e., a diacylglyceride of the above formula, wherein both acyl groups are -C(O)(CH2)i2CH3 (myristoyl). The term "diacylglyceride moiety" means a monovalent radical of diacylglyceride, preferably that in which a hydrogen atom of the hydroxy group has been removed.
The term "DAA" means dialkylamine having the formula HN(alkyl)2 or a salt thereof, wherein each alkyl moiety preferably is straight. In some embodiments, each alkyl moiety has at least 8 carbon atoms. Preferably, each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl moieties include -(CFDnCFE (stearyl), -(CFDisCFE (palmityl), and -(CH2)I3CH3 (myristyl). For example, DMA means 1,2-dimyristylamine, i.e., a dialkylamine of the above formula, wherein both alkyl groups are -(CH2)I3CH3 (myristyl). The term "dialkylamine moiety" means a monovalent radical of dialkylamine, preferably that in which the hydrogen atom of the amino group has been removed.
The term "ceramide" means acyl sphingosine having the following formula: or a salt thereof, wherein -C13H27 refers to the moiety -(CFDnCFE; and acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight). In some embodiments, the acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety may be saturated or unsaturated (such as monounsaturated). Examples of acyl moieties include -C(O)(CH2)ieCH3 (stearoyl), -C(O)(CH2)i4CH3 (palmitoyl), -C(O)(CH2)i2CH3 (myristoyl), and -cA-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). For example, palmitoyl ceramide means a ceramide of the above formula, wherein acyl is -C(O)(CH2)i4CH3 (palmitoyl). The term "ceramide moiety" means a monovalent radical of ceramide, preferably that in which the hydrogen atom of a hydroxy group (preferably the hydrogen of the terminal (primary) hydroxy group) has been removed.
The term "MAA" means monoalkylamine having the formula H2N(alkyl) or a salt thereof, wherein the alkyl moiety preferably is straight. In some embodiments, the alkyl moiety has at least 8 carbon atoms. Preferably, the alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably the alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl moieties include -(CIDnCFE (stearyl), -(CIDisCFE (palmityl), and -(CTDBCTE (myristyl). For example, MMA means myristylamine, i.e., a monoalkylamine of the above formula, wherein the alkyl group is - (CH2)i3CH3 (myristyl). The term "monoalkylamine moiety" means a monovalent radical of monoalkylamine, preferably that in which one of the hydrogen atoms of the amino group has been removed.
Nucleic Acids
The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
The term "nucleoside" (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.
The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.
A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is preferably modified by one or more alkyl groups, more preferably one or more CM alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl- guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N’-CM alkyl- guanine, N6-CM alkyl-adenine, 5-CM alkyl-cytosine, 5-CM alkyl-uracil, and N(1)-CM alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(l)- methyl -uracil.
In some embodiments of all aspects of the disclosure, the nucleic acid is DNA.
Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.
RNA In some embodiments of all aspects of the disclosure, the nucleic acid is RNA.
According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered/modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered/modified nucleotides or nucleosides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule . The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
"RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.
In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding and expressing a pharmaceutically active peptide or protein. In some embodiments, RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein. A cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface. Alternatively, the RNA can be non-coding RNA such as antisense -RNA, micro RNA (miRNA) or siRNA. The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). mRNA
In some embodiments of all aspects of the disclosure, the nucleic acid is mRNA.
According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide, polypeptide or protein. Typically, an mRNA comprises a 5'-UTR, a peptide/protein coding region, and a 3'-UTR. In the context of the present disclosure, mRNA is preferably generated by in vitro transcription (IVT) from a DNA template. As set forth above, the in vitro transcription methodology is known to the skilled person, and a variety of in vitro transcription kits is commercially available. mRNA is single -stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.
According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.
In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.
In some embodiments, the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.
As established in the art, the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide/polypeptide/protein coding region and a 3' untranslated region (3'-UTR). In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non- naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and/or added to the mRNA after transcription.
In some embodiments, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.
In some embodiments of the present disclosure, the RNA (such as mRNA) is "replicon RNA" (such as "replicon mRNA") or simply a "replicon", in particular "self-replicating RNA" (such as "self-replicating mRNA") or "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the replicon or self-replicating RNA (such as self-replicating mRNA) is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5 ’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2: 1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould etal., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non- structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
In some embodiments of the present disclosure, the RNA (such as mRNA) described herein (e.g., contained in the compositions of the present disclosure and/or used in the methods of the present disclosure) contains one or more modifications, e.g., in order to increase its stability and/or increase translation efficiency and/or decrease immunogenicity and/or decrease cytotoxicity. For example, in order to increase expression of the RNA (such as mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein. Such modifications are described, for example, in WO 2007/036366 and PCT/EP2019/056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and/or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the G/C content of the RNA). The term "modification" in the context of modified mRNA according to the present disclosure preferably relates to any modification of an mRNA which is not naturally present in said RNA (such as mRNA).
In some embodiments, the RNA (such as mRNA) described herein comprises a 5'-cap structure. In some embodiments, the mRNA does not have uncapped 5 '-triphosphates. In some embodiments, the RNA (such as mRNA) described herein may comprise a conventional 5'-cap and/or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine 5 '-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'- end of the next nucleotide of the mRNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the mRNA). The guanosine may be methylated at position N7 (resulting in the cap structure m7Gppp). The term "5'-cap analog" refers to a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3 '-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called antireverse cap analogs (ARCAs)). Particularly preferred 5 '-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the P-phosphate (such as m27’2 OG(5')ppSp(5')G (referred to as beta-S-ARCA or P-S-ARCA)), as described in PCT/EP2019/056502. Providing an RNA (such as mRNA) with a 5'- cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5 '-cap compound, wherein said 5 '-cap structure is co-transcriptionally incorporated into the generated RNA (such as mRNA) strand, or the RNA (such as mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the mRNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
In some embodiments, the RNA (such as mRNA) comprises a 5 '-cap structure selected from the group consisting of m27’2 OG(5’)ppSp(5')G (in particular its DI diastereomer), m27’3 OG(5')ppp(5')G, and m27’3 OGppp(mi2 O)ApG. In some embodiments, RNA encoding a peptide, polypeptide or protein comprising an antigen or epitope comprises m27’2 OG(5’)ppSp(5')G (in particular its DI diastereomer) as 5'-cap structure.
In some embodiments, the RNA (such as mRNA) comprises a capO, capl, or cap2, preferably capl or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.
The 5'-cap analog beta-S-ARCA (P-S-ARCA) has the following structure:
The "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S- ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml/min can be applied. In one embodiment, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.
The 5 '-cap analog m27’3 OGppp(mi2 O)ApG (also referred to as m27’3 OG(5')ppp(5')m2 OApG) which is a building block of a capl has the following structure: An exemplary capO mRNA comprising P-S-ARCA and mRNA has the following structure:
An exemplary capO mRNA comprising m27’3 °G(5')ppp(5')G and mRNA has the following structure: An exemplary capl mRNA comprising m27’3 OGppp(mi2 O)ApG and mRNA has the following structure:
In some embodiments, the RNA (such as mRNA) comprises a 3’-poly(A) sequence. As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA (such as mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs (such as mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (such as mRNAs) disclosed herein can have a poly-A tail attached to the free 3 '-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template -dependent RNA polymerase.
It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of mRNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).
The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate. In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly (A) cassette.
In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3 '-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.
In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly- A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides. In some embodiments, the poly-A tail comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the poly-A sequence has a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3.
In some embodiments, RNA (such as mRNA) used in present disclosure comprises a 5'-UTR and/or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5 '-UTR) and/or 3' (downstream) of an open reading frame (3 -UTR). A 5 '-UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3 '-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does preferably not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3 '-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'- UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (preferably mRNA) into which they are introduced. In one particular embodiment the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2 -globin, alpha 1 -globin, or beta-globin, preferably betaglobin, more preferably human beta-globin. For example, the RNA (preferably mRNA) may be modified by the replacement of the existing 3 '-UTR with or the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2 -globin, alpha 1 -globin, beta-globin, preferably beta-globin, more preferably human beta-globin.
In some embodiments, the RNA (such as mRNA) used in present disclosure comprises a 5 ’-UTR comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.
In some embodiments, the RNA (such as mRNA) used in present disclosure comprises a 3 ’-UTR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.
The RNA (such as mRNA) described herein may have modified ribonucleotides in order to increase its stability and/or decrease immunogenicity and/or decrease cytotoxicity. For example, in some embodiments, uridine in the RNA (such as mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.
In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml\|/), 5-methyl-uridine (m5U), and combinations thereof. In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA (such as mRNA) may be any one or more of 3-methyl-uridine (m3U), 5 -methoxy-uridine (mo5U), 5 -aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5 -bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5 -carboxymethyl -uridine (cm5U), 1- carboxymethyl-pseudouridine, 5 -carboxyhydroxymethyl -uridine (chm5U), 5 -carboxyhydroxymethyl - uridine methyl ester (mchm5U), 5 -methoxycarbonylmethyl -uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5 -aminomethyl -2-thio-uridine (nm5s2U), 5- methylaminomethyl-uridine (mnm5U), 1 -ethyl -pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5 -carbamoylmethyl -uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio- uridine (cmnm5s2U), 5 -propynyl -uridine, 1-propynyl -pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(rm5s2U), 1-taurinomethyl -4-thio- pseudouridine), 5 -methyl-2 -thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (m ls4i|/). 4-thio-l- methyl-pseudouridine, 3 -methyl -pseudouridine (m3i|i). 2-thio-l -methyl -pseudouridine, 1-methyl-l- deaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5 -methyl -dihydrouridine (m5D), 2-thio-dihydrouridine, 2- thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine
(acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 \|F), 5-
(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a- thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (\|/m), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um),
5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl- uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl- uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5- (2 -carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.
An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "T-modificd". whereas the term "ml'P-modified" means that the RNA (preferably mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5 -methyluridine (replacing partially or completely, preferably completely, uridine). Such T- or m IT- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(l)-methylpseudouridine replacing completely uridine.
The codons of the RNA (preferably mRNA) described in the present disclosure may further be optimized, e.g., to increase the G/C content of the RNA and/or to replace codons which are rare in the cell (or subject) in which the peptide or protein of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA described in the present disclosure is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In one embodiment, the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.
The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions are preferably codon-optimized for optimal expression in a subject to be treated using the RNA (preferably mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (preferably mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".
In some embodiments, the guanosine/cytosine (G/C) content of the coding region of the RNA (preferably mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA (preferably mRNA) is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA (preferably mRNA). Sequences having an increased G (guanosine )/C (cytosine) content are more stable than sequences having an increased A (adenosine )/U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA (preferably mRNA), there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides. In various embodiments, the G/C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.
A combination of the above described modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and/or 3'- UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5 -methylcytidine for cytidine and/or pseudouridine (T) or N(l)-methylpseudouridine (m I T) or 5 -methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (preferably mRNA) described in the present disclosure, in particular an RNA (preferably mRNA) encoding an antigen or epitope for inducing an immune response disclosed herein, contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure; (ii) incorporation of a poly- A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5 -methylcytidine for cytidine and/or pseudouridine (T) or N(l)- methylpseudouridine (m I T) or 5 -methyluridine (m5U) for uridine); and (v) codon optimization. In some embodiments, the RNA (preferably mRNA) described in the present disclosure comprises a capl or cap2, preferably a capl structure. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the a 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the 3’-UTR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.
Some aspects of the disclosure involve the targeted delivery of the RNA (preferably mRNA) disclosed herein to certain cells or tissues. In some embodiments, the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is in particular preferred if the RNA (preferably mRNA) administered is RNA (preferably mRNA) encoding an antigen or epitope for inducing an immune response. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. The "lymphatic system" is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph. The primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and the bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, which include lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response. In some embodiments, the target cell is a T cell.
Lipid-based RNA (such as mRNA) delivery systems have an inherent preference to the liver. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ is liver and the target tissue is liver tissue. The delivery to such target tissue is preferred, in particular, if presence of mRNA or of the encoded peptide or protein in this organ or tissue is desired and/or if it is desired to express large amounts of the encoded peptide or protein and/or if systemic presence of the encoded peptide or protein, in particular in significant amounts, is desired or required.
In some embodiments, after administration of the RNA (in particular, mRNA) compositions described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is RNA (preferably mRNA) encoding a peptide or protein and the RNA is translated by the target cell to produce the peptide or protein. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell in the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in the lymph nodes. In some embodiments, the target cell is a cell in the lung In some embodiments, the target cell is a cell in the skin. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is aNK cell. In some embodiments, the target cell is a monocyte. Thus, nucleic acid (such as RNA) particle (such as RNA LNP) compositions described herein may be used for delivering nucleic acid (such as RNA, preferably mRNA) to such target cell. Accordingly, the present disclosure also relates to a method for delivering nucleic acid (such as RNA, preferably mRNA) to a target cell in a subject comprising the administration of the nucleic acid (such as RNA, preferably mRNA) compositions described herein to the subject. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is RNA (preferably mRNA) encoding a peptide or protein and the RNA is translated by the target cell to produce the peptide or protein. Inhibitory RNA
In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.
The term "inhibitory RNA" as used herein means RNA which selectively hybridizes to and/or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and/or translation thereof. Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA.
The term "antisense RNA" as used herein refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and/or translation of said mRNA. An antisense transcript of a nucleic acid or of a part thereof may form a duplex with naturally occurring mRNA and thus prevent accumulation of or translation of the mRNA. Another possibility is the use of ribozymes for inactivating a nucleic acid. The antisense RNA may hybridize with an N-terminal or 5' upstream site such as a translation initiation site, transcription initiation site or promoter site. In some embodiments, the antisense RNA may hybridize with a 3 '-untranslated region or mRNA splicing site.
The size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides. In one embodiment, the antisense RNA has a length of at least 2,700 nucleotides (such as at least 2,800, at least 2,900, at least 3,000, at least 3,100, at least 3,200, at least 3,300, at least 3,400, at least 3,500, at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900, at least 5,000 nucleotides).
The stability of antisense RNA may be modified as required. For example, antisense RNA may be stabilized by one or more modifications having a stabilizing effect. Such modifications include modified phosphodiester linkages (such as methylphosphonate, phosphorothioate, phosphorodithioate or phosphoramidate linkages instead of naturally occurring phosphodiester linkages) and 2'-substitutions (e.g., 2'-fluoro, 2'-O-alkyl (such as 2'-O-methyl, 2'-O-propyl, or 2'-O-pentyl) and 2'-O-allyl). For example, in some embodiments of the antisense RNA, phosphorothioate linkages are substituted partially for phosphodiester linkages. Alternatively or additionally, in some embodiments of the antisense RNA, the ribose moiety is substituted partially at the 2'-position with O-alkyl (such as 2'-O- methyl).
An antisense RNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3'-direction) from the start codon. The target sequence can, however, be located in the 5'- or 3 '-untranslated regions, or in the region nearby the start codon.
Antisense RNA can be obtained using a number of techniques known to those of skill in the art. For example, antisense RNA can be chemically synthesized or recombinantly produced using methods known in the art. Preferably, antisense RNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.
Selection of plasmids suitable for expressing antisense RNA, methods for inserting nucleic acid sequences for expressing the antisense RNA into the plasmid, and IVT methods of in vitro transcription of said antisense RNA are within the skill in the art.
By "small interfering RNA" or "siRNA" as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs. Although, in principle, the sense and antisense strands of siRNAs can comprise two complementary, single-stranded RNA molecules, the siRNAs, according to the present disclosure, comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. That is, the sense region and antisense region can be covalently connected via a linker molecule. The linker molecule can be a polynucleotide or non-nucleotide linker, but is preferably a polynucleotide linker. Without wishing to be bound by any theory, it is believed that the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.
The siRNA can also comprise a 3'-overhang. As used herein, a "3'-overhang" refers to at least one unpaired nucleotide extending from the 3'-end of an RNA strand. Thus, in some embodiments, the siRNA comprises at least one 3 '-overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length. In the embodiments in which both strands of the siRNA molecule (i.e., after the siRNA molecule is cleaved intracellularly by the "Dicer" protein) comprise a 3'-overhang, the length of the overhangs can be the same or different for each strand. In some preferred embodiments, the 3'- overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA can comprise 3 '-overhangs of dideoxythymidylic acid ("TT") or diuridylic acid ("uu").
In order to enhance the stability of the siRNA, the 3'-overhangs can be also stabilized against degradation. In some embodiments, the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotides in the 3 '-overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the absence of a 2'- hydroxyl in the 2'-deoxythymidine significantly enhances the nuclease resistance of the 3'-overhang in tissue culture medium.
As used herein, "target mRNA" refers to an RNA molecule that is a target for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.
According to the present disclosure, siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide", revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. "The siRNA User Guide" is available on the world wide web at a website maintained by Dr. Thomas Tuschl, Uaboratory of RNA Molecular Biology, Rockefeller University, New York, USA, and can be found by accessing the website of the Rockefeller University and searching with the keyword "siRNA". Further guidance with respect to the selection of target sequences and/or the design of siRNA can be found on the webpages of Protocol Online (www.protocol-online.com) using the keyword "siRNA". Thus, in some embodiments, the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA. Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3'- direction) from the start codon. The target sequence can, however, be located in the 5'- or 3 '-untranslated regions, or in the region nearby the start codon. siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. application no. 2002/0086356 of Tuschl et al., the entire disclosure of which is herein incorporated by reference. siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
Preferably, siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for transcribing siRNA used in the present disclosure from a plasmid include, for example, the U6 or Hl RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
Selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and IVT methods of in vitro transcription of said siRNA are within the skill in the art.
The term "miRNA" (microRNA) as used herein relates to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradation and/or prevent translation of target mRNAs. miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can be obtained using a number of techniques known to those of skill in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Pharmaceutically active peptides or polypeptides
"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (preferably mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
In some embodiments, RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence (e.g., an ORF) encoding one or more polypeptides, e.g., a peptide or protein, preferably a pharmaceutically active peptide or protein.
In some embodiments, RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence (e.g., an ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein, and is capable of expressing said peptide or protein, in particular if transferred into a cell or subject. Thus, in some embodiments, the RNA (preferably mRNA) described in the present disclosure contains a coding region (ORF) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein. In this respect, an "open reading frame" or "ORF" is a continuous stretch of codons beginning with a start codon and ending with a stop codon. Such RNA (preferably mRNA) encoding a pharmaceutically active peptide or protein is also referred to herein as "pharmaceutically active RNA" (or "pharmaceutically active mRNA"). In some embodiments, RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide, e.g., two, three, four or more peptides or polypeptides.
The term "pharmaceutically active RNA" also encompasses RNA which is pharmaceutically active in its own; thus, pharmaceutically active RNA may alternatively be non-coding RNA such as antisense- RNA, micro RNA (miRNA), siRNA, one or more strands of RNA interference (RNAi), short hairpin RNAs (shRNAs), or precursor of a siRNA or microRNA-like RNA, targeted to a target transcript, e.g., a transcript of an endogenous disease-related transcript of a subject.
According to the present disclosure, the term "pharmaceutically active peptide or protein" means a peptide or protein that can be used in the treatment of an individual where the expression of the peptide or protein would be of benefit, e.g., in ameliorating the symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. In some embodiments, a pharmaceutically active peptide or protein has a positive or advantageous effect on the condition or disease state of an individual when administered to the individual in a therapeutically effective amount. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or disorder or to lessen the severity of such disease or disorder. The term "pharmaceutically active peptide or protein" includes entire proteins or polypeptides, and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of a peptide or protein. The terms "pharmaceutically active peptide or protein" and "therapeutic peptide or protein" are used interchangeable herein.
Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, immunostimulants, e.g., cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins. In some embodiments, the pharmaceutically active peptide and polypeptide includes a replacement protein.
An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing activity of any of the immune system's components, in particular immune effector cells. The immunostimulant may be pro-inflammatory (e.g., when treating infections or cancer), or antiinflammatory (e.g., when treating autoimmune diseases).
In some embodiments, the immunostimulant is a cytokine or a variant thereof. Examples of cytokines include interferons, such as interferon-alpha (IFN-a) or interferon-gamma (IFN-y), interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. According to another aspect, the immunostimulant includes an adjuvant-type immunostimulatory agent such as APC Toll-like Receptor agonists or costimulatory/cell adhesion membrane proteins. Examples of Toll -like Receptor agonists include costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.
The term "cytokines" relates to proteins which have a molecular weight of about 5 to 60 kDa (such as about 5 to 20 kDa) and which participate in cell signaling (e.g., paracrine, endocrine, and/or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they usually act at much more variable concentrations than hormones and (ii) generally are made by a broad range of cells (nearly all nucleated cells can produce cytokines). Interferons are usually characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface, thereby preventing viral replication within the cells. The interferons can be grouped into two types. IFN- gamma is the sole type II interferon; all others are type I interferons. Particular examples of cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte -macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFNa), interferon beta (IFNP), interferon gamma (INFy), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL- 12), and interleukin 21 (IL-21).
According to the disclosure, a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. A cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal. One particularly preferred cytokine is interferon-a.
Immunostimulants may be provided to a subject by administering to the subject nucleic acid (such as DNA or RNA) encoding an immunostimulant in a formulation for preferential delivery of nucleic acid to liver or liver tissue. The delivery of nucleic acid (such as DNA or RNA) to such target organ or tissue is preferred, in particular, if it is desired to express large amounts of the immunostimulant and/or if systemic presence of the immunostimulant, in particular in significant amounts, is desired or required.
Nucleic acid (such as DNA or RNA) delivery systems have an inherent preference to the liver. This pertains to lipid-based particles, cationic and neutral nanoparticles, in particular lipid nanoparticles.
Examples of suitable immunostimulants for targeting liver are cytokines involved in T cell proliferation and/or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.
In some embodiments, nucleic acid (such as DNA or RNA) encoding an immunostimulant may be administered in a formulation for preferential delivery of nucleic acid (such as DNA or RNA) to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. The delivery of an immunostimulant to such target tissue is preferred, in particular, if presence of the immunostimulant in this organ or tissue is desired (e.g., for inducing an immune response, in particular in case immunostimulants such as cytokines are required during T-cell priming or for activation of resident immune cells), while it is not desired that the immunostimulant is present systemically, in particular in significant amounts (e.g., because the immunostimulant has systemic toxicity). Examples of suitable immunostimulants are cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-a, or IFN-P, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.
Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.
Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon, type II interferon, and type III interferon.
All type I interferons bind to a specific cell surface receptor complex known as the IFN-a/p receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.
The type I interferons present in humans are IFNa, IFNp, IFNs, IFNK and IFNco. In general, type I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.
The IFNa proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection. The genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA 14, IFNA 16, IFNA 17, IFNA21. These genes are found together in a cluster on chromosome 9.
The IFNp proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFNp have been described, IFNpi and IFNP3. The natural and recombinant forms of IFNpi have antiviral, antibacterial, and anticancer properties.
Type II interferon (IFNy in humans) is also known as immune interferon and is activated by IL12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells.
Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of virus or fungal infections. In general, type I and II interferons are responsible for regulating and activating the immune response.
According to the disclosure, a type I interferon is preferably IFNa or IFNp, more preferably IFNa.
According to the disclosure, an interferon may be a naturally occurring interferon or a functional fragment or variant thereof. An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.
Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins.
According to the disclosure, an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.
Immunostimulant polypeptides described herein can be prepared as fusion or chimeric polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulant). The immunostimulant may be fused to an extended-PK group, which increases circulation half-life. Non-limiting examples of extended-PK groups are described infra. It should be understood that other PK groups that increase the circulation half-life of immunostimulants such as cytokines, or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended-PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).
As used herein, the term "PK" is an acronym for "pharmacokinetic" and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. As used herein, an "extended-PK group" refers to a protein, peptide, or moiety that increases the circulation half-life of a biologically active molecule when fused to or administered together with the biologically active molecule. Examples of an extended-PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549). Other exemplary extended-PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul; 16(7):903-15 which is herein incorporated by reference in its entirety. As used herein, an "extended-PK" immunostimulant refers to an immunostimulant moiety in combination with an extended-PK group. In some embodiments, the extended-PK immunostimulant is a fusion protein in which an immunostimulant moiety is linked or fused to an extended-PK group. In certain embodiments, the serum half-life of an extended-PK immunostimulant is increased relative to the immunostimulant alone (i.e., the immunostimulant not fused to an extended-PK group). In certain embodiments, the serum half-life of the extended-PK immunostimulant is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer relative to the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended-PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7- fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35- fold, 40-fold, or 50-fold greater than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended-PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.
As used herein, "half-life" refers to the time taken for the serum or plasma concentration of a compound such as a peptide or polypeptide to reduce by 50%, in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms. An extended-PK immunostimulant suitable for use herein is stabilized in vivo and its half-life increased by, e.g., fusion to serum albumin (e.g., HSA or MSA), which resist degradation and/or clearance or sequestration. The half-life can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).
In certain embodiments, the extended-PK group includes serum albumin, or fragments thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "albumin"). Polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Publication No. 20070048282. As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein such as a therapeutic protein, in particular an immunostimulant. The albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding an albumin. The therapeutic protein and albumin, once part of the albumin fusion protein, may each be referred to as a "portion", "region" or "moiety" of the albumin fusion protein (e.g., a "therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin). In some embodiments, an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, e.g. a liver cell, and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and/or assembly into multimeric proteins. An albumin fusion protein is preferably encoded by RNA in a non-processed form which in particular has a signal peptide at its N-terminus and following secretion by a cell is preferably present in the processed form wherein in particular the signal peptide has been cleaved off. In a most preferred embodiment, the "processed form of an albumin fusion protein" refers to an albumin fusion protein product which has undergone N-terminal signal peptide cleavage, herein also referred to as a "mature albumin fusion protein".
In certain embodiments, albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.
As used herein, "albumin" refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules. The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.
In certain embodiments, the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789. The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fusion state.
The albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin. For instance, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.
Generally speaking, an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.
According to the disclosure, albumin may be naturally occurring albumin or a fragment or variant thereof. Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.
In some embodiments, the albumin fusion protein comprises albumin as the N-terminal portion, and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion, and a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N- and C-termini are the same therapeutic proteins. In another preferred embodiment, the therapeutic proteins fused at the N- and C-termini are different therapeutic proteins. In some embodiments, the different therapeutic proteins are both cytokines.
In some embodiments, the therapeutic protein(s) is (are) joined to the albumin through (a) peptide linker(s). A peptide linker between the fused portions may provide greater physical separation between the moieties and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor. The peptide linker may consist of amino acids such that it is flexible or more rigid. The linker sequence may be cleavable by a protease or chemically.
As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain. In certain embodiments, an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain. This includes, but is not limited to, polypeptides comprising the entire CHI, hinge, CH2, and/or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derived from an immunoglobulin of any species and/or any subtype, including, but not limited to, a human IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fc domain encompasses native Fc and Fc variant molecules. As set forth herein, it will be understood by one of ordinary skill in the art that any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcyR binding). The Fc domains of a polypeptide described herein may be derived from different immunoglobulin molecules. For example, an Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
In certain embodiments, an extended-PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "Fc domain"). The Fc domain does not contain a variable region that binds to antigen. Fc domains suitable for use in the present disclosure may be obtained from a number of different sources. In certain embodiments, an Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgGl constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.
Moreover, the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgGl, IgG2, IgG3, and IgG4.
A variety of Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and/or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art recognized techniques.
In certain embodiments, the extended-PK group is a serum albumin binding protein such as those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422,
US2010/0113339, W02009/083804, and W02009/133208, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a serum immunoglobulin binding protein such as those disclosed in US2007/0178082, US2014/0220017, and US2017/0145062, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012/0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012/0094909. A non-limiting example of a Fn3 -based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin.
In certain embodiments, the extended-PK immunostimulant, suitable for use according to the disclosure, can employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended-PK moiety and an immunostimulant moiety) in a linear amino acid sequence of a polypeptide chain. For example, peptide linkers may be used to connect an immunostimulant moiety to an HSA domain.
Linkers suitable for fusing the extended-PK group to, e.g., an immunostimulant are well known in the art. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine- polypeptide linker, i.e., a peptide that consists of glycine and serine residues.
In some embodiments, a pharmaceutically active peptide or protein comprises a replacement protein. In these embodiments, the present disclosure provides a method for treatment of a subject having a disorder requiring protein replacement (e.g., protein deficiency disorders) comprising administering to the subject RNA as described herein encoding a replacement protein. The term "protein replacement" refers to the introduction of a protein (including functional variants thereof) into a subject having a deficiency in such protein. The term also refers to the introduction of a protein into a subject otherwise requiring or benefiting from providing a protein, e.g., suffering from protein insufficiency. The term "disorder characterized by a protein deficiency" refers to any disorder that presents with a pathology caused by absent or insufficient amounts of a protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in a biologically inactive protein product. Protein insufficiency can be involved in infectious diseases, immunosuppression, organ failure, glandular problems, radiation illness, nutritional deficiency, poisoning, or other environmental or external insults.
The term "hormones" relates to a class of signaling molecules produced by glands, wherein signaling usually includes the following steps: (i) synthesis of a hormone in a particular tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) relay and amplification of the signal; and (vi) breakdown of the hormone. Hormones differ from cytokines in that (1) hormones usually act in less variable concentrations and (2) generally are made by specific kinds of cells. In some embodiments, a "hormone" is a peptide or protein hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormones (such as human grown hormone or bovine somatotropin), oxytocin, atrial -natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptins. The term "adhesion molecules" relates to proteins which are located on the surface of a cell and which are involved in binding of the cell with other cells or with the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Particular examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.
Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins modulate cell signaling pathways, e.g., pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTK). Such regulation can lead to cellular growth, division, survival, or differentiation or to apoptosis. Particular examples of integrins include:
The term "immunoglobulins" or "immunoglobulin superfamily" refers to molecules which are involved in the recognition, binding, and/or adhesion processes of cells. Molecules belonging to this superfamily share the feature that they contain a region known as immunoglobulin domain or fold. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD 19), antigen receptor accessory molecules (e.g., CD-3y, CD3-5, CD-3a, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and other.
The term "immunologically active compound" relates to any compound altering an immune response, preferably by inducing and/or suppressing maturation of immune cells, inducing and/or suppressing cytokine biosynthesis, and/or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds possess potent immunostimulating activity including, but not limited to, antiviral and antitumor activity, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2 mediated diseases. Immunologically active compounds can be useful as vaccine adjuvants. Particular examples of immunologically active compounds include interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T cell receptors, chimeric antigen receptors (CARs), immunoglobulins, and antigens, in particular tumor-associated antigens, pathogen- associated antigens (such as bacterial, parasitic, or viral antigens), allergens, autoantigens, hormones (insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthestic or degradative, steriodogenic enzymes, kinases, phosphodiesterases, methylases, de- methylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylaste cyclases, neuramidases, lysosomal enzymes and the like), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins and the like), transcription and translation factors, tumor growth suppressing proteins (e.g., proteins which inhibit angiogenesis), structural proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins (thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Wilebrand factor, antithrombin III, glucocerebrosidase, erythropoietin granulocyte colony stimulating factor (GCSF) or modified Factor VIII, anticoagulants and the like. A preferred immunologically active compound is a vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response.
In some embodiments, nucleic acid (such as DNA or RNA, in particular, mRNA) described in the present disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject. The "peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject" is also designated herein as "vaccine antigen", "peptide and protein antigen" or simply "antigen".
In some embodiments, the RNA (in particular, mRNA) encoding vaccine antigen is a single -stranded, 5' capped mRNA that is translated into the respective protein upon entering cells of a subject being administered the RNA, e.g., antigen-presenting cells (APCs). Preferably, the RNA (i) contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence); (ii) is modified for optimized efficacy of the RNA (e.g., increased translation efficacy, decreased immunogenicity, and/or decreased cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (in particular cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml\|/), and 5-methyl-uridine); and/or codonoptimization), or (iii) both (i) and (ii).
In some embodiments, beta-S-ARCA(Dl) is utilized as specific capping structure at the 5'-end of the RNA. In some embodiments, the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the poly(A) sequence is 110 nucleotides in length and consists of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence was designed to enhance RNA stability and translational efficiency in dendritic cells. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the RNA comprises a modified nucleoside in place of uridine. In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine is selected from the group consisting of pseudouridine (y), N1 -methyl -pseudouridine (ml\|/), and 5-methyl-uridine. In some embodiments, the RNA encoding the vaccine antigen has a coding sequence (a) which is codon-optimized, (b) the G/C content of which is increased compared to the wild type coding sequence, or (c) both (a) and (b).
In some embodiments, the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is expressed in cells of the subject to provide the vaccine antigen. In some embodiments, expression of the vaccine antigen is at the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is transiently expressed in cells of the subject. In some embodiments, the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is administered systemically. In some embodiments, after systemic administration of the nucleic acid (such as DNA or RNA) encoding the vaccine antigen, expression of the nucleic acid encoding the vaccine antigen in spleen occurs. In some embodiments, after systemic administration of the nucleic acid encoding the vaccine antigen, expression of the nucleic acid encoding the vaccine antigen in antigen presenting cells, preferably professional antigen presenting cells occurs. In some embodiments, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells. In some embodiments, after systemic administration of the nucleic acid encoding the vaccine antigen, no or essentially no expression of the nucleic acid encoding the vaccine antigen in lung and/or liver occurs. In some embodiments, after systemic administration of the nucleic acid encoding the vaccine antigen, expression of the nucleic acid encoding the vaccine antigen in spleen is at least 5-fold the amount of expression in lung.
The vaccine antigen comprises an epitope for inducing an immune response against an antigen in a subject. Accordingly, the vaccine antigen comprises an antigenic sequence for inducing an immune response against an antigen in a subject. Such antigenic sequence may correspond to a target antigen or disease-associated antigen, e.g., a protein of an infectious agent (e.g., viral or bacterial antigen) or tumor antigen, or may correspond to an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or the immunogenic variant thereof. Thus, the antigenic sequence may comprise at least an epitope of a target antigen or disease-associated antigen or an immunogenic variant thereof. The antigenic sequences, e.g., epitopes, suitable for use according to the disclosure typically may be derived from a target antigen, i.e. the antigen against which an immune response is to be elicited. For example, the antigenic sequences contained within the vaccine antigen may be a target antigen or a fragment or variant of a target antigen.
The antigenic sequence or a procession product thereof, e.g., a fragment thereof, may bind to the antigen receptor such as TCR or CAR carried by immune effector cells. In some embodiments, the antigenic sequence is selected from the group consisting of the antigen expressed by a target cell to which the immune effector cells are targeted or a fragment thereof, or a variant of the antigenic sequence or the fragment.
A vaccine antigen which is provided to a subject according to the present disclosure by administering nucleic acid (such as DNA or RNA) encoding the vaccine antigen, preferably results in the induction of an immune response, e.g., in the stimulation, priming and/or expansion of immune effector cells, in the subject being provided the vaccine antigen. Said immune response, e.g., stimulated, primed and/or expanded immune effector cells, is preferably directed against a target antigen, in particular a target antigen expressed by diseased cells, tissues and/or organs, i.e., a disease-associated antigen. Thus, a vaccine antigen may comprise the disease-associated antigen, or a fragment or variant thereof. In some embodiments, such fragment or variant is immunologically equivalent to the disease-associated antigen.
In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent which results in the induction of an immune response, e.g., in the stimulation, priming and/or expansion of immune effector cells, which immune response, e.g., stimulated, primed and/or expanded immune effector cells, targets the antigen, i.e. a disease-associated antigen, in particular when presented by diseased cells, tissues and/or organs. Thus, the vaccine antigen may correspond to or may comprise the disease-associated antigen, may correspond to or may comprise a fragment of the disease- associated antigen or may correspond to or may comprise an antigen which is homologous to the disease- associated antigen or a fragment thereof. If the vaccine antigen comprises a fragment of the disease- associated antigen or an amino acid sequence which is homologous to a fragment of the disease- associated antigen said fragment or amino acid sequence may comprise an epitope of the disease- associated antigen to which the antigen receptor of the immune effector cells is targeted or a sequence which is homologous to an epitope of the disease-associated antigen. Thus, according to the disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence being homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against, e.g., stimulating, priming and/or expanding immune effector cells carrying an antigen receptor binding to, the antigen or cells expressing the antigen. It is preferred that the vaccine antigen (similar to the disease-associated antigen) provides the relevant epitope for binding by the antigen receptor present on the immune effector cells. In some embodiments, the vaccine antigen or a fragment thereof (similar to the disease-associated antigen) is expressed on the surface of a cell such as an antigen-presenting cell (optionally in the context of MHC) so as to provide the relevant epitope for binding by immune effector cells. The vaccine antigen may be a recombinant antigen.
In some embodiments of all aspects of the disclosure, the nucleic acid (such as DNA or RNA) encoding the vaccine antigen is expressed in cells of a subject to provide the antigen or a procession product thereof for binding by the antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and/or expansion of the immune effector cells.
In some embodiments, an antigen is presented or present on the surface of cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. An antigen or a procession product thereof such as a T cell epitope is in some embodiments bound by an antigen receptor. Accordingly, an antigen or a procession product thereof may react specifically with immune effector cells such as T- lymphocytes (T cells).
The term "autoantigen" or "self-antigen" refers to an antigen which originates from within the body of a subject (i.e., the autoantigen can also be called "autologous antigen") and which produces an abnormally vigorous immune response against this normal part of the body. Such vigorous immune reactions against autoantigens may be the cause of "autoimmune diseases".
In some embodiments, an antigen is expressed on the surface of a diseased cell (such as tumor cell or an infected cell). In some embodiments, an antigen receptor is a CAR which binds to an extracellular domain or to an epitope in an extracellular domain of an antigen. In some embodiments, a CAR binds to native epitopes of an antigen present on the surface of living cells. In some embodiments, binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells. In some embodiments, binding of a CAR when expressed by T cells and/or present on T cells to an antigen present on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
According to some embodiments, an amino acid sequence enhancing antigen processing and/or presentation is fused, either directly or through a linker, to an antigenic peptide or polypeptide (antigenic sequence). Accordingly, in some embodiments, the nucleic acid (such as DNA or RNA) described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence enhancing antigen processing and/or presentation.
In some embodiments, an antigen for vaccination which may be administered in the form of nucleic acid (such as DNA or RNA) coding therefor comprises a naturally occurring antigen or a fragment such as an epitope thereof.
Such amino acid sequences enhancing antigen processing and/or presentation are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the C-terminus of an amino acid sequence which breaks immunological tolerance), without being limited thereto. Amino acid sequences enhancing antigen processing and/or presentation as defined herein preferably improve antigen processing and presentation. In some embodiments, the amino acid sequence enhancing antigen processing and/or presentation as defined herein includes, without being limited thereto, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof.
In some embodiments, a secretory sequence, e.g., a sequence comprising the amino acid sequence of SEQ ID NO: 4, may be fused to the N-terminus of the antigenic peptide or polypeptide.
In some embodiments, an amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or a functional fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, an amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 5.
Accordingly, in some embodiments, the nucleic acid (such as DNA or RNA) described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence enhancing antigen processing and/or presentation, said amino acid sequence enhancing antigen processing and/or presentation preferably being fused to the antigenic peptide or polypeptide, more preferably to the C-terminus of the antigenic peptide or polypeptide as described herein.
Furthermore, a secretory sequence, e.g., a sequence comprising the amino acid sequence of SEQ ID NO: 4, may be fused to the N-terminus of the antigenic peptide or polypeptide. Amino acid sequences derived from tetanus toxoid of Clostridium tetani may be employed to overcome self-tolerance mechanisms in order to efficiently mount an immune response to self-antigens by providing T-cell help during priming.
It is known that tetanus toxoid heavy chain includes epitopes that can bind promiscuously to MHC class II alleles and induce CD4+ memory T cells in almost all tetanus vaccinated individuals. In addition, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens is known to improve the immune stimulation compared to application of tumor-associated antigen alone by providing CD4+-mediated T-cell help during priming. To reduce the risk of stimulating CD8+ T cells with the tetanus sequences which might compete with the intended induction of tumor antigen-specific T-cell response, not the whole fragment C of tetanus toxoid is used as it is known to contain CD8+ T-cell epitopes. Two peptide sequences containing promiscuously binding helper epitopes were selected alternatively to ensure binding to as many MHC class II alleles as possible. Based on the data of the ex vivo studies the well-known epitopes p2 (QYIKANSKFIGITEL; TT830-844; SEQ ID NO: 9) and pl6 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT578-609; SEQ ID NO: 10) were selected. The p2 epitope was already used for peptide vaccination in clinical trials to boost anti-melanoma activity.
Non-clinical data showed that RNA vaccines encoding both a tumor antigen plus promiscuously binding tetanus toxoid sequences lead to enhanced CD8+ T-cell responses directed against the tumor antigen and improved break of tolerance. Immunomonitoring data from patients vaccinated with vaccines including those sequences fused in frame with the tumor antigen-specific sequences reveal that the tetanus sequences chosen are able to induce tetanus-specific T-cell responses in almost all patients.
According to some embodiments, an amino acid sequence which breaks immunological tolerance is fused, either directly or through a linker, e.g., a linker having the amino acid sequence according to SEQ ID NO: 7, to the antigenic peptide or polypeptide.
Such amino acid sequences which break immunological tolerance are preferably located at the C- terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence enhancing antigen processing and/or presentation, wherein the amino acid sequence which breaks immunological tolerance and the amino acid sequence enhancing antigen processing and/or presentation may be fused either directly or through a linker, e.g., a linker having the amino acid sequence according to SEQ ID NO: 8), without being limited thereto. Amino acid sequences which break immunological tolerance as defined herein preferably improve T cell responses. In some embodiments, the amino acid sequence which breaks immunological tolerance as defined herein includes, without being limited thereto, sequences derived from tetanus toxoid-derived helper sequences p2 and pl6 (P2P16), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 6 or a functional variant thereof.
In some embodiments, an amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6, or a functional fragment of the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, an amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 6.
In the following, embodiments of vaccine RNAs are described, wherein certain terms used when describing elements thereof have the following meanings: cap: 5'-cap structure selected from the group consisting of m27’2 OG(5’)ppSp(5')G (in particular its DI diastereomer), m27’3 °G(5')ppp(5')G, and m27’3 OGppp(mi2 O)ApG. hAg-Kozak: 5'-UTR sequence of the human alpha-globin mRNA with an optimized ‘Kozak sequence’ to increase translational efficiency. sec/MITD: Fusion-protein tags derived from the sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), which have been shown to improve antigen processing and presentation. Sec corresponds to the 78 bp fragment coding for the secretory signal peptide, which guides translocation of the nascent polypeptide chain into the endoplasmatic reticulum. MITD corresponds to the transmembrane and cytoplasmic domain of the MHC class I molecule, also called MHC class I trafficking domain.
Antigen: Sequences encoding the respective vaccine antigen/epitope.
Glycine-serine linker (GS): Sequences coding for short peptide linkers predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.
P2P16: Sequence coding for tetanus toxoid-derived helper epitopes to break immunological tolerance.
FI element: The 3'-UTR is a combination of two sequence elements derived from the “amino terminal enhancer of split” (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.
A30L70: A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells.
In some embodiments, vaccine RNA described herein has one of the following structures: cap-hAg-Kozak-sec-GS(l)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 beta-S-ARCA(Dl)-hAg-Kozak-sec-GS(l)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70
In some embodiments, vaccine antigen described herein has the structure: sec-GS( l)-Antigen-GS(2)-P2P 16-GS(3)-MITD
In some embodiments, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, sec comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, P2P16 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, MITD comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, GS(1) comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, GS(2) comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, GS(3) comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, FI comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, A30L70 comprises the nucleotide sequence of SEQ ID NO: 3.
In some embodiments, the sequence encoding the vaccine antigen/epitope comprises a modified nucleoside replacing (partially or completely, preferably completely) uridine, wherein the modified nucleoside is selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (m I \|/)_ and 5-methyl-uridine.
In some embodiments, the sequence encoding the vaccine antigen/epitope is codon-optimized.
In some embodiments, the G/C content of the sequence encoding the vaccine antigen/epitope is increased compared to the wild type coding sequence.
The term "professional antigen presenting cells" relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.
The term "non-professional antigen presenting cells" relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.
The term "dendritic cell" (DC) refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In some embodiments, the dendritic cells are splenic dendritic cells.
The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are splenic macrophages.
The term "allergen" refers to a kind of antigen which originates from outside the body of a subject (i.e., the allergen can also be called "heterologous antigen") and which produces an abnormally vigorous immune response in which the immune system of the subject fights off a perceived threat that would otherwise be harmless to the subject. "Allergies" are the diseases caused by such vigorous immune reactions against allergens. An allergen usually is an antigen which is able to stimulate a type-I hypersensitivity reaction in atopic individuals through immunoglobulin E (IgE) responses. Particular examples of allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phi p 5), and proteins of dust mites (e.g., Der p 2).
