WO2017117528A1 - Lipids and lipid nanoparticle formulations for delivery of nucleic acids - Google Patents

Lipids and lipid nanoparticle formulations for delivery of nucleic acids Download PDF

Info

Publication number
WO2017117528A1
WO2017117528A1 PCT/US2016/069491 US2016069491W WO2017117528A1 WO 2017117528 A1 WO2017117528 A1 WO 2017117528A1 US 2016069491 W US2016069491 W US 2016069491W WO 2017117528 A1 WO2017117528 A1 WO 2017117528A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
independently
occurrence
integer
composition
Prior art date
Application number
PCT/US2016/069491
Other languages
French (fr)
Inventor
Steven M. Ansell
Xinyao Du
Original Assignee
Acuitas Therapeutics, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Acuitas Therapeutics, Inc. filed Critical Acuitas Therapeutics, Inc.
Priority to EP16829386.8A priority Critical patent/EP3397613A1/en
Priority to US16/067,538 priority patent/US20190022247A1/en
Publication of WO2017117528A1 publication Critical patent/WO2017117528A1/en

Links

Classifications

    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K47/54—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 organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons

Definitions

  • the present invention generally relates to novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g. oligonucleotides, messenger RNA) both in vitro and in vivo.
  • lipid components such as neutral lipids, cholesterol and polymer conjugated lipids
  • oligonucleotides e.g. oligonucleotides, messenger RNA
  • nucleic acid based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism in order to realize this potential.
  • Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense
  • nucleic acids such as mRNA or plasmids
  • mRNA or plasmids can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme.
  • the therapeutic applications of translatable nucleotide delivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether or not indigenous to the system.
  • the expression products of the nucleic acid can augment existing levels of protein, replace missing or non-functional versions of a protein, or introduce new protein and associated functionality in a cell or organism.
  • nucleic acids such as miRNA inhibitors
  • miRNA inhibitors can be used to effect expression of specific cellular products that are regulated by miRNA as would be useful in the treatment of, for example, diseases related to deficiency of protein or enzyme.
  • the therapeutic applications of miRNA inhibition are extremely broad as constructs can be synthesized to inhibit one or more miRNA that would in turn regulate the expression of mRNA products.
  • the inhibition of endogenous miRNA can augment its downstream target endogenous protein expression and restore proper function in a cell or organism as a means to treat disease associated to a specific miRNA or a group of miRNA.
  • nucleic acids can down-regulate intracellular levels of specific mRNA and, as a result, down-regulate the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA.
  • RNA interference RNA interference
  • the therapeutic applications of antisense oligonucleotide and RNAi are also extremely broad, since oligonucleotide constructs can be synthesized with any nucleotide sequence directed against a target mRNA.
  • Targets may include mRNAs from normal cells, mRNAs associated with disease-states, such as cancer, and mRNAs of infectious agents, such as viruses.
  • antisense oligonucleotide constructs have shown the ability to specifically down-regulate target proteins through degradation of the cognate mRNA in both in vitro and in vivo models.
  • antisense oligonucleotide constructs are currently being evaluated in clinical studies.
  • RNAs are susceptible to nuclease digestion in plasma.
  • free RNAs have limited ability to gain access to the intracellular compartment where the relevant translation machinery resides.
  • Lipid nanoparticles formed from cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides.
  • these lipid nanoparticles would provide optimal drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid.
  • these lipid-nucleic acid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.
  • the present invention provides lipid compounds, including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including for example, all sterols) and/or their analogs, and/or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents.
  • the lipid nanoparticles are used to deliver nucleic acids such as antisense and/or messenger RNA. Methods for use of such lipid nanoparticles for treatment of various diseases or conditions, such as those caused by infectious entities and/or insufficiency of a protein, are also provided.
  • R, R 1 , R 2 , L, X, L, G 1 , G 2 , Z, a 1 , a 2 and n are independently as defined herein for each of structures (I) and (II).
  • compositions comprising one or more of the foregoing compounds of structure (I) or (II) and a therapeutic agent are also provided.
  • the pharmaceutical compositions further comprise one or more components selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.
  • Such compositions are useful for formation of lipid nanoparticles for the delivery of the therapeutic agent.
  • the present invention provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition of lipid nanoparticles comprising the compound of structure (I) or (II) and a therapeutic agent and delivering the composition to the patient.
  • Figure 1 shows time course of luciferase expression in mouse liver.
  • Figure 2 illustrates the calculation of pK a for MC3 as a representative example relevant to the disclosed lipids.
  • the present invention is based, in part, upon the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of an active or therapeutic agent such as a nucleic acid into a cell of a mammal.
  • embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo, resulting in a significant increase in the therapeutic index as compared to nucleic acid-lipid nanoparticle compositions previously described.
  • the present invention provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and/or other oligonucleotides.
  • these improved lipid nanoparticle compositions are useful for expression of protein encoded by mRNA.
  • these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA.
  • these improved lipid nanoparticle compositions are useful for down-regulating (e.g., silencing) the protein levels and/or mRNA levels of target genes.
  • the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes.
  • the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antigen or antibody.
  • lipid nanoparticles and compositions of the present invention may be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, both in vitro and in vivo. Accordingly, embodiments of the present invention provide methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel cationic lipids described herein.
  • embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc.
  • nucleic acids including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc.
  • the lipid nanoparticles and compositions of the present invention may be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or inhibit processes that terminate expression of mRNA (e.g., miRNA inhibitors).
  • a desired protein e.g., a messenger RNA or plasmid encoding the desired protein
  • miRNA inhibitors e.g., miRNA inhibitors
  • the lipid nanoparticles and compositions of the present invention may be used to decrease the expression of target genes and proteins both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)).