The term "growth factors" refers to molecules which are able to stimulate cellular growth, proliferation, healing, and/or cellular differentiation. Typically, growth factors act as signaling molecules between cells. The term "growth factors" include particular cytokines and hormones which bind to specific receptors on the surface of their target cells. Examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), such as VEGFA, epidermal growth factor (EGF), insulin-like growth factor, ephrins, macrophage colonystimulating factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, neuregulins, neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti- apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P)), and tumor necrosis factor-alpha (TNF-a). In one embodiment, a "growth factor" is a peptide or protein growth factor.
The term "protease inhibitors" refers to molecules, in particular peptides or proteins, which inhibit the function of proteases. Protease inhibitors can be classified by the protease which is inhibited (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serpins). Particular examples of protease inhibitors include serpins, such as alpha 1 -antitrypsin, aprotinin, and be statin.
The term "enzymes" refers to macromolecular biological catalysts which accelerate chemical reactions. Like any catalyst, enzymes are not consumed in the reaction they catalyze and do not alter the equilibrium of said reaction. Unlike many other catalysts, enzymes are much more specific. In one embodiment, an enzyme is essential for homeostasis of a subject, e.g., any malfunction (in particular, decreased activity which may be caused by any of mutation, deletion or decreased production) of the enzyme results in a disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.
The term "receptors" refers to protein molecules which receive signals (in particular chemical signals called ligands) from outside a cell. The binding of a signal (e.g., ligand) to a receptor causes some kind of response of the cell, e.g., the intracellular activation of a kinase. Receptors include transmembrane receptors (such as ion channel-linked (ionotropic) receptors, G protein-linked (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (such as cytoplasmic receptors and nuclear receptors). Particular examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand- 1 (PSGL-1). The term "growth factor receptors" refers to receptors which bind to growth factors.
The term "apoptosis regulators" refers to molecules, in particular peptides or proteins, which modulate apoptosis, i.e., which either activate or inhibit apoptosis. Apoptosis regulators can be grouped into two broad classes: those which modulate mitochondrial function and those which regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) which act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Also to this first class belong proapoptotic proteins (e.g., BAX, BAK, BIM) which promote release of cytochrome C. The second class includes proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP which block the activation of caspases.
The term "transcription factors" relates to proteins which regulate the rate of transcription of genetic information from DNA to messenger RNA, in particular by binding to a specific DNA sequence. Transcription factors may regulate cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and/or in response to signals from outside the cell, such as a hormone. Transcription factors contain at least one DNA-binding domain which binds to a specific DNA sequence, usually adjacent to the genes which are regulated by the transcription factors. Particular examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.
The term "tumor suppressor proteins" relates to molecules, in particular peptides or proteins, which protect a cell from one step on the path to cancer. Tumor-suppressor proteins (usually encoded by corresponding tumor-suppressor genes) exhibit a weakening or repressive effect on the regulation of the cell cycle and/or promote apoptosis. Their functions may be one or more of the following: repression of genes essential for the continuing of the cell cycle; coupling the cell cycle to DNA damage (as long as damaged DNA is present in a cell, no cell division should take place); initiation of apoptosis, if the damaged DNA cannot be repaired; metastasis suppression (e.g., preventing tumor cells from dispersing, blocking loss of contact inhibition, and inhibiting metastasis); and DNA repair. Particular examples of tumor-suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI/SNF (SWItch/Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3). The term "structural proteins" refers to proteins which confer stiffness and rigidity to otherwise-fluid biological components. Structural proteins are mostly fibrous (such as collagen and elastin) but may also be globular (such as actin and tubulin). Usually, globular proteins are soluble as monomers, but polymerize to form long, fibers which, for example, may make up the cytoskeleton. Other structural proteins are motor proteins (such as myosin, kinesin, and dynein) which are capable of generating mechanical forces, and surfactant proteins. Particular examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.
The term "reprogramming factors" or "reprogramming transcription factors" relates to molecules, in particular peptides or proteins, which, when expressed in somatic cells optionally together with further agents such as further reprogramming factors, lead to reprogramming or de-differentiation of said somatic cells to cells having stem cell characteristics, in particular pluripotency. Particular examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.
The term "genomic engineering proteins" relates to proteins which are able to insert, delete or replace DNA in the genome of a subject. Particular examples of genomic engineering proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9).
The term "blood proteins" relates to peptides or proteins which are present in blood plasma of a subject, in particular blood plasma of a healthy subject. Blood proteins have diverse functions such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), protease inhibitors (e.g., alpha 1 -antitrypsin), etc. Particular examples of blood proteins include thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony stimulating factor (G-CSF), modified Factor VIII, and anticoagulants.
Thus, in some embodiments, the pharmaceutically active peptide or protein is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2), and interleukin 10 (IL- 11), more preferably EPO; (ii) an adhesion molecule, in particular an integrin; (iii) an immunoglobulin, in particular an antibody; (iv) an immunologically active compound, in particular an antigen, such as a viral or bacterial antigen, e.g., an antigen of SARS-CoV-2; (v) a hormone, in particular vasopressin, insulin or growth hormone; (vi) a growth factor, in particular VEGFA; (vii) a protease inhibitor, in particular alpha 1 -antitrypsin; (viii) an enzyme, preferably selected from the group consisting of herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes, and lactase; (ix) a receptor, in particular growth factor receptors; (x) an apoptosis regulator, in particular BAX; (xi) a transcription factor, in particular F0XP3; (xii) a tumor suppressor protein, in particular p53; (xiii) a structural protein, in particular surfactant protein B; (xiv) a reprogramming factor, e.g., selected from the group consisting of 0CT4, SOX2, c- MYC, KLF4, LIN28 and NANOG; (xv) a genomic engineering protein, in particular clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) a blood protein, in particular fibrinogen.
In some embodiments, a pharmaceutically active peptide or protein comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or protein to a subject elicits an immune response against the one or more antigens or one or more epitopes in a subject which may be therapeutic or partially or fully protective.
In certain embodiments, the nucleic acid (such as DNA or RNA, preferably mRNA) encodes at least one epitope, e.g. , at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.
In certain embodiments, the target antigen is a tumor antigen and the antigenic sequence (e.g., an epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen, which is generally known to be expressed in various cancers. The tumor antigen may also be a "neo-antigen", which is specific to an individual’s tumor and has not been previously recognized by the immune system. A neo-antigen or neo-epitope may result from one or more cancer-specific mutations in the genome of cancer cells resulting in amino acid changes. If the tumor antigen is a neo-antigen, the vaccine antigen preferably comprises an epitope or a fragment of said neo-antigen comprising one or more amino acid changes.
Examples of tumor antigens include, without limitation, p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1 , CASP-8, CDC27/m, CDK4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6- AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A 10, MAGE-A11, or MAGE- A12, MAGE-B, MAGE-C, MART-1 /Melan-A, MC1R, Myosin/m, MUC1, MUM-1, MUM-2, MUM- 3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT, and WT-1. Cancer mutations vary with each individual. Thus, cancer mutations that encode novel epitopes (neoepitopes) represent attractive targets in the development of vaccine compositions and immunotherapies. The efficacy of tumor immunotherapy relies on the selection of cancer-specific antigens and epitopes capable of inducing a potent immune response within a host. Nucleic acid (such as DNA or RNA) can be used to deliver patient-specific tumor epitopes to a patient. Dendritic cells (DCs) residing in the spleen represent antigen-presenting cells of particular interest for RNA expression of immunogenic epitopes or antigens such as tumor epitopes. The use of multiple epitopes has been shown to promote therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of the tumor mutanome may provide multiple epitopes for individualized vaccines which can be encoded by nucleic acid (such as DNA or RNA, in particular mRNA) described herein, e.g., as a single polypeptide wherein the epitopes are optionally separated by linkers. In certain embodiments of the present disclosure, the nucleic acid (such as DNA or RNA, in particular mRNA) encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary embodiments include nucleic acid (such as DNA or RNA, in particular mRNA) that encodes at least five epitopes (termed a "pentatope"), nucleic acid (such as DNA or RNA, in particular mRNA) that encodes at least ten epitopes (termed a "decatope"), and nucleic acid (such as DNA or RNA, in particular mRNA) that encodes at least twenty epitopes (termed an "eicosatope").
In certain embodiments, the epitope is derived from a pathogen-associated antigen. In some embodiments, the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof.
In some embodiments, the pathogen is selected from viruses, bacteria, fungi, parasites, and other microorganisms.
Exemplary viruses include, but are not limited to, are severe acute respiratory syndrome coronavirus (SARS-CoV), such as SARS-CoV2, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) (e.g., CMV5), human herpesviruses (HHV) (e.g., HHV6, 7 or 8), herpes simplex viruses (HSV), bovine herpes virus (BHV) (e.g., BHV4), equine herpes virus (EHV) (e.g., EHV2), human T-Cell leukemia viruses (HTLV)5, Varicella-Zoster virus (VZV), measles virus, papovaviruses (JC and BK), hepatitis viruses (e.g., HBV or HCV), myxoma virus, adenoviruses, rhinoviruses, enteroviruses, parvoviruses, polyoma virus, influenza viruses, papillomaviruses (such as human papillomavirus (HPV)), poxviruses such as vaccinia virus, and molluscum contagiosum virus (MCV), lyssaviruses, rotaviruses, noroviruses, rubella viruses, and mumps viruses. Exemplary diseases caused by viral infection include, but are not limited to, SARS, acquired immune deficiency syndrome (AIDS), measles, chicken pox, cytomegalovirus infections, genital herpes, hepatitis (such as hepatitis B or C), influenza (flu, such as human flu, swine flu, dog flu, horse flu, and avian flu), HPV infection, shingles, rabies, common cold, gastroenteritis, rubella, and mumps.
Exemplary bacteria include, but are not limited to, Campylobacter (such as Campylobacter jejuni), Enterobacter species, Enterococcus faecium, Enterococcus faecalis, Escherichia coli (e.g., F. coli O157:H7), Group A streptococci, Haemophilus influenzae, Helicobacter pylori, listeria, Mycobacterium tuberculosis, Pseudomonas aeruginosa, S. pneumoniae, Salmonella, Shigella, Staphylococcus aureus, Staphylococcus epidermidis, Borrelia and Rickettsia, Chlamydiaceae, Neisseria gonorrhoeae, Bordetella pertussis, Clostridium tetani, Neisseria meningitidis, Streptococcus (such as Streptococcus pneumoniae or Streptococcus pyogenes), and Treponema pallidum. Exemplary diseases caused by bacterial infection include, but are not limited to, anthrax, cholera, diphtheria, foodbome illnesses, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, gonorrhea, pertussis, tetanus, meningitis, scarlet fever, and syphilis.
Exemplary parasites include, but are not limited to, Plasmodium, Trypanosoma, Leishmania, Trichomonas, Dientamoeba, Giardia, Entamoeba histolytica, Naegleria, Isospora, Toxoplasma, Sarcocystis, Rhinosporidium seeberi, and Balantidium. Exemplary diseases caused by parasite infection include, but are not limited to, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, dientamoebiasis, giardiasis, amebic dysentery, coccidiosis, toxoplasmosis, sarcocystosis, rhinosporidiosis, and balantidiasis.
In some embodiments, the pathogen is an infectious pathogen, in particular a pathogen causing an infectious disease, such as a viral disease, a bacterial disease, or a parasitic disease. In some embodiments, the pathogen is a virus, bacterium, or parasite. Thus, in these embodiments, the nucleic acid (such as DNA or RNA, in particular mRNA) and/or compositions described herein can be used to prevent and/or treat an infectious disease caused by said pathogen.
In certain embodiments, the epitope is derived from a viral antigen.
In some embodiments, the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof. Thus, in some embodiments, the nucleic acid (such as DNA or RNA, in particular mRNA) used in the present disclosure encodes an amino acid sequence comprising a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof.
In some embodiments, the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or the immunogenic variant thereof. Thus, in some embodiments, the nucleic acid (such as DNA or RNA, in particular, mRNA) described in the present disclosure encodes an amino acid sequence comprising a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or the immunogenic variant thereof. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.
In some embodiments, the nucleic acid (e.g., DNA or RNA, in particular, mRNA) described herein is a modified RNA, in particular a stabilized mRNA. In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (y), Nl-methyl-pseudouridine (ml\|/), and 5-methyl-uridine (m5U).
In some embodiments, the RNA (in particular, mRNA) described herein comprises a modified nucleoside in place of uridine.
In some embodiments, the modified nucleoside is selected from pseudouridine (y), Nl-methyl- pseudouridine (ml\|/), and 5-methyl-uridine (m5U).
In some embodiments, the RNA (in particular, mRNA) described herein comprises a 5’ cap. In some embodiments, rm73 °Gppp(mi2 °) ApG is utilized as specific capping structure at the 5'-end of the mRNA.
In some embodiments, the RNA (in particular, mRNA) encoding an antigen (in particular the vaccine RNA) comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.
In some embodiments, the RNA (in particular, mRNA) encoding an antigen (in particular the vaccine RNA) comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the RNA (in particular, mRNA) encoding an antigen (in particular the vaccine RNA) comprises a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 3.
In some embodiments of the present disclosure, the antigen (such as a tumor antigen or vaccine antigen) is preferably administered as single -stranded, 5' capped RNA (preferably mRNA) that is translated into the respective protein upon entering cells of a subject being administered the RNA. Preferably, the RNA contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence).
In some embodiments, beta-S-ARCA(Dl) is utilized as specific capping structure at the 5'-end of the RNA. In one embodiment, mj7,3 °Gppp(mi2 °)ApG is utilized as specific capping structure at the 5'- end of the RNA. In some embodiments, the 5'-UTR sequence is derived from the human alpha-globin mRNA and optionally has an optimized 'Kozak sequence' to increase translational efficiency. In some embodiments, a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA. In some embodiments, two re-iterated 3'-UTRs derived from the human beta-globin mRNA are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA. In some embodiments, a poly (A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues is used. This poly(A) sequence was designed to enhance RNA stability and translational efficiency.
In general, vaccine RNA described herein may comprise, from 5' to 3', one of the following structures: Cap-5'-UTR-Vaccine Antigen-Encoding Sequence-3 '-UTR-Poly(A) or beta-S-ARCA(D l)-hAg-Kozak-Vaccine Antigen-Encoding Sequence-FI-A30L70.
The above described RNA or RNA encoding the above described vaccine antigen may be non-modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA) or self-amplifying RNA (saRNA). In some embodiments, the above described RNA or RNA encoding the above described vaccine antigen is nucleoside modified mRNA (modRNA).
Non-modified uridine messenger RNA (uRNA) The active principle of the non-modified messenger RNA (uRNA) is a single-stranded mRNA that is translated upon entering a cell. In addition to the sequence encoding the vaccine antigen (i.e. open reading frame), each uRNA preferably contains common structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5 '-cap, 5'-UTR, 3'-UTR, poly(A)-tail). The preferred 5’ cap structure is beta-S-ARCA(Dl) (m27’2 OGppSpG). The preferred 5'- UTR and 3'-UTR comprise the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively. The preferred poly(A)-tail comprises the sequence of SEQ ID NO: 3.
In this respect, "hAg -Kozak" mean the 5'-UTR sequence of the human alpha-globin mRNA with an optimized ‘Kozak sequence’ to increase translational efficiency; "FI element" means that the 3'-UTR is a combination of two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression; "A30L70" means a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells; "GS" means a glycineserine linker, i.e., sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.
Nucleoside modified messenger RNA (modRNA)
The active principle of the nucleoside modified messenger RNA (modRNA) drug substance is as well a single-stranded mRNA that is translated upon entering a cell. In addition to the sequence encoding the vaccine antigen (i.e., open reading frame), each modRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5 '-cap, 5'-UTR, 3'-UTR, poly(A)-tail). Compared to the uRNA, modRNA contains 1-methyl-pseudouridine instead of uridine. The preferred 5’ cap structure is m27’3 °Gppp(mi2 °)ApG. The preferred 5'-UTR and 3 '-UTR comprise the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively. The preferred poly(A)-tail comprises the sequence of SEQ ID NO: 3. An additional purification step is applied for modRNA to reduce dsRNA contaminants generated during the in vitro transcription reaction.
Self-amplifying RNA (saRNA)
The active principle of the self-amplifying mRNA (saRNA) drug substance is a single-stranded RNA, which self-amplifies upon entering a cell, and the vaccine antigen is translated thereafter. In contrast to uRNA and modRNA that preferably code for a single protein, the coding region of saRNA contains two open reading frames (ORFs). The 5’-ORF encodes the RNA-dependent RNA polymerase such as Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase). The replicase ORF is followed 3’ by a subgenomic promoter and a second ORF encoding the antigen. Furthermore, saRNA UTRs contain 5’ and 3’ conserved sequence elements (CSEs) required for selfamplification. The saRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5 '-cap, 5'-UTR, 3'-UTR, poly(A)-tail). The saRNA preferably contains uridine. The preferred 5’ cap structure is beta-S-ARCA(Dl) (m27’2 OGppSpG).
Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle. However, the saRNA does not encode for alphaviral structural proteins that are required for genome packaging or cell entry, therefore generation of replication competent viral particles is very unlikely to not possible. Replication does not involve any intermediate steps that generate DNA. The use/uptake of saRNA therefore poses no risk of genomic integration or other permanent genetic modification within the target cell. Furthermore, the saRNA itself prevents its persistent replication by effectively activating innate immune response via recognition of dsRNA intermediates.
Furthermore, a secretory signal peptide (sec) may be fused to the antigen-encoding regions preferably in a way that the sec is translated as N terminal tag. In some embodiments, sec corresponds to the secretory signal peptide of the S protein. Sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins may be used as GS/Linkers.
In some embodiments, nucleic acid (such as DNA or RNA, preferably mRNA) encoding an antigen (such as a tumor antigen or a vaccine antigen) is expressed in cells of the subject treated to provide the antigen. In some embodiments, the nucleic acid (such as DNA or RNA) is transiently expressed in cells of the subject. In some embodiments, the RNA is in vitro transcribed. In some embodiments, expression of the antigen is at the cell surface. In some embodiments, the antigen is expressed and presented in the context of MHC. In some embodiments, expression of the antigen is into the extracellular space, i.e., the antigen is secreted.
The antigen molecule or a procession product thereof, e.g., a fragment thereof, may bind to an antigen receptor such as a BCR or TCR carried by immune effector cells, or to antibodies.
A peptide and protein antigen which is provided to a subject according to the present disclosure by administering nucleic acid (such as DNA or RNA, in particular mRNA) encoding a peptide and protein antigen, wherein the antigen is a vaccine antigen, preferably results in the induction of an immune response, e.g., a humoral and/or cellular immune response in the subject being provided the peptide or protein antigen. Said immune response is preferably directed against a target antigen. Thus, a vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In one embodiment, such fragment or variant is immunologically equivalent to the target antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent which results in the induction of an immune response which immune response targets the antigen, i.e. a target antigen. Thus, the vaccine antigen may correspond to or may comprise the target antigen, may correspond to or may comprise a fragment of the target antigen or may correspond to or may comprise an antigen which is homologous to the target antigen or a fragment thereof. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence being homologous to an immunogenic fragment of a target antigen. An "immunogenic fragment of an antigen" according to the disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.
The term "immunologically equivalent" means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence. Thus, in some embodiments, a molecule which is immunologically equivalent to an antigen exhibits the same or essentially the same properties and/or exerts the same or essentially the same effects regarding the stimulation, priming and/or expansion of T cells as the antigen to which the T cells are targeted.
In one embodiment, the RNA (preferably mRNA) used in the present disclosure is non-immunogenic. RNA encoding an immunostimulant may be administered according to the present disclosure to provide an adjuvant effect. The RNA encoding an immunostimulant may be standard RNA or non-immunogenic RNA.
The term "non-immunogenic RNA" (such as "non-immunogenic mRNA") as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non- immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA, which is also termed modified RNA (modRNA) herein, is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or limiting the amount of double -stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA), e.g., during in vitro transcription, and/or by removing double -stranded RNA (dsRNA), e.g., following in vitro transcription. In certain embodiments, non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.
For rendering the non-immunogenic RNA (especially mRNA) non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA- mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In one embodiment, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3 -methyl -uridine (m3U), 5- methoxy-uridine (mo5U), 5 -aza-uridine, 6-aza-uridine, 2-thio-5 -aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy-uridine (ho5U), 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5 -iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5 -carboxymethyl -uridine (cm5U), 1 -carboxymethylpseudouridine, 5 -carboxyhydroxymethyl -uridine (chm5U), 5 -carboxyhydroxymethyl -uridine methyl ester (mchm5U), 5 -methoxycarbonylmethyl -uridine (mcm5U), 5 -methoxycarbonylmethyl-2 -thiouridine (mcm5s2U), 5 -aminomethyl -2 -thio-uridine (nm5s2U), 5 -methylaminomethyl -uridine (mnm5U),
1 -ethyl -pseudouridine, 5 -methylaminomethyl -2 -thio-uridine (mnm5s2U), 5 -methylaminomethyl -2- seleno-uridine (mnm5se2U), 5 -carbamoylmethyl -uridine (ncm5U), 5 -carboxymethylaminomethyl - uridine (cmnm5U), 5 -carboxymethylaminomethyl-2 -thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynyl-pseudouridine, 5 -taurinomethyl -uridine (rm5U), I-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(rm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5 -methyl -2 -thiouridine (m5s2U), 1 -methyl -4-thio-pseudouridine (m 's4i|i). 4-thio-l -methyl -pseudouridine, 3-methyl- pseudouridine (m y/). 2 -thio- 1 -methyl -pseudouridine, I -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5- methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy -2-thio-pseudouridine, N1 -methyl - pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3 y), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O- dimethyl -uridine (m5Um), 2'-O-methyl-pseudouridine (y/m). 2-thio-2'-O-methyl -uridine (s2Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3, 2'-O-dimethyl -uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-0H-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E- propenylamino)uridine. In one particularly preferred embodiment, the nucleoside comprising a modified nucleobase is pseudouridine (y), N1 -methyl -pseudouridine (m I \|/) or 5 -methyl -uridine (m5U), in particular N 1 -methyl -pseudouridine .
In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.
During synthesis of RNA (preferably mRNA) by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double -stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. Formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP may be added once or several times during synthesis of mRNA. Also, dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In one embodiment, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524.
As the term is used herein, "remove" or "removal" refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the nonseparated mixture of first and second substances.
In some embodiments, the removal of dsRNA (especially dsmRNA) from non-immunogenic RNA comprises a removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside modified RNA (especially mRNA) is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).
Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with dsRNA-specific antibody and the amount of antibody binding to RNA may be taken as a measure for the amount of dsRNA in the sample. A sample containing a known amount of dsRNA may be used as a reference.
For example, RNA may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v/v) TWEEN-20) containing 5% (w/v) skim milk powder. For detection of dsRNA, the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
In some embodiments, the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3 -fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5 -fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10-fold factor. In some embodiments, translation is enhanced by a 15 -fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In one embodiment, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.
In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non- immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3 -fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5 -fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by a 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15 -fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.
The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non- immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non- immunogenic RNA. In some embodiments, the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA. In some embodiments, the nucleic acid (e.g., DNA or RNA, in particular, mRNA) described herein is formulated or is to be formulated as a liquid, a solid, or a combination thereof.
In some embodiments, the nucleic acid (e.g., DNA or RNA, in particular, mRNA) described herein is formulated or is to be formulated for injection.
In some embodiments, the nucleic acid (e.g., DNA or RNA, in particular, mRNA) described herein is formulated or is to be formulated for intramuscular administration.
In some embodiments, the nucleic acid (e.g., DNA or RNA, in particular, mRNA) described herein is formulated or is to be formulated as a composition, e.g., a nucleic acid composition, in particular a pharmaceutical composition.
In some embodiments, the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-ll), (V-12), (V-13), (V- 14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)).
In some embodiments, the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid selected from the group consisting of DODMA, DOTMA, DPL14, 3D-P- DMA, formula (X), and formula (XI); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (VJ’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-ll), (V-12), (V-13), (V- 14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); and (iv) one or more additional lipids. In some embodiments, the one or more additional lipids are selected from neutral lipids and combinations thereof. In some embodiments, the neutral lipids include phospholipids, steroid lipids, and combinations thereof. In some embodiments, the one or more additional lipids are a combination of a phospholipid and a steroid lipid.
In some embodiments, the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any one of formulas A to G; (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments, the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (X); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (VJ’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V- 12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments, the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (XI) (e.g., any one of formulas (Xlla), (Xllb), (Xllla), (Xlllb), (XIV-1), (XIV-2), and (XIV-3); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V-12), (V-13), (V- 14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments, the composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any one of formulas (XV- 1) to (XV-6); (iii) a polymer-conjugated compound as specified herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22),
(V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35),
(V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48),
(V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61),
(V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments, the nucleic acid is DNA.
In some embodiments, the nucleic acid is RNA, in particular mRNA.
In some embodiments, the composition, in particular the pharmaceutical composition, is a vaccine.
In some embodiments, the composition, in particular the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. In some embodiments, the nucleic acid (e.g., DNA or RNA) and/or the composition, in particular the pharmaceutical composition, is/are a component of a kit.
In some embodiments, the kit further comprises instructions for use of the nucleic acid (e.g., DNA or RNA) for inducing an immune response against a pathogen, e.g., against a virus, such as coronavirus, in a subject. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.
In some embodiments, the kit further comprises instructions for use of the nucleic acid (e.g., DNA or RNA) for therapeutically or prophylactically treating an infection, e.g., a viral infection, such as a coronavirus infection, in a subject. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV- 2.
In some embodiments, the subject is a human.
In certain embodiments of the present disclosure, the nucleic acid (e.g., DNA or RNA) in the nucleic acid (e.g., DNA or RNA) compositions (e.g., in the nucleic acid (e.g., DNA or RNA) particles) described herein is at a concentration from about 0.002 mg/mL to about 5 mg/mL, from about 0.002 mg/mL to about 2 mg/mL, from about 0.005 mg/mL to about 2 mg/mL, from about 0.01 mg/mL to about 1 mg/mL, from about 0.05 mg/mL to about 0.5 mg/mL or from about 0. 1 mg/mL to about 0.5 mg/mL. In specific embodiments, the nucleic acid (e.g., DNA or RNA) is at a concentration from about 0.005 mg/mL to about 0.1 mg/mL, from about 0.005 mg/mL to about 0.09 mg/mL, from about 0.005 mg/mL to about 0.08 mg/mL, from about 0.005 mg/mL to about 0.07 mg/mL, from about 0.005 mg/mL to about 0.06 mg/mL, or from about 0.005 mg/mL to about 0.05 mg/mL.
Particles
Nucleic acids such as DNA or RNA, in particular mRNA, described herein may be present in particles comprising (i) the nucleic acid, (ii) at least one cationic or cationically ionizable compound; and (iii) a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V- 6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V- 5), (V-17), and (V-25)). In some embodiments, the particles comprise (i) the nucleic acid (such as RNA, in particular mRNA); (ii) at least one cationic or cationically ionizable compound as disclosed herein; (iii) a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I); and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VF), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V- 5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31),
(V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44),
(V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57),
(V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-
1), (V-5), (V-17), and (V-25)); (iv) a steroid as disclosed herein; and (v) a neutral lipid as disclosed herein.
Different types of nucleic acid (e.g., DNA or RNA) containing particles have been described previously to be suitable for delivery of nucleic acid (e.g., DNA or RNA) in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid (e.g., DNA or RNA) delivery vehicles, nanoparticle encapsulation of nucleic acid (e.g., DNA or RNA) physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.
Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles.
During the manufacturing process, introduction of an aqueous solution of nucleic acid (such as RNA) to an organic (e.g., ethanolic) solution comprising a lipid mixture containing a cationically ionizable lipid at pH of, e.g., 5 leads to an electrostatic interaction between the negatively charged nucleic acid (e.g., DNA or RNA) drug substance and the positively charged cationically ionizable lipid. This electrostatic interaction leads to particle formation coincident with efficient encapsulation of the nucleic acid (e.g., DNA or RNA) drug substance. After nucleic acid encapsulation, adjustment of the medium surrounding the resulting nucleic acid particle (such as RNA-LNP) to, e.g., pH 7-8 results in neutralization of the surface charge on the particles (e.g., LNPs). When all other variables are held constant, charge -neutral particles display longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are cleared rapidly by the reticuloendothelial system. Upon endosomal uptake, the low pH of the endosome renders the particles (such as LNPs) fusogenic and allows for release of the nucleic acid (such as RNA) into the cytosol of the target cell.
In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids, amphiphilic polymers, and/or amphiphilic proteins/polypeptides). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids and/or additional polymers) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids, amphiphilic polymers, and/or amphiphilic proteins/polypeptides) optionally in combination with additional substances (e.g., additional lipids and/or additional polymers) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. In this respect, the term "micro-sized" means that all three external dimensions of the particle are in the microscale, i.e., between 1 and 5 pm.
According to the present disclosure, the term "particle" includes lipoplex particles (LPXs), lipid nanoparticles (LNPs), polyplex particles, lipopolyplex particles, virus-like particles (VLPs), and mixtures thereof (e.g., a mixture of two or more of particle types, such as a mixture of LPXs and VLPs or a mixture of LNPs and VLPs).
A "nucleic acid particle" can be used to deliver nucleic acid (such as RNA, in particular mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid, a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, liposome-based, polyplex-based, lipopolyplex-based, virus-like (VLP)-based formulations and mixtures thereof. In some embodiments, nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, and/or liposome-based formulations.
Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid and the polymer-conjugated compound as disclosed herein and, if present, additional lipids combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.
In some embodiments, particles comprise an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and nucleic acid (such as RNA, especially mRNA) as described herein. In some embodiments, particles comprise or consist of a cationic/cationically ionizable lipid (in particular, a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein); helper lipids such as a neutral lipid (such as a phospholipid), and a steroid (such as cholesterol), and combinations thereof; and an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22),
(V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35),
(V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48),
(V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61),
(V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25).
In some embodiments, the nucleic acid (such as RNA) particles disclosed herein comprise (i) nucleic acid (such as RNA, especially mRNA) as described herein; (ii) an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, (such as a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; a cationically ionizable lipid of formula (XI) disclosed herein; or a cationic lipid as disclosed herein); (iii) a polymer-conjugated compound as disclosed herein comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V- 6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-
5), (V-17), and (V-25)); (iv) a steroid (such as cholesterol); and (v) a neutral lipid (such as a phospholipid). In some embodiments, the steroid is cholesterol; and the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE. In some embodiments, the amphiphilic OEG- conjugated compound is a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-
6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19),
(V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32),
(V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45),
(V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58),
(V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V- 5), (V-17), and (V-25). In some embodiments, the steroid is cholesterol; the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and the amphiphilic OEG-conjugated compound is a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34),
(V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47),
(V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60),
(V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and
(V-25).
In some embodiments, in the nucleic acid particles (such as RNA particles) described herein the nucleic acid (such as RNA, in particular, mRNA) is bound by cationically ionizable lipid (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein) that, in the case of LNPs, occupies the central core of the LNPs. In some embodiments, the polymer- conjugated compound forms the surface of the particles (such as LNPs), along with phospholipids. In some embodiments, the particles are substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units. In some embodiments, the particles are substantially free of PEG lipids having at least 30 consecutive ethylene glycol repeating units and substantially free of polysarcosine-conjugated lipids. In some embodiments, the particles are substantially free of polymer-conjugated lipids other than the polymer- conjugated compounds comprising (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22),
(V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35),
(V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48),
(V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61),
(V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)). In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and cationically ionizable lipid (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein) in charged and uncharged forms can be distributed throughout the particles such as LNPs.
In general, a lipoplex (LPX) is obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid (in particular RNA) and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes.
In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.
In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.
In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with nucleic acids (such as DNAs or RNAs). In some embodiments, formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid-lipoplexes. In some embodiments, these formulations are characterized by their poor encapsulation of the nucleic acid (such as DNA or RNA) and incomplete entrapment of the nucleic acid.
In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and nucleic acid (such as RNA, especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acid (such as RNA, especially mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as a cationically ionizable lipid of formula (I), DOTMA and/or DODMA, and additional lipids, such as DSPC. In some embodiments, a nucleic acid (such as RNA, especially mRNA) lipoplex particle is a nanoparticle.
In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of nucleic acid (such as RNA) in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water. In some embodiments, the lipids comprise a cationically ionizable lipid (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), a steroid as disclosed herein (such as cholesterol), a neutral lipid as disclosed herein (such as a phospholipid), and an amphiphilic OEG-conjugated compound as specified herein (e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l),
(V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24),
(V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37),
(V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)).
In some embodiments, particles described herein comprise a cationically ionizable lipid as disclosed herein (in particular a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), a steroid as disclosed herein (such as cholesterol), a neutral lipid as disclosed herein (such as a phospholipid), and a polymer-conjugated compound as disclosed herein (z. e. , an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V- 14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25).
In some embodiments, nucleic acid particles (especially RNA particles such as RNA LNPs (e.g., mRNA particles such as mRNA LNPs)) comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features,
In some embodiments, nucleic acid (such as RNA, e.g., mRNA) described herein may be noncovalently associated with a particle as described herein. In some embodiments, the nucleic acid (such as RNA, especially mRNA) may be adhered to the outer surface of the particle (surface nuclei acid (such as surface RNA, especially surface mRNA)) and/or may be contained in the particle (encapsulated nucleic acid (such as surface RNA, especially encapsulated mRNA)).
As used in the present disclosure, "nanoparticle" refers to a particle comprising nucleic acid (especially RNA such as mRNA) as described herein and at least one cationic lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm (preferably, between 10 and 990 nm, such as between 15 and 900 nm, between 20 and 800 nm, between 30 and 700 nm, between 40 and 600 nm, or between 50 and 500 nm). Preferably, the longest and shortest axes do not differ significantly. Preferably, the size of a particle is its diameter. Preferably, the particle has an average diameter suitable for intravenous administration.
Nucleic acid particles described herein (especially RNA particles, such as mRNA particles) may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the nucleic acid particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0. 1 to about 0.3.
In the context of the present disclosure, the term "lipoplex particle" relates to a particle that contains an amphiphilic lipid, in particular cationic amphiphilic lipid, and nucleic acid (especially RNA such as mRNA) as described herein. Electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic amphiphilic lipids) and negatively charged nucleic acid (especially RNA such as mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic amphiphilic lipid, such as DOTMA, and additional lipids, such as DOPE or DSPC. In one embodiment, a nucleic acid (especially RNA such as mRNA) lipoplex particle is a nanoparticle.
The term "lipid nanoparticle" relates to a nano-sized lipid containing particle.
In the context of the present disclosure, the term "polyplex particle" relates to a particle that contains an amphiphilic polymer, in particular a cationic amphiphilic polymer, and nucleic acid (especially RNA such as mRNA) as described herein. Electrostatic interactions between positively charged cationic amphiphilic polymers and negatively charged nucleic acid (especially RNA such as mRNA) results in complexation and spontaneous formation of nucleic acid polyplex particles. Positively charged amphiphilic polymers suitable for the preparation of polyplex particle include protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine and histone. In one embodiment, a nucleic acid (especially RNA such as mRNA) polyplex particle is a nanoparticle.
The term "lipopolyplex particle" relates to particle that contains amphiphilic lipid (in particular cationic amphiphilic lipid) as described herein, amphiphilic polymer (in particular cationic amphiphilic polymer) as described herein, and nucleic acid (especially RNA such as mRNA) as described herein. In one embodiment, a nucleic acid (especially RNA such as mRNA) lipopolyplex particle is a nanoparticle.
The term "virus-like particle" (abbreviated herein as VLP) refers to a molecule that closely resembles a virus, but which does not contain any genetic material of said virus and, thus, is non-infectious. Preferably, VLPs contain nucleic acid (preferably RNA) as described herein, said nucleic acid (preferably RNA) being heterologous to the virus(es) from which the VLPs are derived. VLPs can be synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. In one embodiment, combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs can be produced from components of a wide variety of virus families including Hepatitis B virus (HBV) (small HBV derived surface antigen (HBsAg)), Parvoviridae (e.g., adeno-associated virus), Papillomaviridae (e.g., HPV), Retroviridae (e.g., HIV), Flaviviridae (e.g., Hepatitis C virus) and bacteriophages (e.g. QP, AP205).
The term "nucleic acid containing particle" relates to a particle as described herein to which nucleic acid (especially RNA such as mRNA) is bound. In this respect, the nucleic acid (especially RNA such as mRNA) may be adhered to the outer surface of the particle (surface nucleic acid (especially surface RNA such as surface mRNA)) and/or may be contained in the particle (encapsulated nucleic acid (especially encapsulated RNA such as encapsulated mRNA)).
In one embodiment, the particles described herein have a size (preferably a diameter, i.e., double the radius such as double the radius of gyration (Rg) value or double the hydrodynamic radius) in the range of about 10 to about 2000 nm, such as at least about 15 nm (preferably at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most 1900 nm (preferably at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), preferably in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, or about 50 to 500 nm or about 100 to 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, or 70 to 150 nm.
In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm.
With respect to nucleic acid particles (such as RNA lipid particles, especially RNA LNPs such as mRNA LNPs), the N/P ratio or N/P value gives the ratio of the nitrogen groups (in particular positively- chargeable polymer amine (N = nitrogen) groups) in the lipid to the number of negatively-charged phosphate (P) groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid (such as DNA or RNA) is considered to be completely bound to nanoparticles.
Nucleic acid particles (especially RNA particles such as mRNA particles) described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles.
The term "colloid" as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term "colloid" only refers to the particles in the mixture and not the entire suspension.
For the preparation of colloids comprising at least one cationic or cationically ionizable lipid methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media). In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included.
Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.
The term "ethanol injection technique" refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, the nucleic acid (especially RNA such as mRNA) lipoplex particles described herein are obtainable by adding nucleic acid (especially RNA such as mRNA) to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationically ionizable lipids (like a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of the structures A to G disclosed herein; a cationically ionizable lipid of formula (XI) disclosed herein; DOTMA and/or DODMA) and additional lipids (such as the polymer-conjugated lipid described herein (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VF), (VII), (VII’), (VIII), (VIII ), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24),
(V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37),
(V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50),
(V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-
63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)); a neutral lipid (such as a phospholipid); a steroid (such as cholesterol); and combinations), is injected into an aqueous solution under stirring. In some embodiments, the nucleic acid (especially RNA such as mRNA) lipoplex particles described herein are obtainable without a step of extrusion. The term "extruding" or "extrusion" refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.
Other methods having organic solvent free characteristics may also be used according to the present disclosure for preparing a colloid.
Particles (such as LNPs) typically comprise four particle forming components: one or more cationically ionizable or cationic lipids; one or more neutral lipids such as phospholipids; one or more steroids such as cholesterol; and a polymer-conjugated lipid (which is sometimes called "stealth lipid" or "sterically stabilizing lipid"), such as a PEG lipid or a polysarcosine-conjugated lipid. Each component is responsible for payload protection, and enables effective intracellular delivery. However, due to the disadvantages of PEG lipids (which are the most common polymer-conjugated lipids), particles (such as LNPs) of the present disclosure have a slightly different composition: one or more cationically ionizable or cationic lipids; one or more neutral lipids such as phospholipids; one or more steroids such as cholesterol; and a polymer-conjugated compound disclosed herein which comprises (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII ), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V- 1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V-12), (V-13), (V-14), (V- 15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)). In some embodiments, particles (such as LNPs) of the present disclosure are substantially free of a PEG lipid having at least 30 consecutive ethylene glycol repeating units. In some embodiments, particles (such as LNPs) of the present disclosure are substantially free of a polysarcosine-conjugated lipid. In some embodiments, particles (such as LNPs) of the present disclosure are substantially free of a PEG lipid having at least 30 consecutive ethylene glycol repeating units and substantially free of a polysarcosine-conjugated lipid. In some embodiments, particles (such as LNPs) of the present disclosure are substantially free of polymer-conjugated lipids other than the amphiphilic OEG-conjugated compound disclosed herein. Particles (such as LNPs) may be prepared by first mixing lipids (including the amphiphilic OEG- conjugated compound) dissolved in an organic (e.g., ethanolic) solution rapidly with nucleic acid (such as RNA or DNA) in an aqueous buffer. Different types of nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.
In certain embodiments, the compositions (in particular particles, such as LNPs) comprising nucleic acid (e.g., DNA or RNA), at least one cationic or cationically ionizable lipid described herein, and the polymer-conjugated compound disclosed herein which comprises (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V- 6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19),
(V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32),
(V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45),
(V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58),
(V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V- 5), (V-17), and (V-25)) further comprise one or more additional lipids.
In some embodiments, the compositions (in particular particles, such as LNPs) comprising nucleic acid (such as RNA), at least one cationic or cationically ionizable lipid described herein, and the amphiphilic OEG-conjugated compound are prepared by (a) providing (e.g., preparing) a nucleic acid solution containing water and a first buffer system; (b) providing (e.g., preparing) an organic (e.g. ethanolic) solution comprising the at least one cationic or cationically ionizable lipid, the amphiphilic OEG- conjugated compound disclosed herein, and, if present, one or more additional lipids (such as a steroid and/or a neutral lipid; (c) mixing the nucleic acid solution provided (e.g., prepared) under (a) with the organic (e.g., ethanolic) solution provided (e.g., prepared) under (b), thereby preparing a first intermediate formulation comprising the particles (such as LNPs) dispersed in a first aqueous phase comprising the first buffer system; and (d) filtrating (e.g., dialyzing, tangential flow filtrating, or diafiltrating) and/or diluting the first intermediate formulation prepared under (c) using a final aqueous buffer solution comprising the final buffer system, thereby preparing the formulation comprising particles (such as LNPs) dispersed in a final aqueous phase comprising the final buffer system. After step (c) and/or (d) one or more steps selected from diluting and filtrating, such as dialyzing, tangential flow filtrating or diafiltrating, can follow. In some embodiments, the compositions (in particular particles, such as LNPs) comprising nucleic acid (such as RNA), at least one cationic or cationically ionizable lipid described herein, and the polymer- conjugated compound disclosed herein which comprises (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V- 6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19),
(V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32),
(V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45),
(V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58),
(V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V-l), (V- 5), (V-17), and (V-25)) are prepared by (a’) providing (e.g., preparing) liposomes or a colloidal preparation of the at least one cationic or cationically ionizable lipid, the polymer-conjugated compound disclosed herein, and, if present, one or more additional lipids in an aqueous phase; (b’) providing (e.g., preparing) a nucleic acid (such as RNA) solution containing water and a buffering system; and (c’) mixing the liposomes or colloidal preparation provided (e.g., prepared) under (a’) with the nucleic acid (such as RNA) solution provided (e.g., prepared) under (b’). After step (c’) one or more steps selected from diluting and fdtrating, such as dialyzing, tangential flow filtrating, or diafiltrating, can follow.
The present disclosure describes compositions which comprise particles comprising nucleic acid (such as DNA or RNA, especially LNPs comprising RNA), at least one cationic or cationically ionizable lipid, and the polymer-conjugated compound disclosed herein which comprises (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG-conjugated compound as specified herein, e.g., a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V- 5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any one of formulas (V- 1), (V-5), (V-17), and (V-25)) which associate with the nucleic acid (such as DNA or RNA) to form nucleic acid particles. The nucleic acid (such as DNA or RNA) particles may comprise nucleic acid which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells.
Suitable cationic or cationically ionizable lipids are those that form nucleic acid particles and are included by the term "particle forming components" or "particle forming agents". The term "particle forming components" or "particle forming agents" relates to any components which associate with nucleic acid to form nucleic acid particles. Such components include any component which can be part of nucleic acid particles.
In some embodiments, nucleic acid (such as RNA) particles (especially mRNA particles) comprise more than one type of nucleic acid (such as RNA) molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.
In particulate formulation, it is possible that each nucleic acid (such as DNA or RNA) species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one nucleic acid (such as DNA or RNA) species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each nucleic acid (such as DNA or RNA) species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid (such as DNA or RNA) species that is being provided when the particles are formed (individual particulate formulations). In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that all nucleic acid (such as DNA or RNA) species of the pharmaceutical composition are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of all nucleic acid (such as DNA or RNA) species together with a particle -forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one nucleic acid (such as DNA or RNA) species. In a combined particulate composition different nucleic acid (such as DNA or RNA) species are typically present together in a single particle.
Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains (the amphiphilic OEG-conjugated compound)
One or more of the particle -forming components described herein such as polymers, lipids or lipid-like materials used in the compositions and particles described herein comprise a polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester, preferably substituted amide, optionally substituted thioamide, or ester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.
This polymer-conjugated compound comprises oligo ethylene glycol (OEG) stretches (because in formula (I) z is 2 to 24, such as 2 to 10, 2 to 7, or 2 to 5, in particular 2 or 3) which are separated from each other by the moiety -X2-X1-Y-, i.e., by an optionally substituted amide-C1-3 alkylene, optionally substituted thioamide-C1-3 alkylene or ester-C1-3 alkylene moiety. Furthermore, besides the hydrophilic component of formula (I), this polymer-conjugated compound comprises one or more hydrophobic chains. Thus, due to these characteristics, this polymer-conjugated compound is also referred to herein as "amphiphilic OEG-conjugated compound".
Surprisingly, the present inventors found that anti-PEG antibodies do not bind to a polymer comprising the structure of formula (I); cf., e.g., Example 14. Thus, without wishing to be bound to a certain theory, it is believed that the specific structure of formula (I) (i.e., OEG stretches separated from each other by the moiety -X2-X1-Y-) prevents the binding of anti-PEG antibodies to this structure and, likewise, to the amphiphilic OEG-conjugated compound of the present disclosure. Furthermore, the present inventors found that amphiphilic OEG-conjugated compounds of the present disclosure can be synthesized in monodisperse form (by, e.g., SPPS), unlike polydisperse PEG. In particular, a structure of formula (I), wherein X1 is -C(O)-, X2 is -NH-, z is 2, and n is 14, can be easily synthesized in monodisperse form using SPPS and has a molecular weight of 2050 Da. In contrast, PEK2k can only be synthesized in polydisperse form and, thus, has an average molecular weight around 2000 Da.
In addition, it has been found that, e.g., structures of formula (I), wherein X1 is -C(O)-, X2 is -NH-, z is 2, and n is 14, 15, 16, or 17, have properties (e.g., length and hydrophobicity) which are similar to PEK2k. Thus, in some embodiments, the polymer portion of the amphiphilic OEG-conjugated compound contributes to conferring stealth properties on particles containing said amphiphilic OEG- conjugated compound (and, therefore, such amphiphilic OEG-conjugated compounds can replace PEG lipids which have been widely used as stealth lipids).
In some embodiments of formula (I), X2 and X1 taken together are an optionally substituted amide. Thus, in some embodiments, the polymer in the amphiphilic OEG-conjugated compound comprises the structure of the following general formula (la) or (lb): wherein Y, z and n are as defined above or below; and each R1 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), and -N(CI-3 alkyl)2. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)). In some embodiments of formula (la), each R1 is independently hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl). In some embodiments of formula (lb), each R1 is independently hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).
In some embodiments of formula (I), X2 and X1 taken together are an optionally substituted thioamide. Thus, in some embodiments, the polymer in the amphiphilic OEG-conjugated compound comprises the structure of the following general formula (Ic) or (Id): wherein Y, z and n are as defined above or below; and each R1 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), and -N(CI-3 alkyl)2. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)). In some embodiments of formula (Ic), each R1 is independently hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl). In some embodiments of formula (Id), each R1 is independently hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).
In some embodiments of formula (I), X2 and X1 taken together are an ester. Thus, in some embodiments, the polymer in the amphiphilic OEG-conjugated compound comprises the structure of the following general formula (le) or (If): wherein Y, z and n are as defined above or below.
In some embodiments of formula (I), X2 and X1 taken together are a thioester. Thus, in some embodiments, the polymer in the amphiphilic OEG-conjugated compound comprises the structure of the following general formula (Ig), (Ih), (li), or (Ij): wherein Y, z and n are as defined above or below.
In certain embodiments, the polymer in the amphiphilic OEG-conjugated compound comprises the structure of formula (la), wherein R1 at each occurrence (i.e., in each repeating unit) is the same and is hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl). In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same and is hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments of any one of the formulas (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), and (Ij), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.
In some embodiments of any one of the formulas (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), and (Ij), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of any one of the formulas (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), and (Ij), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of any one of the formulas (I), (la), (lb), (Ic), (Id), (le), (If), Ig), (Ih), (li), and (Ij), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (such as 2 or 3); and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16).
In some embodiments of the amphiphilic OEG-conjugated compound, the polymer comprises the structure of the following general formula (II): wherein z and n are as defined above or below; and R1 is hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).
In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)). In some embodiments of formula (II), R1 at each occurrence (i.e., in each repeating unit) is the same and is hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments of formula (II), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of formula (II), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of formula (II), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (such as 2 or 3); and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16).
In some embodiments of the amphiphilic OEG-conjugated compound, the polymer comprises the structure of the following general formula (III): wherein n is as defined above; and R1 is hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl).
In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)). In some embodiments of Formula (III), R1 at each occurrence (i.e., in each repeating unit) is the same and is hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments of formula (III), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the amphiphilic OEG-conjugated compound, the polymer comprises the structure of the following general formula (IV):
In some embodiments of the amphiphilic OEG-conjugated compound, the polymer comprises the structure of the following general formula (IVa):
In some embodiments of any one of formulas (IV) and (IVa), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the amphiphilic OEG-conjugated compound (in particular with respect to any one of formulas (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), (Ij), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are located at either the X1 end or the X2 end of the polymer.
In some embodiments of the amphiphilic OEG-conjugated compound (in particular with respect to any one of formulas (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), (Ij), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are independently selected from non-cyclic, preferably straight, hydrocarbyl groups, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the hydrocarbyl groups have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl groups may be saturated or unsaturated. If the amphiphilic OEG-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different. For example, if the amphiphilic OEG-conjugated compound comprises two hydrophobic chains, in some embodiments said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated. Examples of the one or more hydrophobic chains include the hydrocarbyl chains of fatty acids, in particular the hydrocarbyl chains of naturally occurring fatty acids, such as the hydrocarbyl chains of naturally occurring fatty acids and having at least 8 carbon atoms. Specific examples the one or more hydrophobic chains include the hydrocarbyl chains of caprylic alcohol, capric alcohol, lauric alcohol, myristic alcohol, palmitic alcohol, stearic alcohol, arachidic alcohol, behenic alcohol, lignoceric alcohol, cerotic alcohol, oleic alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.
In some embodiments of the amphiphilic OEG-conjugated compound, the polymer comprising the structure of formula (I) (e.g., the polymer comprising the structure of any one of formulas (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), (Ij), (II), (III), (IV), and (IVa)) and the one or more hydrophobic chains are linked to each other via a linker which comprises at least one functional moiety. In some embodiments, said linker comprises a heterocycloalkylene moiety which is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, said linker comprises an alkylene group and a divalent functional moiety, wherein the divalent functional moiety links the alkylene group to the one or more hydrophobic chains, and the alkylene group is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, said linker comprises an alkylene group and a divalent functional moiety, wherein the divalent functional moiety links the alkylene group to the one or more hydrophobic chains, the alkylene group is substituted with at least one monovalent functional moiety, and the alkylene group is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, said linker comprises a divalent functional moiety, to which the one or more hydrophobic chains are attached and which is attached to the polymer (either directly or via at least one additional difunctional moiety).
In some embodiments, each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties. In some embodiments, each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.
In some embodiments, the amphiphilic OEG-conjugated compound comprises one of the following structures (wherein the index "0" means that the respective group is absent, whereas the index " 1 " means that said group is present):
(hydrophobic chain) ^-(optionally substituted heterocycloalkylene moiety)-(at least one additional difimctional moiety)o or i-(polymer)
[(hydrophobic chain) -(divalent functional moiety)] i-2-(alkylene moiety)-(at least one additional difimctional moiety)o or i-(polymer)
(hydrophobic chain) i-2-(difimctional moiety)-(at least one additional difunctional moiety )o or i -(polymer) [(hydrophobic chain) -(divalent functional moiety)o or i] i-2-(alkylene moiety substituted with at least one monovalent functional moiety)-(at least one additional difimctional moiety)oor i-(polymer)
In some embodiments, the amphiphilic OEG-conjugated compound has one of the following formulas: (hydrophobic chain) i-2-(optionally substituted heterocycloalkylene moiety)-(at least one additional difimctional moiety)o Ori-(polymer)-(end group)
[(hydrophobic chain) -(divalent functional moiety)] i-2-(alkylene moiety)-(at least one additional difimctional moiety)o Ori-(polymer)-(end group)
(hydrophobic chain) i-2-(difimctional moiety)-(at least one additional difimctional moiety)o or i- (polymer)-(end group)
[(hydrophobic chain) -(divalent functional moiety)o or i] i-2-(alkylene moiety substituted with at least one monovalent functional moiety)-(at least one additional difimctional moiety)o0r i-(polymer)-(end group)
In some embodiments, the amphiphilic OEG-conjugated compound comprises an end group at that end of the polymer opposite to the end at which the one or more hydrophobic chains are located. I.e., if the one or more hydrophobic chains are located at the X1 end of the polymer, the end group is located at the X2 end of the polymer, whereas if the one or more hydrophobic chains are located at the X2 end of the polymer, the end group is located at the X1 end of the polymer. In some embodiments, the end group is a neutral end group (such as H or unsubstituted alkyl) or a functionalized end group (e.g., an alkyl group substituted by one or more of hydroxy, thiol, cyano, azido, and amino). In some embodiments, the end group is R3 as defined herein (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), and (IXp)).
In some embodiments, the alkylene moiety substituted with at least one monovalent functional moiety is substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected monovalent functional moieties.
In some embodiments, the alkylene moiety is C1-6-alkylene, such as C 1-3 -alkylene, e.g., methylene, ethylene, or trimethylene.
In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted O-heterocycloalkylene, such as an optionally substituted 4- to 14-membered O-heterocycloalkylene. In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted bicyclic O-heterocycloalkylene. In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted 10-membered O-heterocycloalkylene. In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted chromendiyl moiety or a partially or completely hydrogenated form thereof, such as 3,4-dihydro-2H-chromendiyl, in particular 3,4-dihydro-2H-chromen-2,6-diyl.
In some embodiments, the heterocycloalkylene moiety is substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the heterocycloalkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) substituents independently selected from 1st level substituents, 2nd level substituents, and 3rd level substituents as specified herein. In some embodiments, the heterocycloalkylene moiety is substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the heterocycloalkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) substituents independently selected from C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=0)0CH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, such substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3.
In some embodiments, the linker linking the polymer comprising the structure of formula (I) (e.g., the polymer comprising the structure of any one of formulas (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), (Ij), (II), (III), (IV), and (IVa)) and the one or more hydrophobic chains further comprises at least one additional difunctional moiety via which the linker together with the one or more hydrophobic chains is attached to either the X1 end or the X2 end. In some embodiments, the at least one additional difunctional moiety may additionally comprise an alkylene moiety (such as a C1-6 alkylene moiety, e.g., a C1-3 alkylene moiety). In some embodiments, the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if the linker further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6-alkylene group from each other.
In the case of nucleic acid (e.g., DNA or RNA)-lipid particles, the polymer comprising the structure of formula (I) (e.g., the polymer comprising the structure of any one of formulas (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), (Ij), (II), (III), (IV), and (IVa)) is conjugated to, preferably covalently bound to one or more hydrophobic chains.
In certain embodiments, the end group of the amphiphilic OEG-conjugated compound may be functionalized with one or more molecular moieties conferring certain properties, such as positive or negative charge, or a targeting agent that will direct the particle to a particular cell type, collection of cells, or tissue.
A variety of suitable targeting agents are known in the art. Non-limiting examples of targeting agents include a peptide, a protein, an enzyme, a nucleic acid, a fatty acid, a hormone, an antibody, a carbohydrate, mono-, oligo- or polysaccharides, a peptidoglycan, a glycopeptide, or the like. In some embodiments, targeting agents include the members of targeting pairs, such as the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels- Alder reaction); antigen - antibody specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific (e.g., pegaptanib - VEGF receptor); arginine-glycine-aspartic acid (RGD) peptide - av[k integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor. For example, any of a number of different materials that bind to antigens on the surfaces of target cells can be employed. Antibodies to target cell surface antigens will generally exhibit the necessary specificity for the target. In addition to antibodies, suitable immunoreactive fragments or derivates can also be employed, such as the Fab, Fab', F(ab')2 or scFv fragments or singledomain antibodies (e.g. camelids VHH fragments or nanobodies). Many antibody fragments suitable for use in forming the targeting mechanism are already available in the art. Similarly, ligands for any receptors on the surface of the target cells can suitably be employed as targeting agent. These include any small molecule or biomolecule, natural or synthetic, which binds specifically to a cell surface receptor, protein or glycoprotein found at the surface of the desired target cell.
In some embodiments, the amphiphilic OEG-conjugated compound comprises the following general formula (V) or (V’): wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-;
R2 is a moiety comprising the one or more hydrophobic chains;
R3 is selected from the group consisting of H, C1-6 alkyl, C2.g alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of atargeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, - SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.
In formula (V) R2 is attached to the X1 end of the polymer and R3 is attached to the X2 end of the polymer, whereas in formula (V’) R2 is attached to the X2 end of the polymer and R3 is attached to the XI end of the polymer.
In some embodiments of Formula (V) or (V’), X2 and X1 taken together are an optionally substituted amide. Thus, in some embodiments, the amphiphilic OEG-conjugated compound has one of the followi wherein R2, R3, Y, z and n are as defined above or below; and each R1 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), and -N(CI-3 alkyl)2. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments of any one of formulas (Va), (Va’), (Vb), and (Vb’), R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)). In some embodiments of any one of formulas (Va), (Va’), (Vb), and (Vb’), each R1 is independently hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).
In some embodiments of Formula (V) or (V’), X2 and X1 taken together are an optionally substituted thioamide. Thus, in some embodiments, the amphiphilic OEG-conjugated compound has one of the following general formulas (Vc), (Vc’), (Vd), and (Vd’):
(Vd) (Vd’) wherein R2, R3, Y, z and n are as defined above or below; and each R1 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), and -N(CI-3 alkyl)2. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments of any one of formulas (Vc), (Vc’), (Vd), and (Vd’), R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)). In some embodiments of any one of formulas (Vc), (Vc’), (Vd), and (Vd’), each R1 is independently hydrogen or Cus alkyl (such as hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).
In some embodiments of Formula (V) or (V’), X2 and X1 taken together are an ester. Thus, in some embodiments, the amphiphilic OEG-conjugated compound has one of the following general formulas
(Vf) (Vf) wherein R2, R3, Y, z and n are as defined above or below.
In some embodiments of Formula (V) or (V’), X2 and X1 taken together are a thioester. Thus, in some embodiments, the amphiphilic OEG-conjugated compound has one of the following general formulas wherein R2, R3, Y, z and n are as defined above or below.
In some preferred embodiments, the amphiphilic OEG-conjugated compound has formula (Va) or (Va’), wherein each R1 at each occurrence (i.e., in each repeating unit) is the same and is hydrogen or C1-8 alkyl (such as hydrogen, methyl, or ethyl). In some embodiments, R1 at each occurrence (z. e., in each repeating unit) is the same and is hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments of any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), and (Vj’), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-. In some embodiments of any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), and (Vj’), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), and (Vj’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), and (Vj’), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (such as 2 or 3); and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16).
In some embodiments of any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), and (Vj’), Y is -CH2-; z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments,
Y is -CH2-; z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16). In some embodiments,
Y is -CH2-; z is 2 to 5 (such as 2 or 3); and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16).
In some embodiments, the amphiphilic OEG-conjugated compound has the following general formula
(VI) (VI ) wherein R2, R3, z, and n are as defined for formula (V) and (V’); and R1 is hydrogen or C1-8 alkyl.
In some embodiments of formula (VI) or (VI’), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of formula (VI) or (VI’), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments of Formula (VI) or (VI’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of Formula (VI) or (VF), z is 2 to 20, such as 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (such as 2 or 3); and n is 7 to 16, e.g., 7 to 14 (such as 8, 10, 12, 14, or 16).
In some embodiments, the amphiphilic OEG-conjugated compound has the following general formula wherein R2, R3, and n are as defined for Formula (V) and (V’); and R1 is hydrogen or Cus alkyl.
In some embodiments of formula (VII) or (VIF), R1 is hydrogen or methyl. For example, R1 may be hydrogen. In some embodiments, R1 at each occurrence (i.e., in each repeating unit) is the same (e.g., R1 may be H or methyl in each repeating unit). In some embodiments, R1 in at least one repeating unit differs from R1 in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1 is a different specific alkyl (such as methyl)).
In some embodiments of formula (VII) or (VIF), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16. In some embodiments, the amphiphilic OEG-conjugated compound has one of the following general formulas (VIII), (VIII’), (Villa), and (Villa’): wherein R2, R3, and n are as defined for formula (V) and (V’).
In some embodiments of any one of formulas (VIII), (VIII’), (Villa), and (Villa’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.
In some embodiments of the amphiphilic OEG-conjugated compound (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), the one or more hydrophobic chains are independently selected from non-cyclic, preferably straight, hydrocarbyl groups, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the hydrocarbyl groups have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl groups may be saturated or unsaturated. If the amphiphilic OEG-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different. For example, if the polymer-conjugated compound comprises two hydrophobic chains, in some embodiments said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated. Examples of the one or more hydrophobic chains include the hydrocarbyl chains of fatty acids, in particular the hydrocarbyl chains of naturally occurring fatty acids, such as the hydrocarbyl chains of naturally occurring fatty acids and having at least 8 carbon atoms. Specific examples the one or more hydrophobic chains include the hydrocarbyl chains of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol. In some embodiments of the amphiphilic OEG-conjugated compound having any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’), R2 is R4 or -L1(R4)P, wherein each R4 is independently a hydrophobic chain (i.e., one of the one or more hydrophobic chains comprised in R2), such as a hydrocarbyl group; L1 is a linker; and p is 1 or 2.
In some embodiments, L1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), L1 comprises an optionally substituted heterocycloalkylene moiety which is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted O-heterocycloalkylene, such as an optionally substituted 4- to 14-membered O- heterocycloalkylene. In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted bicyclic O-heterocycloalkylene. In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted 10-membered O-heterocycloalkylene. In some embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted chromendiyl moiety which is partially or completely hydrogenated, such as 3,4-dihydro-2H- chromendiyl, in particular 3,4-dihydro-2H-chromen-2,6-diyl. In some embodiments, the heterocycloalkylene moiety is substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the heterocycloalkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) substituents independently selected from 1st level substituents, 2nd level substituents, and 3rd level substituents as specified herein. In some embodiments, the heterocycloalkylene moiety is substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the heterocycloalkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) substituents independently selected from C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=0)0CH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, such substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3. In some embodiments, the optionally substituted heterocycloalkylene moiety is 3,4-dihydro-2H-chromen-2,6-diyl substituted with a methyl group at (i) each of positions 2, 5, 7, and 8 (corresponding to the methyl substitution pattern of a-tocopherol), (ii) each of positions 2, 5, and 8 (corresponding to the methyl substitution pattern of P-tocopherol), (iii) each of positions 2, 7, and 8 (corresponding to the methyl substitution pattern of y-tocopherol), or (iv) each of positions 2 and 8 (corresponding to the methyl substitution pattern of 5-tocopherol).
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), L1 comprises an alkylene group and a divalent functional moiety, wherein the divalent functional moiety links the alkylene group to the one or more hydrophobic chains, and the alkylene group is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, the alkylene moiety is C1-6 -alkylene, such as C1-3-alkylene, e.g., methylene, ethylene, or trimethylene. In some preferred embodiments, the alkylene moiety is trimethylene. In some embodiments, the divalent functional moiety is selected from the group consisting of ester, amide, sulfide, disulfide, sulfone, orthoester, acylhydrazone, hydrazine, oxime, acetal, and ketal. In some embodiments, the divalent functional moiety is selected from the group consisting of ester and amide.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), L1 comprises an alkylene group and a divalent functional moiety, wherein the divalent functional moiety links the alkylene group to the one or more hydrophobic chains, the alkylene group is substituted with at least one monovalent functional moiety, and the alkylene group is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, the alkylene moiety substituted with at least one monovalent functional moiety is substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected monovalent functional moieties. In some embodiments, the alkylene moiety is C1-6 -alkylene, such as C1-3-alkylene, e.g., methylene, ethylene, or trimethylene. In some preferred embodiments, the alkylene moiety is trimethylene. In some embodiments, the alkylene moiety is trimethylene substituted with one OH moiety and a hydrophobic chain which is directly attached to the trimethylene moiety (preferably the OH moiety and the hydrophobic chain are attached to the same carbon atom (e.g., to one of the terminal carbon atoms) of the trimethylene moiety). In some embodiments, the hydrophobic chain which is directly attached to the trimethylene moiety is unsaturated, preferably monounsaturated. In some embodiments, the hydrophobic chain which is directly attached to the trimethylene moiety (and which is preferably monounsaturated) has at least 8 carbon atoms, such as at least 10 carbon atoms.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), L1 comprises a divalent functional moiety, to which the one or more hydrophobic chains are attached and which is attached to the polymer (either directly or via at least one additional difunctional moiety). In some embodiments, the divalent functional group is an amino group. In some embodiments, the divalent functional moiety is an unsubstituted amino group (i.e., resulting in an R2 substituent being -N(R4)2).
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), L1 comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-, [*-NHC(O)]p(Ci- 6-alkylene)-, [*-C(O)NH]P(CI-6 -alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6 -alkylene)-, [*- S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r](C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*- C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p(C1-6-alkylene)-, [*=C(=N- N(R26)C(O)(R25))]p(Ci-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-, [*=C(=N(OH))]P(C1-6- alkylene)-, [*-OC(R25)(R26)O]p(C1-6-alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-)pN(R26)2.p, and [*-C(O)NH](C1-6-alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25 is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), L1 further comprises at least one additional difunctional moiety via which R2 is attached to either X1 in formula (V) (or to the carbonyl group of any one of formulas (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj), (VI), (VII), (VIII), and (Villa)) or X2 in formula (V’) (or the N atom of any one of formulas (Va’), (Vb’), (Vc’), (Vd’), (Ve’), (Vf ), (Vg’), (Vh’), (Vi’), (Vj’), (VI’), (VII’), (VIII’), and (Villa’)). In some embodiments, the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of ether, amino, phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl. In some embodiments, the at least one additional difunctional moiety is selected from the group consisting of ether and amino. If L1 further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated from each other by a C1-6 -alkylene group.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)-NR26-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-
OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Ci-6-alkylene)-, [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)NR26-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)-NR26-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6 -alkylene)©-, [*-OC(O)]p(C1-6- alkylcncjO-. (*-)pN(R26)2-p, and [*-C(O)NH](C1-6-alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6 -alkylene), [*-OC(O)]p(C1-6 -alkylene), [*- NHC(O)]p(C1-6-alkylene), and [*-C(O)NH]p(C1-6-alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27 is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen- 6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(Ci-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*- OC(O)]p(C1-6-alkylene)-OP(O)(OR27)-O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(Ci- 6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)NH-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)NH-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H- chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*- C(O)NH](C1-6-alkyltriyl)O- or L1 is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6 -alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(0)]P(CI-6- alkylene), and [*-C(O)NH]p(C 1-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NHZ); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), R2 is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)- O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)C(O)-,
[R4NHC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4NHC(O)]p(C2-3-alkylene)-
OP(O)(OR27)O(C1-3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)NH]p(C2-3-alkylene)-
OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(C1-3-alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2 is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); 2-R4-3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2 is a monoalkylamine moiety .
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VF), (VII), (VIE), (VIII), (VIII’), (Villa), and (Villa’)), each of the one or more hydrophobic chains (i.e., each of R4) is independently a non-cyclic, preferably straight, hydrocarbyl group, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the one or more hydrocarbyl groups independently have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The one or more hydrocarbyl groups may be saturated or unsaturated. If the amphiphilic OEG-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different. For example, if the amphiphilic OEG-conjugated compound comprises two hydrophobic chains, in some embodiments said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated and/or said two hydrophobic chains differ in their length. Examples of the one or more hydrophobic chains include the hydrocarbyl chains of fatty acids, in particular the hydrocarbyl chains of naturally occurring fatty acids, such as the hydrocarbyl chains of naturally occurring fatty acids and having at least 8 carbon atoms. Specific examples the one or more hydrophobic chains include the hydrocarbyl chains of caprylic alcohol, capric alcohol, lauric alcohol, myristic alcohol, palmitic alcohol, stearic alcohol, arachidic alcohol, behenic alcohol, lignoceric alcohol, cerotic alcohol, oleic alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl (e.g., a-tocopheryl, P-tocopheryl, y-tocopheryl, or 5-tocopheryl), DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties, or R2 is a monomyristylamine moiety.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, - SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. a sugar, an amino acid, a peptide, and a member of a targeting pair.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), R3 is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2. and a member of a targeting pair.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (Vlir), (Villa), and (Villa’)), R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(0)NH2, -C(0)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair.
In some embodiments (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), and (Villa’)), the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide - av[k integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglycoprotein receptor. In some embodiments, the member of a targeting pair is selected from the group consisting of a maleimide moiety, a thiol moiety, a halogenated (in particular, brominated) alkyl moiety, an azide moiety, an alkynyl moiety, an antigen, and an antibody (including fragments or derivatives thereof). In some embodiments, the amphiphilic OEG-conjugated compound has one of the following formulas
wherein n is 5 to 25; R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair; R27 is H or a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH/); or an organic cation, e.g., a quaternary ammonium or amine cation); and in each case -C(O)CI2H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)Ci5H3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)I2CH3 (myristoyl), in each case -Ci4H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -CBH27 refers to the moiety -(CH2)I2CH3, and in each case -C(O)CI?H33 refers to the moiety -czs-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl).
In some embodiments of any one of formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12. 14, or 16.
In some embodiments, of any one of formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr)the member of a targeting pair is selected from the group consisting of a maleimide moiety, a thiol moiety, a halogenated (in particular, brominated) alkyl moiety, an azide moiety, an alkynyl moiety, an antigen, and an antibody (including fragments or derivatives thereof). In some embodiments of any one of formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, and C2-6 alkynyl.
In some embodiments of any one of formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), n is 7 to 16, e.g., 7 to 14 (preferably 8, 10, 12, 14, or 16); and R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5 -dioxo-2, 5 -dihydro- IH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, and C2-6 alkynyl.
Particular examples of the amphiphilic OEG-conjugated compound are the following compounds (V-l) to (V-63):
08 - 10 (V-57)
Particularly preferred examples of the amphiphilic OEG-conjugated compound are those having any one of formulas (V-l), (V-5), (V-17), and (V-25) and their salts.
It is to be understood that any reference to an amphiphilic OEG-conjugated compound or a polymer- conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains (in particular, to a compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi),
(Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V- 2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V- 16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably to a compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)) also includes the salts (in particular pharmaceutically acceptable salts), tautomers, stereoisomers, solvates (e.g., hydrates), and isotopically labeled forms thereof.
In certain instances, the amphiphilic OEG-conjugated compound (in particular, the compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably the compound of any one of formulas (V-l), (V-5), (V-17), and (V-25)) may comprise from about 0.1 mol % to about 20 mol %, such as from about 0.2 mol % to about 15 mol %, from about 0.5 mol % to about 10 mol %, from about 1 mol % to about 5 mol %, or from about 1.5 mol % to about 2.5 mol %, or from about 2 mol % to about 6 mol %, or from about 2 mol % to about 5 mol %, of the total lipid present in the composition/particles. In certain instances, the amphiphilic OEG-conjugated compound (in particular, the compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), wherein n is 14, such as (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), and (V-20), preferably the compound of any one of formulas (V-l), (V- 5), and (V-17)) may comprise from about 0.1 mol % to about 2.5 mol %, such as from about 0.2 mol % to about 2.4 mol %, from about 0.5 mol % to about 2.2 mol %, or from about 1 mol % to about 2 mol %, of the total lipid present in the composition/particles. In certain instances, the amphiphilic OEG- conjugated compound (in particular, the compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), wherein n is 8, such as (V- 21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), and (¥-40), preferably the compound of formula (V- 25)) may comprise from about 2 mol % to about 5 mol %, such as from about 2.5 mol % to about 5 mol %, in particular from about 3 mol % to about 5 mol %, from about 3.5 mol % to about 4.5 mol %, from about 3.6 mol % to about 4.4 mol %, from about 3.8 mol % to about 4.2 mol %, or about 4 mol, of the total lipid present in the composition/particles. In certain instances, the amphiphilic OEG-conjugated compound (in particular, the compound of any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VIE), (VIII), (VIIF), (Villa), and (Villa’), wherein R2 comprises a ceramide moiety, such as (IXo), (IXp), (V-13), (V-14), (V-15), (V-16), (V-33), (V-34), (V-35), (V-36), (V-53), (V-54), (V-55), and (V-56), preferably the compound of formula (V-13)) may comprise from about 2 mol % to about 5 mol %, such as from about 2.5 mol % to about 5 mol %, in particular from about 3 mol % to about 5 mol %, of the total lipid present in the composition/particles.
The amphiphilic OEG-conjugated compounds can be prepared using conventional synthesis methods known to the skilled person. For example, intermediate compounds (such as Ac-(AEEA)n-OH or H- (AEEA)n-OH; AEEA = (2-(2-(2-aminoethoxy)ethoxy)acetic acid) may be prepared using solid phase peptide synthesis (SPPS). After isolation and purification of the intermediate compounds they can be conjugated with organic molecules (such as a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety) resulting in the amphiphilic OEG-conjugated compounds. Finally, the amphiphilic OEG-conjugated compounds may be purified and isolated. In the following, the above steps are described in more detail.
Synthesis of intermediate compounds (such as Ac-(AEEA)n-OH or H-(AEEA)n-OH)
Synthesis of intermediate compounds (such as Ac-(AEEA)i4-OH or Ac-(AEEA)8-OH) can be performed using solid phase peptide synthesis (SPPS). The solid support - also referred to as resin - is chosen to obtain a free acid at the C-terminus. The resin is suspended in organic solvent to ensure an appropriate swelling. Synthesis scale depends on the resin substitution and may incorporate multiple replicates. The synthesis is initiated by an optional deprotection of the resin (e.g., if the amino groups of the resin are protected) followed by a coupling with the amino-acid of interest (e.g., Fmoc-AEEA-OH). The coupling occurs in an appropriate solvent mixture containing coupling reagents such as but not limited to carbodiimides and benzotriazoles. After the coupling, the solution is drained and the peptidyl -resin washed to undergo the Fmoc deprotection/next amino-acid coupling until the expected sequence is completed. If applicable (for obtaining N-terminally acetylated intermediates), the final N-terminal Fmoc is first deprotected (to obtain the peptidyl resin H-(AEEA)n-resin) and then acetylated (to obtain the acetylated peptidyl resin Ac-(AEEA)n-resin). The intermediate compound is then cleaved from the resin in acidic conditions to yield the crude Ac-(AEEA)n-OH or H-(AEEA)n-OH. The solution may then be evaporated and the crude intermediate may be resolubilized, e.g., in an aqueous and organic solvent mixture. The solubilized crude intermediate can be further purified, e.g., using a chromatography system (without or with prepurification, wherein the prepurification may include a solvent exchange and/or a salt exchange (e.g., by using lyophilization)), in particular a liquid chromatography system (such as HPLC), wherein the chromatography system preferably comprises an appropriate detector (such as a mass detector). The purity relevant fractions can be collected, pooled and lyophilized.
Conjugation of intermediate compounds (in particular Ac-(AEEA)n-OH) with organic molecules to obtain amphiphilic OEG-conjugated compounds
Lyophilized Ac-(AEEA)n-OH is solubilized in an organic solvent and reacted with an organic molecule (such as a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety), preferably in the presence of a coupling reagent (such as but not limited to carbodiimides and/or benzotriazoles) to obtain an amphiphilic OEG-conjugated compound (such as Ac-(AEEA)i4-DSPE or Ac-(AEEA)i4-a-tocopherol). If applicable, the amphiphilic OEG-conjugated compound is purified, e.g., using a chromatography system (without or with prepurification, wherein the prepurification may include a solvent exchange and/or a salt exchange (e.g., by using lyophilization)), in particular a liquid chromatography system (such as HPLC), wherein the chromatography system preferably comprises an appropriate detector (such as a mass detector). The purity relevant fractions can be collected, pooled and lyophilized.
Conjugation intermediate compounds (in particular H-(AEEA)n-OH) with organic molecules to obtain amphiphilic OEG-conjugated compounds
Preferably, the N-terminus of H-(AEEA)n-OH is first subjected to a protection reaction (e.g., a BOC protection reaction) and optionally purified (e.g., using a chromatographic system) to obtain PG- (AEEA)n-OH, wherein PG is an amino protecting group (e.g., PG-(AEEA)n-OH can be B0C-(AEEA)i4- OH). Thereafter, PG-(AEEA)n-OH is reacted with an organic molecule (such as a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety), preferably in the presence of a coupling reagent (such as but not limited to carbodiimides and/or benzotriazoles) to obtain PG-(AEEA)n-organic molecule, i.e., an N-terminally protected version of an amphiphilic OEG-conjugated compound (e.g., Boc-(AEEA)n-organic molecule such as Boc-(AEEA)i4-DSPE or Boc-(AEEA)i4-a-tocopherol). Preferably, the PG-(AEEA)n-organic molecule is then subjected to a deprotection reaction to remove the amino protecting group and to obtain the final amphiphilic OEG-conjugated compound (such as H- (AEEA)i4-DSPE or H-(AEEA)i4-a-tocopherol). If applicable, the amphiphilic OEG-conjugated compound is purified, e.g., using a chromatography system (without or with prepurification, wherein the prepurification may include a solvent exchange and/or a salt exchange (e.g., by using lyophilization)), in particular a liquid chromatography system (such as HPLC), wherein the chromatography system preferably comprises an appropriate detector (such as a mass detector). The purity relevant fractions can be collected, pooled and lyophilized.
Thus, a method for preparing an amphiphilic OEG-conjugated compound may comprise the following steps: (a) providing an intermediate compound having the formula (VII) or (VII’), wherein, for formula (VII), R2 is OH, and R3 is H, acetyl, or Fmoc; and, for formula (VII’), R2 is H, acetyl, or Fmoc, and R3 is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, in particular a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, thereby obtaining the amphiphilic OEG-conjugated compound.
In some embodiments of the method for preparing an amphiphilic OEG-conjugated compound, in step (a) the intermediate compound having the formula (VII) or (VII’) is prepared by using solid phase peptide synthesis (SPPS) and a corresponding N-terminally protected AEEA-containing compound (such as Fmoc-AEEA-OH). For example, this preparation by SPPS may include the following steps: (al) suspending the solid phase (such as a resin) having amino groups in a suitable organic solvent (preferably to ensure an appropriate swelling of the solid phase); (a2) optionally conducting a deprotection reaction in case the amino groups of the solid phase are protected (e.g., to convert PG-solid phase (wherein PG is an amino protecting group) into H2N-solid phase or an Fmoc-(AEEA)x-loaded solid phase into an H-(AEEA)x-loaded solid phase); (a3) reacting the solid phase containing free amino groups obtained in step (al) or (a2) with an N-terminally protected AEEA-containing compound (such as Fmoc-AEEA-OH), preferably in the presence of a coupling reagent (such as a carbodiimide or benzotriazole); (a4) draining the solution from the solid phase; (a5) optionally washing the solid phase obtained after step (a4); (a6) optionally repeating steps (a2) to (a5) one or more times in order to obtain the desired number of repeating units (n) of AEEA; (a7) optionally subjecting the PG-(AEEA)n-loaded solid phase (such as Fmoc-(AEEA)n-loaded solid phase) to a final deprotection reaction (such as a final Fmoc deprotection reaction in case PG is Fmoc) thereby obtaining an H-(AEEA)n-loaded solid phase; (a8) optionally subjecting the H-(AEEA)n-loaded solid phase to an acetylating reaction thereby obtaining an Ac-(AEEA)n-loaded solid phase; and (a9) cleaving the AEEA-polymer obtained in step (a3), (a4), (a5), (a6), (a7), or (a8) from the solid support, preferably in the presence of an acid, thereby providing the intermediate compound having the formula (VII) or (VIE), wherein, for formula (VII), R2 is OH, and R3 is H, acetyl, or Fmoc; and, for formula (VIE), R2 is H, acetyl, or Fmoc, and R3 is OH. In some embodiments, the intermediate compound having the formula (VII) or (VIE) may be purified, e.g., using a chromatography system, without or with prepurification. In some embodiments, the prepurification includes a solvent exchange and/or a salt exchange (e.g., by using lyophilization). In some embodiments, the chromatography system is a liquid chromatography system (such as HPLC), wherein the chromatography system preferably comprises an appropriate detector (such as a mass detector). In some embodiments, after separation using the chromatography system, the fractions containing the intermediate compound having the formula (VII) or (VII’) (preferably containing the intermediate compound having the formula (VII) or (VII’) in a sufficient purity (such as > 90%)) can be collected, pooled and lyophilized.
In some embodiments of the method for preparing an amphiphilic OEG-conjugated compound, in particular those, where the intermediate compound having the formula (VII) or (VII’) has an acetylated N-terminus, step (b) comprises the following step: (bl) reacting Ac-(AEEA)n-OH with an organic molecule, in particular a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, preferably in the presence of a coupling reagent (such as a carbodiimide or benzotriazole), thereby obtaining an Ac-(AEEA)n-organic molecule, i.e., an amphiphilic OEG-conjugated compound having an acetylated N-terminus.
In some embodiments of the method for preparing an amphiphilic OEG-conjugated compound, in particular those, where the intermediate compound having the formula (VII) or (VIE) is N-terminally protected by Fmoc, prior to step (b) said compound is subjected to Fmoc deprotection reaction, thereby obtaining the intermediate compound having the formula (VII) or (VIE) and having a free N-terminus.
In some embodiments of the method for preparing an amphiphilic OEG-conjugated compound, in particular those, where the intermediate compound having the formula (VII) or (VIE) has a free N- terminus (i.e., an NEE group), step (b) comprises the following steps: (bl’) subjecting the N-terminus of H-(AEEA)n-OH to a protection reaction (e.g., by introducing a BOC protecting group at the N- terminus) thereby obtaining PG-(AEEA)n-OH, wherein PG is amino protecting group; (b2’) optionally purifying PG-(AEEA)n-OH (e.g., using a chromatographic system); (b3’) reacting PG-(AEEA)n-OH with an organic molecule, in particular a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, preferably in the presence of a coupling reagent (such as a carbodiimide or benzotriazole), thereby obtaining a PG-(AEEA)n-organic molecule; and (b4’) subjecting the PG- (AEEA)n-organic molecule to a deprotection reaction, thereby obtaining the amphiphilic OEG- conjugated compound having a free N-terminus. Optionally, the N-terminus may be further derivatized in order to obtain other amphiphilic OEG-conjugated compounds.
In some embodiments, the amphiphilic OEG-conjugated compound may be purified, e.g., using a chromatography system, without or with prepurification. In some embodiments, the prepurification includes a solvent exchange and/or a salt exchange (e.g., by using lyophilization). In some embodiments, the chromatography system is a liquid chromatography system (such as HPLC), wherein the chromatography system preferably comprises an appropriate detector (such as a mass detector). In some embodiments, after separation using the chromatography system, the fractions containing the amphiphilic OEG-conjugated compound (preferably containing the amphiphilic OEG-conjugated compound in a sufficient purity (such as > 90%)) can be collected, pooled and lyophilized.
Lipids
The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino (e.g., tertiary amino), sulfhydryl, nitro, hydroxyl, and other like groups.
The term "hydrophobic" as used herein with respect to a compound, group or moiety means that said compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, aggregates and excludes water molecules. In some embodiments, the term "hydrophobic" refers to any compound, group or moiety which is substantially immiscible or insoluble in aqueous solution. In some embodiments, a hydrophobic compound, group or moiety is substantially nonpolar. Examples of hydrophobic groups are hydrocarbyl groups and fluorinated (e.g., perfluorinated) hydrocarbyl groups. In some embodiments, a hydrophobic compound, group or moiety is lipophilic. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.
The term "lipophilic" as used herein with respect to a compound, group or moiety means that said compound, group or moiety is soluble in nonpolar solvents (such as hexane, tetrahydrofuran (THF), and/or chloroform). In some embodiments, the term "lipophilic" refers to any compound, group or moiety which is soluble in nonpolar solvents (such as hexane, tetrahydrofuran (THF), and/or chloroform) and which is substantially immiscible or insoluble in aqueous solution. Examples of lipophilic groups are hydrocarbyl groups, such as non-cyclic, preferably straight, hydrocarbyl groups (such as hydrocarbyl groups having at least 10 carbon atoms), e.g., the lipophilic chain of a natural lipid.
The term "perfluorinated" as used herein with respect to a compound, group or moiety means that in said compound, group or moiety all C-H moieties have been replaced with C-F moieties. For example, perfluorinated n-octanoic acid has the formula F3C(CF2)eCOOH.
The term "hydrocarbon" includes non-cyclic, e.g., linear (straight) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogen atoms in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.
As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. Examples of lipid- like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules. Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble. Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.
Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.
In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds that are characterized by being insoluble in water, but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. G.s-doublc bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide- linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.
Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.
Cationic or cationically ionizable lipids
The nucleic acid (such as DNA or RNA) compositions described herein and the nucleic acid particles (especially RNA LNPs) described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
As used herein, a "cationic lipid" or "cationic lipid-like material" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., a lipid which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, such "cationically ionizable" lipids are comprised by the term "cationic lipid" unless contradicted by the circumstances.
Examples of cationic lipids include, but are not limited to N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3 -trimethylammonium propane (DOTAP); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3 -dimethylammonium propanes; l,2-dialkyloxy-3 -dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1.2-dimyristoyl-sn-glycero-3 -ethylphosphocholine (DMEPC), l,2-dimyristoyl-3 -trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3 -dimethyl -hydroxyethyl ammonium bromide (DORIE), and
2.3 -dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate
(DOSPA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2- (cholest-5-en-3-beta-oxybutan-4-oxy)- l-(cis,cis-9, 12-oc-tadecadienoxy)propane (CLinDMA), 2- [5 '- (cholest-5 -en-3 -beta-oxy)-3 '-oxapentoxy)-3 -dimethyl- 1 -(cA,cA-9', 12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2- N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin- K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl- 4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31 -tetraen- 19-yl - 4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(cA-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(3P)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (octyl- CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), N 1 -[2-(( 1 S)- 1 - [(3 -aminopropyl)amino] -4- [di( 3 -amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero- 3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-l- amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)- dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3- bis(tetradecyloxy)propan- 1 -amine (DMDMA), Di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)- oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl- ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethyl- amino)propionamide (lipidoid 98N12-5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200), and the following structures (XV- 1) to (XV-6):
Preferred are DODMA, DOTMA, DOTAP, DODAC, and DOSPA. In specific embodiments, the cationic or cationically ionizable lipid is DODMA.
DOTMA is a cationic lipid with a quaternary amine headgroup. The structure of DOTMA may be represented as follows:
DODMA is an ionizable cationic lipid with a tertiary amine headgroup. The structure of DODMA may be represented as follows:
In certain embodiments, the composition comprises a cationically ionizable lipid. In some embodiments, the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, preferably under physiological conditions. In some embodiments, the cationically ionizable lipid comprises a head group which includes at least one tertiary amine moiety.
Examples of cationically ionizable lipids are disclosed, for example, in WO 2016/176330 and WO 2018/078053. In some embodiments, the cationically ionizable lipid has the structure of formula (X): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L10 and L20 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L10 and L20 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;
G1 and G2 are each independently unsubstituted C1-C12 alkylene or C2-12 alkenylene;
G3 is C 1-24 alkylene, C2-24 alkenylene, C3-8 cycloalkylene, or C3-8 cycloalkenylene;
Ra is H or C1-12 alkyl;
R35 and R36 are each independently C6-24 alkyl or C6-24 alkenyl;
R37 is H, OR50, CN, -C(=O)OR40, -OC(=O)R40 or -NR50C(=O)R40;
R40 is C1-12 alkyl;
R50 is H or C1-6 alkyl; and x is 0, 1 or 2.
In some of the foregoing embodiments of formula (X), the lipid has one of the following structures (XA) or (XB):
(XA) (XB) wherein:
A is a 3 to 8-membered cycloalkyl or cycloalkylene group; R60 is, at each occurrence, independently H, OH or C1-C24 alkyl; nl is an integer ranging from 1 to 15.
In some of the foregoing embodiments of formula (X), the lipid has structure (XA), and in other embodiments, the lipid has structure (XB).
In other embodiments of formula (X), the lipid has one of the following structures (XC) or (XD): wherein y and z are each independently integers ranging from 1 to 12.
In any of the foregoing embodiments of formula (X), one of L10 and L20 is -O(C=O)-. For example, in some embodiments each of L10 and L20 are -O(C=O)-. In some different embodiments of any of the foregoing, L10 and L20 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L10 and L20 is -(C=O)O-.
In some different embodiments of formula (X), the lipid has one of the following structures (XE) or
In some of the foregoing embodiments of formula (X), the lipid has one of the following structures
In some of the foregoing embodiments of formula (X), nl is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, nl is 3, 4, 5 or 6. In some embodiments, nl is 3. In some embodiments, nl is 4. In some embodiments, nl is 5. In some embodiments, nl is 6.