  • a nucleic acid that reduces target gene expression e.g., an antisense oligonucleotide or small interfering RNA (siRNA)
  • the lipid nanoparticles and compositions of the present invention may also be used for co- delivery of different nucleic acids (e.g.
  • mRNA and plasmid DNA separately or in combination, such as may be useful to provide an effect requiring colocalization of different nucleic acids (e.g. mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome).
  • nucleic acids e.g. mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome.
  • Nucleic acids for use with this invention may be prepared according to any available technique.
  • the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA.
  • In vitro transcription describes a process of template-directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g. including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest.
  • an upstream bacteriophage promoter sequence e.g. including but not limited to that from the T7, T3 and SP6 coliphage
  • Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L. and Conn, G.L., General protocols for preparation of plasmid DNA template and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012)
  • RNA polymerase adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts.
  • rNTPs ribonucleoside triphosphates
  • In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs.
  • the methodology for in vitro transcription of mRNA is well known in the art. (see, e.g.
  • the desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including
  • RNA transcripts unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.).
  • Techniques for the isolation of the mRNA transcripts are well known in the art. Well known procedures include phenol/chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.
  • RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012).
  • Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).
  • RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation.
  • RNA impurities include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self- complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses.
  • dsRNA double-stranded RNA
  • HPLC purification eliminates immune activation and improves translation of nucleoside- modified, protein-encoding mRNA, Nucl Acid Res, v.
  • Endogenous eukaryotic mRNA typically contain a cap structure on the 5'- end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts.
  • CBP mRNA Cap Binding Protein
  • the 5 '-cap contains a 5 '-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide.
  • the conjugated guanine nucleotide is methylated at the N7 position.
  • modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'-hydroxyl group.
  • 5 '-capping of synthetic mRNA can be performed co- transcriptionally with chemical cap analogs (i.e. capping during in vitro transcription).
  • the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3'-0-methyl group.
  • ARC A Anti -Reverse Cap Analog
  • the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability.
  • synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5 '-cap structure that more closely mimics, either structurally or functionally, the endogenous 5 '-cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping. Numerous synthetic 5 '-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see e.g.
  • poly-A tail On the 3 '-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3 ' end of the transcript is cleaved to free a 3' hydroxyl to which poly- A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation.
  • the poly-A tail has been extensively shown to enhance both translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J.
  • Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase.
  • the first case allows in vitro transcription of mRNA with poly (A) tails of defined length, depending on the size of the poly (T) tract, but requires additional manipulation of the template.
  • the latter case involves the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 'termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length.
  • 5'- capping and 3 '-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.
  • modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see e.g. Kariko, K. And Weissman, D.
  • modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art.
  • nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA (see e.g. US2012/0251618).
  • In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.
  • mRNA which can be modified to provide benefit in terms of translatability and stability
  • 5' and 3' untranslated regions include the 5' and 3' untranslated regions (UTR). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA (see e.g. Pardi, N.,
  • RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).
  • other nucleic acid payloads may be used for this invention.
  • methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g. Gait, M. J.
  • plasmid DNA preparation for use with this invention commonly utilizes but is not limited to expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest.
  • a gene in the plasmid of interest that encodes resistance to a particular antibiotic penicillin, kanamycin, etc.
  • penicillin, kanamycin, etc. allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures.
  • Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g. Heilig, J., Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology.
  • Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents.
  • lipid nanoparticles and compositions comprising the same and their use to deliver active (e.g. therapeutic agents), such as nucleic acids, to modulate gene and protein expression, are described in further detail below.
  • active e.g. therapeutic agents
  • nucleic acids such as nucleic acids
  • a test sample e.g. a sample of cells in culture expressing the desired protein
  • a test mammal e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or a non-human primate (e.g., monkey) model
  • a nucleic acid e.g. nucleic acid in combination with a lipid of the present invention.
  • expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g.
  • a sample of cells in culture expressing the desired protein or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model) that is not contacted with or administered the nucleic acid.
  • a control mammal e.g., a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model
  • the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1.0.
  • inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1, for example, about 1.1, 1.5, 2.0. 5.0 or 10.0.
  • inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected.
  • the phrase "inhibiting expression of a target gene” refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene.
  • a test sample e.g. a sample of cells in culture expressing the target gene
  • a test mammal e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or a non-human primate (e.g. monkey) model
  • a nucleic acid that silences, reduces, or inhibits expression of the target gene.
  • Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g.
  • a sample of cells in culture expressing the target gene or a control mammal (e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model) that is not contacted with or administered the nucleic acid.
  • a control mammal e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model