In some other of the foregoing embodiments of formula (X), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
In some of the foregoing embodiments of formula (X), R60 is H. In other of the foregoing embodiments, R60 is C1-C24 alkyl. In other embodiments, R60 is OH.
In some embodiments of formula (X), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is linear C1-C24 alkylene or linear C2-C24 alkenylene.
In some other foregoing embodiments of formula (X), R35 or R36, or both, is C6-C24 alkenyl. For example, in some embodiments, R35 and R36 each, independently have the following structure: wherein:
R7a and R7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7b and a are each selected such that R35 and R36 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.
In some of the foregoing embodiments of formula (X), at least one occurrence of R7a is H. For example, in some embodiments, R7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7b is Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl. In different embodiments of formula (X), R35 or R36, or both, has one of the following structures:
In some of the foregoing embodiments of formula (X), R37 is OH, CN, -C(=O)OR40, -OC(=O)R40 or -NHC(=O)R40. In some embodiments, R40 is methyl or ethyl.
In various different embodiments, the cationically ionizable lipid of formula (X) has one of the structures set forth below.
In various different embodiments, the cationically ionizable lipid has one of the structures set forth in the table below.
In some embodiments, the cationically ionizable lipid has the structure of formula (XI): wherein each of Ri and R2 is independently R5 or -G1-L1-R5, wherein at least one of Ri and R2 is -G1-L1-R5; each of R3 and R4 is independently selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, aryl, and C3-10 cycloalkyl; each of R5 and Rs is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms; each of Gi and G2 is independently unsubstituted C1-12 alkylene or C2-12 alkenylene; each of Li and L2 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-;
Ra is H or C1-12 alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1 or 2.
In some of the foregoing embodiments of formula (XI), Gi is independently unsubstituted C1-C12 alkylene or unsubstituted C2-12 alkenylene, e.g., unsubstituted, straight C1-12 alkylene or unsubstituted, straight C2-12 alkenylene. In some embodiments, each Gi is independently unsubstituted Cg-12 alkylene or unsubstituted Cg-12 alkenylene, e.g., unsubstituted, straight Cg-12 alkylene or unsubstituted, straight C6-12 alkenylene. In some embodiments, each Gi is independently unsubstituted Cs-i2 alkylene or unsubstituted Cs-i2 alkenylene, e.g., unsubstituted, straight Cs-i2 alkylene or unsubstituted, straight Cs-i2 alkenylene. In some embodiments, each Gi is independently unsubstituted Ce-io alkylene or unsubstituted Ce-io alkenylene, e.g., unsubstituted, straight Ce-io alkylene or unsubstituted, straight Ce-io alkenylene. In some embodiments, each Gi is independently unsubstituted alkylene having 8, 9 or 10 carbon atoms, e.g., unsubstituted, straight alkylene having 8, 9 or 10 carbon atoms. In some embodiments, where Ri and R2 are both independently -G1-L1-R5, Gi for Ri may be different from Gi for R2. In some of these embodiments, for example, Gi for Ri is unsubstituted, straight C1-12 alkylene and Gi for R2 is unsubstituted, straight C2-12 alkenylene; or Gi for Ri is an unsubstituted, straight C1-12 alkylene group and Gi for R2 is a different unsubstituted, straight C1-12 alkylene group. In some embodiments, where Ri and R2 are both independently -G1-L1-R5, Gi for Ri may be identical to Gi for R2. In some of these embodiments, for example, each Gi is the same unsubstituted, straight Cs-i2 alkylene, such as unsubstituted, straight Cs-io alkylene, or each Gi is the same unsubstituted, straight C6-12 alkenylene.
In some of the foregoing embodiments of formula (XI), each Li is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In some embodiments, Ra of Li is H or C1-12 alkyl. In some embodiments, Ra of Li is H or C1-6 alkyl, e.g., H or C1-3 alkyl. In some embodiments, Ra of Li is H, methyl, or ethyl. In some embodiments, each Li is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, and -SC(=O)-. In some embodiments, each Li is independently -O(C=O)- or -(C=O)O-. In some embodiments, where Ri and R2 are both independently -G1-L1-R5, Li for Ri may be different from Li for R2. In some of these embodiments, for example, Li for Ri is one moiety selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa- (e.g., Li for Ri is -O(C=O)-), and Li for R2 is a different moiety selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa- (e.g., Li for R2 is -(C=O)O-). In some embodiments, where Ri and R2 are both independently -G I-L|-R6. Li for Ri may be identical to Li for R2. In some of these embodiments, for example, each Li is the same moiety selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-, e.g., each Li is -O(C=O)- or each Li is -(C=O)O-.
In some of the foregoing embodiments of formula (XI), each Rs is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms, e.g., a straight hydrocarbyl group having at least 10 carbon atoms. In some embodiments, each Rs has independently at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments, each Rs is independently a non-cyclic hydrocarbyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight hydrocarbyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments, each Rs is attached to Li via an internal carbon atom of Rs. In some embodiments, each Rs has independently at most 30 carbon atoms (such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms), and each Rs is attached to Li via an internal carbon atom of Rs. In some embodiments, each Rs is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms, e.g., a straight hydrocarbyl group having at least 10 carbon atoms, and each Rs is attached to Li via an internal carbon atom of Rs. In some embodiments, each Rs is independently a non-cyclic hydrocarbyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight hydrocarbyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and each Rs is attached to Li via an internal carbon atom of Rs. In some embodiments, the hydrocarbyl group of Rs is an alkyl or alkenyl group, e.g., a C10-30 alkyl or alkenyl group. Thus, in some embodiments, each Rs is independently a non-cyclic alkyl group having at least 10 carbon atoms or a non-cyclic alkenyl group having at least 10 carbon atoms, e.g., a straight alkyl group having at least 10 carbon atoms or a straight alkenyl group having at least 10 carbon atoms. In some embodiments, each Rs is independently a non-cyclic alkyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a non-cyclic alkenyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight alkyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a straight alkenyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments, each Re is independently anon-cyclic alkyl group having 11 to 19 carbon atoms (such as 11, 13, 15, 17, or 17 carbon atoms), e.g., a straight alkyl group having 11 to 19 carbon atoms (such as 11, 13, 15, 17, or 17 carbon atoms). In some embodiments, each Re is independently a non-cyclic alkyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a non-cyclic alkenyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight alkyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a straight alkenyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and each Re is attached to Li via an internal carbon atom of Re. In some embodiments, each Re is independently a non-cyclic alkyl group having 11 to 19 carbon atoms (such as 11, 13, 15, 17, or 17 carbon atoms), e.g., a straight alkyl group having 11 to 19 carbon atoms (such as 11, 13, 15, 17, or 17 carbon atoms), and each Re is attached to Li via an internal carbon atom of Re. The expression "internal carbon atom" means that the carbon atom of Re by which Re is attached to Li is directly bonded to at least 2 other carbon atoms of Re. For example, for the following Cn alkyl group, each carbon atom at any one of positions 2, 3, 4, 5, and 7 qualifies as "internal carbon atom" according to the present disclosure, whereas the carbon atoms at positions 1, 6, 8, 9, 10, and 11 do not.
Consequently, Re being a Cn alkyl group attached to Li via an internal carbon of Re includes the following groups: wherein ww represents the bond by which Re is bound to Li. Furthermore, for a straight alkyl group, e.g., a straight Cn alkyl group, each carbon atom except for the first and last carbon atoms of the straight alkyl group (i.e., except the carbon atoms at positions 1 and 11 of the straight Cn alkyl group) qualifies as "internal carbon atom" . Thus, in some embodiments, Re being a straight alkyl group having p carbon atoms and being attached to Li via an internal carbon atom of Re means that Re is attached to Li via a carbon atom of Re at any one of positions 2 to (p-1) (thereby excluding the terminal C atoms at positions 1 and p). In some embodiments, where Re is a straight alkyl group having p’ carbon atoms (wherein p’ is an even number) and being attached to Li via an internal carbon atom of Re, Re is attached to Li via a carbon at any one of positions (p72 - 1), (p72), and (p72 + 1) of Re (e.g., if p’ is 10, Re is attached to Li via a carbon atom at any one of positions 4, 5, and 6 of Re). In some embodiments, where Re is a straight alkyl group having p” carbon atoms (wherein p” is an uneven number) and being attached to Li via an internal carbon atom of Re, Re is attached to Li via a carbon atom at any one of positions (p” - l)/2 and (p” + l)/2 of Re (e.g., if p” is 11, Re is attached to Li via a carbon at any one of positions 5 and 6 of Re). Generally, it is to be understood that if both Ri and R2 are -Gi-Li-Re and each Re is attached to Li via an internal carbon atom of Re, Re of Ri is attached to Li of Ri (and not to Li of R2) via an internal carbon atom of Re of Ri and Re of R2 is attached to Li of R2 (and not to Li of Ri) via an internal carbon atom of Re of R2. In some embodiments, each Re is independently selected from the group consisting of: , p y e to Li. In some embodiments, where Ri and R2 are both independently -Gi-Li-Re, Re for Ri is different from Re for R2. In some of these embodiments, for example, Re for Ri may be a non-cyclic, preferably straight, hydrocarbyl group having at least 10 carbon atoms (e.g., Re for Ri is and Re for R2 may be a different non-cyclic, preferably straight, hydrocarbyl group having at least 10 carbon atoms (e.g., Re for R2 is ). In some embodiments, where Ri and R2 are both independently -Gi-Li-Re, Re for Ri is identical to Re for R2. In some of these embodiments, for example, each Re is the same non-cyclic, preferably straight, hydrocarbyl group having at least 10 carbon atoms (e.g., each Re is
In some of the foregoing embodiments of formula (XI), R5 is a non-cyclic hydrocarbyl group having at least 10 carbon atoms, e.g., a straight hydrocarbyl group having at least 10 carbon atoms. In some embodiments, R5 is a non-cyclic hydrocarbyl group having at least 12 carbon atoms, such as at least 14, at least 16, or at least 18 carbon atoms, e.g., a straight hydrocarbyl group having at least 12, at least 14, at least 16, or at least 18 carbon atoms. In some embodiments, Rs has at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments, Rs is a non-cyclic hydrocarbyl group, e.g., a straight hydrocarbyl group, wherein each hydrocarbyl group has 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, or 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments, the hydrocarbyl group of Rs is an alkyl or alkenyl group, e.g., a C10-30 alkyl or alkenyl group. Thus, in some embodiments, Rs is a non-cyclic alkyl group having at least 10 carbon atoms (such as at least 12, at least 14, at least 16, or at least 18 carbon atoms) or a non-cyclic alkenyl group having at least 10 carbon atoms (such as at least 12, at least 14, at least 16, or at least 18 carbon atoms), e.g., a straight alkyl group having at least 10 carbon atoms (such as at least 12, at least 14, at least 16, or at least 18 carbon atoms) or a straight alkenyl group having at least 10 carbon atoms (such as at least 12, at least 14, at least 16, or at least 18 carbon atoms). In some embodiments, R5 is a non-cyclic alkyl group or a non-cyclic alkenyl group, e.g., a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, or 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments, the alkenyl group has at least 2 carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds, such as 2 carbon-carbon double bonds. In some embodiments, the alkenyl group has at least 1 carbon-carbon double bond in cis configuration, e.g., 1, 2 or 3, such as 2, carbon-carbon double bonds in cis configuration. Thus, in some embodiments, R5 is a non-cyclic alkyl group or a non-cyclic alkenyl group, e.g., a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, or 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has at least 2 carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds. In some embodiments, R5 is a non-cyclic alkyl group or a non-cyclic alkenyl group, e.g., a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms, or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms, or 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has at least 1 carbon-carbon double bond, such as 1, 2, or 3 carbon-carbon double bonds, in cis configuration. In some embodiments, R5 has the following structure: wherein ww represents the bond by which R5 is bound to the remainder of the compound.
In some of the foregoing embodiments of formula (XI), L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-. In some embodiments, L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In some embodiments, Ra of L2 is H or C1-12 alkyl. In some embodiments, Ra of L2 is H or C1-6 alkyl, e.g., H or C1-3 alkyl. In some embodiments, Ra of L2 is H, methyl, or ethyl. In some embodiments, L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, and -SC(=O)-. In some embodiments, L2 is -O(C=O)- or -(C=O)O-.
In some of the foregoing embodiments of formula (XI), G2 is unsubstituted C1-12 alkylene or unsubstituted C2-12 alkenylene, e.g., unsubstituted, straight C1-12 alkylene or unsubstituted, straight C2-12 alkenylene. In some embodiments, G2 is unsubstituted C2-10 alkylene or unsubstituted C2-10 alkenylene, e.g., unsubstituted, straight C2-10 alkylene or unsubstituted, straight C2-10 alkenylene. In some embodiments, G2 is unsubstituted C2-6 alkylene or unsubstituted C2-6 alkenylene, e.g., unsubstituted, straight C2-6 alkylene or unsubstituted, straight C2-6 alkenylene. In some embodiments, G2 is unsubstituted C2-4 alkylene or unsubstituted C2-4 alkenylene, e.g., unsubstituted, straight C2-4 alkylene or unsubstituted, straight C2-4 alkenylene. In some embodiments, G2 is ethylene or trimethylene.
In some of the foregoing embodiments of formula (XI), each of R3 and R4 is independently C1-6 alkyl or C2-6 alkenyl. In some embodiments, each of R3 and R4 is independently C1-4 alkyl or C2-4 alkenyl. In some embodiments, each of R3 and R4 is independently C1-3 alkyl. In some embodiments, each of R3 and R4 is independently methyl or ethyl. In some embodiments, each of R3 and R4 is methyl.
In some of the foregoing embodiments of formula (XI), m is 0, 1, 2 or 3. In some embodiments, m is 0 or 2. In some embodiments, m is 0. In some embodiments, m is 2.
In some of the foregoing embodiments of formula (XI), the cationically ionizable lipid has the structure of formula (Xlla) or (Xllb): (Xllb), wherein each of R3 and R4 is independently Ci-Ce alkyl or C2-6 alkenyl;
Rs is a straight hydrocarbyl group having at least 14 carbon atoms (such as at least 16 carbon atoms), wherein the hydrocarbyl group preferably has at least 2 carbon-carbon double bonds; each Rs is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms and/or each Rs is attached to Li via an internal carbon atom of Rs, preferably each Rs is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms and each Rs is attached to Li via an internal carbon atom of Rs; each Gi is independently unsubstituted, straight C4-12 alkylene or C4-12 alkenylene, e.g., unsubstituted, straight Cg-12 alkylene or Cg-12 alkenylene, such as unsubstituted, straight Cs-i2 alkylene or unsubstituted, straight Cs-i2 alkenylene;
G2 is unsubstituted C2-C10 alkylene or C2-10 alkenylene, preferably unsubstituted C2-C6 alkylene or C2-6 alkenylene; each of Li and L2 is independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2 or 3, preferably 0 or 2.
In some of the foregoing embodiments of formula (Xlla), R5 has at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments of formulas (Xlla), R5 is a straight hydrocarbyl group having 14 to 30 carbon atoms (such as 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments of formula (Xlla), R5 is a straight alkyl or alkenyl group having 14 to 30 carbon atoms (such as 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments of formula (Xlla), the alkenyl group has at least 2 carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds, such as 2 carbon-carbon double bonds. In some embodiments, the alkenyl group has at least 1 carbon-carbon double bond in cis configuration, e.g., 1, 2 or 3, such as 2, carbon-carbon double bonds in cis configuration. Thus, in some embodiments of formula (Xlla), R5 is a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 14 to 30 carbon atoms (such as 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has at least 2 carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds. In some embodiments of formula (Xlla), R5 is a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 14 to 30 carbon atoms (such as 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has at least 1 carbon-carbon double bond, such as 1, 2, or 3 carbon-carbon double bonds, in cis configuration. In some embodiments of formula (Xlla), Rs is a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 14 to 30 carbon atoms (such as 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has 2 or 3 carbon-carbon double bonds, wherein at least 1 carbon-carbon double bond, such as 1, 2, or 3 carbon-carbon double bonds, is in cis configuration. In some embodiments of formula (Xlla), R5 has the following structure: wherein ww represents the bond by which Rs is bound to the remainder of the compound. In some embodiments of formula (Xlla), R5 has at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments of formula (Xlla), R5 is a non-cyclic hydrocarbyl group (e.g., a non-cyclic alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight hydrocarbyl group (e.g., a straight alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments of formula (Xlla), R5 is a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms and R6 is attached to Li via an internal carbon atom of R5. In some embodiments of formula (Xlla), R5 is a non-cyclic hydrocarbyl group (e.g., a non-cyclic alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight hydrocarbyl group (e.g., a straight alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and R5 is attached to Li via an internal carbon atom of R5. In some embodiments of formula (Xlla), Gi is independently unsubstituted, straight C4-12 alkylene or C4-12 alkenylene, e.g., unsubstituted, straight Cg-12 alkylene or Cg-12 alkenylene. In some embodiments of formula (Xlla), R5 is a straight hydrocarbyl group, e.g., a straight alkenyl group, having at least 14 carbon atoms (such as 14 to 30 carbon atoms) and 2 or 3 carbon-carbon double bonds; R5 is a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms (e.g., having 10 to 30 carbon atoms) and R5 is attached to Li via an internal carbon atom of R5; and Gi is independently unsubstituted, straight C4-12 alkylene or C4-12 alkenylene, e.g., unsubstituted, straight Cg-12 alkylene or C6-12 alkenylene.
In some of the foregoing embodiments of formula (Xllb), each R5 has independently at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments of formula (Xllb), each R5 is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, such as 11, 13, 15, 17, or 17 carbon atoms). In some embodiments of formula (Xllb), each Re is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, such as 11, 13, 15, 17, or 17 carbon atoms) and each Re is attached to Li via an internal carbon atom of Re. In some embodiments of formula (Xllb), each Re is independently selected from the group consisting of: , p y e to Li. In some embodiments of formula (Xllb), each Gi is independently unsubstituted, straight Ce-i2 alkylene or Ce-i2 alkenylene. In some embodiments of formula (Xllb), each Gi is independently unsubstituted, straight Cs-i2 alkylene or Cs-i2 alkenylene. In some embodiments of formula (Xllb), each Re is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, such as 11, 13, 15, 17, or 17 carbon atoms) and is attached to Li via an internal carbon atom of Re; and each Gi is independently unsubstituted, straight Cs-i2 alkylene or Cs-i2 alkenylene.
In some of the foregoing embodiments of formula (XI), the cationically ionizable lipid has the structure of formula (Xllla) or (Xlllb): , wherein each of R3 and R4 is independently CM alkyl or C2-4 alkenyl, more preferably C1-3 alkyl, such as methyl or ethyl; Rs is a straight alkyl or alkenyl group having at least 16 carbon atoms, wherein the alkenyl group preferably has at least 2 carbon-carbon double bonds; each R5 is independently a straight hydrocarbyl group having at least 10 carbon atoms, wherein R5 is attached to Li via an internal carbon atom of R5; each Gi is independently unsubstituted, straight C6-12 alkylene or unsubstituted, straight Cg-12 alkenylene, e.g., unsubstituted, straight Cs-i2 alkylene or unsubstituted, straight Cs-i2 alkenylene, such as unsubstituted, straight Cs-io alkylene or unsubstituted, straight Cs-io alkenylene, such as unsubstituted, straight Cx alkylene;
G2 is unsubstituted C2-6 alkylene or C2-6 alkenylene, preferably unsubstituted C2-4 alkylene or C2-4 alkenylene, such as ethylene or trimethylene; each of Li and L2 is independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2 or 3, preferably 0 or 2.
In some of the foregoing embodiments of formula (Xllla), R5 has at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments of formulas (Xllla), R5 is a straight alkyl or alkenyl group having 16 to 30 carbon atoms (such as 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In some embodiments of formula (Xllla), the alkenyl group has at least 2 carboncarbon double bonds, e.g., 2 or 3 carbon-carbon double bonds, such as 2 carbon-carbon double bonds. In some embodiments, the alkenyl group has at least 1 carbon-carbon double bond in cis configuration, e.g., 1, 2 or 3, such as 2, carbon-carbon double bonds in cis configuration. Thus, in some embodiments of formula (Xllla), R5 is a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 16 to 30 carbon atoms (such as 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has at least 2 carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds. In some embodiments of formula (Xllla), R5 is a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 16 to 30 carbon atoms (such as 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has at least 1 carbon-carbon double bond, such as 1, 2, or 3 carbon-carbon double bonds, in cis configuration. In some embodiments of formula (Xllla), R5 is a straight alkyl group or a straight alkenyl group, wherein each of the alkyl and alkenyl groups has independently 16 to 30 carbon atoms (such as 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms) and the alkenyl group has 2 or 3 carbon-carbon double bonds, wherein at least 1 carbon-carbon double bond, such as 1, 2, or 3 carbon-carbon double bonds, is in cis configuration. In some embodiments of formula (Xllla), R5 has the following structure: wherein ww represents the bond by which R5 is bound to the remainder of the compound. In some embodiments of formula (Xllla), Re has at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments of formula (Xllla), Re is a straight hydrocarbyl group (e.g., a straight alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) and Re is attached to Li via an internal carbon atom of Re. In some embodiments of formula (Xllla), Re is a straight alkyl group having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) and Re is attached to Li via an internal carbon atom of Re. In some embodiments of formula (Xllla), Gi is independently unsubstituted, straight C4-12 alkylene or C4-12 alkenylene, e.g., unsubstituted, straight Ce-i2 alkylene or Ce-i2 alkenylene. In some embodiments of formula (Xllla), R5 is a straight hydrocarbyl group, e.g., a straight alkenyl group, having at least 16 carbon atoms (such as 16 to 30 carbon atoms) and 2 or 3 carbon-carbon double bonds; Re is a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms (e.g., having 10 to 30 carbon atoms) and Re is attached to Li via an internal carbon atom of Re; and Gi is independently unsubstituted, straight C4-12 alkylene or C4-12 alkenylene, e.g., unsubstituted, straight Ce-i2 alkylene or Ce-i2 alkenylene.
In some of the foregoing embodiments of formula (Xlllb), each Re has independently at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, or at most 20 carbon atoms. In some embodiments of formula (Xlllb), each Re is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having 10 to 30 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, such as 11, 13, 15, 17, or 17 carbon atoms) and each Re is attached to Li via an internal carbon atom of Re. In some embodiments of formula (Xlllb), each Re is attached to Li via an internal carbon atom of Re and is independently selected from the group consisting of: , p y e to Li. In some embodiments of formula (Xlllb), each Gi is independently unsubstituted, straight Cs-i2 alkylene or Cs-i2 alkenylene, e.g., unsubstituted, straight Cs-io alkylene or Cs-io alkenylene. In some embodiments of formula (Xlllb), each Re is independently a straight hydrocarbyl group (e.g., a straight alkyl group) having at least 10 carbon atoms (such as 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms, or 11 to 19 carbon atoms, such as 11, 13, 15, 17, or 17 carbon atoms) and is attached to Li via an internal carbon atom of Re; and each Gi is independently unsubstituted, straight Cs-i2 alkylene or Cs-i2 alkenylene, e.g., unsubstituted, straight Cs-io alkylene or Cs-io alkenylene.
In some of the foregoing embodiments of formula (XI), the cationically ionizable lipid has one of the following formulas (XIV-1), (XIV-2), and (XIV-3):
In some embodiments, the cationically ionizable lipid is (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16- dien-l l-yl 5-(dimethylamino)pentanoate (3D-P-DMA). The structure of 3D-P-DMA may be represented as follows: In various different embodiments, the cationically ionizable lipid is selected from the group consisting of N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), and 4-((di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)oxy)-N,N-dimethyl-4-oxobutan- 1 -amine (DPL- 14).
Further examples of cationically ionizable lipids include, but are not limited to, 3-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3 -dimethylammonium propanes; l,2-dialkyloxy-3 -dimethylammonium propanes, l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)- l-(cis,cis-9, 12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethyl-l-(cA,cA-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin- K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl- 4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31 -tetraen- 19-yl - 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), 1,2- dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3 -dimethylammoniumpropane (DPDAP), N 1 -[2-(( 1 S)- 1 -[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan- 1 -amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan- 1 -amine (DMDMA), di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N- dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2 -dodecylcarbamoyl- ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12- 5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l- yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200).
In certain embodiments, the cationically ionizable lipid has the structure X-3.
In some embodiments, the cationic lipid for use herein is or comprises DPL-14. As used herein, "DPL- 14" is a lipid comprising the following general formula:
It is to be understood that any reference to a cationically ionizable lipid disclosed herein also includes the salts (in particular pharmaceutically acceptable salts), tautomers, stereoisomers, solvates (e.g., hydrates), and isotopically labeled forms thereof.
In some embodiments, the cationically ionizable lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the composition/particle. In some embodiments, the cationically ionizable lipid comprises from about 40 mol % to about 75 mol %, preferably from about 40 mol % to about 70 mol %, more preferably from about 45 mol % to about 65 mol %, of the total lipid present in the composition/particles.
In some embodiments, wherein the nucleic acid compositions/particles (in particular the RNA compositions/particles) described herein comprise a cationically ionizable lipid and one or more additional lipids, the cationically ionizable lipid comprises from about 10 mol % to about 80 mol %, from about 20 mol % to about 75 mol %, from about 20 mol % to about 70 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 50 mol %, from about 35 mol % to about 45 mol %, or from about 40 mol % to about 55 mol % of the total lipid present in the composition/particles.
In some embodiments of the nucleic acid (such as DNA or RNA) compositions (especially the mRNA compositions) described herein, where at least a portion of (i) the nucleic acid and (ii) the cationic or cationically ionizable lipid form particles (e.g., LNPs), the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 80 mol %, from about 20 mol % to about 75 mol %, from about 20 mol % to about 70 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 50 mol %, from about 35 mol % to about 45 mol %, or from about 40 mol % to about 55 mol % of the total lipid present in the particles.
In some embodiments, the N/P value is at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.
Additional lipids
In some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles) described herein may also comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids, i.e., non-cationic lipids or lipid-like materials (including non- cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationically ionizable lipid may enhance composition and/or particle stability and efficacy of nucleic acid (such as DNA or RNA) delivery.
One or more additional lipids may be incorporated which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the one or more additional lipids are a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
In certain embodiments, the nucleic acid (such as DNA or RNA) compositions/particles (especially the mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and one or more additional lipids.
Without wishing to be bound by theory, the amount of the cationic/cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some embodiments, the molar ratio of the cationic/cationically ionizable lipid to the one or more additional lipids is from about 10 : 0 to about 1:9, about 4 : 1 to about 1 : 2, or about 3 : 1 to about 1: 1.
In some embodiments, the one or more additional lipids comprised in the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially in the mRNA compositions/particles) described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof. In some embodiments, the one or more additional lipids comprised in the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially in the mRNA compositions/particles) described herein comprise a combination of a neutral lipid and a steroid.
Neutral lipids
In some embodiments, the one or more additional lipids comprise a neutral lipid which is preferably a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1- oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl- phosphatidylethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac -glycerol) (DPPG), 1 -palmitoyl -2 -oleoyl-sn-glycero-3- phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.
Thus, in some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and a phospholipid. In some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and a phospholipid selected from the group consisting of DSPC, DPPC, DSPE, and DPPE. In some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and DSPC.
In some embodiments, the neutral lipid is present in the nucleic acid compositions (such as DNA or RNA compositions, in particular the mRNA compositions) described herein in a concentration ranging from about 5 mol % to about 40 mol %, such as from about 5 mol % to about 25 mol %, from about 5 mol % to about 20 mol %, from about 5 mol % to about 15 mol %, or from about 5 mol % to about 10 mol %, of the total lipids present in the nucleic acid compositions (such as DNA or RNA composition, especially the mRNA composition) described herein.
In some embodiments of the nucleic acid (such as DNA or RNA) compositions (especially the mRNA compositions) described herein, where at least a portion of (i) the nucleic acid (such as RNA), (ii) the cationic/cationically ionizable lipid, and (iii) the neutral lipid form particles (e.g., LNPs), the neutral lipid (e.g., one or more phospholipids) may comprise from about 5 mol % to about 40 mol %, such as from about 5 mol % to about 25 mol %, from about 5 mol % to about 20 mol %, from about 5 mol % to about 15 mol %, or from about 5 mol % to about 10 mol %of the total lipid present in the particles.
Steroid
In some embodiments, the steroid is cholesterol. Thus, in some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and cholesterol.
In some embodiments, the steroid is present in the nucleic acid compositions (such as DNA or RNA compositions, in particular the mRNA compositions) described herein in a concentration ranging from about 10 mol % to about 65 mol %, such as from about 20 mol % to about 60 mol %, from about 30 mol % to about 50 mol %, or from about 25 mol % to about 35 mol % of the total lipids present in the compositions (especially the mRNA compositions) described herein.
In some embodiments of the nucleic acid (such as DNA or RNA) compositions (especially the mRNA compositions) described herein, where at least a portion of (i) the nucleic acid (such as RNA), (ii) the cationic/cationically ionizable lipid, and (iii) the steroid (e.g., cholesterol) form particles (e.g., LNPs), the steroid may comprise from about 10 mol % to about 65 mol %, such as from about 20 mol % to about 60 mol %, from about 30 mol % to about 50 mol %, or from about 25 mol % to about 35 mol % of the total lipid present in the particles.
In certain preferred embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise a phospholipid and cholesterol, preferably in the concentrations given above. In some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise a phospholipid selected from the group consisting of DSPC, DPPC, DSPE, and DPPE, and cholesterol, preferably in the concentrations given above. In some embodiments, the nucleic acid compositions/particles (such as DNA or RNA compositions/particles, especially the mRNA compositions/particles) described herein comprise DSPC and cholesterol, preferably in the concentrations given above. In some embodiments, the combined concentration of the neutral lipid (in particular, one or more phospholipids) and steroid (in particular, cholesterol) may comprise from about 0 mol % to about 70 mol %, such as from about 2 mol % to about 60 mol %, from about 5 mol % to about 55 mol %, from about 5 mol % to about 50 mol %, from about 5 mol % to about 40 mol %, or from about 20 mol % to about 40 mol % of the total lipids present in the nucleic acid compositions (such as DNA or RNA compositions, especially the mRNA compositions) described herein.
In some embodiments of the nucleic acid (such as DNA or RNA) compositions (especially the mRNA compositions) described herein, where at least a portion of (i) the nucleic acid (such as DNA or RNA, especially mRNA), (ii) the cationic/cationically ionizable lipid, and (iii) the one or more additional lipids (e.g., one or more phospholipids and/or steroids) form particles (e.g., LNPs), the additional lipid (e.g., one or more phospholipids and/or steroids) may comprise from about 0 mol % to about 70 mol %, such as from about 2 mol % to about 60 mol %, from about 5 mol % to about 55 mol %, from about 5 mol % to about 50 mol %, from about 5 mol % to about 40 mol %, or from about 20 mol % to about 40 mol %of the total lipid present in the particles.
Polymer-conjugated lipids
A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.
An example of a polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as pegylated lipid or PEG-lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
Another example of a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine) portion.
Another example of a polymer-conjugated lipid is a polyoxazoline (POX)-conjugated and/or polyoxazine (POZ)-conjugated lipid, also referred to herein as a conjugate of a POX and/or POZ polymer and one or more hydrophobic chains or as oxazolinylated and/or oxazinylated lipid or POX- and/or POZ-lipid. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule comprising both a lipid portion and a polyoxazoline portion. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule comprising both a lipid portion and a polyoxazine portion. The term "oxazolinylated/oxazinylated lipid" or "POX/POZ -lipid" or "POXZ -lipid" refers to a molecule comprising both a lipid portion and a portion of a copolymer of polyoxazoline and poly oxazine. A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers), connected by covalent bonds. The repeating units can all be identical, or in some cases, there can be more than one type of repeating unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties, such as those described herein. If more than one type of repeating unit is present within the polymer, then the polymer is said to be a "copolymer." The repeating units forming the copolymer can be arranged in any fashion. For example, the repeating units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeating unit (e.g., a first block), and one or more regions each comprising a second repeating unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
In some embodiments, a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a polymer-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.
In some embodiments, the nucleic acid (such as DNA or RNA) compositions described herein are substantially free of a sarcosinylated lipid.
In some embodiments, the nucleic acid (such as DNA or RNA) compositions described herein are substantially free of a pegylated lipid having at least 30 consecutive ethylene glycol repeating units.
In some embodiments, the nucleic acid (such as DNA or RNA) compositions described herein are substantially free of a (POX)-conjugated and/or polyoxazine (POZ)-conjugated lipid.
In some embodiments, the nucleic acid (such as DNA or RNA) compositions described herein are substantially free of pegylated lipid having at least 30 consecutive ethylene glycol repeating units, substantially free of sarcosinylated lipid, and substantially free of a (POX) -conjugated and/or polyoxazine (POZ) -conjugated lipid (or do not include a pegylated lipid having at least 30 consecutive ethylene glycol repeating units, a sarcosinylated lipid, or a oxazolinylated and/or oxazinylated lipid).
In some embodiments, the nucleic acid (such as RNA) compositions described herein are substantially free of any polymer-conjugated lipid other than the amphiphilic OEG-conjugated compound (or do not include any polymer-conjugated lipid other than the amphiphilic OEG-conjugated compound). PEG-conjugated lipids
In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein comprise a cationic/cationically ionizable lipid as described herein and a PEG-conjugated lipid. In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein may further comprise a neutral lipid (e.g., a phospholipid, cholesterol or a derivative thereof) or a combination of neutral lipids (e.g., a phospholipid, and cholesterol or a derivative thereof). In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein comprise a cationic/cationically ionizable lipid as described herein, a PEG-conjugated lipid, a neutral lipid (e.g., a phospholipid), and cholesterol or a derivative thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the cationic/cationically ionizable lipid is a cationically ionizable lipid of formula (X) (such as a cationically ionizable lipid of formula (X-3) or (X-45)). In some embodiments, the cationic/cationically ionizable lipid is a cationically ionizable lipid of formula (XI) (such as a cationically ionizable lipid of formula (XIV-1), (XIV-2), or (XIV-3)). In some embodiments, the cationic/cationically ionizable lipid is DPL14, EA-2, or 3D-P-DMA.
In some embodiments, the PEG-conjugated lipid is a lipid having the structure of the following general formula (XVI): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: each of R 12 and R13 is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
In some embodiments of formula (XVI), each of R12 and R13 is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.
In some embodiments of formula (XVI), R12 and R13 are identical. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 16 carbon atoms. In some embodiments of formula (XVI), R12 and R 13 are different. In some embodiments, one of R12 and R13 is a straight alkyl chain containing 12 carbon atoms and the other of R12 and R13 is a straight alkyl chain containing 14 carbon atoms.
In some embodiments of formula (XVI), w has a mean value ranging from 40 to 50, such as a mean value of 45.
In some embodiments of formula (XVI), w is within a range such that the PEG portion of the pegylated lipid of formula (XVI) has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol.
Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as 1 -(monomethoxy -poly ethylene glycol)-2, 3 -dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG- S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(a>-methoxy(polyethoxy)ethyl)- butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as a>-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)- propyl-N-(a> methoxy(polyethoxy)ethyl)carbamate, and the like.
In some embodiments, the PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide. In some embodiments, the pegylated lipid has the following structure:
In some embodiments, the PEG-conjugated lipid is DMG-PEG 2000, e.g., having the following structure:
In some embodiments, the PEG-conjugated lipid has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50. In one embodiment, the PEG- conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(co-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3 -bis(tetradecyloxy)propylcarbamate (2000) .
In some embodiments, the PEG-conjugated lipid is or comprises one or more PEG-conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
Sarcosinylated lipids
In some embodiments, the sarcosinylated lipid comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.
In some embodiments, the sarcosinylated lipid comprises the structure of the following general formula (XVII): wherein s is the number of sarcosine units.
In some embodiments, the sarcosinylated lipid comprises the structure of the following general formula (XVIII): wherein one of R21 and R22 comprises a hydrophobic group and the other is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and x is the number of sarcosine units.
In some embodiments of formula (XVIII), R21 is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and R22 comprises one or two straight alkyl or alkenyl groups each having at least 12 carbon atoms, such as at least 14 carbon atoms. In some embodiments, each of the straight alkyl and alkenyl groups has at most 30 carbon atoms, such as at most 28, at most 26, at most 24, at most 22, at most 20, or at most 18 carbon atoms. In some embodiments, R22 comprises one or two straight alkyl or alkenyl groups each having 12 to 30 carbon atoms (such as 12 to 28 carbon atoms, 12 to 26 carbon atoms, 12 to 24 carbon atoms, 12 to 22 carbon atoms, 12 to 20 carbon atoms, or 12 to 18 carbon atoms).
In some embodiments, the sarcosinylated lipid has the structure of the following general formula (IXX): wherein R is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and s is the number of sarcosine units.
In some embodiments, the sarcosinylated lipid has the structure of the following formula (IXX-1): wherein si is 23. The sarcosinylated lipid of formula (IXX-1) is also referred to herein as "C14pSar23".
Oxazolinylated and/or oxazinylated lipids
The oxazolinylated and/or oxazinylated lipid is a conjugate comprising (i) a POX and/or POZ polymer and (ii) one or more hydrophobic chains.
In some embodiments, the POX and/or POZ polymer in the oxazolinylated and/or oxazinylated lipid comprises the following general formula (XX): wherein a is an integer between 1 and 2; Rn is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, isopropyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of POX and/or POZ repeating units.
In some embodiments of the oxazolinylated and/or oxazinylated lipid, the POX and/or POZ polymer is a polymer of POX and comprises repeating units of the following general formula (XXa):
In some embodiments of the oxazolinylated and/or oxazinylated lipid, the POX and/or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula (XXb):
In any of the above embodiments of formulas (XX), (XXa), and (XXb), m (i.e., the number of repeating units of formula (XXa) or formula (XXb) in the polymer) preferably is between 2 and 200.
In some embodiments of the oxazolinylated and/or oxazinylated lipid, the POX and/or POZ polymer is a copolymer comprising repeating units of the following general formulas (XXa) and (XXb): wherein the number of repeating units of formula (XXa) in the copolymer is 1 to 199; the number of repeating units of formula (XXb) in the copolymer is 1 to 199; and the sum of the number of repeating units of formula (XXa) and the number of repeating units of formula (XXb) in the copolymer is 2 to 200.
In some embodiments, the oxazolinylated and/or oxazinylated lipid has the following general formula wherein: a is an integer between 1 and 2;
R11 is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; m is 2 to 200;
R12 is R14 or -Ln(Ri4)p, wherein each R14 is independently a hydrocarbyl group; Ln is a linker; and p is 1 or 2; and
R13 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R2I, -C(O)R2I, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R2i, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, -C(O)NR22R23, -NR22C(O)R2i, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair. In formula (XXI) R12 is attached to the N-end (i.e., the terminal N atom) of the POX and/or POZ polymer and R13 is attached to the C-end (i.e., the terminal C atom) of the POX and/or POZ polymer, whereas in formula (XXI’) R12 is attached to the C-end (i.e., the terminal C atom) of the POX and/or POZ polymer and R13 is attached to the N-end (i.e., the terminal N atom) of the POX and/or POZ polymer.
In some embodiments, the oxazolinylated and/or oxazinylated lipid has the following general formula (XXIII):
Ri5-POXZ-Ri6 wherein:
R15 is R17 or -Li2(Ri?)q, wherein each R17 is independently a hydrocarbyl group; L12 is a linker; and q is 1 or 2;
POXZ is a copolymer containing repeating units of the following general formulas (XXa) and (XXb): wherein each of Rn is independently alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; the number of repeating units of formula (XXa) in the copolymer is 1 to 199; the number of repeating units of formula (XXb) in the copolymer is 1 to 199; the sum of the number of repeating units of formula (XXa) and the number of repeating units of formula (XXb) in the copolymer is 2 to 200; and the repeating units of formulas (XXa) and (XXb) are arranged in a random, periodic, alternating or block wise manner; and
Ri6 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R2i, -C(O)R2i, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R2i, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, -C(O)NR22R23, -NR22C(O)R2i, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6- membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2 . a sugar, an amino acid, a peptide, and a member of a targeting pair.
Conjugate of (a) an amphiphilic OEG-conjugated compound and (b) a compound comprising the other member of the targeting pair
The amphiphilic OEG-conjugated compound of the present disclosure containing a member of a targeting pair may be used to prepare a conjugate comprising (a) said amphiphilic OEG-conjugated compound and (b) a compound comprising the other member of the targeting pair.
Thus, the present disclosure also relates to a conjugate of (a) an amphiphilic OEG-conjugated compound of the disclosure (in particular having any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’j, (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI ), (VII), (VII’), (VIII), (VIII ), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V- 6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably having any one of formulas (IXa), (IXi), (IXm), (V-l), (V-5), (V-17), and (V-25), such as any one of formulas (IXa), (IXi), and (IXm) or any one of formulas (V-l), (V-5), (V-17), and (V-25)) which contains a member of a targeting pair and (b) a compound comprising the other member of the targeting pair.
In some embodiments of the amphiphilic OEG-conjugated compound (in particular having any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIIF), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l 1), (V- 12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V- 63), preferably having any one of formulas (IXa), (IXi), (IXm), (V-l), (V-5), (V-17), and (V-25), such as any one of formulas (IXa), (IXi), and (IXm) or any one of formulas (V-l), (V-5), (V-17), and (V-25)) containing a member of a targeting pair, the targeting pair is selected from the group consisting of the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody (including immunoreactive fragments or derivatives thereof, such as Fab, Fab', F(ab')2 or scFv fragments or single-domain antibodies, e.g. VHH fragments or nanobodies) specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific (e.g., pegaptanib - VEGF receptor); arginine -glycine -aspartic acid (RGD) peptide - avp3 integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglycoprotein receptor.
For example, if the amphiphilic OEG-conjugated compound contains a maleimide moiety as a member of a targeting pair (e.g., as component of R3), then the compound comprising the other member of the targeting pair comprises a thiol moiety. Reaction of the maleimide moiety with the thiol moiety results in the covalent attachment of the amphiphilic OEG-conjugated compound to the compound comprising the other member of the targeting pair via a succinimidyl-S- moiety, i.e., providing a conjugate between the amphiphilic OEG-conjugated compound and the compound comprising the other member of the targeting pair. An exemplary reaction scheme for this reaction using an amphiphilic OEG-conjugated compound of formula (V) and a thiol compound ("HS-compound") is shown below: wherein R2, X1, X2, Y, z, and n are as defined above (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (Vf), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and
(IXr), preferably with respect to any one of formulas (IXa), (IXi), and (IXm)). In some embodiments, n is 5 to 25, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In another example, the amphiphilic OEG-conjugated compound contains a thiol moiety as a member of a targeting pair (e.g., as component of R3) and the compound comprising the other member of the targeting pair comprises a halogenated (in particular, brominated) alkyl group. Reaction of the thiol moiety with the halogenated (in particular, brominated) alkyl group results in the covalent attachment of the amphiphilic OEG-conjugated compound to the compound comprising the other member of the targeting pair via a thioether moiety, i.e., providing a conjugate between the amphiphilic OEG- conjugated compound and the compound comprising the other member of the targeting pair. An exemplary reaction scheme for this reaction using an amphiphilic OEG-conjugated compound of formula (V) and a brominated alkan ("Br-alkyl compound") is shown below: wherein R2, X1, X2, Y, z, and n are as defined above (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’X (Vi), (Vi’), (Vj), (Vj’), (VI), (VF), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), preferably with respect to any one of formulas (IXa), (IXi), and (IXm)). In some embodiments, n is 5 to 25, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2. An exemplary reaction scheme for a reaction using inverted targeting members (i.e., an amphiphilic OEG-conjugated compound containing a halogenated (in particular, brominated) alkyl group; and a thiol compound ("HS-compound")) is shown below:
In another example, the amphiphilic OEG-conjugated compound contains an antigen (e.g., peptide antigen) as a member of a targeting pair (e.g., as component of R3) and the compound comprising the other member of the targeting pair comprises an antibody (including immunoreactive fragments or derivatives thereof, such as Fab, Fab', F(ab')2 or scFv fragments or single-domain antibodies, e.g. VHH fragments or nanobodies) specific for said antigen. Reaction of the antigen with the antibody results in the non-co valent attachment of the amphiphilic OEG-conjugated compound to the compound comprising the other member of the targeting pair via a thioether moiety, i.e., providing a conjugate between the amphiphilic OEG-conjugated compound and the compound comprising the other member of the targeting pair. An exemplary reaction scheme for this reaction using an amphiphilic OEG- conjugated compound of formula (V) containing an antigen and an antibody specific for said antigen is shown below: wherein R2, X1, X2, Y, z, and n are as defined above (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’X (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), preferably with respect to any one of formulas (IXa), (IXi), and (IXm)); Pept is an antigen; antibody is an antibody specific for said antigen; and the dashed lines represent the non-covalent attachment between the antigen and the antibody. In some embodiments, n is 5 to 25, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments, the conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair has one of the following formulas (CI), (CII), and (CIII): wherein R2, X1, X2, Y, z, and n are as defined above (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’X (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), preferably with respect to any one of formulas (IXa), (IXi), and (IXm)); each ml is independently 1, 2, 3, 4, or 5 (such as 1, 2, 3, 4, preferably 1, 2 or 3, more preferably 1 or 2); compound is any compound (e.g., antigen or an antibody specific for said antigen); Pept is an antigen; and antibody is any compound comprising an antibody specific for said antigen. In some embodiments, the antigen is a peptide, e.g., a peptide having one of the following sequences: PSRLEEELRRRLTE-NH2 (SEQ ID NO: 11), SRLE- cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 12), PSRLE-cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 13) (in SEQ ID NOs: 12 and 13 "cyclo" means that the side chains of the two amino acids at positions 1 and 5 in the brackets (i.e., E and K) are taken together to form an amide bond). In some embodiments, n is 5 to 25, preferably 8, 10, or 14. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments, the conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair has one of the following formulas (Ca), (Cb), and (Cc): wherein R2, X1, X2, Y, z, and n are as defined above (in particular with respect to any one of formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’X (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), preferably with respect to any one of formulas (IXa), (IXi), and (IXm)); and compound is any compound (e.g., antigen or an antibody specific for said antigen). In some embodiments, the compound is an antigen, preferably a peptide, e.g., a peptide having one of the following sequences: PSRLEEELRRRLTE-NH2 (SEQ ID NO: 11), SRLE-cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 12), PSRLE-cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 13) (in SEQ ID NOs: 12 and 13 "cyclo" means that the side chains of the two amino acids at positions 1 and 5 in the brackets (i.e., E and K) are taken together to form an amide bond). In some embodiments, n is 5 to 25, preferably 8, 10, 12. 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments, the conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair has one of the following formulas (CIV) and (CV): or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12. 14, or 16; in each case -QOjCnHss refers to the moiety -C(O)(CH2)igCH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen. In some embodiments of formula (CIV), ml is 2, 3, or 4 (preferably 2); and m2 is 2, 3, or 4 (preferably 2). In some embodiments of formula (CV), ml is 1 is and m2 is 2; or ml is 2 and m2 is 1. In some embodiments, the antigen is a peptide, e.g., a peptide having one of the following sequences: PSRLEEELRRRLTE-NH2 (SEQ ID NO: 11), SRLE- cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 12), PSRLE-cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 13). In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8.
In some embodiments, the conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair has one of the following formulas (Ce), (Cf), and (Cg): or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12. 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen. In some embodiments, the antigen is a peptide, e.g., a peptide having one of the following sequences: PSRLEEELRRRLTE-NH2 (SEQ ID NO: 11), SREE-cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 12), PSRLE-cyclo(EELRK)-RLTE-NH2 (SEQ ID NO: 13). In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8.
A particular example of the conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair is the following compound (Cl):
Pept (Cl) or a salt thereof, wherein n is 14; and Pept is a peptide having the sequence PSRLEEELRRRLTE-NH2. In a further aspect, the present disclosure provides a nucleic acid conjugate composition comprising (i) nucleic acid (such as DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)).
It is understood that any embodiment described herein in the context of the nucleic acid composition comprising the amphiphilic OEG-conjugated compound (including uses of the nucleic acid composition comprising the amphiphilic OEG-conjugated compound) may also apply to any embodiment of the nucleic acid conjugate composition.
Thus, in some embodiemts, the nucleic acid conjugate composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)).
In some embodiments, the nucleic acid conjugate composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid selected from the group consisting of DODMA, DOTMA, DPL14, 3D-P-DMA, formula (X), and formula (XI); (iii) a conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)); and (iv) one or more additional lipids. In some embodiments, the one or more additional lipids are selected from neutral lipids and combinations thereof. In some embodiments, the neutral lipids include phospholipids, steroid lipids, and combinations thereof. In some embodiments, the one or more additional lipids are a combination of a phospholipid and a steroid lipid.
In some embodiments, the nucleic acid conjugate composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any one of formulas A to G; (iii) a conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments, the nucleic acid conjugate composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (X); (iii) a conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments, the nucleic acid conjugate composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (XI) (e.g., any one of formulas (Xlla), (Xllb), (Xllla), (Xlllb), (XIV-1), (XIV-2), and (XIV-3); (iii) a conjugate of (a) the amphiphilic OEG- conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)).
In some embodiments, the nucleic acid conjugate composition comprises (i) nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any one of formulas (XV- 1) to (XV-6); (iii) a conjugate of (a) the amphiphilic OEG-conjugated compound containing a member of a targeting pair and (b) a compound comprising the other member of the targeting pair as disclosed herein (e.g., a conjugate of any one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl)); (iv) a phospholipid; and (v) cholesterol.
In some embodiments of the nucleic acid conjugate composition, the nucleic acid is DNA.
In some embodiments of the nucleic acid conjugate composition, the nucleic acid is RNA, in particular mRNA.
In some embodiments of the nucleic acid conjugate composition, the composition is a pharmaceutical composition.
In some embodiments of the nucleic acid conjugate composition, the composition, in particular the pharmaceutical composition, is a vaccine.
In some embodiments of the nucleic acid conjugate composition, the composition, in particular the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
In some embodiments of the nucleic acid conjugate composition, the nucleic acid (e.g., DNA or RNA) and/or the composition, in particular the pharmaceutical composition, is/are a component of a kit. In some embodiments of the nucleic acid conjugate composition, the kit further comprises instructions for use of the nucleic acid (e.g., DNA or RNA) for inducing an immune response against a pathogen, e.g., against a virus, such as coronavirus, in a subject. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.
In some embodiments of the nucleic acid conjugate composition, the kit further comprises instructions for use of the nucleic acid (e.g., DNA or RNA) for therapeutically or prophylactically treating an infection, e.g., a viral infection, such as a coronavirus infection, in a subject. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.
In some embodiments of the nucleic acid conjugate composition, the subject is a human.
In certain embodiments of the nucleic acid conjugate composition, the nucleic acid (e.g., DNA or RNA) in the nucleic acid (e.g., DNA or RNA) compositions (e.g., in the nucleic acid (e.g., DNA or RNA) particles) described herein is at a concentration from about 0.002 mg/mL to about 5 mg/mL, from about 0.002 mg/mL to about 2 mg/mL, from about 0.005 mg/mL to about 2 mg/mL, from about 0.01 mg/mL to about 1 mg/mL, from about 0.05 mg/mL to about 0.5 mg/mL or from about 0. 1 mg/mL to about 0.5 mg/mL. In specific embodiments, the nucleic acid (e.g., DNA or RNA) is at a concentration from about 0.005 mg/mL to about 0.1 mg/mL, from about 0.005 mg/mL to about 0.09 mg/mL, from about 0.005 mg/mL to about 0.08 mg/mL, from about 0.005 mg/mL to about 0.07 mg/mL, from about 0.005 mg/mL to about 0.06 mg/mL, or from about 0.005 mg/mL to about 0.05 mg/mL.
In a further aspect, the present disclosure provides a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject the nucleic acid conjugate composition as specified herein.
In a further aspect, the present disclosure provides to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a nucleic acid conjugate composition as specified herein, wherein the nucleic acid encodes the therapeutic peptide or protein.
In a further aspect, the present disclosure provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid conjugate composition as specified herein, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. In a related aspect, the present disclosure provides a nucleic acid conjugate composition as specified herein for use in a method for treating or preventing a disease or disorder in a subject, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.
In a further aspect, the present disclosure provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid conjugate composition as specified disclosed herein, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. In a related aspect, the present disclosure provides a nucleic acid conjugate composition as specified disclosed herein for use in a method for treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
Embodiments of nucleic acid (such as DNA or RNA) compositions
In some embodiments, the one or more additional lipids comprise one of the following components: (1) a neutral lipid; (2) a steroid; or (3) a mixture of a neutral lipid and a steroid; preferably each in the concentration given above. In some embodiments, the one or more additional lipids comprise (3) a mixture of a neutral lipid and a steroid (and preferably do not comprise a PEG-lipid having at least 30 consecutive ethylene glycol repeating units, more preferably do not comprise any polymer-conjugated lipid other than the amphiphilic OEG-conjugated compound). In some embodiments, the one or more additional lipids comprise one of the following components: (1) a phospholipid; (2) cholesterol; (3) a mixture of a phospholipid and cholesterol; preferably each in the concentration given above. In some embodiments, the one or more additional lipids comprise (3) a mixture a phospholipid and cholesterol (and preferably do not comprise a PEG-lipid having at least 30 consecutive ethylene glycol repeating units, more preferably do not comprise any polymer-conjugated lipid).
Thus, in preferred embodiments, the nucleic acid compositions described herein comprise (i) nucleic acid (such as DNA or RNA, in particular mRNA), (ii) a cationic or cationically ionizable lipid; (iiia) an amphiphilic OEG-conjugated compound or (iiib) a conjugate of the amphiphilic OEG-conjugated compound containing a member of a targeting pair and a compound comprising the other member of the targeting pair as disclosed herein; (iv) a steroid; and (v) a neutral lipid (in particular, a phospholipid), preferably each in the concentration given above.
In specific embodiments, the amphiphilic OEG-conjugated compound has any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V-12), (V-13), (V- 14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63).
In specific preferred embodiments, the amphiphilic OEG-conjugated compound has any one of the formulas (V-l), (V-5), (V-17), and (V-25).
The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N/P value is about 6.
In some embodiments, the nucleic acid (such as DNA or RNA) is present in the nucleic acid compositions (such as DNA or RNA compositions) described herein in a concentration from about 1 mg/1 to about 500 mg/1, such as from about 1 mg/1 to about 100 mg/1, about 5 mg/1 to about 100 mg/1, or about 10 mg/1 to about 100 mg/1.
In some embodiments, the steroid is cholesterol; the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and the amphiphilic OEG-conjugated compound has any one of the formulas (V), (V’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf ), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (Villa), (Villa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (1X1), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-l), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-l l), (V- 12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37),
(V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50),
(V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-
63), preferably any one of formulas (V-l), (V-5), (V-17), and (V-25).
In specific embodiments of the nucleic acid conjugate composition, the conjugate has any one one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl).
In some embodiments of the nucleic acid conjugate composition, the steroid is cholesterol; the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and the conjugate has any one one of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (Cl). of the nucleic acid conjugate composition, the N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N/P value is about 6.
In some embodiments of the nucleic acid conjugate composition, the nucleic acid (such as DNA or RNA) is present in the nucleic acid compositions (such as DNA or RNA compositions) described herein in a concentration from about 1 mg/1 to about 500 mg/1, such as from about 1 mg/1 to about 100 mg/1, about 5 mg/1 to about 100 mg/1, or about 10 mg/1 to about 100 mg/1.
Compositions comprising nucleic acid (such as DNA or RNA, preferably mRNA) particles
The nucleic acid (such as DNA or RNA) compositions described herein (including the nucleic acid conjugate composition described herein) may comprise nucleic acid particles (such as DNA or RNA particles, like RNA LNPs), preferably a plurality of nucleic acid particles (such as a plurality of DNA or RNA particles). The term "plurality of nucleic acid particles" or "plurality of nucleic acid-lipid particles" refers to a population of a certain number of particles. In certain embodiments, the term refers to a population of more than 10, 102, 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 102°, 1021, 1022, or 1023 or more particles.
Nucleic acid particles (such as DNA or RNA particles, like LNPs comprising RNA (or "RNA LNPs")) described herein have an average diameter that in some embodiments ranges from about 30 nm to about 1000 nm, from about 30 nm to about 800 nm, from about 30 nm to about 700 nm, from about 30 nm to about 600 nm, from about 30 nm to about 500 nm, from about 30 nm to about 450 nm, from about 30 nm to about 400 nm, from about 30 nm to about 350 nm, from about 30 nm to about 300 nm, from about 30 nm to about 250 nm, from about 30 nm to about 200 nm, from about 30 nm to about 190 nm, from about 30 nm to about 180 nm, from about 30 nm to about 170 nm, from about 30 nm to about 160 nm, from about 30 nm to about 150 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 50 nm to about 190 nm, from about 50 nm to about 180 nm, from about 50 nm to about 170 nm, from about 50 nm to about 160 nm, or from about 50 nm to about 150 nm.
In certain embodiments, nucleic acid particles (such as DNA or RNA particles, like RNA LNPs) described herein have an average diameter that ranges from about 40 nm to about 800 nm, from about 50 nm to about 700 nm, from about 60 nm to about 600 nm, from about 70 nm to about 500 nm, from about 80 nm to about 400 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, or from about 200 nm to about 400 nm. In certain embodiments, nucleic acid particles (such as DNA or RNA particles, like RNA LNPs) described herein may have an average diameter that in some embodiments ranges from about 30 nm to about 500 nm, such as from about 30 nm to about 200 nm, or from about 50 nm to about 150 nm.
In some embodiments, the nucleic acid (such as DNA or RNA) compositions described herein comprise particles with a size of at least 10 pm in an amount of less than 4000/ml, preferably at most 3500/ml, such as at most 3400/ml, at most 3300/ml, at most 3200/ml, at most 3100/ml, or at most 3000/ml.
It will be apparent to those of skill in the art that the plurality of particles can include any fraction of the foregoing ranges or any range therein.
Nucleic acid particles (such as DNA or RNA particles, like RNA LNPs) described herein, e.g. prepared by the methods described herein, exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1 or about 0.05 or less. By way of example, the nucleic acid particles (such as DNA or RNA LNPs) can exhibit a polydispersity index in a range of about 0.05 to about 0.2, such as about 0.05 to about 0.1.
Generally, the nucleic acid particles (such as DNA or RNA particles, like RNA LNPs) described herein are "nucleic acid-lipid particles" (such as "DNA-lipid particles" or "RNA-lipid particles") that can be used to deliver the nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid-lipid particle is typically formed from a cationic or cationically ionizable lipid (such as the cationic or cationically ionizable lipids disclosed herein), an amphiphilic OEG-conjugated compound (or a conjugate of the amphiphilic OEG-conjugated compound containing a member of a targeting pair and a compound comprising the other member of the targeting pair) as disclosed herein, and one or more additional lipids (such as a phospholipid as disclosed herein (e.g., DSPC), and a steroid (e.g., cholesterol or analogues thereof)).
Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid, the amphiphilic OEG-conjugated compound (or its conjugate) and the one or more additional lipids combine together with the nucleic acid (such as DNA or RNA) to form particles, which become colloidally stable due to the presence of the amphiphilic OEG-conjugated compound (or its conjugate), in particular due to the stealth properties of the amphiphilic OEG-conjugated compound (or its conjugate).
In some embodiments, nucleic acid-lipid particles (such as DNA- or RNA-lipid particles) comprise more than one type of nucleic acid molecules (such as more than one type of DNA or RNA molecules), where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.
In some embodiments, the composition described herein is a liquid or a solid, with a solid referring to a dried, lyophilized or frozen form.
Compositions described herein may also comprise a cryoprotectant and/or a surfactant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of nucleic acid (e.g., DNA or RNA) activity during storage and/or freezing, for example to reduce or prevent aggregation, particle collapse, nucleic acid (such as DNA or RNA) degradation and/or other types of damage.
In some embodiments, the cryoprotectant is a carbohydrate. The term "carbohydrate", as used herein, refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.
In some embodiments, the cryoprotectant is a monosaccharide. The term "monosaccharide", as used herein refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose and the like.
In some embodiments, the cryoprotectant is a disaccharide. The term "disaccharide", as used herein refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose and the like.
The term "trisaccharide" means three sugars linked together to form one molecule. Examples of a trisaccharides include raffinose and melezitose.
In some embodiments, the cryoprotectant is an oligosaccharide. The term "oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, preferably 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. An oligosaccharide can be oxidized or reduced. In some embodiments, the cryoprotectant is a cyclic oligosaccharide. The term "cyclic oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, preferably 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, p cyclodextrin, or y cyclodextrin.
Other exemplary cyclic oligosaccharide cryoprotectants include compounds which include a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. A cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms. The term "cyclodextrin moiety", as used herein refers to cyclodextrin (e.g., an a, P, or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer. A cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker. A cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.
Carbohydrate cryoprotectants, e.g., cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates. For example, in an embodiment, the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-P-cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified P cyclodextrins).
An exemplary cryoprotectant is a polysaccharide. The term "polysaccharide", as used herein refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin and the like.
In some embodiments, the cryoprotectant is a sugar alcohol. The term "sugar alcohol", as used herein, refers to organic compounds containing at least two carbon atoms and one hydroxyl group attached to each carbon atom. Typically, sugar alcohols are derived from sugars (e.g., by hydrogenation of sugars) and are water-soluble solids. The term "sugar", as used herein, refers sweet-tasting, soluble carbohydrates. Examples of sugar alcohols include ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol. In one embodiment, the sugar alcohol has the formula HOCH2(CHOH)nCH2OH, wherein n is 0 to 22 (e.g., 0, 1, 2, 3, or 4), or a cyclic variant thereof (which can formally be derived by dehydration of the sugar alcohol to give cyclic ethers; e.g. isosorbide is the cyclic dehydrated variant of sorbitol).
In some embodiments, the cryoprotectant is glycerol and/or sorbitol.
In some embodiments, the nucleic acid (such as DNA or RNA) compositions (such as RNA LNP compositions) described herein may include sucrose as cryoprotectant. Without wishing to be bound by theory, sucrose functions to promote cryoprotection of the compositions, thereby preventing nucleic acid (especially DNA or RNA) particle aggregation and maintaining chemical and physical stability of the composition. Certain embodiments contemplate alternative cryoprotectants to sucrose in the present disclosure. Alternative stabilizers include, without limitation, glucose, glycerol, and sorbitol.
A preferred cryoprotectant is selected from the group consisting of sucrose, glucose, glycerol, sorbitol, and a combination thereof. In a preferred embodiment, the cryoprotectant comprises sucrose and/or glycerol. In a more preferred embodiment, the cryoprotectant is sucrose.
In some alternative embodiments, the nucleic acid (such as DNA or RNA) compositions (such as RNA LNP compositions) described herein are substantially free of a cryoprotectant, for example they do not contain any cryoprotectant.
Certain embodiments of the present disclosure contemplate the use of a chelating agent in a nucleic acid (such as DNA or RNA) composition (such as an RNA LNP composition) described herein. Chelating agents refer to chemical compounds that are capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), a salt of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTP A), and bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid. In certain embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is EDTA disodium dihydrate. In some embodiments, the EDTA is at a concentration from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM or from about 0.25 mM to about 1 mM.
In some embodiments, the aqueous phase of the nucleic acid (such as DNA or RNA) compositions (such as RNA LNP compositions) described herein do not comprise a chelating agent. For example, it is preferred that if the nucleic acid (such as DNA or RNA) compositions (such as RNA LNP compositions) described herein comprise a chelating agent, said chelating agent is only present in the particles, if present.
In some embodiments, the composition described herein comprises water as the main component and/or the total amount of solvent(s) other than water contained in the composition is less than about 1.0% (v/v). For example, the amount of water contained in the composition may be at least 50% (w/w), such as at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), at least 90% (w/w), or at least 95% (w/w). In particular, if the composition comprises a cryoprotectant, the amount of water contained in the composition may be at least 50% (w/w), such as at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), or at least 90% (w/w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition may be at least 95% (w/w). Additionally or alternatively, the total amount of solvent(s) other than water contained in the composition may be less than about 1.0% (v/v), such as less than about 0.9% (v/v), less than about 0.8% (v/v), less than about 0.7% (v/v), less than about 0.6% (v/v), less than about 0.5% (v/v), less than about 0.4% (v/v), less than about 0.3% (v/v), less than about 0.2% (v/v), less than about 0.1% (v/v), less than about 0.05% (v/v), or less than about 0.01% (v/v). In this respect, a cryoprotectant which is liquid under normal conditions will not be considered as a solvent other than water but as cryoprotectant. In other words, the above optional limitation that the total amount of solvent(s) other than water contained in the composition may be less than about 1.0% (v/v) does not apply to cryoprotectants which are liquids under normal conditions.
Pharmaceutical compositions
The compositions comprising nucleic acid (such as DNA or RNA), such as nucleic acid (such as DNA or RNA) particles (in particular RNA LNPs), described herein are useful as or for preparing pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments. Thus, in some embodiments, a composition described herein, in particular a nucleic acid composition described herein, is a pharmaceutical composition.
The nucleic acid (such as DNA or RNA) compositions described herein may be administered in the form of any suitable pharmaceutical composition.
The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. In the context of the present disclosure, the pharmaceutical composition comprises nucleic acid (such DNA or RNA), such as nucleic acid (such as DNA or RNA) particles (in particular RNA LNPs) as described herein.
The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund’s adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune -stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).
The pharmaceutical compositions of the present disclosure may be in a storable form (e.g., in a frozen or dried or lyophilized/freeze-dried form) or in a "ready -to-use form" (i.e., in a form, in particular a liquid form, which can be immediately administered to a subject, e.g., without any processing such as thawing, reconstituting or diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready -to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented in containers such as vials, ampoules or syringes wherein the container may contain one or more doses.
The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".
The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.
The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses . In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the particles or pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the particles or pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
In particular embodiments, a pharmaceutical composition of the present disclosure (e.g., an immunogenic composition, i.e., a pharmaceutical composition which can be used for inducing an immune response) is formulated as a single-dose in a container, e.g., a vial. In some embodiments, the immunogenic composition is formulated as a multi -dose formulation in a vial. In some embodiments, the multi-dose formulation includes at least 2 doses per vial. In some embodiments, the multi-dose formulation includes a total of 2-20 doses per vial, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in the vial is equal in volume. In some embodiments, a first dose is a different volume than a subsequent dose.
A "stable" multi -dose formulation preferably exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). As used herein, a "stable" immunogenic composition includes a formulation that remains capable of eliciting a desired immunologic response when administered to a subject.
The pharmaceutical compositions of the present disclosure may contain buffers (in particular, derived from the nucleic acid (such as DNA or RNA) compositions with which the pharmaceutical compositions have been prepared), preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure, in particular the ready-to-use pharmaceutical compositions, comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.
Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.
The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants. "Pharmaceutically acceptable salts" comprise, for example, acid addition salts which may, for example, be formed by using a pharmaceutically acceptable acid such as hydrochloric acid, acetic acid, lactic acid, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) or benzoic acid. Furthermore, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH/); and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations). Illustrative examples of pharmaceutically acceptable salts can be found in the prior art; see, for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)). Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically acceptable salts and are included in the present disclosure.
The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol and water.
The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.
Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.
Routes of administration of pharmaceutical compositions
In some embodiments, the compositions described herein, such as the pharmaceutical compositions, in particular the ready-to-use pharmaceutical compositions described herein, may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly or intratumorally. In certain embodiments, the (pharmaceutical) composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the (pharmaceutical) compositions, in particular the ready-to-use pharmaceutical compositions, are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) compositions, in particular the ready-to-use pharmaceutical compositions, are formulated for intramuscular administration.
Use of pharmaceutical compositions
Nucleic acid (such as RNA) compositions described herein may be used in the therapeutic or prophylactic treatment of various diseases, in particular diseases in which provision of a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, provision of an antigen or epitope which is derived from a virus may be useful in the treatment or prevention of a viral disease caused by said virus. Provision of a tumor antigen or epitope may be useful in the treatment of a cancer disease wherein cancer cells express said tumor antigen. Provision of a functional protein or enzyme may be useful in the treatment of genetic disorder characterized by a dysfunctional protein, for example in lysosomal storage diseases (e.g. Mucopolysaccharidoses) or factor deficiencies. Provision of a cytokine or a cytokine-fusion may be useful to modulate tumor microenvironment.
The term "disease" (also referred to as "disorder" herein) refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.
The term "disease involving an antigen" refers to any disease which implicates an antigen, e.g. a disease which is characterized by the presence of an antigen. The disease involving an antigen can be an infectious disease, or a cancer disease or simply cancer. The antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In some embodiments, a disease involving an antigen is a disease involving cells expressing an antigen, and preferably presenting the antigen on the cell surface, e.g., in the context of MHC.
The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, sexually transmitted diseases (e.g., chlamydia, gonorrhea, or syphilis), SARS, acquired immune deficiency syndrome (AIDS), measles, chicken pox, cytomegalovirus infections, genital herpes, hepatitis (such as hepatitis B or C), influenza (flu, such as human flu, swine flu, dog flu, horse flu, and avian flu), HPV infection, shingles, rabies, common cold, gastroenteritis, rubella, mumps, anthrax, cholera, diphtheria, foodbome illnesses, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, pertussis, tetanus, meningitis, scarlet fever, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, dientamoebiasis, giardiasis, amebic dysentery, coccidiosis, toxoplasmosis, sarcocystosis, rhinosporidiosis, and balantidiasis.
The terms "cancer disease" or "cancer" refer to or describe the physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the disclosure also comprises cancer metastases.
In some embodiments, nucleic acid (such as DNA or RNA) compositions described herein (including nucleic acid conjugate compositrions described herein) may be used in the therapeutic or prophylactic treatment of an infectious disease.
In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.
The term "therapeutic treatment" relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.
The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.
The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other nonmammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer, infectious diseases) but may or may not have the disease or disorder, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".
The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.
In some embodiments of the disclosure, the aim is to provide protection against an infectious disease by vaccination.
In some embodiments of the disclosure, the aim is to deliver nucleic acid (such as DNA or RNA) to cells of a subject. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to a subject to induce an immune response by providing a vaccine. A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, nucleic acids (such as DNA or RNA) described herein are applicable for inducing or enhancing an immune response. Nucleic acids described herein are thus useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.
In some embodiments of the disclosure, nucleic acid (such as DNA or RNA) may be administered to a subject for delivering a therapeutic or prophylactic peptide or polypeptide (e.g., a pharmaceutically active peptide or polypeptide) to the subject, wherein the nucleic acid encodes a therapeutic or prophylactic peptide or polypeptide.
In some embodiments of the disclosure, nucleic acid (such as DNA or RNA) may be administered to a subject for treating or preventing a disease in a subject, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease. In some embodiments, the nucleic acid encodes a therapeutic or prophylactic peptide or polypeptide, wherein delivering the therapeutic or prophylactic peptide or polypeptide to the subject is beneficial in treating or preventing the disease.
In some embodiments of the disclosure, the aim is to provide an immune response against diseased cells expressing an antigen such as cancer cells expressing a tumor antigen, and to treat a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to a subject to provide an immune response against diseased cells expressing an antigen such as cancer cells expressing a tumor antigen, and to treat a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen.
In some embodiments of the disclosure, the aim is to treat cancer by vaccination. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to a subject to treat cancer by vaccination.
In some embodiments of the disclosure, the aim is to provide an immune response against cancer cells expressing a tumor antigen and to treat a cancer disease involving cells expressing a tumor antigen. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to a subject to provide an immune response against cancer cells expressing a tumor antigen and to treat a cancer disease involving cells expressing a tumor antigen. In some embodiments of the disclosure, the aim is to provide protection against an infectious disease by vaccination. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to a subject to provide protection against an infectious disease by vaccination.
In some embodiments of the disclosure, nucleic acid (such as DNA or RNA) may be administered to a subject to provide secreted therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes, to a subject, in particular a subject in need thereof.
In some embodiments of the disclosure, the aim is to provide one or more cytokines or cytokine fusions which modulate tumor microenvironment to a subject, in particular a subject in need thereof. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to provide one or more cytokines or cytokine fusions which modulate tumor microenvironment to a subject, in particular a subject in need thereof.
In some embodiments of the disclosure, the aim is to provide one or more cytokines or cytokine fusions which have antitumoral activity to a subject, in particular a subject in need thereof. Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to provide one or more cytokines or cytokine fusions which have antitumoral activity to a subject, in particular a subject in need thereof.
In some embodiments of the disclosure, nucleic acid (such as DNA or RNA) may be administered to a subject to provide a protein replacement therapy, such as production of erythropoietin, Factor VII, Von Willebrand factor, p-galactosidase, Alpha-N-acetylglucosaminidase, to a subject, in particular a subject in need thereof.
In some embodiments of the disclosure, nucleic acid (such as DNA or RNA) may be administered to a subject to modulate/reprogram immune cells in the blood of the subject.
In some embodiments of the disclosure (in particular those relating to inhibitory RNA), the aim is to reduce or inhibit the expression of a peptide or polypeptide (such as the transcription and/or translation of a target mRNA). Thus, in some embodiments, nucleic acid (such as DNA or RNA) may be administered to reduce or inhibit the expression of a peptide or polypeptide (such as the transcription and/or translation of a target mRNA). In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, in particular a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.
In some embodiments, the nucleic acid is present in a composition as described herein.
In some embodiments, the nucleic acid is administered in a pharmaceutically effective amount.
In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.
A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Pharmaceutical compositions described herein are thus useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.
The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.
Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.
The description (including the following examples) is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims. Itemized embodiments
1. A composition comprising (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and
(iii) a polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.
2. The composition of item 1, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-; or
(vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl.
3. The composition of item 1 or 2, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl. 4. The composition of any one of items 1 to 3, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl.
5. The composition of any one of items 1 to 4, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.
6. The composition of any one of items 1 to 5, wherein Y is -CH2- or
7. The composition of any one of items 1 to 6, wherein Y is -CH2-.
8. The composition of any one of items 1 to 7, wherein the polymer comprises the following general formula (II): wherein R1 is hydrogen or C1-8 alkyl.
9. The composition of any one of items 1 to 8, wherein z is 2 to 10, such as 2 to 7.
10. The composition of any one of items 1 to 9, wherein z is 2 to 5.
11. The composition of any one of items 1 to 10, wherein z is 2 or 3.
12. The composition of any one of items 1 to 11, wherein z is 2.
13. The composition of any one of items 1 to 12, wherein the polymer comprises the following general formula (III): wherein R1 is hydrogen or Cus alkyl.
14. The composition of any one of items 8 to 13, wherein R1 is hydrogen or methyl. 15. The composition of any one of items 8 to 14, wherein R1 is hydrogen.
16. The composition of any one of items 1 to 15, wherein the polymer comprises the following general formula (IV):
17. The composition of any one of items 1 to 16, wherein n is 5 to 50.
18. The composition of any one of items 1 to 17, wherein n is 5 to 25.
19. The composition of any one of items 1 to 18, wherein n is 7 to 16, such as 7 to 14, preferably 8,
10, 12, 14, or 16.
20. The composition of any one of items 1 to 19, wherein the one or more hydrophobic chains are located at either the X1 end or the X2 end of the polymer.
21. The composition of any one of items 1 to 20, wherein the one or more hydrophobic chains are non-cyclic, preferably straight, hydrocarbyl groups, more preferably those having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.
22. The composition of any one of items 1 to 21, wherein the polymer-conjugated compound comprises the following general formula (V) or (V’): wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-;
R2 is a moiety comprising the one or more hydrophobic chains; R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.
23. The composition of item 22, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-; (iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(0)- then X2 is -0-; or
(vi) when X1 is -0- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl.
24. The composition of item 22 or 23, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or C1-8 alkyl.
25. The composition of any one of items 22 to 24, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl.
26. The composition of any one of items 22 to 25, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.
27. The composition of any one of items 22 to 26, wherein Y is -CH2- or -(CILh-.
28. The composition of any one of items 22 to 27, wherein Y is -CH2-.
29. The composition of any one of items 22 to 28, wherein the polymer-conjugated compound comp
(VI) (VT) wherein R1 is hydrogen or C1-8 alkyl.
30. The composition of any one of items 22 to 29, wherein z is 2 to 10, such as 2 to 7.
31. The composition of any one of items 22 to 30, wherein z is 2 to 5.
32. The composition of any one of items 22 to 31, wherein z is 2 or 3.
33. The composition of any one of items 22 to 32, wherein z is 2.
34. The composition of any one of items 22 to 33, which comprises the following general formula (VII) or (VIT): wherein R1 is hydrogen or Cus alkyl.
35. The composition of any one of items 29 to 34, wherein R1 is hydrogen or methyl.
36. The composition of any one of items 29 to 35, wherein R1 is hydrogen.
37. The composition of any one of items 22 to 36, wherein the polymer-conjugated compound comp
38. The composition of any one of items 22 to 37, wherein n is 5 to 50.
39. The composition of any one of items 22 to 38, wherein n is 5 to 25.
40. The composition of any one of items 22 to 39, wherein n is 7 to 16, such as 7 to 14, preferably
8, 10, 12, 14, or 16.
41. The composition of any one of items 22 to 40, wherein R2 is R4 or -L1(R4)P, wherein each R4 is independently a hydrophobic chain, such as a hydrocarbyl group; L1 is a linker; and p is 1 or 2.
42. The composition of item 41, wherein L1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.
43. The composition of item 41 or 42, wherein L1 comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6 -alkylene)-, [*-OC(O)]p(C1-6 -alkylene)-, [*-NHC(O)]p(C1-6- alkylene)-, [*-C(O)NH]p(C1-6 -alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p( C1-6 -alkylene)-, [*- S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r]( C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*- C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p( C1-6-alkylene)-, [*=C(=N- N(R26)C(O)(R25))]p(C1-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-, [*=C(=N(OH))]P(C1-6- alkylene)-, [*-OC(R25)(R26)O]p(C1-6-alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6 -alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6 -alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25 is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
44. The composition of any one of items 41 to 43, wherein L1 further comprises at least one additional difunctional moiety via which R2 is attached to either X1 in formula (V) or X2 in formula (V’).
45. The composition of item 44, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1 further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6-alkylene group from each other.
46. The composition of any one of items 41 to 45, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-
OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)NR26-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)NR26-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6- alkylene)O-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6-alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6 -alkylene), [*-OC(O)]p(C1-6 -alkylene), [*- NHC(O)]p(C1-6-alkylene), and [*-C(O)NH]p(C1-6-alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27 is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation; 3,4-dihydro-2H- chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
47. The composition of any one of items 41 to 46, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*- OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-
OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*- NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Ci-6-alkylene), [*-NHC(O)]P(CI_6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)NH-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)NH-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6- alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6-alkyltriyl)O- or L1 is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C 1-6 -alkylene), [*-OC(O)]p(C1-6- alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*-C(O)NH]p(C 1-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
48. The composition of any one of items 22 to 47, wherein R2 is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3-alkylene)-
OP(O)(OR27)O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene), [R4NHC(O)]P(C2-3- alkylene)OP(O)(OR27)O(Ci.3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(Ci.3- alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)O(Ci.3-alkylene), [R4C(O)NH]P(C2-3- alkylene)OP(O)(OR27)O(Ci.3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(Ci.3- alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2. 3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2 is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H- chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
49. The composition of any one of items 22 to 48, wherein R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2 is a monoalkylamine moiety.
50. The composition of any one of items 41 to 49, wherein each R4 is independently a non-cyclic, preferably straight, hydrocarbyl group.
51. The composition of any one of items 41 to 50, wherein each R4 is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.
52. The composition of any one of items 22 to 51, wherein R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2 is a monomyristylamine moiety. 53. The composition of any one of items 22 to 52, wherein R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair.
54. The composition of any one of items 22 to 53, wherein R3 is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2. and a member of a targeting pair.
55. The composition of any one of items 22 to 54, wherein R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair.
56. The composition of any one of items 22 to 55, wherein the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide - avp3 integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglycoprotein receptor.
57. The composition of any one of items 1 to 56, wherein the polymer-conjugated compound has one of the following formulas:
wherein n is 5 to 25;
R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair;
R27 is H or a countercation; and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)I2CH3 (myristoyl), in each case -CUHM refers to the moiety -(CH2)I3CH3 (myristyl), in each case -CBH27 refers to the moiety -(CH2)I2CH3, and in each case -C(O)CI7H33 refers to the moiety -czs-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl).
58. The composition of item 57, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. 59. The composition of item 57 or 58, wherein R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, C2-6 alkynyl, an antigen, or an antibody.
60. The composition of any one of items 1 to 59, wherein the polymer-conjugated compound has one of the following formulas:
wherein in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)i6CH3 (stearoyl), in each case -C(O)CI5H3I refers to the moiety (palmitoyl), in each case refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety (myristyl), in each case -C13H27 refers to the moiety -(CEDnCEL, and in each case n is 8, 10, 12, 14, or 16.
61. The composition of any one of claims 1 to 60, wherein the polymer-conjugated compound has one of the following formulas: wherein n is 14, and in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl); wherein n is 14, and in each case -C14H29 refers to the moiety -(CEDBCEL (myristyl); wherein n is 8, 12, 14, ro 16.
62. The composition of any one of items 1 to 61, wherein the composition is substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units.
63. The composition of any one of items 1 to 62, wherein water is the main component in the composition and/or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v/v).
64. The composition of any one of items 1 to 63, wherein the concentration of the nucleic acid in the composition is about 1 mg/1 to about 500 mg/1, such as about 1 mg/1 to about 100 mg/1, about 5 mg/1 to about 100 mg/1, or about 10 mg/1 to about 100 mg/1. 65. The composition of any one of items 1 to 64, wherein the cationically ionizable lipid comprises a head group which includes at least one tertiary amine moiety.
66. The composition of any one of items 1 to 65, wherein the cationic or cationically ionizable lipid has the structure of Formula (X) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L10 and L20 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L10 and L20 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;
G1 and G2 are each independently unsubstituted C1-C12 alkylene or C2-12 alkenylene;
G3 is C 1-24 alkylene, C2-24 alkenylene, C3-8 cycloalkylene, or C3-8 cycloalkenylene;
Ra is H or C1-12 alkyl;
R35 and R36 are each independently C6-24 alkyl or C6-24 alkenyl;
R37 is H, OR50, CN, -C(=O)OR40, -OC(=O)R40 or -NR50C(=O)R40;
R40 is C1-12 alkyl;
R50 is H or C1-6 alkyl; and x is 0, 1 or 2.
67. The composition of any one of items 1 to 65, wherein the cationic or cationically ionizable lipid has the structure of Formula (XI): wherein each of Ri and R2 is independently R5 or -G1-L1-R5, wherein at least one of Ri and R2 is -G1-L1-R5; each of R3 and R4 is independently selected from the group consisting of C1-6 alkyl, C2-g alkenyl, aryl, and C3-10 cycloalkyl; each of R5 and Rg is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms; each of Gi and G2 is independently unsubstituted C1-12 alkylene or C2-12 alkenylene; each of Li and L2 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-,
-NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-;
Ra is H or C1-12 alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1 or 2.
68. The composition of any one of items 1 to 65, wherein the cationic or cationically ionizable lipid comprises 2,3-dioleyloxy-l-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N- (l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.
69. The composition of any one of items 1 to 68, wherein the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol % of the total lipid present in the composition.
70. The composition of any one of items 1 to 69, further comprising one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of a phospholipid and a steroid.
71. The composition of item 70, wherein the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1- oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl- phosphatidylethanolamine (DPyPE). 72. The composition of item 70 or 71, wherein the phospholipid comprises from about 5 mol % to about 30 mol % of the total lipid present in the composition.
73. The composition of any one of items 70 to 72, wherein the steroid comprises a sterol such as cholesterol.
74. The composition of any one of items 70 to 73, wherein the steroid comprises from about 10 mol % to about 60 mol % of the total lipid present in the composition.
75. The composition of any one of items 70 to 74, wherein the cationic or cationically ionizable lipid comprises from about 20 mol % to about 70 mol % of the total lipid present in the composition; the polymer-conjugated compound comprises from about 0.5 mol %to about 15 mol % of the total lipid present in the composition; the phospholipid comprises from about 5 mol % to about 25 mol % of the total lipid present in the composition; and the steroid comprises from about 20 mol % to about 55 mol % of the total lipid present in the composition.
76. The composition of any one of items 1 to 75, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the cationic or cationically ionizable lipid, and at least a portion of the polymer- conjugated compound.
77. The composition of item 76, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof.
78. The composition of item 76 or 77, wherein the particles comprise at least 50%, preferably at least 75%, more preferably at least 85%, of the nucleic acid present in the composition.
79. The composition of any one of items 76 to 78, wherein the particles have a size of from about 30 nm to about 500 nm.
80. The composition of any one of items 1 to 79, wherein the nucleic acid is RNA, preferably mRNA.
81. The composition of item 80, wherein the RNA (1) comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (y), Nl-methyl- pseudouridine (ml\|/), and 5 -methyl -uridine (m5U); (2) has a coding sequence which is codon- optimized; and/or (3) has a coding sequence whose G/C content is increased compared to the wild-type coding sequence.
82. The composition of item 80 or 81, wherein the RNA comprises at least one of the following, preferably all of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly-A sequence.
83. The composition of item 82, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence preferably is an interrupted sequence of A nucleotides.
84. The composition of item 82 or 83, wherein the 5’ cap is a capl or cap2 structure.
85. The composition of any one of items 80 to 84, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and/or comprise an epitope for inducing an immune response against an antigen in a subject.
86. The composition of item 85, wherein the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof.
87. A method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a composition of any one of items 1 to 86.
88. A method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a composition of any one of items 1 to 86, wherein the nucleic acid encodes the therapeutic peptide or protein.
89. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of any one of items 1 to 86, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.
90. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of any one of items 1 to 86, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
91. The method of any one of items 87 to 90, wherein the subject is a mammal. 92. The method of item 91, wherein the mammal is a human.
93. A polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.
94. The polymer-conjugated compound of item 93, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-; or
(vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl.
95. The polymer-conjugated compound of item 93 or 94, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or C1-8 alkyl. 96. The polymer-conjugated compound of any one of items 93 to 95, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl.
97. The polymer-conjugated compound of any one of items 93 to 96, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.
98. The polymer-conjugated compound of any one of items 93 to 97, wherein Y is -CH2- or -(CH2)2-.
99. The polymer-conjugated compound of any one of items 93 to 98, wherein Y is -CH2-.
100. The polymer-conjugated compound of any one of items 93 to 99, wherein the polymer comprises the following general formula (II): wherein R1 is hydrogen or C1-8 alkyl.
101. The polymer-conjugated compound of any one of items 93 to 100, wherein z is 2 to 10, such as 2 to 7.
102. The polymer-conjugated compound of any one of items 93 to 101, wherein z is 2 to 5.
103. The polymer-conjugated compound of any one of items 93 to 102, wherein z is 2 or 3.
104. The polymer-conjugated compound of any one of items 93 to 103, wherein z is 2.
105. The polymer-conjugated compound of any one of items 93 to 104, wherein the polymer comprises the following general formula (III): wherein R1 is hydrogen or Cus alkyl. 106. The polymer-conjugated compound of any one of items 100 to 105, wherein R1 is hydrogen or methyl.
107. The polymer-conjugated compound of any one of items 100 to 106, wherein R1 is hydrogen.
108. The polymer-conjugated compound of any one of items 93 to 107, wherein the polymer comprises the following general formula (IV):
109. The polymer-conjugated compound of any one of items 93 to 108, wherein n is 5 to 50.
110. The polymer-conjugated compound of any one of items 93 to 109, wherein n is 5 to 25.
111. The polymer-conjugated compound of any one of items 93 to 110, wherein n is 7 to 16, such as
7 to 14, preferably 8, 10, 12, 14, or 16.
112. The polymer-conjugated compound of any one of items 93 to 111, wherein the one or more hydrophobic chains are located at either the X1 end or the X2 end of the polymer.
113. The polymer-conjugated compound of any one of items 93 to 112, wherein the one or more hydrophobic chains are non-cyclic, preferably straight, hydrocarbyl groups, more preferably those having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.
114. The polymer-conjugated compound of any one of items 93 to 113, comprising the following general formula (V) or (V’): wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester; Y is -CH2-, -(CH2)2-> or -(CH2)3-;
R2 is a moiety comprising the one or more hydrophobic chains;
R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2.g alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.
115. The polymer-conjugated compound of item 114, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl; preferably (i) when X1 is -C(0)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(0)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(0)- then X2 is -0-; or
(vi) when X1 is -0- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl
116. The polymer-conjugated compound of item 114 or 115, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or C1-8 alkyl.
117. The polymer-conjugated compound of any one of items 114 to 116, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl.
118. The polymer-conjugated compound of any one of items 114 to 117, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.
119. The polymer-conjugated compound of any one of items 114 to 118, wherein Y is -CH2- or -(CH2)2-.
120. The polymer-conjugated compound of any one of items 114 to 119, wherein Y is -CH2-.
121. The polymer-conjugated compound of any one of items 114 to 120, comprising the following general formula (VI) or (VI’): wherein R1 is hydrogen or C1-8 alkyl.
122. The polymer-conjugated compound of any one of items 114 to 121, wherein z is 2 to 10, such as 2 to 7.
123. The polymer-conjugated compound of any one of items 114 to 122, wherein z is 2 to 5.
124 The polymer-conjugated compound of any one of items 114 to 123, wherein z is 2 or 3. 125. The polymer-conjugated compound of any one of items 114 to 124, wherein z is 2.
126. The polymer-conjugated compound of any one of items 114 to 125, comprising the following general formula (VII) or (VIT): wherein R1 is hydrogen or Cus alkyl.
127. The polymer-conjugated compound of any one of items 121 to 126, wherein R1 is hydrogen or methyl.
128. The polymer-conjugated compound of any one of items 121 to 127, wherein R1 is hydrogen.
129. The polymer-conjugated compound of any one of items 114 to 128, comprising the following general formula (VIII) or (VIIT):
130. The polymer-conjugated compound of any one of items 114 to 129, wherein n is 5 to 50.
131. The polymer-conjugated compound of any one of items 114 to 130, wherein n is 5 to 25.
132. The polymer-conjugated compound of any one of items 114 to 131, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16.
133. The polymer-conjugated compound of any one of items 114 to 132, wherein R2 is R4 or -L1(R4)P, wherein each R4 is independently a hydrophobic chain, such as a hydrocarbyl group; L1 is a linker; and p is 1 or 2.
134. The polymer-conjugated compound of item 133, wherein L1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.
135. The polymer-conjugated compound of item 133 or 134, wherein L1 comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6 -alkylene)-, [*-OC(O)]p(C1-6-alkylene)-, [* -NHC(O)]P(C i-6-alkylene)-, [* -C(O)NH]P(C i-6 -alkylene)-, [* -S]P(C i-6 -alkylene)-, [* -S S]P(C i-6- alkylene)-, [*-S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r](C1-6-alkylene)-, [*-C(OR25)2O]P(C1-6- alkylene)-, [*-C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p(C1-6-alkylene)-
[*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-,
[*=C(=N(OH))]P(C i-6 -alkylene)-, [*-OC(R25)(R26)O]P(CI-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6- yl)-, (*-)PN(R26)2-P, and [*-C(0)NH](CI-6 -alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25 is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
136. The polymer-conjugated compound of any one of items 133 to 135, wherein L1 further comprises at least one additional difunctional moiety, via which R2 is attached to either X1 in formula (V) or X2 in formula (V’). 137. The polymer-conjugated compound of item 136, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1 further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6-alkylene group from each other.
138. The polymer-conjugated compound of any one of items 133 to 137, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6- alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)O]p(Cw-alkylene)-OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6- alkylenejO-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6-alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6 -alkylene), [*-OC(O)]p(C1-6 -alkylene), [*- NHC(O)]p(C1-6-alkylene), and [*-C(O)NH]p(C1-6-alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27 is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation; 3,4-dihydro-2H- chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
139. The polymer-conjugated compound of any one of items 133 to 138, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6- alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*- NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6 -alkylene)©-, [*-OC(O)]p(C1-6- alkylene)©-, (*-)2N-, and [*-C(O)NH](C1-6-alkyltriyl)O- or L1 is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6 -alkylene), [*-OC(O)]p(C1-6- alkylene), [*-NHC(0)]p(C1-6-alkylene), and [*-C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OCi- 3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
140. The polymer-conjugated compound of any one of items 114 to 139, wherein R2 is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3- alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3- alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O-(C1-3-alkylene), [R4NHC(O)]p(C2-3- alkylene)OP(O)(OR27)O(C1-3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene), [R4C(O)NH]p(C2-3- alkylene)OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(C1-3- alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2- 3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2 is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H- chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.
141. The polymer-conjugated compound of any one of items 114 to 140, wherein R2 is selected from a phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2 is a monoalkylamine moiety.
142. The polymer-conjugated compound of any one of items 133 to 141, wherein each R4 is independently a non-cyclic, preferably straight, hydrocarbyl group. 143. The polymer-conjugated compound of any one of items 133 to 142, wherein each R4 is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.
144. The polymer-conjugated compound of any one of items 114 to 143, wherein R2 is selected from DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), or POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2 is a monomyristylamine moiety.
145. The polymer-conjugated compound of any one of items 114 to 144, wherein R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair.
146. The polymer-conjugated compound of any one of items 114 to 145, wherein R3 is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair.
147. The polymer-conjugated compound of any one of items 114 to 146, wherein R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), and -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair.
148. The polymer-conjugated compound of any one of items 114 to 147, wherein the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide - av[T integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor.
149. The polymer-conjugated compound of any one of items 93 to 148, having one of the following formulas: wherein n is 5 to 25;
R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair;
R27 is H or a countercation; and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)16CH3 (stearoyl), in each case -C(O)C15H3i refers to the moiety -C(O)(CH2)14CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)I2CH3 (myristoyl), in each case -CUHM refers to the moiety -(CH2)i3CH3 (myristyl), in each case -Ci3H27 refers to the moiety -(CH2)I2CH3, and in each case -C(O)Ci7H33 refers to the moiety -cis- C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl) .
150. The polymer-conjugated compound of item 149, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16.
151. The polymer-conjugated compound of item 149 or 150, wherein R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, and C2-6 alkynyl.
152. The polymer-conjugated compound of any one of items 93 to 151, having one of the following formulas:
wherein in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl), in each case -C(O)CI5H3I refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(ClDnCTL, and in each case n is 8, 10, 12, 14, .
153. The polymer-conjugated compound of any one of items 93 to 152, having one of the following formulas: wherein n is 14, and in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl); wherein n is 14, and in each case -C14H29 refers to the moiety -(CH2)I3CH3 (myristyl); wherein n is 8, 12, 14, or 16.
154. A conjugate of (a) the polymer-conjugated compound of any one of items 93 to 153 containing a member of a targeting pair; and (b) a compound comprising the other member of the targeting pair.
155. The conjugate of item 154, wherein the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (such as an antigen or epitope), or an antibody.
156. The conjugate of item 154 or 155, having one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, r 14, or 16; in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)I6CH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen.
157. The conjugate of item 156, having one of the following formulas: wherein ml is 2, 3, or 4, preferably 2; and m2 is 2, 3, or 4, preferably 2; wherein each of ml and m2 is independently 1, 2, or 3, preferably ml is 1 is and m2 is 2 or ml is 2 and m2 is 1.
158. The conjugate of any one of items 154 to 157, having one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)I6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen.
159. The conjugate of item 154, wherein the polymer-conjugated compound containing a member of a targeting pair has the following formula: wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)i6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen; the compound comprising the other member of the targeting pair is a compound comprising (i) an antibody specific for said antigen if Pept is said antigen; or (ii) an antigen if Pept is an antibody specific for said antigen; and the polymer-conjugated compound containing a member of a targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) said antibody specific for said antigen and (2) said antigen. Further aspects of the present disclosure are disclosed herein.
Examples
Materials
Cholesterol and distearoyl-sn-glycerol-3 -phosphocholine (DSPC) were supplied from Avanti (Alabama, USA). The cationically ionizable lipid HY-501 (also referred to herein as the cationically ionizable lipid of formula XIV-3) was purchased from NucleoSyn (Olivet, France). Ethanol absolute, citrate buffer, and OmniPur® Water (WFI Quality, sterile purified water) were purchased from Merck Millipore. The RNase-free TE buffer concentrate, 20X used for the RiboGreen® RNA quantitation assays, as well as the RiboGreen dye were purchased from Invitrogen. Triton X-100 was purchased from VWR International GmbH, Darmstadt, Germany. (+)-a-tocopherol and DMA (dimyristylamine) were purchased from Sigma- Aldrich, Inc. DMG (1,2-dimyristoylglycerol) was purchased from Biosynth International, Inc. Fmoc-AEEA-OH was purchased from Ambeed Inc. DSPE was purchased from Echelon Biosciences, Inc. All materials in contact with the solutions and LNP preparations were sterile. V09 mRNA (modified mRNA encoding the P2 S protein of the SARS-CoV-2) which was used in the LNP experiments has been synthesized in house using internal protocols. Alternatively, luciferase mRNA (LUC mRNA, modified mRNA encoding firefly luciferase) or Thy 1. 1 RNA (encoding Thymus Cell Antigen 1.1) was used.
Methods
All methods including the use of kits and reagents are carried out according to the manufacturers information unless specifically indicated.
Salt exchange and purification of amphiphilic compounds
The lyophilized composition comprising the amphiphilic compound to be purified is resuspended in ammonium acetate (10 g/L) in 90%water, 10% acetonitrile and loaded onto a preparative HPLC column (XBridge Protein BEH Ci OBD Prep Column, 300 A, 5 pm, 10 mm X 50 mm) with automatic injector through a 10 mb sample loop. Sample loading is done in ammonium acetate (10 g/L) in 90% water, 10% acetonitrile, which washes over the sample for 15 min. Then mobile phase A is switched to 1% TFA (or 1% acetic acid) in water, and run through the column for 15 min, followed by a sharp increase to 95% B (Acetonitrile) in 0.1 min. Fraction elution is triggered by the same masses used in the purification step (or by a mass spectrometer). Eluted fractions are pooled together. An aliquot is injected on the UPLC-MS for purity and ID. The pooled sample is lyophilized to dryness.
For some amphiphilic compounds (such as those comprising a DSPE moiety), the following adapted procedure may be used. The lyophilized composition comprising the amphiphilic compound to be purified is resuspended in ammonium acetate (10 g/L) in 90%water, 10% acetonitrile and loaded onto a preparative HPLC column (XBridge Protein BEH Ci OBD Prep Column, 300 A, 5 pm, 10 mm X 50 mm) with automatic injector through a 10 mL sample loop. Sample loading is done in ammonium acetate (10 g/L) in 90% water, 10% acetonitrile, which washes over the sample for 15 min. Then mobile phase A is switched to 95% water, 5% acetonitrile + 1% acetic acid, and mobile phase B is 100% acetonitrile. Due to the hydrophobicity of the amphiphilic compound, a sharp gradient is used for elution, as shown in the following table.
Using this gradient, e.g., Ac-(AEEA)i4-DSPE elutes between 60 and 95% B, while more hydrophilic impurities elute earlier. Due to the sharpness of the gradient at elution, close eluting impurities will likely be collected with the main peak. Thus, the DPSE starting lipid should be completely consumed during the reaction. Fractions are collected by MS using the M+2, M+3, and M+4 values of each compound; however, absorbance at 214 nm may be used for collection as well. Fractions with estimated or measured UV214 purity (preferably >95%) are pooled into polypropylene vials and lyophilized. They are then resuspended in 50/50 water/acetonitrile + 0.5% acetic acid, transferred into a glass vial and lyophilized for at least 2 days. Fraction and final QC are performed in accordance with the procedure described in section "Determination of purity and identity of amphiphilic compounds", below.
Alternative purification of amphiphilic compounds
Solvent (e.g., chloroform), if still present in the composition comprising the amphiphilic compound to be purified, is removed (e.g., using Biotage V10 rotary evaporator), and samples are resuspended in methanol. Purification may be performed using reverse phase HPLC. The preparative column is Waters XB ridge Protein BEH C4 OBD Prep Column, 300 A, 5 pm, 19 mm X 100 mm. Mobile Phase A is 1% acetic acid in water and mobile phase B is 100% acetonitrile. The flow rate is 15 mL/min. Exemplary values for gradient and elution point of certain amphiphilic compounds are shown in the following table.
Fractions are collected by MS using the M+2, M+3, and M+4 values of each compound; however, absorbance at 214 nm may be used for collection as well. Fractions with estimated or measured UV214 purity are pooled into polypropylene vials and lyophilized. They are then resuspended in 50/50 water/acetonitrile + 0.5% acetic acid, transferred into a glass vial and lyophilized for at least 2 days. Fraction and final QC are performed in accordance with the procedure described in section "Determination of purity and identity of amphiphilic compounds", below.
Further alternative purification of amphiphilic compounds
Solvent, if still present in the composition comprising the amphiphilic compound to be purified, is removed (e.g., using Biotage V10 rotary evaporator), and samples are resuspended in chloroform. Purification may be performed using size exclusion HPLC. The preparative columns in series are JAIGEL 2.5 HR and JAIGEL 2HR. The mobile phase is 100% chloroform, or if needed for solubility, 90% methanol, 10% chloroform. The flow rate is 10 mL/min. Recycling can be performed to resolve impurities. Fractions are collected by UV absorption. Purity relevant fractions are pooled, and solvent is removed (e.g., using rotovap). The amphiphilic compound is then resuspended in 50/50 water/acetonitrile + 0.5% acetic acid and lyophilized for at least 2 days. Fraction and final QC are performed in accordance with the procedure described in section "Determination of purity and identity of amphiphilic compounds".
When applicable, coeluting hydrophilic impurities from preparative size exclusion HPLC can be removed by water extraction. The sample is resuspended in chloroform, water is added and the sample is vortexed. The aqueous layer separates from the organic layer, and the aqueous layer containing hydrophilic impurities is removed. The chloroform is then removed from the organic layer (e.g. using Biotage V10 rotary evaporator). The sample is resuspended in 50/50 water/acetonitrile + 0.5% acetic acid and lyophilized for at least 2 days.
Determination of purity and identity of amphiphilic compounds
The purity and identity of an amphiphilic compound is measured on a Waters H-Class UPLC/MS system containing a Waters H-Class UPLC with PDA UV Detector and QDa mass detector and using a linear gradient from 10 to 80% B in 8 min with flow rate of 0.5mL/min, where mobile phase A is 0.100% TFA in water and mobile phase B is 0.085% TFA in acetonitrile. The column (ACQUITY UPLC Protein BEH C4 Column, 300 A, 1.7 pm, 2.1 mm X 100 mm) is heated to 60 °C. The following table shows the parameters used for this analysis. Integration was performed automatically with Empower 3 software.
Passing criteria for the final conjugates is 95% UV214 purity and molecular weight (MW) within 1 amu of the theoretical value.
UPLC-MS
A crude sample (e.g., a crude peptide) is dissolved 50/50 water/acetonitrile + 0.05% TFA to about 30 mg/mL. The solution is vortexed and sonicated until fully dissolved (no visual presence of solid material). The solution is loaded onto the preparative HPLC column (Phenomenex Luna C18(2) LC Column, 100 A pore size, 10 pm particle size, 30 mm x 250 mm) with an automatic injector through a 10 mL sample loop. Sample loading is done in 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Peptide elution is performed in a 36-min linear gradient of 20 - 35%B. The flow rate is 30 mL/min. A Waters SQD2 mass spectrometer is used to trigger fraction collection automatically based on the M+1H, M+2H, M+3H values for the target compound (such as the target peptide). UV214 absorption is monitored but not used for fraction triggering. Aliquots of selected compound fractions are injected on a Waters H-Class UPLC/MS system using a linear gradient from 10 to 80% B in 8 min with flow rate of 0.5 mL/min, where mobile phase A is 0. 100% TFA in water and mobile phase B is 0.085% TFA in acetonitrile. The column (ACQUITY UPLC Peptide BEH C18 Column, 130 A, 1.7 pm, 2.1 mm X 100 mm) is heated to 40 °C. For some compounds (e.g., peptides), in-process purity measurements are omitted by visually monitoring the preparative HPLC UV214 absorption and MS to estimate which fractions were high purity. Fractions with measured or estimated purity >90% are combined into tared polypropylene tubes and lyophilized. If the pure fraction volume exceeds the tube capacity, multiple polypropylene tubes are fdled and lyophilized. Then each compound (e.g., peptide) is resuspended in 50/50 water/acetonitrile + 0.05% TFA and combined into one tared polypropylene tube and lyophilized to dryness. About 1 mg of the peptide is dissolved to 1 mg/mL in 50/50 water/acetonitrile + 0.05% TFA and injected on the UPLC/MS for purity and MW.
Resin loading of a peptide to 2-Cltrt polystyrene resin
One gram of peptide (e.g., 2.59 mmol of Fmoc-AEEA-OH) is weighed into a 50 ml conical tube and dissolved with enough DCM to allow for complete dissolution of the amino acid material as well as sufficient volume to provide coverage and mobility to the resin while loading. In order to provide the ideal resin loading substitution the millimole amount of the peptide is doubled (e.g., 5.19 mmol for Fmoc-AEEA-OH) and used to calculate the necessary amount of 2-cltrt resin (e.g., the final amount of 2-cltrt resin used for loading Fmoc-AEEA-OH was roughly 4.63 g). Once the necessary amount of resin is calculated and weighed into two 20 ml syringes enough DCM is added to pre-swell the resin in preparation for loading. The resin pre-swelling continues for roughly 30 min to 1 hour, and after that time is completed 10 equivalents (eq.) of neat DIEA is added to the amino acid solution. Once the DIEA has been added the amino acid solution is added into the resin syringes acting as a solvent for transfer into a 250 ml flat bottom centrifuge bottle. The 250 ml centrifuge bottle is capped and secured on an orbital shaker for a reaction time of 2 hrs minimum to overnight maximum. Once the reaction time has been completed the resin and amino acid solution is filtered back through the 20 ml syringes used for swelling and the resin is rinsed 3-5 times with DCM to remove any excess amino acid solution. After rinsing is completed a solution containing a mixture ratio of 17:2: 1 (DCM:methanol:DIEA) is added to each syringe to cap non reacted sites on the resin. This capping reaction is performed twice at 15minutes each. Once the second capping reaction is completed the resins are rinsed 5 times with DCM and dried on a vacuum manifold or within a vacuum desiccator for 30 minutes to 1 hour.
Synthesis of peptide intermediates (such as Ac-(AEEA)i4-OH or Ac-(AEEA)s-OH)
A number of exemplary protocols are described for the synthesis of peptide intermediates:
Protocol 1
Synthesis of peptide intermediates (such as Ac-(AEEA)i4-OH) can be performed at 0.1 mmol scale using the Liberty Blue HT24 synthesizer. The preloaded 2-cltrt resins from the method "Resin loading of a peptide to 2-Cltrt polystyrene resin" disclosed above are added onto the Liberty HT suspended in 10 ml of 1 : 1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. Synthesis may incorporate multiple replicates at 0.1 mmol scale for added material. The synthesis of, e.g., Ac- (AEEA)i4-OH or AC-(AEEA)8-OH may be as follows. The synthesis begins with deprotection of the N- terminal a Fmoc protecting group of the Fmoc-(AEEA)-OH using 4 mb of 20% piperidine in DMF heated by microwave for 3 min at 60°C with nitrogen dispensing every three seconds to mix. After draining, the resin is washed three times with 5 mb DMF at five seconds per wash. Then for the coupling reaction 2.5 ml of Fmoc-AEEA-OH 0.3 M amino acid solution in DMF (7.5 eq.), 1 mb 1 M DIC (10 eq.), and 0.5 mb 1 M Oxyma + 0.1 M diisopropylethylamine (DIEA) (5 eq.) is added to the reaction vessel (RV). The single or double coupling steps proceed with microwave heating for a 10 min cycle at 50°C with nitrogen dispensing every three seconds to mix during the coupling cycles. After the coupling time is completed the resins are drained and 20% piperidine in DMF is added to the reaction vessel (RV) to start deprotection (preferably at 60°C) in preparation for the next incoming amino acid. This cycle of Fmoc removal and coupling is repeated for every amino acid sequentially, ending with a final Fmoc deprotection cycle which preferably results in a free N-terminus for acetyl capping (no final deprotection of the amino acid sequence is performed for the Fmoc-(AEEA)i4-OH linker). After the instrument synthesis run is completed, the resins are transferred to a 24 ml fritted syringe using DMF. Roughly 10 ml of capping solution containing 6.25% acetic anhydride, 12.5% 2 M diisopropylethylamine (DIEA) in N-methylpyrrolidone (NMP), 81.25% DMF is added to each syringe. The capping reaction proceeds for 15 min at room temperature (RT). After 15 min the capping solution is evacuated from the syringes and the resins are rinsed once with DMF and the capping reaction is repeated for an additional 15 min. Once the second capping reaction is completed the resins are rinsed five times with 5 ml DMF and then five times with 5 ml DCM in preparation for cleavage from the resin.
The cleavage solution is prepared containing 92.5% trifluoroacetic acid (TFA), 3.75% water, and 3.75% triisopropyl silane (TIPS). 10 mL of cleavage solution is added to each syringe (for peptides made at 0. 1 mmol scale). The peptides are cleaved for at least 2 h at room temperature on a shaker. The cleaved peptides are then ejected from the syringe. We found it to be more efficient to combine all peptides in cleavage solution into one large 250 mL bottle, so that this can be evaporated under nitrogen at the same time. The pAEEA peptides will not crash out of solution using ether or hexane, so the cleavage solution is evaporated under nitrogen (air or rotovap can potentially be used as an alternative to nitrogen) in a fume hood. After evaporating, the 250 mL bottle usually contains a gel-like substance of crude peptide material. This is resuspended in enough 50/50 water/acetonitrile + 0.05% TFA so that each replicate made can be transferred back into a respective conical tube with about 10 mL of resuspension solution.
Purification of peptide intermediates (such as Ac-(AEEA) u-OH or Ac-(AEEA)g-OH)
After cleavage from the resin, each synthesis replicate is resuspended in 50/50 water/acetonitrile + 0.05% TFA, frozen, and lyophilized for 1-2 days. Following lyophilization, samples are dissolved in 50/50 water/acetonitrile + 0.05% TFA to about 50 mg/mL. The solution is vortexed and sonicated until fully dissolved (no visual presence of solid material). An aliquot is taken for crude QC (see section "UPLC-MS analytical QC method for peptide intermediates" described below). Purification is performed on a Waters Autopurification System with SQD2 mass detector. The samples are loaded onto the preparative HPLC column (Phenomenex Luna Cl 8(2) LC Column, 100 A pore size, 10 pm particle size) with an automatic injector through a 10 mL sample loop. Sample loading is done in 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Elution may be performed in a 36-min linear gradient (optionally, the duration is adaped to the sacle such that, e.g., for a 0.3 mmol scale a 42-min linear gradient is used). Column size, flow rate, and gradient may also be adapted to scale. For example, the following table provides exemplary purification gradients for the peptide intermediates Ac-(AEEA)i4-OH and Ac-(AEEA)8-OH.
A Waters SQD2 mass spectrometer is used to trigger fraction collection automatically based on the M+1H, M+2H, M+3H values for the target peptide. UV214 absorption is monitored but not used for fraction triggering. Aliquots of selected fractions are run on the QC method described in section "UPLC- MS analytical QC method for peptide intermediates". For some fractions, in process purity measurements may be omitted by visually monitoring the preparative HPLC MS data to estimate which fractions are high purity. Fractions with measured or estimated purity >90% are combined into tared polypropylene tubes and lyophilized. If the pure fraction volume exceeds the tube capacity, multiple polypropylene tubes are filled and lyophilized. Then each peptide is resuspended in 50/50 water/acetonitrile + 0.05% TFA and combined into one tared polypropylene tube and lyophilized for at least two days. An aliquot is taken from the combined pool and diluted in 50/50 water/acetonitrile + 0.05% TFA for QC.
Protocol 2
Synthesis of peptide intermediates (such as Ac-(AEEA)i4-OH) can be performed at 36.6 mmol scale using a semi-automated or automated synthesizer with no microwave assistance. The 2-cltrt resin is loaded with the first Fmoc-AEEA-OH of the synthesis using PyBOP and DIPEA for 3 h at room temperature. Synthesis may incorporate multiple replicates at 0.2 mmol scale for added material. The synthesis of the peptide intermediate may be as follows: deprotection of the a Fmoc protecting group of the Fmoc-(AEEA)-OH residue attached to the resin followed by the coupling of the next Fmoc-AEEA- OH until the desired sequence is reached. The deprotection of the a Fmoc protecting group is performed using 30% piperidine in DMF 2 times during 15 min. After draining the Fmoc deprotection solution, the resin is washed with DMF. Then the coupling with the next Fmoc-AEEA-OH is performed using PyBOP and DIPEA during 3 h at room temperature. After the coupling time is reached the resin is drained and 20% piperidine in DMF is added to the reaction vessel (RV) for the next coupling cycle. After the completion of all the coupling/deprotection cycles leading to the fully assembled sequence, the final Fmoc deprotection to have a free N-terminus for acetyl capping is performed. The free N-terminal amine is then acetylated at room temperature. After the capping, the solution is evacuated from the resin and rinsed with DMF. Syringes and the resins are rinsed with DMF. The cleavage solution to remove the peptide from the resin is prepared containing 25% HFIP in DCM during Ih at room temperature. The cleaved peptides are then ejected from the syringe, evaporated and injected for purification.
Purification of peptide intermediates Ac-(AEEA) u-OH
After cleavage from the resin, the crude peptide is solubilized and purified by RP-HPLC using an Agilent PLRP-S lOOA, 50 x 300 mm, 8 pm, 50 x 300mm system. Elution is performed with Eluent A: 1% AcOH in H2O and Eluent B: ACN with a linear 0-45% B in 140 min. This choice of eluents allowed to combine purification and salt exchange in the same process step. Fractions presenting a purity >90% are combined and lyophilized.
UPLC-MS analytical QC method for peptide intermediates (such as AC-(AEEA)I4-OH or Ac-(AEEA)g- OH)
Purity and identity of the peptide intermediates is determined by UPLC-UV-MS using a Waters H-Class UPLC with PDA UV Detector and QDa mass detector. The following table shows the UPLC parameters used for this analysis. Integration is performed automatically with Empower 3 software.
Analytical method 1
Passing criteria for the peptide intermediates is 90% UV214 purity and MW within 1 amu of the theoretical value.
Analytical method 2
Anti-PEG ELISA assay for polyclonal anti-PEG antibody binding This assay was used to determine the binding of anti-PEG polyclonal antibodies to different antigens (PEG, AEEA, pSAR).
Reagents:
• Pierce Neutravidin high binding plates, 8-well strips, 15508
• Assay Buffer: 1XDPBS, 2%BSA, 0.1% CHAPS
• Wash buffer: 1XDPBS, 0.1% CHAPS
• Rabbit polyclonal anti PEG, Life diagnostics #PEGPAB-01, 1 mg/mL
• Rabbit polyclonal anti PEG, cloud-clone corp #PAX163Ge01, 1 mg/mL
• Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP, Thermo A18817, 0.5 mg/mL
• "QUANTARED ENHANCED, Pierce 15159CHEMIFLUORESCENT HRP SUBSTRATE"
Procedure
Biotin-labeled antigens (biotin-PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped- pSAR, or biotin-NH2-pSAR) are synthesized and captured on Neutravidin-coated plates in wash buffer (IxPBS, 0.1% CHAPS) for 2 h at RT and under slow shaking. After washing with wash buffer (4x), the plates are incubated with different amounts of rabbit anti-PEG polyclonal sera (7 ng/ml, 3 ng/ml, 167 ng/ml, or 830 ng/ml) in assay buffer (IxPBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT and under slow shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody is added (diluted at 1:5000) and the plates are incubated for 1 h at RT and under slow shaking. After washing with wash buffer (5x), HRP substrate is added and the fluorescence signal was measured.
Detailed procedure
• Add 100 pL of 1 pM antigen to respective wells. Shake at 350 RPM for 2 hours.
• Wash 4 x 200 pL of wash buffer.
• Prepare respective antibody dilutions in assay buffer and add 100 pL to respective wells.
• Shake the plate at 350 RPM for 2 hours.
• Wash 5 x 200 pL of wash buffer. Wait for 1 min after adding wash buffer and then discard.
• Add 100 pL of 1 : 5000 dilution of Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (diluted in assay buffer) to respective well.
• Shake the plate at 350 RPM for 1 hour.
• Wash 5 x 200 pL of wash buffer. Wait for 1 min after adding wash buffer and then discard.
• Mix 50 parts QuantaRed Enhancer Solution with 50 parts QuantaRed Stable Peroxide and 1 part of the QuantaRedADHP Concentrate.
• Add 100 pl of QuantaRed Working Solution to each microplate well and incubate for 10 min at room temperature.
• Stop peroxidase activity by adding 10 pl of QuantaRed Stop Solution and shake plate for 30 seconds. • Measure relative fluorescence units (RFU) of each well. The excitation and emission maxima for QuantaRed Substrateare 570 nm and 585 nm, respectively.
Anti-PEG ELISA assay for monoclonal anti-PEG antibody binding
This assay was used to determine the binding of anti-PEG monoclonal antibodies to different antigens (PEG, AEEA).
Reagents:
• Pierce Neutravidin high binding plates, 8-well strips, 15508
• Assay Buffer: 1 XDPB S, 2%B SA, 0. 1 % CHAPS
• Wash buffer: 1XDPBS, 0.1% CHAPS
• Anti-PEG IgG (monoclonal) , Creative diagnostics CABT-L3140, 190ug/mL
• Anti-PEG IgM (monoclonal), Creative diagnostics CABT-L3141, 240 ug/mL
• Goat anti-Human IgG Fc Secondary Antibody, HRP, Thermo A18817, 0.5mg/mL
• Goat anti-Human IgM (Heavy chain) Secondary Antibody, HRP, Thermo A18835, 0.5mg/mL
• "QUANTARED ENHANCED, Pierce 15159CHEMIFLUORESCENT HRP SUBSTRATE"
Procedure
Biotin-labeled antigens (biotin-PEG36K, biotin-capped-AEEA14 or biotin-NH2-AEEA14) are synthesized and captured on Neutravidin-coated plates in wash buffer (IxPBS, 0.1% CHAPS) for 2 h at RT and under slow shaking. After washing with wash buffer (4x), the plates are incubated with different amounts of anti-PEG monoclonal IgG or IgM (25 ng/ml, 250 ng/ml, 500 ng/ml, or 1000 ng/ml) in assay buffer (IxPBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT and under slow shaking. After washing with wash buffer (5x), anti-human-HRP secondary antibody is added (diluted at 1:5000) and the plates are incubated for 1 h at RT and under slow shaking. After washing with wash buffer (5x), HRP substrate is added, and the fluorescence signal was measured.
Detailed procedure
• Add 100 pL of 1 pM antigen to respective wells. Shake at 350 RPM for 2 hours.
• Wash 4 x 200 pL of wash buffer.
• Prepare respective dilutions of anti-PEG monoclonal IgG and IgM in assay buffer and add 100 pL to respective wells.
• Shake the plate at 350 RPM for 2 hours.
• Wash 5 x 200 pL of wash buffer. Wait for 1 min after adding wash buffer and then discard.
• Add 100 pL of 1:5000 dilution of Goat anti-Human IgG Fc Secondary Antibody, HRP (diluted in assay buffer) or Goat anti-Human IgM (Heavy chain) Secondary Antibody, HRP (diluted in assay buffer) to respective well.
• Shake the plate at 350 RPM for 1 hour. • Wash 5 x 200 pL of wash buffer. Wait for 1 min after adding wash buffer and then discard.
• Mix 50 parts QuantaRed Enhancer Solution with 50 parts QuantaRed Stable Peroxide and 1 part of the QuantaRedADHP Concentrate.
• Add 100 pl of QuantaRed Working Solution to each microplate well and incubate for 10 min at room temperature.
• Stop peroxidase activity by adding 10 pl of QuantaRed Stop Solution and shake plate for 30 seconds.
• Measure relative fluorescence units (RFU) of each well. The excitation and emission maxima for QuantaRed Substrateare 570 nm and 585 nm, respectively.
Hydrophobicity Comparison
Compounds to be compared are dissolved in acetonitrile and water (ratio 2: 1) to a final concentration of 10 pM. To test their relative hydrophobicity, the samples are injected into a liquid chromatographymass spectrophotometer system (LC-MS) system consisting of a Thermo Scientific Vanquish LTQ XL linear ion trap. The analysis is performed using an electrospray ion source in positive ion mode. LC separation is carried out on a Waters Cortex T3 50 mm x 3 column with a gradient decreasing from 95% A to 100% B for 2 min at a flow rate of 0.7 mL/min. A similar retention time indicates that the compounds have similar hydrophobicity properties.
Plasma stability assay
Compounds are tested separately, and are added to 750 pL of human or mouse plasma at a final concentration of 10 pM. The final mixtures are vortexed briefly and incubated at 37°C. At various time intervals (0, 1, 3, 6, 24, 48 and 72 hours) 100 pL of acetonitrile containing 1% formic acid is added to the compound-plasma mixture in a microcentrifuge tube. The mixture is vortexed, centrifuged for 10 min at 2900 relative centrifugal force (RCF), and 100 pl of supernatant is transferred to an injection vial. The recovered supernatant is analyzed by using a LC-MS system consisting of a Shimadzu 8060 NX triple quadrupole mass spectrometer with an electrospray in positive ion mode. LC separation is carried out on a Waters Cortex T3 50 mm x 3 column with a gradient decreasing from 95% A to 100% B for 2 min at a flow rate of 0.8 mL/min. Buffer A is 0.1% formic acid in water and buffer B is 0.1% formic acid.
Particle size measurement
Size analysis is performed by dynamic light scattering using a DynaPro Plate Reader II (Wyatt, Dembach, Germany). LNP formulations are diluted to 0.005 mg/mL in IX PBS and 120 pL of diluted sample are measured in triplicate in a 96-well plate. From the measurements, size (Zaverage), and polydispersity indices (PDI) are calculated from the cumulant analysis using Dynamics 7.8.1.3 software. Measurement of Zeta potential (electrophoretic mobility)
Zeta potential of particles is determined by photon correlation spectroscopy using particles sizer (zeta potential/particle sizer, NicompTM 380 ZLS, Santa Barbara, CA, USA) with an E-field strength of 4 V/cm and an electrode spacing of 0.4 cm in a plastic cuvette. LNP formulations are diluted 1:26 in 0.1XPBS for zeta potential measurement. The electrostatic mobility is converted to the zeta potential using the Helmholtz-Smoluchowski equation. All measurements are carried out at a temperature of 23°C. mRNA accessibility method
RNA accessibility and total RNA concentration in the formulations is measured by a modified Quant- iT RiboGreen RNA assay (Invitrogen, Carlsbad, CA). LNP samples are diluted in lx TE buffer pH 7.4 to a mRNA concentration between 2 and 5 ng/pL. Accessible mRNA is measured by diluting the sample in lx TE, and the total RNA amount is quantified by diluting the sample in 2% Triton X-100 (VWR International GmbH, Darmstadt, Germany). RiboGreen reagent is added to each sample, and the fluorescent signal is quantified in an Infinite F200PRO microplate reader (Tecan, Mannedorf, Switzerland). For the determination of total RNA concentration, a standard curve of RiboGreen fluorescence versus mRNA is used between 0 - 2.5 pg/mL mRNA in 1% Triton X-100 (pH 7.4).
Agarose gel electrophoresis
Agarose gel electrophoresis is performed to evaluate free RNA. The gel is poured by using 1 g agarose dissolved in 100 mb of lx TAE Buffer pH 7.4 (Tris-acetate-EDTA) (Rotiphorese® 50X TAE, Carl Roth, Karlsruhe, Germany), 1 mb of 5% Sodium hypochlorite, and 10 pL of GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). The gel is allowed to set for at least 25 min at room temperature. The gel is then placed in a gel electrophoresis tank and lx TAE running buffer (pH 7.4) is used. Before loading, the samples are incubated at 40°C with or without 2% of Triton X-100, for total and free RNA, respectively. The gel is run at 80 V for 40 min. Gel images are taken on a Chemidoc XRS imaging system (Bio-Rad, Berkeley, CA, USA).
Hemolysis
A hemolysis assay is performed to study the hemolytic properties of the formulations. For this goal human blood is diluted in I xPBS to a 20% vol/vol blood solution. In a 96-round bottom plate, 100 pL LNPs diluted at an equivalent concentration of 0.05 mg mRNA/mL are added to 100 pL blood solution in PBS and incubated at 37°C for 1 h (triplicates). After incubation, the plate is centrifuged at 23°C at 500 x g for 5 min. Then, the supernatant is transferred into a clear 96-well plate and UV absorption is read at 540 nm using Tecan Pro200 Plate Reader. Positive and negative controls are carried out with 0.2% Triton-X and buffer alone, respectively. Complement activation
In vitro SC5b-9 levels are determined by Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). Briefly, samples as well as positive (Cremophor El, Merck, Darmstadt, Germany; Cobra venom factor; Quidel Co., SanDiego, CA, USA) and negative (lx DPBS and Intralipid) controls are incubated with Normal Human Serum Complement (Innovative Research, Michigan, USA) at the ratio of 20:80 (specimen : serum) for 1 h at 37 °C. LNP formulations are incubated with human serum at a final mRNA concentration of 0.01 mg/ml, the theoretical concentration (corresponding to a dose of 0.49 mg/kg). SC5b-9 EIA kit (Quidel, San Diego, USA) is used.
Cells and cell lines in Potency Assay
HEK 293T-17 cells are seeded into 12-well plates (4xl05 cells/well in DMEM (#31966-021/ 2293715 Gibco) + 10% FCS (S 0615/BS. 1640839, Sigma)) 6 h prior to transfection. Formulations are diluted in Opti-MEM (#51985-026, Gibco) and 0.3 pg/well are used to transfect the cells in triplicates. Afterwards, cells are centrifuged at 300xg for 4 min. As a transfection and/or staining control, BM LNPs containing PEG lipid and RNA coding for eGFP (LNP-315-dO45, 29th APR 2021, BNT Delivery Technologies GmbH, Halle) or the S protein of SARS-CoV-2 (RBP020.LNP, COVVAC/270320, Polymun, Klosterneuburg) are used. Cells are incubated at 37°C/5% CO2 for 18 h. For FACS analysis, cells are detached using PBS ((#2339094, Gibco), 4°C) and transferred into a 96-well plate. Cells are stained for viability with a 1:500 dilution of the fixable viability dye eFluor 450 (#65-0863-14, eBioscience), followed by fixation (#42081, Biolegend) and permeabilization (lx dilution, #00-8333-56, eBioscience). For the quantification of SI protein expression, cells are stained with a 1:2000 dilution of the primary antibody (#40150-R007, Sino Biological) followed by secondary antibody staining (1: 1000, #P -277 IMP, ThermoFischer). Cells are resuspended in FACS buffer (PBS, EDTA, BSA) for acquisition. To that end, cell viability, transfection frequencies and MFI of the over-all and the Sl- poitive population are determined and compared.
LNP preparation
LNPs are prepared by mixing an aqueous phase (0.15 mg/mL mRNA diluted in 0.1 M citrate buffer, pH 4.0) and an organic phase (a lipid mixture of cationic lipid:DSPC:cholesterol:stealth component dissolved in ethanol at a molar fraction of 47.5:42.5 - X: 10:X, respectively, with N/P=6 and a total concentration of 17.05 mM) at a 3: 1 volume ratio and 12 mL/min, using a microfluidic instrument (NanoAssemblr® Benchtop, Precision NanoSystems, Vancouver, Canada). The mixture is dialyzed against lx DPBS (GIBCO, pH 7.4) for 3 h in a Slide-A-Lyser 10K MWCO dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA.) In LNP experiments, residual ethanol is regularly controlled by osmolality measurements. The physicochemical characterization (size, polydispersity, zeta potential, RNA accessibility and total RNA concentration) is performed on the day of preparation. After complete characterization, formulations are stored at 4°C for not more than 1 day. Lipid nanoparticles are diluted in PBS to the desired RNA concentration prior to in vitro testing.
Example 1 - Preparation of Ac-(AEEA)i4-DSPE
Step A: Synthesis of Ac-(AEEA)i4-OH linker
Synthesis of the Ac-(AEEA)i4-OH linker was performed at 0.1 mmol scale using the Liberty Blue HT24 synthesizer. The preloaded 2-cltrt resins from the method "Resin loading of a peptide to 2-Cltrt polystyrene resin" disclosed above were added onto the Liberty HT suspended in 10 ml of 1: 1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. Synthesis incorporated multiple replicates at 0.1 mmol scale for added material. The synthesis methods began with deprotection of the N-terminal a Fmoc protecting group of Fmoc-(AEEA)i4-OH using 4 mL of 20% piperidine in DMF heated by microwave for 3 min at 60°C with nitrogen dispensing every three seconds to mix. After draining, the resin was washed three times with 5 mL DMF at five seconds per wash. Then for the coupling reaction 2.5 ml of Fmoc-AEEA-OH 0.3 M amino acid solution in DMF (7.5 eq.), 1 mL 1 M DIC (10 eq.), and 0.5 mL 1 M Oxyma + 0.1 M diisopropylethylamine (DIEA) (5 eq.) was added to the reaction vessel (RV). The single or double coupling steps proceeded with microwave heating for a 10 min cycle at 50°C with nitrogen dispensing every three seconds to mix during the coupling cycles. After the coupling time completed the resins were drained and 20% piperidine in DMF was added to the reaction vessel (RV) to start deprotection (preferably at 60°C) in preparation for the next incoming amino acid. This cycle of Fmoc removal and coupling was repeated for every amino acid sequentially, ending with a final Fmoc deprotection cycle (which preferably results in a free N-terminus for acetyl capping; no final deprotection of the amino acid sequence was performed for the Fmoc-(AEEA)i4-OH linker). After the instrument synthesis run was completed, the resins were transferred to a 24 ml fritted syringe using DMF. Roughly 10 ml of capping solution containing 6.25% acetic anhydride, 12.5% 2 M diisopropylethylamine (DIEA) in N-methylpyrrolidone (NMP), 81.25% DMF was added to each syringe. The capping reaction proceeded for 15 min at room temperature (RT). After 15 min the capping solution was evacuated from the syringes and the resins were rinsed once with DMF and the capping reaction was repeated for an additional 15 min. Once the second capping reaction had completed the resins were rinsed five times with 5 ml DMF and then five times with 5 ml DCM in preparation for cleavage from the resin. The cleavage solution was prepared containing 92.5% trifluoroacetic acid (TFA), 3.75% water, and 3.75% triisopropyl silane (TIPS). 10 mL of cleavage solution was added to each syringe (for peptides made at 0.1 mmol scale). The peptides were cleaved for at least 2 h at room temperature on a shaker. The cleaved peptides were then ejected from the syringe. We found it to be more efficient to combine all peptides in cleavage solution into one large 250 mL bottle, so that this can be evaporated under nitrogen at the same time. The pAEEA peptides will not crash out of solution using ether or hexane, so the cleavage solution was evaporated under nitrogen (air or rotovap can potentially be used as an alternative to nitrogen) in a fume hood. After evaporating, the 250 mL bottle contained a gel-like substance of crude peptide material. This was resuspended in enough 50/50 water/acetonitrile + 0.05% TFA so that each replicate made could be transferred back into a respective conical tube with about lOmL of resuspension solution.
After cleavage, each synthesis replicate was resuspended in 50/50 water/acetonitrile + 0.05% TFA, frozen, and lyophilized for 1-2 days. Following lyophilization, samples were dissolved in 50/50 water/acetonitrile + 0.05% TFA to about 50 mg/mL. The solution was vortexed and sonicated until fully dissolved (no visual presence of solid material). An aliquot was taken for crude QC (see section "UPLC- MS analytical QC method for peptide intermediates", above). Purification was performed on a Waters Autopurification System with SQD2 mass detector. The samples were loaded onto the preparative HPLC column (Phenomenex Luna Cl 8(2) LC Column, 100 A pore size, 10 pm particle size) with an automatic injector through a 10 mL sample loop. Sample loading was done in 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Elution was performed in a 36- minute linear gradient. Column size and flow rate corresponded to scale (see the following table). A Waters SQD2 mass spectrometer was used to trigger fraction collection automatically based on the M+1H, M+2H, M+3H values for the target peptide. UV214 absorption was monitored but not used for fraction triggering.
Aliquots of selected fractions were run on the QC method (as described in the section "UPLC-MS analytical QC method for peptide intermediates", above). For some fractions, s-process purity measurements were omitted by visually monitoring the preparative HPLC MS data to estimate which fractions were high purity. Fractions with measured or estimated purity >90% were combined into tared polypropylene tubes and lyophilized. If the pure fraction volume exceeded the tube capacity, multiple polypropylene tubes were fdled and lyophilized. Then each peptide was resuspended in 50/50 water/acetonitrile + 0.05% TFA and combined into one tared polypropylene tube and lyophilized for at least two days. An aliquot was taken from the combined pool and diluted in 50/50 water/acetonitrile + 0.05% TFA for QC.
Step B: Con jugation
Ac-(AEEA)i4-OH (AEEA = (2-(2-(2-aminoethoxy)ethoxy)acetic acid); Ac = acetyl) was dissolved in chloroform at a concentration of ~30 mg/mL. To this solution, 5 molar equivalents each of (7- azabenzotriazol-l-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyAOP) and l-hydroxy-7- azabenzotriazole (HO At) were added followed by 1.1 equivalents (or 1 equivalent) of DSPE, and then 5 equivalents of DIEA. The solution was stirred at 50°C in a closed flask for 18-24 hours until UPLC- MS reaction monitoring suggested all Ac-(AEEA)i4-OH was consumed. Upon complete conversion, solvent was removed in vacuo and product was redissolved in methanol for salt exchange and purification by LC-MS using the method "Salt exchange and purification of amphiphilic compounds" or "Alternative purification of amphiphilic compounds" described above. Pure product was lyophilized to give a dry white sticky powder. The UPLC-UV214 purity of the sample was 96% and the MW determined by MS (electrospray) was 2822.55.
Example 2 - Preparation of H-(AEEA)i4-DSPE
To a stirring solution of H-(AEEA)i4-OH in MeCN (~20 mg/mL) was added 2 molar equivalents of BOC2O and 5 equivalents of DIEA. The reaction was stirred at 50°C for 4h or until complete conversion was observed by UPLC-MS. Product was crashed in cold (-20°C) diethyl ether, centrifuged, and decanted to give the final product as a pellet. Product was continued to the next step without further purification. The MW as determined by MS (electrospray) was 2150.91. Boc-(AEEA)i4-OH was dissolved in chloroform at a concentration of ~30 mg/mL. To this solution, 5 molar equivalents each of PyAOP and HO At were added followed by 1. 1 equivalents of DSPE, and then 5 equivalents of DIEA. The solution was stirred at 50°C in a closed flask for 18-24 hours until UPLC- MS reaction monitoring suggested all Ac-(AEEA)i4-0H was consumed. Upon complete conversion, solvent was removed in vacuo and redissolved in MeOH to a concentration of ~30 mg/mL. To this solution was added 10 molar equivalents of HC1 (4 M in dioxane) to remove the Boc protecting group. After 1 hour the deprotection was complete. Reaction solution may be salt exchanged and purified using the method "Salt exchange and purification of amphiphilic compounds" described above. Pure product may be lyophilized to give a dry white sticky powder. The MW as determined by MS (electrospray) was 2780.46.
Example 3 - Preparation of Ac-(AEEA)i4-a-tocoDherol
Ac-(AEEA)i4-OH was dissolved in chloroform to a concentration of ~12 mg/mL. To this solution was added 1.3 equivalents of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), then 0.5 equivalents of 4-dimethylaminopyridine (DMAP), then 2.2 equivalents (or 1.2 equivalents) of a-tocopherol. Reaction vial was wrapped in tin foil to minimize light exposure and allowed to stir at room temperature for 4 hours or until reaction was judged complete by UPLC-MS. Upon completion, volatiles were evaporated in vacuo and product was redissolved in MeOH for salt exchange and purification. Salt exchange and purification were performed simultaneously using the method "Salt exchange and purification of amphiphilic compounds" described above with elution on a linear gradient of 52 - 67%B. The MW as determined by MS (electrospray) was 2504.84.
In an alternative reaction, Ac-(AEEA)i4-OH was dissolved in chloroform to a concentration of ~12 mg/mL. To this solution was added 2 equivalents of EDC, then 1 equivalent of DMAP, then 2.2 equivalents (or 1.2 equivalents) of a-tocopherol. Reaction vial was wrapped in tin foil to minimize light exposure and allowed to stir at room temperature for 4 hours or until reaction was judged complete by UPLC-MS. Upon completion, volatiles were evaporated in vacuo and product was redissolved in MeOH for salt exchange and purification as described in "Salt exchange and purification of amphiphilic compounds" or "Alternative purification of amphiphilic compounds". In a further alternative reaction, Ac-(AEEA)i4-0H was dissolved in dichloromethane to a concentration of 275 mg/mL in presence of DIC and DMAP. Then a-tocopherol was added to perform the conjugation and the mixture was stirred overnight (approx. 16 h) at room temperature protected from light. Upon completion, the solvent was removed by evaporation and the resulting conjugated crude was resuspended before purification. After the re-suspension, the conjugated product is purified by RP- HPLC using an Agilent PLRP-S lOOA, 50 x 300mm, 8 pm, 50 x 300 mm system. Elution was performed with Eluent A: 1% AcOH in H2O and Eluent B: ACN with a linear 30-90% B in 120 min. This choice of eluents allowed to combine purification and salt exchange in the same process step. Fractions presenting a purity >95% were combined and lyophilized.
Purity and identity were determined using the following method:
Example 4 - Preparation of H-(AEEA)i4-a-tocopherol
Boc-(AEEA)i4-OH was prepared as described in Example 2, Step A.
Step B: Conjugation of a-tocopherol to Boc-(AEEA) u-OH and Boc deprotection a-tocopherol is conjugated to HO-(NMe-AEEA)i4-Boc (or HO-(AEEA)i4-Boc) using the method described in Example 3. Boc deprotection is carried out according to the procedure described in Example 2, Step B. Purification and salt exchange is done using the using the "Salt exchange and purification of amphiphilic compounds" described above.
Example 5 - Preparation of (NMeAEEA) building block
An amount of 10 g (0.026M) Fmoc-AEEA-OH monomer was weighed into a 50 ml conical tube and dissolved with 10 ml 1: 1 chloroform :TFA. Once all material had been dissolved a small sample was taken for QC analysis by UPLC. Roughly 1.5 equivalents of formaldehyde was added to this mixture and another small sample was taken for QC analysis by UPLC to assess the intermediate formation of an Imine group on the nitrogen atom of the Fmoc-AEEA-OH after reaction with formaldehyde. After addition of formaldehyde 6 ml of triethylsilane(TES) or roughly 1.46 equivalents was added to reduce the Imine to yield the desired Fmoc-NMeAEEA-OH monomer (NMeAEEA = (2-(2-(2- methylaminoethoxy)ethoxy)acetic acid)). After addition of the TES several QC samples of the reaction mixture were run by UPLC where it was observed that successful reduction had occurred. The mixture was then diluted with 1 : 1 acetonitrile: water to a rough concentration of 250 mg/ml and stored at -80°C until purification was performed.
The crude sample was dissolved in a mixture of 1 : 1 : 1 : 1 TFA:chloroform:acetonitrile:water to about 250 mg/mL. Material was sonicated and vortexed until fully dissolved (no visual presence of solid material). The sample was loaded onto the preparative HPLC column (Phenomenex Luna Cl 8(2) LC Column, 100 A pore size, 10 pm particle size, 50 mm x 250 mm) with an automatic injector through a 10 mb sample loop. Sample loading was done in 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Peptide elution was performed in a 85-min linear gradient of 45 - 55%B. The flow rate was 30 mL/min. The SQD2 mass spectrometer was used to trigger fraction collection automatically based on the M+1H and M+1H+22 (sodium adduct) values for the target compound. UV214 absorption was monitored but not used for fraction triggering. Aliquots of selected fractions were injected on the UPLC-MS using the method described above for "UPLC-MS". For some fractions, in- process QC was omitted by visually monitoring the preparative HPLC UV214 absorption and MS to estimate which fractions were high purity. Fractions with measured or estimated purity >95% were combined into tared polypropylene tubes and dried down in the rotary drier. If the pure fraction volume exceeded the tube capacity, multiple polypropylene tubes were filled and dried. Then, each tube was resuspended in 50/50 water/acetonitrile + 0.05% TFA and combined into one tared polypropylene tube and lyophilized to dryness. About 1 mg of NMeAEEA was dissolved to 1 mg/mL in 50/50 water/acetonitrile + 0.05% TFA and injected on the UPLC/MS. The MW of the peptide as determined by MS was 399. 13 m/z, consistent with the theoretical MW of 399.17 m/z.
Example 6 - Synthesis of Ac-(NMeAEEA)i4-a-tocopherol
Step A: Synthesis of (NMeAEEA) linkers
Resin loading of (NMeAEEA) building block was performed using the method "Resin loading of_a peptide to 2-Cltrt polystyrene resin" described above. Synthesis of the Fmoc-(NMeAEEA)i4-OH, Ac- (NMeAEEA)i4-OH, and Ac-(NMeAEEA)8-OH linkers was performed at O. lmmol scale using the Liberty Blue HT24 synthesizer. The preloaded 2-cltrt resins from resin loading method 3 were added onto the Liberty HT suspended in lOmls of 1: 1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. Synthesis incorporated multiple replicates at O.lmmol scale for added material. The synthesis methods began with deprotection of the N-terminal a Fmoc protecting group using 4 mL of 20% piperidine in DMF heated by microwave for 3minutes at 60°C with nitrogen dispensing every three seconds to mix. After draining, the resin was washed three times with 5 mL DMF at five seconds per wash. Then for the coupling reaction 2.5ml of Fmoc-NMeAEEA-OH 0.3M amino acid solution in DMF (7.5eq), 1 mL IM DIC (lOeq), and 0.5 mL IM Oxyma + 0.1 M diisopropylethylamine (DIEA) (5eq) was added to the reaction vessel (RV). The double coupling steps proceeded with microwave heating for two 20 min cycles at 50°C with nitrogen dispensing every three seconds to mix during the coupling cycles. After the coupling time completed the resins were drained and 20% piperidine in DMF was added to the RV to start deprotection for the next incoming amino acid. This cycle of Fmoc removal and coupling was repeated for every amino acid sequentially. No final deprotection of the amino acid sequence was performed for the Fmoc-(NMeAEEA)14-OH linker, whereas a final deprotection of the amino acid sequence was performed for the H(NMeAEEA)14-OH linker. After the instrument synthesis run was completed for each compound needing N-terminal acetylation the resins were transferred to a 24 ml fritted syringe using DMF. Roughly 10 ml of capping solution containing 6.25% acetic anhydride, 12.5% 2M diisopropylethylamine (DIEA) in N- methylpyrrolidone (NMP), 81.25% DMF was added to each syringe. The capping reaction proceeded for 15 min at room temperature (RT). After 15 min the capping solution was evacuated from the syringes and the resins were rinsed once with DMF and the capping reaction was repeated for an additional 15 min. Once the second capping reaction had completed the resins were rinsed five times with 5 ml DMF and then five times with 5ml DCM in preparation for cleavage from the resin.
Step C: Conjugation of Ac-(NMe-AEEA)i4-OH to a-tocopherol a-tocopherol is conjugated to Ac-(NMe-AEEA)i4-OH using the method described in Example 3 above.
Example 7 - Preparation of H-(NMeAEEA)i4-q-tocopherol a-tocopherol is conjugated to Boc-(NMeAEEA)i4-OH using the method described in Example 3 above. Boc deprotection is done using the method described in Example 2, Step B. Salt exchange and purification were done using the method "Salt exchange and purification of amphiphilic compounds" described above. Conjugation of DMG to Ac-(AEEA)i4-OH is performed according to the protocol described in Example 3. Upon completion, solvents are removed in vacuo and product is redissolved in MeOH for salt exchange and purification using the method "Salt exchange and purification of amphiphilic compounds" described above. Alternatively, the product is purified using the methods described above under the heading "Alternative purification of amphiphilic compounds" or "Further alternative purification of amphiphilic compounds" (e.g. by redissolving the product in chloroform and purifying the redissolved product by a size exclusion method).
Example 9 - Preparation of Ac-(NMeAEEA)i4-DSPE
DSPE is conjugated to Ac-(NMeAEEA)i4-OH using the method described in Example 1. Salt exchange and purification are done using the method "Salt exchange and purification of amphiphilic compounds" described above.
Example 10 - Preparation of further amphiphilic OEG-conjugated compounds
The following amphiphilic OEG-conjugated compounds are synthesized using the methods indicated in the following table:
Ceramide: N-palmitoyl -ceramide
For example, Ac-(AEEA)8-a-tocopherol was prepared according to the following scheme: In particular, Ac-(AEEA)8-0H was dissolved in chloroform to a concentration of ~12 mg/mL. To this solution was added 2 equivalents of EDC, then 1 equivalent of DMAP, then 2.2 equivalents (or 1.2 equivalents) of a-tocopherol. Reaction vial was wrapped in tin foil to minimize light exposure and allowed to stir at room temperature for 4 hours or until reaction was judged complete by UPLC-MS. Upon completion, volatiles were evaporated in vacuo and product was redissolved in MeOH. Purification was carried out as described in "Salt exchange and purification of amphiphilic compounds" or "Alternative purification of amphiphilic compounds".
AC-(AEEA)8-DMG was prepared according to the following scheme:
Conjugation of DMG to Ac-(AEEA)8-OH was performed according to the synthesis of Ac-(AEEA)8-a- tocopherol without wrapping reaction vial in aluminum foil (not light sensitive). Purification was carried out as described in "Salt exchange and purification of amphiphilic compounds" or "Alternative purification of amphiphilic compounds".
Ac-(AEEA)i4-DMA was prepared according to the following scheme:
DMA
Ac-(AEEA)i4-OH was dissolved in chloroform at a concentration of ~30 mg/mL. To this solution, 5 molar equivalents each of PyAOP and HOAt were added followed by 1.2 equivalents of DMA, and then 5 equivalents of DIEA. Reaction mixture was stirred at 50°C in a closed flask for 18-24 hours until UPLC-MS reaction monitoring suggests all Ac-(AEEA)i4-OH was consumed. Upon completion, purification was carried out as described in "Salt exchange and purification of amphiphilic compounds" or "Alternative purification of amphiphilic compounds".
Further amphiphilic OEG-conjugated compounds (such as those, wherein n is 10) can be synthesized by using the methods described above.
Example 11 - Preparation of the control lipid DSPE-PEGZK-cvcloALFA-NHz
Step A: Synthesis ofMPA-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Ars.-Lys)-Arg-Leu-Thr-Glu-NH2
Synthesis of the Lactam ALFA peptide sequence was performed at 0.1 mmol scale using the Liberty
Blue HT24 synthesizer. Sieber Amide resins were loaded onto the Liberty HT suspended in 10 ml of 1: 1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The synthesis methods began with deprotection of the N-terminal a Fmoc protecting group using 4 mL of 20% piperidine in DMF heated by microwave for 3 min at 60°C with nitrogen dispensing every three seconds to mix. After draining, the resin was washed three times with 5mL DMF at five seconds per wash. Then for the coupling reaction 2.5 ml of the next 0.3 M amino acid solution (7.5 eq.), 1 mL 1 M DIC (10 eq.), and 0.5 mL IM Oxyma + 0.1 M diisopropylethylamine (DIEA) (5 eq.) was added to the reaction vessel (RV). The double coupling steps proceeded with microwave heating for an initial 6 min cycle at 50°C and an additional 10 min cycle at 50°C with nitrogen dispensing every three seconds to mix during both coupling cycles. After the second coupling time completed the resins were drained and 20% piperidine in DMF was added to the RV to start deprotection for the next incoming amino acid. This cycle of Fmoc removal and coupling was repeated for every amino acid sequentially. The Liberty Blue external position #6 was used for addition of Fmoc-Glu(O-2-PhiPr)-OH #7 was used for addition of the Fmoc-Lys(Mmt)-OH amino acid into the peptide sequence at the corresponding cyclization points for Lactam formation. For peptides containing N-terminal Mpa(Trt)-OH no final deprotection of the peptides was needed due to no presence of Fmoc protection of the Mpa(Trt)-OH amino acid, peptide resins were washed four times with 4 mL DMF followed by transfer from the RV back to the starting HT position and the next peptide in the queue began synthesis. After the instrument synthesis run was completed for each peptide, resins were transferred to a 24 ml fritted syringe using DCM and rinsed 3 times with DCM to remove excess DMF.
After washing the resins were treated 6 times with 5 ml of 2% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS) in DCM for roughly 5 min each reaction. The flow through solution from each reaction which contained the semi-protected peptides was collected in a separate 50 ml conical tube for each peptide. The semi -protected peptide solutions were then evaporated under a gentle nitrogen (N2) blanket until the total volume remaining in each tube was less than 5 ml. Once the volume in each conical tube was less than 5 ml the peptides were re-suspended in 20 ml of 1: 1 acetonitrile :t-butanol (AcN:tBu-OH) and sonicated until the solutions were homogenous. The peptide tubes were then frozen at -80°C and placed on a lyophilizer for drying.
Once the semi-protected peptides were dry roughly 5 eq. relative to the 0.1 mmol scale of synthesis of PyAOP and Ho At was weighed out and dissolved in 4 ml of DMF. Once the reagents were completely dissolved this solution was added to each of the peptide tubes. After the semi-protected peptide material was dissolved roughly 10 eq. relative to the 0.1 mmol scale of synthesis of DIEA was added to each solution and a slight color change where the solution turned a yellow color was observed. The solutions were then left to react for 2-3 hrs at RT. After 2-3 hrs of reaction time at RT the protected peptides were precipitated by adding roughly 45 ml of chilled HPLC water to each 50 ml conical tube and the tubes were then centrifuged at 1000-1200 rpm for roughly 5 min. This was repeated 2 times to remove any excess DMF and after the second precipitation the peptide pellets were resuspended in 1 : 1 (AcN:tBu-OH) frozen at -80°C and lyophilized overnight.
After the peptides completed drying on lyo roughly 10 ml of cleavage cocktail containing 92.5% TFA, 2.5% water (H2O), 2.5% TIS, and 2.5% thioanisole was added to each 50 ml conical tube and left to shake at RT for 2-3 hours. The 10 mb of cleavage cocktail was split into two 50 mb conical tubes at 5 ml each. Roughly 45 ml cold (-20°C) 1 : 1 hexane :diethylether was added to each tube. Then the mixture was centrifuged at 3500 rpm for 10 min. After decanting the ether, another 40 mb cold 1: 1 hexane: diethylether was added to further wash the peptide pellets. The container was agitated to break up the peptide pellets and then centrifuged again at 3500 rpm for 10 min. After decanting the ether a second time the tubes we placed on their side within a fume hood and allowed to dry. The peptide pellets were then reconstituted in roughly 10 ml of 1: 1 acetonitrile/water (AcbkFFO), frozen at -80°C and lyophilized. After the peptides completed drying small sample of the dried crude peptide powder was transferred to a 1.5 mb Eppendorf tube, and then dissolved with enough 3: 1 dimethyl sulfoxide/water (DMSOFEO) in order to make a approximate 2 mg/ml peptide sample concentration. Then 50 pL of the peptide sample was transferred to a plastic vial and loaded onto a Waters Acuity UPLC-MS for crude analysis. Once confirmation of the molecular weight and column retention time was acquired the crude peptide was submitted for purification in preparation for conjugation to DSPE-PEG2K-Malimide.
The crude peptide was subjected UPLC-MS as described above. About 1 mg of the UPLC-purified peptide was dissolved to 1 mg/mL in 50/50 water/acetonitrile + 0.05% TFA and injected on the UPLC/MS for purity and MW. The MW of the peptide as determined by MS (electrospray) was 1728.2, consistent with the theoretical MW of 1728.0
Step B : Conjugation of MPA-Ser-Arg-Leu-Glu-cyclofGlu-Glu-Leu-Arg-Lysj-Arg-Leu-Thr-Glu-NH? to DSPE-PEG2K
Once purification of the cyclic ALFA peptide was completed the final weight of the purified material was used to calculate the millimolar amount of cyclic ALFA to be used in conjugation to the DSPE- PEG2K-Mal compound. Once the amount of cyclic ALFA peptide was assessed 1.2 equivalents of DSPE-PEG2K-Mal relative to the amount of cyclic ALFA was weighed into a 15 ml conical tube and dissolved with DMSO to a rough concentration of 4-5 mg/ml. A small sample of the dissolved DSPE- PEG2K-Mal was taken for QC analysis by UPLC to assess initial retention time. Once the DSPE- PEG2K-Mal material was dissolved this solution was then used to dissolve the cyclic ALFA peptide which resulted in a rough concentration of 5-8 mg/ml of the cyclic ALFA peptide in this mixture. A small sample was taken of this mixture for QC analysis by UPLC to assess retention time of both components in the mixture . Once all material had been fully dissolved adjustment of the reaction mixture pH was performed using a solution of Trizma Base at 2.5 mg/ml concentration prepared in water which was added to the reaction mixture resulting in a dilution of the reaction mixture total volume by roughly 30-40%. A small sample of the reaction mixture was taken for QC analysis at time intervals of 15 and 30 min to assess the reaction progress. Once it was verified that all cyclic ALFA starting material was removed from the reaction the mixture was submitted for purification and isolation of the conjugate material.
The title compound (control lipid DSPE-PEG2K-cycloALFA-NH2) was loaded onto the preparative HPLC column (Waters XSelect Peptide CSH Cl 8 OBD Prep Column, 130 A, 5 pm, 19 mm X 100 mm) with an automatic injector through a 10 mb sample loop. Sample loading was done in 85% mobile phase A (0.05% TFA in water), 15% mobile phase B (0.05% TFA in acetonitrile). Peptide elution was performed in a 19-minute linear gradient of 75 - 95%B. The flow rate was 15 mL/min. The SQD2 mass spectrometer was used to trigger fraction collection automatically. For the first purification run, the triggering masses were selected by using the highest intensity MS peaks seen in the crude reaction UPLC-MS trace. After one round of purification, the masses were adjusted to the highest intensity masses that spanned the collection range of the conjugate on the preparative system. UV214 absorption was monitored but not used for fraction triggering. Fractions with measured or estimated purity >90% were combined into tared polypropylene tubes and lyophilized.
For salt exchange and purification, the lyophilized title compound was subjected to the method "Salt exchange and purification of amphiphilic compounds" as described above.
The purity and identity of the purified and lyophilized title compound were measured on a Waters H- Class UPLC/MS system using a linear gradient from 10 to 80% B in 8 minutes with flow rate of 0.5 mL/min, where mobile phase A was 0.100% TFA in water and mobile phase B was 0.085% TFA in acetonitrile. The column (ACQUITY UPLC Protein BEH C4 Column, 300 A, 1.7 pm, 2.1 mm X 100 mm) was heated to 60 °C. The UPLC-UV214 purity of the sample was 98% and the MW determined by MS (electrospray) was 4399.2. Due to the polydisperse nature of PEG2K, the MW is based on the most abundant of many ions.
The title compound (DSPE-PEG2K-cycloALFA-NH2; also referred to herein as DSPE-PEG2K-succ- MPA-cycloALFA-NH2) has the following formula: Example 12 - Preparation of the control lipid PEGZK-DSPE-Pro-cycloALFA-NHz
The titled compound was synthesized substantially according to the procedure described in Example 11.
Step C: Resin loading of Fmoc-AEEA-OH to 2-Cltrt polystyrene resin
The resin loading was done using the method "Resin loading of a peptide to 2-Cltrt polystyrene resin" described above and Fmoc-AEEA-OH.
Step D: Synthesis of the Ac-(AEEA)s-OH linker
Synthesis of the Ac-(AEEA)X-OH linker was according to Example 1, Step A.
The title compound (PEG2K-DSPE-Pro-cycloALFA-NH2; also referred to herein as DSPE-PEG2K- succ-MPA-Pro-cycloALFA-NEE) has the following formula:
Example 13 - Preparation of the conjugate H2N-(rvALFA-Pro-MPA)-succ-(AEEA)i4-DSPE
H-(AEEA) 14-OH is dissolved in DMF at a concentration of ~45 mg/mL. To this solution is added 1.3 eq. of 3-maleimidopropionic acid N-hydroxy succinimide ester and 4 equivalents of DIEA. Solution is stirred at room temperature for 2-4 hours until reaction is complete by UPLC-MS monitoring. Upon completion, reaction solution is precipitated in diethyl ether in a conical tube, centrifuged, and the supernatant is decanted to give the product as a viscous pellet. The product is then brought to the next step without further purification. The MW as determined by MS (electrospray) was 2201.73.
Conjugation of DSPE to maleimide-(AEEA)i4-OH was carried out using the procedure described in Example 1. Volatiles were removed in vacuo and product was used for conjugation to ALFA without purification. A yield was not obtained for this step. The MW as determined by MS (electrospray) was 2930.38.
Maleimide-(AEEA)i4-DSPE was dissolved in DMSO at ~5 mg/mL and stirred in a round bottom flask at room temperature. To this solution was added MPA-Pro-ALFA (e.g., in an amount of 1.1 molar equivalents). A solution of Trizma base in water was prepared until pH was approximately 8.5 (~2.5 mg/mL). This solution was added to the reaction flask until 5.5 molar eq. of trizma base was reached. The reaction solution was allowed to stir for 4 hours or until all starting material was consumed as indicated by UPLC-MS. The product solution was injected on HPLC for salt exchange and purification using the method "Salt exchange and purification of amphiphilic compounds" as described above. Pure product was lyophilized to give a dry white sticky powder. The UPLC-UV214 purity of the sample was 95% and the MW determined by MS (electrospray) was 4802.75.
The title compound (conjugate H2N-(rvALFA-Pro-MPA)-succ-(AEEA)i4-DSPE) has the following formula: Example 14 - Determination of the binding of anti-PEG antibodies to different antigens (PEG, AEEA, pSAR) using the anti-PEG ELISA assay
The "anti-PEG ELISA assay" as described above (cf., also Fig. 1) was used to determine binding of anti- PEG polyclonal antibodies to different antigens (PEG, AEEA, pSAR). Briefly, Biotin-labeled antigens (biotin-PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NFE- pSAR (wherein pSAR is a compound of formula XVIII disclosed herein, wherein R21 is H or acetyl (Ac); and R22 is Lys-biotin) were synthesized and captured on Neutravidin-coated plates in wash buffer (IxPBS, 0.1% CHAPS) for 2 h at RT and under slow shaking. After washing with wash buffer (4x), the plates were incubated with different amounts of rabbit anti-PEG polyclonal sera (7 ng/ml, 3 ng/ml, 167 ng/ml, or 830 ng/ml) in assay buffer (IxPBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT and under slow shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody was added (diluted at 1 :5000) and the plates were incubated for 1 h at RT and under slow shaking. After washing with wash buffer (5x), HRP substrate was added and the fluorescence signal was measured. The results are shown in Fig. 2.
As can be seen from Fig. 2, rabbit polyclonal anti-PEG antibodies bind to PEG36k, whereas they do not bind to a polymer comprising the structure of formula (I) or negative controls (biotin-labelled polysarcosine structures). Thus, without wishing to be bound to a certain theory, it is believed that the specific structure of formula (I) (i.e., OEG stretches separated from each other by the moiety -X2-X1-Y- ) prevents the binding of anti-PEG antibodies to this structure and, likewise, to the amphiphilic OEG- conjugated compound of the present disclosure.
The anti-PEG ELISA assay was repeated using anti-PEG IgG and anti-PEG IgM antibodies, respectively. The results of these experiments are shown in Fig. 3.
According to the data presented in Fig. 3, neither anti-PEG IgG antibodies nor anti-PEG IgM antibodies bind to a polymer comprising the structure of formula (I) at the antibody concentrations tested, whereas both types of antibodies bind to PEG36k. Thus, Fig. 3 confirms the results of Fig. 2.
Example 15 - Comparison of properties (length, molecular weight, and hydrophobicity) of PEG structures and structures of formula (I)
The structures (AEEA) 14.17 have lengths which are similar to the length of PEG2k. Furthermore, structure (AEEA) 14 can be synthesized in monodisperse form (MW: 2050 Da), whereas PEG2k can only be synthesized in poly disperse form (average MW: about 2000 Da).
In order to determine whether structures of formula (I) differ from PEG structures with respect to hydrophobicity, H-(AEEA)i4-OH and Fmoc-(PEG)36 were subjected to the method "Hydrophobicity Comparison" as described above. The retention times of the compounds H-(AEEA)i4-0H and Fmoc- (PEG)36 as determined by this method were E03 and E18 min, respectively. As similar retention times of different compounds indicate that the compounds have similar hydrophobicity properties, it can be concluded that the structures of formula (I) exhibit hydrophobicity properties which are similar to PEG structures.
Example 16 - Determination of plasma stability of structures of formula (I)
The plasma stability of Ac-(AEEA)i4-0H and H-(AEEA)i4-0H was tested using the "Plasma stability assay" described above. The results of these experiments are shown in Fig. 4.
As can been seen from Fig. 4, both compounds are stable in human and mouse plasma for at least 72 hours.
Example 17 - Manufacturing of lipid nanoparticles using HY501 as cationically ionizable lipid and different fractions of DSPE-AEEA14-AC
The objective of this example was the investigation of DSPE-AEEA14-AC (i.e. the amphiphilic OEG- conjugated compound of formula (V-l) of the present disclosure) as stealth moiety for engineering mRNA nanoparticles using the LNP technology comprising HY501 (i.e., the cationically ionizable lipid of formula XIV-3) as a cationically ionizable lipid. The DSPE-AEEA14-AC containing LNPs were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fraction of DSPE-AEEA14-AC lipid (1 - 5%) were used for LNP formulations. The ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where X is the fraction of the DSPE-AEEA14-AC. N/P ratio of 6 was selected for the experiments and the RNA construct for all LNP formulations was V09 DS (P020.2) (modified mRNA encoding the P2 S protein of the SARS-CoV-2). For all LNP formulations the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Moreover, the physicochemical properties and biological performance of these DSPE- AEEA14-AC containing formulations were compared with two PEG containing formulations (BM_1 and BM_2). A summary of all the formulations used in this study is given in table 2.
Tiihic 2 Ee^n i '! c\ peri mein ior the prcfhiiMlion o' JJYyi i -EYPE-AEE. I / 4-A( cuimniiiiip J V/X.
The evolution of size and size distribution of DSPE-AEEA14-AC containing LNPs (based on HY-501) manufactured with different concentration of DSPE-AEEA14-AC in the formulation are shown in Figure 5. As can be seen, the particle sizes decreased sharply from 300 to 101 nm with the increasing amounts of DSPE-AEEA14-AC from 1 to 2 mol% in LNP compositions. Moreover, the particle sizes decreased gently from 101 to 64 nm with the increasing amounts of DSPE-AEEA14-AC lipid from 2 to 5 mol%. On the other hand, all the formulations exhibited a PDI inferior to 0.30.
Figure 6 shows the ^-potential of all the DSPE-AEEA14-AC containing LNPs in this example measured in 0.1XPBS at pH 7. The result of BM formulations is also included. As can be seen, slight negative surface charge was obtained for all the formulations.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 7 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, the inaccessibility of RNA in the formulations increased with the concentration of DSPE-AEEA14-AC in the formulation. On the other hand, the agarose gel electrophoresis images show almost no to negligible free RNA in the formulations (free RNA < 1% for each formulation). These results from the agarose gel measurements show that virtually almost all of RNA is bound to the particles for the formulations containing DSPE-AEEA14-AC.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). Figure 8 shows the terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations. The concentration of formed SC5b-9 complex corresponding to the control items are also included. As can be seen, no to negligible complement activation was observed for all the formulations.
Figure 9 presents the hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations. The hemolysis analysis of control items is also included. As can be seen, negligible hemolysis was observed for all formulations tested. Figure 10 represents cell viability after incubation with LNP formulations and was investigated by FACS (Fluorescent Activated Cell Sorting) analysis. As can be seen, a good viability (>90%) was observed for all the tested formulations.
Example 18 - Manufacturing of lipid nanoparticles using HY501 as cationically ionizable lipid and different fractions of VE-AEEA14-AC
The objective of this example was the investigation of VE-AEEA14-AC (i.e. the amphiphilic OEG- conjugated compound of formula (V-5) of the present disclosure) as stealth moiety for engineering mRNA nanoparticles using the LNP technology comprising HY501 as a cationically ionizable lipid. The VE-AEEA14-AC containing LNPs were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fraction of VE-AEEA14-AC lipid (2 - 6 %) were used for LNP formulations. The cationically ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where x is the fraction of the VE- AEEA14-AC. N/P ratio of 6 was selected for the experiments and the RNA construct for all LNP formulations was V09 DS (P020.2) (modified mRNA encoding the P2 S protein of the SARS-CoV-2). For all LNP formulations the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Moreover, the physicochemical properties and biological performance of these VE-AEEA14-AC containing formulations were compared with other PEG containing formulations (BM_1 and BM_2). A summary of all the formulations used in this study is given in the following table 3.
Table 3. Design of experiment for the preparation ofVE-AEEA 14-AC and other PEG containing LNPs. The evolution of size and size distribution of VE-AEEA14-AC containing LNPs (based on HY-501) manufactured with different concentration of VE-AEEA14-AC in the formulation are shown in Figure 11. As can be seen, small particles (d < 100 nm) were obtained by using 2 - 6 mol% VE-AEEA14-AC lipid in the formulations. Furthermore, all the formulations exhibited a PDI inferior to 0.32.
Figure 12 shows the ^-potential of all the VE-AEEA14-AC containing LNPs in this example measured in 0.1XPBS at pH 7. The result of BM formulations is also included. As can be seen, neutral to slight negative surface charge was obtained for all the formulations.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 13 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, high inaccessible RNA was observed for all the formulations. On the other hand, the agarose gel electrophoresis images show almost no to negligible free RNA in the formulations (free RNA < 1% for each formulation with the exception of the BM_1 formulation having free RNA < 5%). These results from the agarose gel measurements show that virtually almost all of RNA is bound to the particles for the formulations containing VE-AEEA14-AC.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). No complement activation was observed for all the formulations.
Hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations was determined. Negligible hemolysis was observed for all formulations tested.
Figure 14 represents the SI protein expression in HEK 293T-17 cells after incubation with LNP formulations. Mean fluorescent intensities (MFI) (cf., Figure 14A) and cell viability (cf., Figure 14B) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. As can be seen, the expression was observed for all of the tested LNP formulations. Moreover, a clear VE-AEEA14-AC-mol% dependent activity upon LNP transfection was observed by maintaining a good viability (>90%). As can be seen, for the VE-AEEA14-AC containing formulations the highest expression was observed for the formulations containing 2 mol% VE-AEEA14-AC and the expression decreased with increasing VE- AEEA14-AC concentration.
Example 19 - Yield and purity of peptide intermediates and amphiphilic compounds
The peptide intermediates Ac-(AEEA)8-OH and Ac-(AEEA)i4-OH were synthesized as disclosed above and samples of these intermediates for UPLC and mass spectrometry were taken either directly after cleavage of the respective intermediate from the resin, i.e., prior to the QC method (crude purities), or after the QC method (final purities). Figures 15 A-D represent the results for Ac-(AEEA)8-0H, whereas Figures 15 E-H represent the results for Ac-(AEEA)i4-0H. Figures 15 A, B, E, and F show respective UPLC chromatograms and Figures 15 C, D, G, and H show respective mass spectra, recorded using either samples which have not been subjected to the QC method (Figures 15 A, C, E, G) or samples which have been subjected to the QC method (Figures 15 B, D, F, H).
From these data, the yields and purities (crude purities (i.e., prior to the QC method) and final purities (i.e., after the QC method)) have been calculated which are shown in table 4.
Table 4. Yields and purities of peptide intermediates
In addition, several amphiphilic compounds have been synthesized as disclosed above. During their synthesis as well as after their purification samples have been taken and analyzed using the methods described in "Salt exchange and purification of amphiphilic compounds" or "Alternative purification of amphiphilic compounds". Figure 16 shows UPLC chromatograms of samples for the synthesis of Ac- (AEEAjs-a-tocopherol taken (A) after 5 minutes and (B) after 4 h (i.e., at the end) of the reaction. As can be seen, the staring material Ac-(AEEA)8-OH was present after a reaction time of 5 min, but could not be detected at the end of the reaction. Figure 17 shows exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J) for the amphiphilic compounds Ac-(AEEA)8-a-tocopherol (A, B), Ac-(AEEA)i4-a-tocopherol (C, D), AC-(AEEA)I4-DMA (E, F), Ac-(AEEA)8-DMG (G, H), and Ac- (AEEA)i4-DSPE (I, J).
From these data, the yields and purities (crude purities (i.e., prior to the QC method) and final purities (i.e., after the QC method)) have been calculated which are shown in table 5. All purities have calculated based on UV absorption at 214 nm.
Table 5. Yields and purities of peptide intermediates of amphiphilic compounds
*60% yield for Ac-(AEEA)8-a-tocopherol resulted from purification by size exclusion chromatography (SEC). All others were purified by RP-HPLC. Crude purities are low for reactions with lipid starting materials that absorb strongly at 214 nm, and for any reactions that use PyAOP/HOAt/DIEA because some of these reagents and their biproducts can be observed on UPLC.
Example 20 - Exploration of the effect of various amphiphilic OEG-conjugated compounds on the engineering and biological behavior of LNP formulation Potency formulations
The objective of this example was the investigation of various amphiphilic OEG-conjugated compounds as stealth moiety for engineering mRNA nanoparticles using the LNP technology comprising HY501 as a cationically ionizable lipid. The LNPs containing amphiphilic OEG-conjugated compounds were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fractions of amphiphilic OEG-conjugated compounds were used for LNP formulations. The cationically ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where X is the fraction of the respective amphiphilic OEG- conjugated compound. V09 mRNA (modified mRNA encoding the P2 S protein of the SARS-CoV-2) was used in this example. N/P ratio of 6 was selected for the experiments. Moreover, the physicochemical properties and biological performance of these formulations containing amphiphilic OEG-conjugated compounds were compared with two PEG containing formulations (BM_1 and BM_2). A summary of all the formulations used in this example is given in table 6.
Table 6. Design of experiment for the preparation of LNPs containing amphiphilic OEG- conjugated compounds and other PEG compounds.
The evolution of size and size distribution of LNPs containing amphiphilic OEG-conjugated compounds (based on HY -501) manufactured with different type and concentration of amphiphilic OEG-conjugated compound in the formulation are shown in Figure 18. The size and size distribution of BM formulations are also included. As can be seen, the size of LNP formulations containing amphiphilic OEG-conjugated compounds decreased with increasing stealth moiety concentration. Moreover, small particles (d < 100 nm) were obtained for most of the formulations. On the other hand, all the formulations exhibited a PDI inferior to 0.25.
Figure 19 shows the ^-potential of all the LNPs containing amphiphilic OEG-conjugated compounds measured in 0. 1 XPBS at pH 7. The result of BM formulations is also included. As can be seen, neutral to slight negative surface charge was obtained for all the formulations. The slight negative surface charge in formulations containing amphiphilic OEG-conjugated compounds is most likely due to the acetylated functionality in these molecules.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 20 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, independent of the type of of amphiphilic OEG-conjugated compound the level of inaccessible RNA increased with increasing concentration of the amphiphilic OEG-conjugated compound in the formulation. Moreover, inaccessible RNA was observed for almost all of the formulations. On the other hand, the agarose gel electrophoresis images show almost no to negligible free RNA in the formulations. These results from the agarose gel measurements show that virtually almost all of RNA is bound to the particles for the LNP formulations containing amphiphilic OEG-conjugated compounds.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). As can be seen in Figure 21, no complement activation was observed for all the formulations.
Figure 22 presents the hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations. The hemolysis analysis of control items is also included. As can be seen negligible hemolysis was observed for all other formulations.
Figure 23 represents the SI protein expression in HEK 293T-17 cells after incubation with LNP formulations. Mean fluorescent intensities (MFI) (cf., Figure 23A) and cell viability (cf., Figure 23B) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. As can be seen, the expression was observed for all of the tested LNP formulations. Moreover, a clear amphiphilic OEG-conjugated compound-mol% dependent activity upon LNP transfection was observed by maintaining a good viability (>90%). For the VE-AEEA14-AC containing formulations the highest expression was observed for the formulations containing 1 mol% VE-AEEA14-AC and the expression decreased with increasing VE-AEEA14-AC concentration. Moreover, for VE-(AEEA)8_AC containing formulations the highest level of expression was observed for HY501-VE-(AEEA)8-AC_3. The comparison of VE- (AEEA)14-AC and VE-(AEEA)8_AC containing formulations shows higher performance for the formulations containing shorter stealth moiety. In addition, for DMG-(AEEA)8_AC containing formulations the highest level of expression was observed for HY501-DMG-(AEEA)8-AC_4. The lower level of activity for HY501-DMG-(AEEA)8-AC_3 is most likely due to bigger particle size for this formulation. The comparison of all tested LNP formulations containing amphiphilic OEG- conjugated compounds showed that the type of hydrophobic tail may also play a role on the activity of the formulations and the following trend was observed for the expression efficiency: VE > DMG > DMA.
Example 21 - Exploration of the effect of various amphiphilic OEG-conjugated compounds on the engineering and biological behavior of LNP formulation Luciferase formulations
The objective of this example was the investigation of various amphiphilic OEG-conjugated compounds as stealth moiety for engineering mRNA nanoparticles using the LNP technology comprising HY501 as a cationically ionizable lipid. The LNPs containing amphiphilic OEG-conjugated compounds were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fractions of amphiphilic OEG-conjugated compounds were used for LNP formulations. The cationically ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where X is the fraction of the respective amphiphilic OEG- conjugated compound. Luciferase mRNA (modified mRNA encoding firefly luciferase) was used in this example. N/P ratio of 6 was selected for the experiments. Moreover, the physicochemical properties and biological performance of these formulations containing amphiphilic OEG-conjugated compounds were evaluated. A summary of all the formulations used in this example is given in table 7.
Table 7. Design of experiment for the preparation of LNPs containing amphiphilic OEG- conjugated compounds. The evolution of size and size distribution of LNPs containing amphiphilic OEG-conjugated compounds are shown in Figure 24. As can be seen, the size of LNP formulations containing amphiphilic OEG- conjugated compounds decreased with increasing stealth moiety concentration. Moreover, small particles (<100 nm) were obtained for all the formulations except HY501-DMG-(AEEA)8-AC_3 formulation. In addition, most of the formulations exhibited a Pdl inferior to 0.3.
Figure 25 shows the ^-potential of all the LNPs containing amphiphilic OEG-conjugated compounds measured in 0.1 XPBS at pH 7. As can be seen, neutral to slight negative surface charge was obtained for all the formulations. The slight negative surface charge in formulations containing amphiphilic OEG- conjugated compounds is most likely due to the acetylated functionality in these molecules.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 26 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, inaccessible RNA was observed for almost all of the formulations. On the other hand, the agarose gel electrophoresis images show almost no to negligible free RNA in the formulations. These results from the agarose gel measurements show that virtually almost all of RNA is bound to the particles for the of LNP formulations containing amphiphilic OEG- conjugated compounds.
To investigate the impact of various amphiphilic OEG-conjugated compounds as stealth moiety on the biological behavior of LNP formulations, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), ahepatocarcinoma cell line (HepG2) and a murine macrophage cell line (Raw cell). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well, and luciferase expression was assessed 24 h post-incubation. Mean fluorescent intensities (MFI) (Figures 27A, C, E) and cell viability (Figures 27B, D, F) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. Figures 27A, C, and E show that the luciferase expression was observed for all the formulations in the tested cell lines C2C12 (Figures 27A, B), Raw (Figures 27C, D), and HepG2 (Figures 27E, F), respectively. Moreover, the performance and transfection efficiency of LNP formulations containing amphiphilic OEG-conjugated compounds depended on the type and concentration of stealth moiety in the formulations. For the VE-AEEA14-AC containing formulations the highest expression was observed for the formulations containing 2 mol% VE-AEEA14-AC and the expression decreased with increasing VE-AEEA14-AC concentration. Moreover, for VE-(AEEA)8_AC containing formulations the level of expression was in the same level forHY501-VE-(AEEA)8-AC_3 and HY501-VE-(AEEA)8-AC_2. The comparison of VE-(AEEA)14-AC and VE-(AEEA)8_AC containing formulations shows higher performance for the formulations containing shorter stealth moiety. The same trend was observed for the formulations containing DMA-(AEEA)14-AC and DMA-(AEEA)8_AC. The comparison of all tested LNP formulations showed that the type of hydrophobic tail may also play a role on the activity of the formulations and the following trend was observed for the expression efficiency: VE > DMG > DMA. The same trend was observed for V09 mRNA containing formulations (see Example 20).
Example 22 - T-cell targeting using LNPs with different stealth lipids and Alfa lipids
LNPs were formulated with different lipid compositions (Cargo: Thy 1.1 RNA/Luc RNA/Np proxy Venus 1: 1:2 w/w; N/P ratio: 6; Lipid mix: HY501/Cholesterol/DSPC/stealth lipid/Alfa lipid. The lipid ratio has been selected as [47.5/40.5/10/1.8/0.2] for the following combinations of stealth lipid and Alfa lipid: C16 PEG2k Ceramide/DSPE PEG2k Alfa, DSPE PEG2k/DSPE PEG2k Alfa, DSPE- AEEA14/DSPE-AEEA14-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa. The lipid ratio has been selected as [47.5/38.5/10/3.8/0.2] for the following combinations of stealth lipid and Alfa lipid: VE- PEGlk/DSPE-PEG2k-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa. The LNPs were equipped with aCD3 VHH X NbAlfa ligand via post functionalization [w/w* = ligand to cargo ratio 0.48]; RNA concentration: 0.1 pg/pl). Diameter of all LNPs is between 100 to 170 nm with a PDI below 0.4 as determined via DLS measurement.
For transfection studies, 10 pl (1000 ng dose) of respective formulations were pre-diluted in 50 pl X- Vivo 15 in an ultra-low adhesion 96 well plate. 106 thawed human PBMC were diluted in 50 pl of 100 % clotted PHS and added to nanoparticle dilution. After 30 min of incubation (37°C, 5% CO2) 30 pl of each transfection reaction was transferred to second ultra-low adhesion 96 well plate and 170 pl of X- Vivo 15 medium + 100 U/ml IL2 was added per well. Cell dilutions were cultivated for additional 18 h (37°C, 5% CO2). In the following cell-type specific transfection (Thy 1.1) was analyzed via flowcytometry. Depicted are the percentages of transfected cell (CD2 negative cells, CD 19+ B cells, CD4+ T cells and CD8+ T cells) within all transfected PBMCs (Transfection, y-axes) per tested formulation condition.
As shown in Figure 28, the experiment demonstrates the modular nature of the conjugation approach. As shown for LNPs, ligands can be attached to nanoparticles using Alfa lipids with varying lipid backbone and spacer type as well as spacer length. Good physicochemical properties of the nanoparticles (size, PDI and RNA encapsulation) can be achieved with all tested stealth/Alfa lipid combinations. The selection of the stealth lipid anchor has an impact on the T cell transfection efficiency. DSPE has a tendency towards lower transfection efficiency.
Example 23 - Variation of DMG-AEEA14-AC and C14-AEEA14-AC fraction for manufacturing of nanoparticles using HY501 as ionizable lipid
The objective of this example was the investigation of DMG-AEEA14-AC and C14-AEEA14-AC lipid (compound of formula (V-63)) as stealth moiety for engineering mRNA nanoparticles using the LNP technology comprising HY501 as an ionizable lipid. The LNPs were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fraction of stealth-grafted lipids (1 - 3%) were used for LNP formulations. The ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where x is the fraction of the stealth-grafted lipid. Luciferase mRNA was used in this example. N/P ratio of 6 was selected for the experiments. For all LNP formulations the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Moreover, the physicochemical properties and biological performance of these DMG-AEEA14-AC and C14- AEEA14-AC containing formulations were compared with other PEG containing BM_1 and BM_2 formulations. In addition, 2 LNP formulations containing VE-AEEA14-AC and DMA-AEEA14-AC as stealth moiety were used in this study for comparison purpose. A summary of all the formulations used in this study is given in Table 8.
Table 8. Design of experiment for the preparation of LNPs containing amphiphilic OEG- conjugated compounds.
The evolution of size and size distribution of LNPs containing amphiphilic OEG-conjugated compounds are shown in Figure 29. As can be seen, the size of LNP formulations containing amphiphilic OEG- conjugated compounds decreased with increasing stealth moiety concentration. Moreover, small particles (d < 100 nm) were obtained by using > 2 mol% DMG-AEEA14-AC or C14-AEEA14-AC lipid in the formulations. On the other hand, all the formulations exhibited a Pdl inferior to 0.3. Figure 30 shows the ^-potential of all the LNPs containing amphiphilic OEG-conjugated compounds measured in 0.1 XPBS at pH 7. As can be seen, neutral to slight negative surface charge was obtained for all the formulations. The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 31 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, RNA inaccessibility increased with stealth lipid concentration and high amount of inaccessible RNA was observed for HY501-DMG-(AEEA)14-AC_3 and HY501-C14-(AEEA)14-AC_3 formulations. On the other hand, the agarose gel electrophoresis image shows almost no free RNA in the formulations. These results from the agarose gel measurements show that virtually almost all of RNA is bound to the particles for LNP formulations containing amphiphilic OEG-conjugated compounds.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). As can be seen in Figure 32, no complement activation was observed for all the formulations.
Figure 33 presents the hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations. The hemolysis analysis of control items is also included. As can be seen HY501-C14-(AEEA)14-AC_l LNP shows high level of hemolysis, most likely due to the big size and low amount of C14-AEEA14-AC in the formulation. Moreover, negligible hemolysis was observed for the rest of the formulations.
To investigate the impact of various amphiphilic OEG-conjugated compounds as stealth moiety on the biological behavior of LNP formulations, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), ahepatocarcinoma cell line (HepG2) and a murine macrophage cell line (Raw cell). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well, and luciferase expression was assessed 24 h post-incubation. Mean fluorescent intensities (MFI) (Figures 34A, C, E) and cell viability (Figures 34B, D, F) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. Figures 34A, C, and E show that the luciferase expression was observed for all the formulations (except for HY501-C14-(AEEA)14-AC_l LNP in C2C12 cell line)) in the tested cell lines C2C12 (Figures 34A, B), Raw (Figures 34C, D), and HepG2 (Figures 34E, F), respectively. Furthermore, a clear OEG-mol% dependent activity upon LNP transfection was observed by maintaining a good viability (>90%). Moreover, the performance and transfection efficiency of LNP formulations containing amphiphilic OEG-conjugated compounds depended on the type of stealth moiety in the formulations. For the DMG- AEEA14-AC containing formulations the highest expression was observed for the formulations containing 2 and 3 mol% DMG-AEEA14-AC. On the other hand, for C14-AEEA14-AC containing formulations the highest expression was observed for the formulations containing 2 mol% C14- AEEA14-AC. The comparison of DMG-AEEA14-AC and C14-AEEA14-AC containing formulations shows higher performance for the formulations containing DMG-AEEA14-AC stealth moiety. In addition, the transfection efficiency of DMG-AEEA14-AC containing formulation is slightly higher than VE-AEEA14-AC and DMA-AEEA14-AC containing LNPs.
Example 24 - Exploration of the effect of poly(AEEA)-grafted lipid length on the engineering and biological behavior of LNP formulation
The objective of this example was the investigation of various lengths of poly (AEEA) -grafted lipids as stealth moiety for engineering mRNA nanoparticles using the LNP technology comprising HY501 as an ionizable lipid. VE-(AEEA)n-AC was used in this example, where n is the number of AEEA repeating unit in the hydrophilic block of the stealth grafted lipid. The VE-(AEEA)n-AC containing LNPs were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fraction of VE-(AEEA)n-AC-grafted lipids were used for LNP formulations. The ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where x is the fraction of the VE-(AEEA)n-AC-grafted Lipids. Luciferase mRNA was used in this study. N/P ratio of 6 was selected for the experiments. Lor all LNP formulations the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Moreover, the physicochemical properties and biological performance of these VE-(AEEA)n-AC-grafted Lipids containing formulations were compared with other PEG containing BM_1 and BM_2 formulations. A summary of all the formulations used in this study is given in Table 9.
Table 9. Design of experiment for the preparation of LNPs containing amphiphilic VE-(AEEA)n- AC-conjugated compounds.
The evolution of size and size distribution of LNPs containing amphiphilic VE-(AEEA)n-AC- conjugated compounds are shown in Figure 35. The size and size distribution of BM formulations are also included. As can be seen, the size of LNP formulations containing amphiphilic VE-(AEEA)n-AC- conjugated compounds decreased with increasing stealth moiety concentration. Moreover, small particles (d < 100 nm) were obtained for all the formulations. On the other hand, all the formulations exhibited a PDI inferior to 0.3.
Figure 36 shows the ^-potential of all the LNPs containing amphiphilic VE-(AEEA)n-AC-conjugated compounds measured in 0.1 XPBS at pH 7. The result of BM formulations is also included. As can be seen, neutral to slight negative surface charge was obtained for all the formulations. The slight negative surface charge in VE-(AEEA)n-AC containing formulations is most likely due to the acetylated functionality in these molecules.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 37 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, independent of the length and composition of VE-(AEEA)n- AC high level of inaccessible RNA was observed in the formulations. On the other hand, the agarose gel electrophoresis image shows almost negligible free RNA in the formulations. These results from the agarose gel measurements show that virtually almost all of RNA is bound to the particles for LNP formulations containing amphiphilic VE-(AEEA)n-AC-conjugated compounds.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). Figure 38 shows the terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations. The concentration of formed SC5b-9 complex corresponding to the control items is also included. As can be seen in Figure 38, no to negligible complement activation was observed for all the formulations. Figure 39 presents the hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations. The hemolysis analysis of control items is also included. As can be seen negligible hemolysis was observed for all other formulations.
To investigate the impact of VE-(AEEA)n-AC length as stealth moiety on the biological behavior of LNP formulations, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2) and a murine macrophage cell line (Raw cell). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well, and luciferase expression was assessed 24 h post-incubation. Mean fluorescent intensities (MFI) (Figures 40A, C, E) and cell viability (Figures 40B, D, F) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. Figure 40 shows that the luciferase expression was observed for all the formulations in the tested cell lines by maintaining a good viability (>80%; except for some of the formulations with 50 ng/well dose in C2C12 cell line). Moreover, the comparison of the performance and transfection efficiency of the LNP formulations containing VE- (AEEA)n- AC -conjugated compounds having different length of (AEEA)n are in the same level and HY501-VE-(AEEA)12-AC_3 and HY501-VE-(AEEA)12-AC_4 LNPs showed the highest performance in HepG2 and Raw cell lines.
Example 25 - Exploration of the effect of OEG-conjugated compounds with a ceramide as lipid component (C16-Cr-AEEA14-AC) on the engineering and biological behavior of LNP formulation The objective of this example was the investigation of OEG-conjugated compounds with a ceramide as lipid component (C16-Cr-AEEA14-AC) for engineering mRNA nanoparticles using the LNP technology comprising HY501 as an ionizable lipid. The LNPs were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. Different mole fractions of OEG-conjugated compounds were used for LNP formulations. The ionizable lipid HY501, the helper lipid DSPC, and cholesterol at respective molar fractions 47.5:42.5 - X: 10 was used, where x is the fraction of the OEG-conjugated compounds. Luciferase mRNA was used in this study. N/P ratio of 6 was selected for the experiments. For all LNP formulations the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Moreover, the physicochemical properties and biological performance of these LNP formulations containing OEG- conjugated compounds were compared with other PEG containing BM_1 and BM_2 formulations. A VE-(AEEA)14-AC containing LNP was used as well for comparison purposes. A summary of all the formulations used in this study is given in Table 10. Table 10. Design of experiment for the preparation of LNPs containing amphiphilic OEG- conjugated compounds.
The evolution of size and size distribution of LNPs containing amphiphilic OEG-conjugated compounds are shown in Figure 41. The size and size distribution of BM formulations are also included. As can be seen, the size of LNP formulations containing amphiphilic OEG-conjugated compounds decreased with increasing stealth moiety concentration. Moreover, small particles (d < 100 nm) were obtained for several formulations. On the other hand, all the formulations exhibited a PDI inferior to 0.3.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 42 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, high amount of inaccessible RNA was observed for C16- Cr-AEEA 14-AC containing LNPs and the amount of inaccessible RNA increased with increasing stealth lipid concentration. On the other hand, some free RNA was also observed for HY501-C16-Cr- (AEEA)14-AC_1 and HY501-C16-Cr-(AEEA)14-AC_1.5 LNPs and negligible free RNA was observed for the formulations containing > 2 mol% C16-Cr-(AEEA)14-AC.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). Figure 43 shows the terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations. The concentration of formed SC5b-9 complex corresponding to the control items is also included. As can be seen in Figure 43, no to negligible complement activation was observed for all the formulations. Figure 44 presents the hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations. The hemolysis analysis of control items is also included. As can be seen the hemolytic properties of LNP formulations decreased with increasing stealth lipid concentration in the formulations, most likely due to the lower interaction of particles containing higher amount of stealth lipid with red blood cells. Moreover, the interaction of smaller particles with red blood cells is lower than bigger particles.
To investigate the impact of C16-Cr-AEEA14-AC as stealth moiety on the biological behavior of LNP formulations, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2) and a murine macrophage cell line (Raw cell). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well, and luciferase expression was assessed 24 h post-incubation. Mean fluorescent intensities (MFI) (Figures 45A, C, E) and cell viability (Figures 45B, D, F) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. Figure 45 shows that low luciferase expression was observed for the LNP formulations containing 1 and 1.5 mol% ofC16-Cr-AEEA14-AC, most likely due to the big size for these formulations. Moreover, the luciferase expression of LNP formulations containing 3 mol% of C16-Cr-AEEA14-AC is in the range of BM and control LNPs. On the other hand, all the formulations in the tested cell lines maintained a good viability (>80%).
Example 26 - Exploration of the engineering and biological feasibility of cationic LNPs containing VE-AEEA14-AC conjugated compound
The objective of this example was the investigation of VE-AEEA14-AC conjugated compound for engineering mRNA nanoparticles using the LNP technology comprising DOTMA and DOTAP as cationic lipids. The LNPs were prepared by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic instrument. The cationic lipids (DOTMA and DOTAP), cholesterol, the helper lipid DSPC and VE-AEEA14-AC at respective molar fractions 47.5:40.7: 10:2 was used. Luciferase mRNA was used in this study. N/P ratio of 6 was selected for the experiments. For all LNP formulations the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Moreover, the physicochemical properties and biological performance of these LNP formulations containing VE-AEEA14-AC conjugated compound and DOTMA or DOTAP were compared with LNP formulations containing their corresponding ionizable lipids (DODMA and DODAP). In addition, a PEG containing BM_1 formulation was used as control in this experiment. A summary of all the formulations used in this study is given in Table 11. Table 11. Design of experiment for the preparation ofLNPs containing amphiphilic VE-AEEA14- AC conjugated compound and DOTMA, DODMA, DOTAP, or DODAP as cationic/cationically ionizable lipid.
The evolution of size and size distribution of the LNPs are shown in Figure 46. The size and size distribution of BM formulation are also included. As can be seen, small particles (d < 100 nm) were obtained for all the formulations. On the other hand, all the formulations exhibited a PDI inferior to 0.25.
Figure 47 shows the ^-potential of all the LNPs in this example measured in 0.1XPBS at pH 7. The result of BM formulation is also included. As can be seen, neutral to slight negative surface charge was obtained for BM, DODMA-VE-AEEA 14-AC 2 and DODAP -VE-AEEA 14-AC 2 formulations . On the other hand, high positive surface charge has been observed for DOTMA-VE-AEEA14-AC_2 and DOTAP-VE-AEEA14-AC 2 formulations due to permanently positive charge for these cationic lipids.
The accessibility of RNA and the amount of free RNA in the LNP formulations was measured by RiboGreen Assay and agarose gel electrophoresis methods, respectively. Figure 48 presents the RNA accessibility of the formulations as measured by the RiboGreen Assay method (A) or the agarose gel electrophoresis method (B). As can be seen, all LNPs showed high amount of inaccessible RNA. The highest RNA inaccessibility has been observed for DOTMA-VE-AEEA14-AC_2 and DOTAP-VE- AEEA14-AC_2 formulations due to permanently positive charge for these cationic lipids.. On the other hand, no to negligible free RNA for the formulations was observed.
Complement activation of the formulations were studied by in vitro quantification of SC5b-9 level using Microvue SC5b-9 Plus ELISA kit (Quidel Co., SanDiego, CA, USA). Figure 49 shows the terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations. The concentration of formed SC5b-9 complex corresponding to the control items is also included. As can be seen in Figure 49, no to negligible complement activation was observed for all the formulations. In addition, no significant difference in the complement activation of LNPs formulated with ionizable and permanent cationic lipid was observed (comparison of DODMA with DOTMA or DODAP with DOTAP LNPs).
Figure 50 presents the hemolysis analysis after incubation (in neutral pH condition) of the whole human blood with LNP formulations. The hemolysis analysis of control items is also included. As can be seen negligible hemolysis was observed for all other formulations.
To investigate the impact of permanently charged cationic lipids on the biological behavior of VE- AEEA14-AC containing LNP formulations, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C 12), a hepatocarcinoma cell line (HepG2) and a murine macrophage cell line (Raw cell). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well, and luciferase expression was assessed 24 h post-incubation. Mean fluorescent intensities (MFI) (Figures 51 A, C, E) and cell viability (Figures 5 IB, D, F) were investigated by FACS (Fluorescent Activated Cell Sorting) analysis. Figure 51 shows that the level of expression for DOTMA containing LNP is much lower than the corresponding ionizable (DODMA) based LNP in all the tested cell lines. Moreover, the level of expression for DODAP and DODAP containing LNPs is very low. On the other hand, all the formulations in the tested cell lines maintained a good viability (>90%).
Example 27 - Preparation, characterization and biological tests of functionalized LNPs containing OEG-conjugated compounds
Description of the methods
Preparation of functionalized LNPs containing OEG-conjugated (pAEEA) compounds
Alfa tagged lipid particles can be prepared using a known aqueous/organic manufacturing protocol. For preparing alfa-tagged lipid nanoparticles a lipid mix consisting of HY501, DSPC, cholesterol, DSPE- pAEEA14-a//a peptide and an OEG-conjugated compound was dissolved in organic solvent at 22.8 mM total lipid concentration, with composition reported in Table 12. The lipid mix was mixed with an aqueous phase containing the relevant cargos (Thyl.l/Luc RNA/DNA 1: 1:2 w/w) at an N/P ratio of 12 using a standard syringe pump-based set-up and a T mixing element.
The LNPs were equipped with aCD3 VHH X NbAlfa ligand via post functionalization (w/w* = ligand to cargo ratio 0.48) and further diluted with respective buffer of choice supplemented with sucrose so that final cone, of total cargos in the lipid nanoparticles was 0.1 mg/ml and sucrose content in the final matrix was 10% w/v. All the formulations were sterile filtered with 0.22 pm PES filter and stored at -80°C until use. Table 12: Composition and molar ratios for the 5 formulations prepared.
Characterization of the RNA formulations
Particle size was determined by dynamic light scattering using a DynaPro Plate Reader II (Wyatt, Dembach, Germany). From the measurements, size (Z-average) and polydispersity indices (Pdl) were calculated from cumulant analysis using Dynamics 7.8.1.3 software. For the measurement, samples were diluted 1: 10 in water and analysis was performed in triplicates. pH was measured on 150 pL sample after calibration of the device with pH 4, 7, and 10 standards.
Successful cargos incorporation was verified via Agarose Gel Electrophoresis.
Freeze thaw studies
Freeze thaw studies were conducted by cycling the formulations from -20°C or -80°C (overnight) to +25 °C (2 h) for at least three times. The particles size and polydispersity index were measured for freeze - thaw samples.
In vitro tests
For transfection studies, 5 pl (500 ng dose) of respective formulations were pre-diluted in 50 pl X-Vivo 15 in an ultra-low adhesion 96 well plate. 3e5 thawed human PBMCs were diluted in 100 % PHS and added to nanoparticle dilution. Plates were incubated for 30 minutes (37°C, 5% CO2). 100 pl X-Vivo 15 containing 200IU/ml IL-2 was added on each well. Plates were incubated for 4 days (37°C, 5% CO2). Transfection (Surface Thy 1.1 expression) was analyzed via flowcytometry after labelling of samples with cell type specific antibodies and Thy 1.1 detection antibody.
In vivo administration
1 pg of the respective formulation was injected into the Thigh muscle of both hind legs of a B6- hCD3EDG transgenic mice. Therefore, a total dose of 2 pg per mouse was used. 18h after LNP injection, animals were intraperitoneally injected with Luciferin and whole-body bioluminescence imaging was performed. Consequently, mice were sacrificed, peripheral lymph nodes, spleens and livers were isolated. Organs were placed on a black well plate with 500ul PBS, added luciferin and imaged for bioluminescence. Lymph nodes and spleens were further processed to obtain single cell suspensions, stained with cell type specific antibodies and Thy 1.1 antibody and analyzed for Thy 1.1 expression via flowcytometry.
Experimental Results.
Size and PDI was determined for functionalized sterile filtered lipid nanoparticles stored at final RNA concentration of 0.1 mg/mL via DLS measurement. It was observed that aCD3-VHH-functionalized lipid nanoparticles can be successfully manufactured with pAEEAs-based compounds using different anchoring moiety and different pAEEAs length, resulting in particles of around -90-100 nm in size and a PDI < 0.3 (Figure 52).
Successful cargo incorporation was verified via Agarose gel electrophoresis: no RNA or DNA bands are present in the samples, indicating that the formulation did not have any free cargo. Moreover, all the respective RNA and DNA bands were clearly visible when the samples were treated with a release solution (controls), confirming the successful encapsulation of all the three cargos (Figure 53).
Figure 54A-E show the results from freezing of aCD3-LNPs at -20°C and -80°C, respectively, and storage for at least 3 freeze/thaw cycles. As can be seen, no significant changes in size and PDI could be observed at both freezing temperatures tested.
All tested LNPs containing OEG-conjugated (pAEEA) compounds show an RNA transfection between 20-30% of all cells. Percentage of non-specific transfection on B cells or monocytes was less than 1% within this transfected population for all particles (Figure-55A). Total counts of cells after 4-day incubation indicate survival and expansion of all cell subsets (Figure 55B). All particles except C provided an expansion or survival benefit to T cells relative to without particle condition (Figure 55B).
Highest Luciferase bioluminescence was observed from popliteal lymph nodes followed by inguinal, axillary and brachial nodes of the mice indicating efficient drainage of particles and transfection to lymph nodes (Figure 56A). Differences between particles were not statistically significant. Accumulation in the Liver was overall higher in group E. Groups A and C demonstrated the lowest Liver signal (Figure 56A). Groups A, C and E demonstrated lower T cell transfection than groups B and D in LNs and Spleens of the mice. Group A demonstrated significantly low T cell transfection than all other groups in popliteal LN and group B and D in inguinal LN (Figure 56B). Groups B and D demonstrated 1.6 and 0.9% B cell transfection respectively and group D and E demonstrated 1 and 0.8% PMN transfection in the in popliteal LNs respectively (Table 13). Off target transfection was less than 1% for Inguinal LN and spleen. Table 13: Mean percentages of transfected cell subsets within all cells
Popliteal Lymph node
F4/80
PMN Macro DC Macro/Mono B cells NK cells T cells
A 0,15 0,00 0,06 0,26 0,14 0,26 0,93
B 0,23 0,03 0,06 0,10 1,61 0,08 4,03
C 0,68 0,00 0,07 0,10 0,75 0,10 3,29
D 1,04 0,00 0,06 0,18 0,94 0,20 5,14
E 0,82 0,00 0,08 0,12 0,30 0,15 3,15
Ctrl 0,00 0,00 0,00 0,01 0,02 0,01 0,02
Inguinal Lymph node
A 0,02 0,00 0,05 0,10 0,12 0,17 0,31
B 0,20 0,02 0,09 0,07 0,67 0,07 3,06
C 0,21 0,01 0,04 0,08 0,59 0,05 1,64
D 0,38 0,01 0,06 0,08 0,51 0,13 3,06
E 0,22 0,00 0,09 0,06 0,28 0,15 1,48
Ctrl 0,00 0,00 0,01 0,00 0,02 0,01 0,02
Spleen
A 0,05 0,02 0,02 0,00 0,09 0,05 0,03
B 0,08 0,11 0,03 0,11 0.34 0,06 6,09
C 0,05 0,05 0,01 0,04 0.14 0,04 1,72
D 0,13 0,06 0,02 0,08 0.25 0,09 4,02
E 0,04 0,01 0,02 0,02 0.20 0,08 3,23
Ctrl 0,02 0,03 0,01 0,01 0,11 0,03 0,03

Claims

CLAIMS A composition comprising (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100. The composition of claim 1, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-; or
(vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl.
3. The composition of claim 1 or 2, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Ci-8 alkyl.
4. The composition of any one of claims 1 to 3, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl.
5. The composition of any one of claims 1 to 4, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.
6. The composition of any one of claims 1 to 5, wherein Y is -CH2- or -(CfT^-.
7. The composition of any one of claims 1 to 6, wherein Y is -CH2-.
8. The composition of any one of claims 1 to 7, wherein the polymer comprises the following general formula (II): wherein R1 is hydrogen or C1-8 alkyl.
9. The composition of any one of claims 1 to 8, wherein z is 2 to 10, such as 2 to 7.
10. The composition of any one of claims 1 to 9, wherein z is 2 to 5.
11. The composition of any one of claims 1 to 10, wherein z is 2 or 3.
12. The composition of any one of claims 1 to 11, wherein z is 2.
13. The composition of any one of claims 1 to 12, wherein the polymer comprises the following general formula (III): wherein R1 is hydrogen or C1-8 alkyl.
14. The composition of any one of claims 8 to 13, wherein R1 is hydrogen or methyl.
15. The composition of any one of claims 8 to 14, wherein R1 is hydrogen.
16. The composition of any one of claims 1 to 15, wherein the polymer comprises the following general formula (IV):
17. The composition of any one of claims 1 to 16, wherein n is 5 to 50.
18. The composition of any one of claims 1 to 17, wherein n is 5 to 25.
19. The composition of any one of claims 1 to 18, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. 0. The composition of any one of claims 1 to 19, wherein the one or more hydrophobic chains are located at either the X1 end or the X2 end of the polymer. 1. The composition of any one of claims 1 to 20, wherein the one or more hydrophobic chains are non-cyclic, preferably straight, hydrocarbyl groups, more preferably those having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. 2. The composition of any one of claims 1 to 21, wherein the polymer-conjugated compound c wherein X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-;
R2 is a moiety comprising the one or more hydrophobic chains;
R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6- membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100. The composition of claim 22, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-; (viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-; or
(vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl. The composition of claim 22 or 23, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or C1-8 alkyl. The composition of any one of claims 22 to 24, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl. The composition of any one of claims 22 to 25, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen. The composition of any one of claims 22 to 26, wherein Y is -CH2- or -(CTh^-. The composition of any one of claims 22 to 27, wherein Y is -CH2-. The composition of any one of claims 22 to 28, wherein the polymer-conjugated compound comprises the following general formula (VI) wherein R1 is hydrogen or C1-8 alkyl. The composition of any one of claims 22 to 29, wherein z is 2 to 10, such as 2 to 7. The composition of any one of claims 22 to 30, wherein z is 2 to 5. The composition of any one of claims 22 to 31, wherein z is 2 or 3. The composition of any one of claims 22 to 32, wherein z is 2. The composition of any one of claims 22 to 33, which comprises the following general formula wherein R1 is hydrogen or Cus alkyl. The composition of any one of claims 29 to 34, wherein R1 is hydrogen or methyl. The composition of any one of claims 29 to 35, wherein R1 is hydrogen. The composition of any one of claims 22 to 36, wherein the polymer-conjugated compound c The composition of any one of claims 22 to 37, wherein n is 5 to 50. The composition of any one of claims 22 to 38, wherein n is 5 to 25. The composition of any one of claims 22 to 39, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. The composition of any one of claims 22 to 40, wherein R2 is R4 or -L1(R4)P, wherein each R4 is independently a hydrophobic chain, such as a hydrocarbyl group; L1 is a linker; and p is 1 or 2. The composition of claim 41, wherein L1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties. The composition of claim 41 or 42, wherein L1 comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)-, [*-OC(O)]p(C1-6 -alkylene)-, [*-NHC(0)]P(CI-6- alkylene)-, [*-C(O)NH]p(C1-6-alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6 -alkylene)-, [*- S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r](C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*- C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p(C1-6-alkylene)-,
[*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-,
[*=C(=N(OH))]p(C1-6-alkylene)-, [*-OC(R25)(R26)O]p(C1-6-alkylene)-, *-(3,4-dihydro-2H- chromen-6-yl)-, (*-)pN(R26)2.p, and [*-C(0)NH](CI-6 -alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25 is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The composition of any one of claims 41 to 43, wherein L1 further comprises at least one additional difunctional moiety via which R2 is attached to either X1 in formula (V) or X2 in formula (V’). The composition of claim 44, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1 further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6 -alkylene group from each other. The composition of any one of claims 41 to 45, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(Ci-6- alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(Ci- 6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6 -alkylene)©-, [*- OC(O)]p(C1-6-alkylene)O-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6-alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6-alkylene), [*-OC(O)]p(C i-6 -alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*-C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27 is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The composition of any one of claims 41 to 46, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(Ci-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(Ci-6- alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(Cw- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*- OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6-alkyltriyl)O- or L1 is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(Ci- 6-alkylene), [*-OC(O)]p(C1-6 -alkylene), [*-NHC(0)]P(CI-6 -alkylene), and [*-C(O)NH]p(C1-6- alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The composition of any one of claims 22 to 47, wherein R2 is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3- alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene), [R4NHC(O)]p(C2-3- alkylene)OP(O)(OR27)O(C1-3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)O(Ci.3-alkylene), [R4C(O)NH]P(C2-3- alkylene)OP(O)(OR27)O(Ci.3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(Ci.3- alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*- OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2 is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and - OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The composition of any one of claims 22 to 48, wherein R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2 is a monoalkylamine moiety. The composition of any one of claims 41 to 49, wherein each R4 is independently a non-cyclic, preferably straight, hydrocarbyl group. The composition of any one of claims 41 to 50, wherein each R4 is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The composition of any one of claims 22 to 51, wherein R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE
(palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2 is a monomyristylamine moiety. The composition of any one of claims 22 to 52, wherein R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2. -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair. The composition of any one of claims 22 to 53, wherein R3 is selected from the group consisting ofH, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2. -NH(CI-3 alkyl), -N(CI-3 alkyl )2. and a member of a targeting pair. The composition of any one of claims 22 to 54, wherein R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair. The composition of any one of claims 22 to 55, wherein the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine-glycine- aspartic acid (RGD) peptide - av[T integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor. The composition of any one of claims 1 to 56, wherein the polymer-conjugated compound has one of the following formulas:
wherein n is 5 to 25;
R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair;
R27 is H or a countercation; and in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(CH2)i2CH3, and in each case -C(O)Ci7H33 refers to the moiety -cw-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The composition of claim 57, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. The composition of claim 57 or 58, wherein R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, C2-6 alkynyl, an antigen, or an antibody. The composition of any one of claims 1 to 59, wherein the polymer-conjugated compound has one of the following formulas:
wherein in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl), in each case -C(O)CI5H3I refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(CfDnCHs, and in each case n is 8, 10, or 14.
61. The composition of any one of claims 1 to 60, wherein the polymer-conjugated compound has one of the following formulas: wherein n is 14, and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); wherein n is 14, and in each case -C14H29 refers to the moiety -(CFDBCFE (myristyl); wherein n is 8, 12, 14, or 16.
62. The composition of any one of claims 1 to 61, wherein the composition is substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units.
63. The composition of any one of claims 1 to 62, wherein water is the main component in the composition and/or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v/v).
64. The composition of any one of claims 1 to 63, wherein the concentration of the nucleic acid in the composition is about 1 mg/1 to about 500 mg/1, such as about 1 mg/1 to about 100 mg/1, about 5 mg/1 to about 100 mg/1, or about 10 mg/1 to about 100 mg/1.
65. The composition of any one of claims 1 to 64, wherein the cationically ionizable lipid comprises a head group which includes at least one tertiary amine moiety.
66. The composition of any one of claims 1 to 65, wherein the cationic or cationically ionizable lipid has the structure of Formula (X) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L10 and L20 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L10 and L20 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;
G1 and G2 are each independently unsubstituted C1-C12 alkylene or C2-12 alkenylene;
G3 is C 1-24 alkylene, C2-24 alkenylene, C3-8 cycloalkylene, or C3-8 cycloalkenylene;
Ra is H or C1-12 alkyl;
R35 and R36 are each independently C6-24 alkyl or C6-24 alkenyl;
R37 is H, OR50, CN, -C(=O)OR40, -OC(=O)R40 or -NR50C(=O)R40;
R40 is C1-12 alkyl;
R50 is H or C1-6 alkyl; and x is 0, 1 or 2.
The composition of any one of claims 1 to 65, wherein the cationic or cationically ionizable lipid has the structure of Formula (XI): wherein each of Ri and R2 is independently R5 or -G1-L1-R5, wherein at least one of Ri and R2 is -G1-L1- each of R3 and R4 is independently selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, aryl, and C3-10 cycloalkyl; each of R5 and Rs is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms; each of Gi and G2 is independently unsubstituted C1-12 alkylene or C2-12 alkenylene; each of Li and L2 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-;
Ra is H or C1-12 alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1 or 2. The composition of any one of claims 1 to 65, wherein the cationic or cationically ionizable lipid comprises 2,3-dioleyloxy-l-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(l- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy- N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof. The composition of any one of claims 1 to 68, wherein the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol % of the total lipid present in the composition. The composition of any one of claims 1 to 69, further comprising one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of a phospholipid and a steroid. The composition of claim 70, wherein the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl -phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3 - phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl- phosphatidylethanolamine (DPyPE).
72. The composition of claim 70 or 71, wherein the phospholipid comprises from about 5 mol % to about 30 mol % of the total lipid present in the composition.
73. The composition of any one of claims 70 to 72, wherein the steroid comprises a sterol such as cholesterol.
74. The composition of any one of claims 70 to 73, wherein the steroid comprises from about 10 mol % to about 60 mol % of the total lipid present in the composition.
75. The composition of any one of claims 70 to 74, wherein the cationic or cationically ionizable lipid comprises from about 20 mol % to about 70 mol % of the total lipid present in the composition; the polymer-conjugated compound comprises from about 0.5 mol % to about 15 mol % of the total lipid present in the composition; the phospholipid comprises from about 5 mol % to about 25 mol % of the total lipid present in the composition; and the steroid comprises from about 20 mol % to about 55 mol % of the total lipid present in the composition.
76. The composition of any one of claims 1 to 75, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the cationic or cationically ionizable lipid, and at least a portion of the polymer-conjugated compound.
77. The composition of claim 76, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof.
78. The composition of claim 76 or 77, wherein the particles comprise at least 50%, preferably at least 75%, more preferably at least 85%, of the nucleic acid present in the composition.
79. The composition of any one of claims 76 to 78, wherein the particles have a size of from about 30 nm to about 500 nm.
80. The composition of any one of claims 1 to 79, wherein the nucleic acid is RNA, preferably mRNA.
81. The composition of claim 80, wherein the RNA (1) comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (y), Nl- methyl-pseudouridine (ml\|/), and 5 -methyl -uridine (m5U); (2) has a coding sequence which is codon-optimized; and/or (3) has a coding sequence whose G/C content is increased compared to the wild-type coding sequence.
82. The composition of claim 80 or 81, wherein the RNA comprises at least one of the following, preferably all of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly -A sequence.
83. The composition of claim 82, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence preferably is an interrupted sequence of A nucleotides.
84. The composition of claim 82 or 83, wherein the 5’ cap is a capl or cap2 structure.
85. The composition of any one of claims 80 to 84, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and/or comprise an epitope for inducing an immune response against an antigen in a subject.
86. The composition of claim 85, wherein the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof.
87. A method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a composition of any one of claims 1 to 86.
88. A method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a composition of any one of claims 1 to 86, wherein the nucleic acid encodes the therapeutic peptide or protein.
89. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of any one of claims 1 to 86, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.
90. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of any one of claims 1 to 86, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
91. The method of any one of claims 87 to 90, wherein the subject is a mammal. The method of claim 91, wherein the mammal is a human. A polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100. The polymer-conjugated compound of claim 93, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or Cus alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl.
95. The polymer-conjugated compound of claim 93 or 94, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cus alkyl.
96. The polymer-conjugated compound of any one of claims 93 to 95, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl.
97. The polymer-conjugated compound of any one of claims 93 to 96, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.
98. The polymer-conjugated compound of any one of claims 93 to 97, wherein Y is -CH2- or
-(CH2)2-.
99. The polymer-conjugated compound of any one of claims 93 to 98, wherein Y is -CH2-.
100. The polymer-conjugated compound of any one of claims 93 to 99, wherein the polymer comprises the following general formula (II): wherein R1 is hydrogen or C1-8 alkyl.
101. The polymer-conjugated compound of any one of claims 93 to 100, wherein z is 2 to 10, such as 2 to 7.
102. The polymer-conjugated compound of any one of claims 93 to 101, wherein z is 2 to 5.
103. The polymer-conjugated compound of any one of claims 93 to 102, wherein z is 2 or 3.
104. The polymer-conjugated compound of any one of claims 93 to 103, wherein z is 2.
105. The polymer-conjugated compound of any one of claims 93 to 104, wherein the polymer comprises the following general formula (III): wherein R1 is hydrogen or Cus alkyl. The polymer-conjugated compound of any one of claims 100 to 105, wherein R1 is hydrogen or methyl. The polymer-conjugated compound of any one of claims 100 to 106, wherein R1 is hydrogen. The polymer-conjugated compound of any one of claims 93 to 107, wherein the polymer comprises the following general formula (IV): The polymer-conjugated compound of any one of claims 93 to 108, wherein n is 5 to 50. The polymer-conjugated compound of any one of claims 93 to 109, wherein n is 5 to 25. The polymer-conjugated compound of any one of claims 93 to 110, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. The polymer-conjugated compound of any one of claims 93 to 111, wherein the one or more hydrophobic chains are located at either the X1 end or the X2 end of the polymer. The polymer-conjugated compound of any one of claims 93 to 112, wherein the one or more hydrophobic chains are non-cyclic, preferably straight, hydrocarbyl groups, more preferably those having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The polymer-conjugated compound of any one of claims 93 to 113, comprising the following general formula (V) or (V’): wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-;
R2 is a moiety comprising the one or more hydrophobic chains;
R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20 is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2.g alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6- membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100. The polymer-conjugated compound of claim 114, wherein:
(i) when X1 is -C(O)- then X2 is -NR1-; (ii) when X1 is -NR1- then X2 is -C(0)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(0)- then X2 is -0-;
(vi) when X1 is -O- then X2 is -C(O)-;
(vii) when X1 is -C(S)- then X2 is -O-;
(viii) when X1 is -O- then X2 is -C(S)-;
(ix) when X1 is -C(O)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl; preferably
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-; or
(vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl. The polymer-conjugated compound of claim 114 or 115, wherein X1 is -C(O)- and X2 is -NR1- , wherein R1 is hydrogen or C1-8 alkyl. The polymer-conjugated compound of any one of claims 114 to 116, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl. The polymer-conjugated compound of any one of claims 114 to 117, wherein X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen. The polymer-conjugated compound of any one of claims 114 to 118, wherein Y is -CH2- or -(CH2)2-. The polymer-conjugated compound of any one of claims 114 to 119, wherein Y is -CH2-. The polymer-conjugated compound of any one of claims 114 to 120, comprising the following general formula (VI) or (VI’): wherein R1 is hydrogen or C1-8 alkyl. The polymer-conjugated compound of any one of claims 114 to 121, wherein z is 2 to 10, such as 2 to 7. The polymer-conjugated compound of any one of claims 114 to 122, wherein z is 2 to 5. The polymer-conjugated compound of any one of claims 114 to 123, wherein z is 2 or 3. The polymer-conjugated compound of any one of claims 114 to 124, wherein z is 2. The polymer-conjugated compound of any one of claims 114 to 125, comprising the following general formula (VII) or (VIT): wherein R1 is hydrogen or Cus alkyl. The polymer-conjugated compound of any one of claims 121 to 126, wherein R1 is hydrogen or methyl. The polymer-conjugated compound of any one of claims 121 to 127, wherein R1 is hydrogen. The polymer-conjugated compound of any one of claims 114 to 128, comprising the following general formula (VIII) or (VIIT): The polymer-conjugated compound of any one of claims 114 to 129, wherein n is 5 to 50. The polymer-conjugated compound of any one of claims 114 to 130, wherein n is 5 to 25. The polymer-conjugated compound of any one of claims 114 to 131, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. The polymer-conjugated compound of any one of claims 114 to 132, wherein R2 is R4 or -L1(R4)P, wherein each R4 is independently a hydrophobic chain, such as a hydrocarbyl group; L1 is a linker; and p is 1 or 2. The polymer-conjugated compound of claim 133, wherein L1 comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and/or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functional moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties. The polymer-conjugated compound of claim 133 or 134, wherein L1 comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6 -alkylene)-, [*-OC(O)]p(C1-6- alkylene)-, [*-NHC(O)]P(CI-6 -alkylene)-, [*-C(O)NH]P(CI-6 -alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6-alkylene)-, [*-S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r](C1-6-alkylene)-, [*- C(OR25)2O]p(C1-6-alkylene)-, [*-C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)- N=)C(R25)-]p(C1-6-alkylene)-, [*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-,
[*-N(R26)N(R26)]p(C1-6-alkylene)-, [*=C(=N(0H))]p(C1-6-alkylene)-, [*-OC(R25)(R26)O]P(C1-6- alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-)pN(R26)2-p, and [*-C(0)NH](C1-6-alkyltriyl)- , wherein * represents the attachment point to R4; p is 1 or 2; C1-6 -alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25 is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, - CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 133 to 135, wherein L1 further comprises at least one additional difunctional moiety, via which R2 is attached to either X1 in formula (V) or X2 in formula (V’). The polymer-conjugated compound of claim 136, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1 further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6-alkylene group from each other. The polymer-conjugated compound of any one of claims 133 to 137, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*- C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)- , [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*- C(0)0]p(C1-6-alkylene)0-, [*-0C(0)]p(C1-6-alkylene)0-, (*-)pN(R26)2.p, and [*-C(O)NH](C1-6- alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C 1-6 -alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(0)]P(CI-6 -alkylene), and [*- C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27 is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation; 3,4-dihydro-2H-chromen- 6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 133 to 138, wherein L1 is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*- C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)- , [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(0)]P(CI-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(0)NH]P(CI-6- alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*- C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6- alkyltriyl)O- or L1 is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C 1-6 -alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(0)]P(CI-6- alkylene), and [*-C(O)NH]p(C 1-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 114 to 139, wherein R2 is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3- alkylene)-OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O- (C1-3-alkylene), [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)NH-,
[R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4C(O)NH]p(C2-3- alkylene)OP(O)(OR27)O(C1-3-alkylene), [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)O(C1-3- alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(C1-3-alkylene)C(O)-, (2-R4-3,4- dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2 is (R4)(R26)N-, wherein p is 1 or 2; the C2-3- alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26 is selected from the group consisting of H and C1-6 alkyl; R27 is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 114 to 140, wherein R2 is selected from a phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2 is a monoalkylamine moiety. The polymer-conjugated compound of any one of claims 133 to 141, wherein each R4 is independently a non-cyclic, preferably straight, hydrocarbyl group. The polymer-conjugated compound of any one of claims 133 to 142, wherein each R4 is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The polymer-conjugated compound of any one of claims 114 to 143, wherein R2 is selected from DSPE (distearoylphosphatidylethanolamine), DPPE
(dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), or POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2 is a monomyristylamine moiety. The polymer-conjugated compound of any one of claims 114 to 144, wherein R3 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. a sugar, an amino acid, a peptide, and a member of a targeting pair. The polymer-conjugated compound of any one of claims 114 to 145, wherein R3 is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21 is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 alkyl and 3- to 6- membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22 and R23 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22 and R23 may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. and a member of a targeting pair. The polymer-conjugated compound of any one of claims 114 to 146, wherein R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), and -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair. The polymer-conjugated compound of any one of claims 114 to 147, wherein the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said antigen; biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide - avp3 integrin; asparagine-glycine- arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor. The polymer-conjugated compound of any one of claims 93 to 148, having one of the following formulas: wherein n is 5 to 25;
R3 is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair;
R27 is H or a countercation; and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)I2CH3 (myristoyl), in each case -CUHM refers to the moiety -(CIDBCHS (myristyl), in each case -C13H27 refers to the moiety -(CThjnClT, and in each case -C(O)CI7H33 refers to the moiety -cw-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The polymer-conjugated compound of claim 149, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. The polymer-conjugated compound of claim 149 or 150, wherein R3 is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, and C2-6 alkynyl. The polymer-conjugated compound of any one of claims 93 to 151, having one of the following formulas:
wherein in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CI5H3I refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(ClDnCTL, and in each case n is 8, 10, 12, 14, or 16. 153. The polymer-conjugated compound of any one of claims 93 to 152, having one of the following formulas: wherein n is 14, and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); wherein n is 14, and in each case -C14H29 refers to the moiety -(CIDBCIT (myristyl); wherein n is 8, 12, 14, or 16. A conjugate of (a) the polymer-conjugated compound of any one of claims 93 to 153 containing a member of a targeting pair; and (b) a compound comprising the other member of the targeting pair. The conjugate of claim 154, wherein the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (such as an antigen or epitope), or an antibody. The conjugate of claim 154 or 155, having one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen. The conjugate of claim 156, having one of the following formulas: wherein ml is 2, 3, or 4, preferably 2; and m2 is 2, 3, or 4, preferably 2; wherein each of ml and m2 is independently 1, 2, or 3, preferably ml is 1 is and m2 is 2 or ml is 2 and m2 is 1. The conjugate of any one of claims 154 to 157, having one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen. The conjugate of claim 154, wherein the polymer-conjugated compound containing a member of a targeting pair has the following formula: wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)i6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen; the compound comprising the other member of the targeting pair is a compound comprising (i) an antibody specific for said antigen if Pept is said antigen; or (ii) an antigen if Pept is an antibody specific for said antigen; and the polymer-conjugated compound containing a member of a targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) said antibody specific for said antigen and (2) said antigen.
EP23753840.0A 2022-08-01 2023-08-01 Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions Pending EP4565275A1 (en)

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