  • the expression of the target gene in a control sample or a control mammal may be assigned a value of 100%.
  • silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid.
  • Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g. an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid.
  • An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid.
  • an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater.
  • Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%), 15%), 10%), 5%), or 0%.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors.
  • RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl
  • nucleic acids Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.
  • Nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
  • Gene product refers to a product of a gene such as an RNA transcript or a polypeptide.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) "derived lipids” such as steroids.
  • a "steroid” is a com ound comprising the following carbon skeleton:
  • Non-limiting examples of steroids include cholesterol, and the like.
  • a “cationic lipid” refers to a lipid capable of being positively charged.
  • Exemplary cationic lipids include one or more amine group(s) which bears the positive charge.
  • Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH.
  • the ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) critical to the intracellular delivery of nucleic acids.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion.
  • An example of a polymer conjugated lipid is a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
  • 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.
  • lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-s «-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-5 «-glycero-3- phosphocholine (DPPC), l,2-Dimyristoyl-s «-glycero-3-phosphocholine (DMPC), 1- Palmitoyl-2-oleoyl-s «-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), phophatidylethanol amines such as l,2-Dioleoyl-s «-glycero-3- phosphoethanolamine (DOPE), s
  • DOPE phop
  • charged lipid refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g. pH ⁇ 3 to pH ⁇ 9.
  • Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl
  • dimethylaminopropanes ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Choi).
  • lipid nanoparticle refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of structure (I) or (II) or other specified cationic lipids.
  • lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like).
  • an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA)
  • a target site of interest e.g., cell, tissue, organ, tumor, and the like.
  • the lipid nanoparticles of the invention comprise a nucleic acid.
  • Such lipid nanoparticles typically comprise a compound of structure (I) or (II) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.
  • the active agent or therapeutic agent such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 n
  • nucleic acids when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.
  • Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub. Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • lipid encapsulated refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both.
  • a nucleic acid e.g., mRNA
  • the nucleic acid is fully encapsulated in the lipid nanoparticle.
  • aqueous solution refers to a composition comprising water.
  • “Serum-stable” in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA.
  • Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.
  • Systemic delivery refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism.
  • Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body.
  • Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.
  • Local delivery refers to delivery of an active agent directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.
  • Amino refers to the - H 2 radical.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double (alkenyl) and/or triple bonds (alkynyl)), having, for example, from one to twenty-four carbon atoms (Ci-C 24 alkyl), four to twenty carbon atoms (C 4 -C 20 alkyl), six to sixteen carbon atoms (C 6 -C 16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl),one to twelve carbon atoms (Ci-Ci 2 alkyl), one to eight carbon atoms (Ci-C 8 alkyl) or one to six carbon atoms (Ci-C 6 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl,
  • Alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (Ci-C 24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene),one to twelve carbon atoms (Ci-Ci 2 alkylene), one to eight carbon atoms (Ci- C 8 alkylene), one to six carbon atoms (Ci-C 6 alkylene), two to four carbon atoms (C 2 -C 4 alkylene), one to two carbon atoms (C 1 -C 2 alkylene), e.g., methylene, ethylene, propylene, «-butylene, ethenylene, propenylene, «-
  • the alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond.
  • the points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
  • an alkylene chain may be optionally substituted.
  • Alkoxy refers to a radical of the formula -OR a where R a is an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.
  • Alkylaminyl refers to a radical of the formula -NHR a or - R a R a where each R a is, independently, an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkylaminyl group is optionally substituted.
  • Aryl refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring.
  • the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
  • “Arylene” is a divalent aryl radical. Unless stated otherwise specifically in the specification, the term “arylene,” “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant to include aryl radicals that are optionally substituted.
  • a "carbocyclic ring” is a ring wherein each ring atom is carbon.
  • Carbocyclic rings may saturated or unsaturated, including aromatic rings. Unless stated otherwise specifically in the specification, a carbocylic group is optionally substituted.
  • Cycloalkyl or “carbocyclic ring” refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond.
  • Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl,
  • Cycloalkylene is a divalent cycloalkyl radical. Unless otherwise stated specifically in the specification, cycloalkyl and cycloalkylene groups are optionally substituted.
  • Heterocyclyl or “heterocyclic ring” refers to a stable 3- to 18-membered (e.g., 5, 6 or 7-membered) non-aromatic ring radical, wherein at least one ring atom is a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur, and the remaining ring atoms are selected from the group consisting of carbon nitrogen, oxygen and sulfur.
  • the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated.
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,
  • heterocyclene is a divalent heterocycle radical. Unless stated otherwise specifically in the specification, heterocyclyl and heterocylene groups are optionally substituted.
  • Heteroaryl refers to a 5- to 14-membered ring system radical comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring.
  • the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized.
  • Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[£][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl,
  • fused refers to any ring structure described herein which is fused to an existing ring structure in the compounds of the invention.
  • the fused ring is a heterocyclyl ring or a heteroaryl ring
  • any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.
  • Halo or halogen refers to bromo, chloro, fluoro or iodo.
  • Hydroxylalkyl is an alkyl group comprising at least one hydroxyl substituent. Unless stated otherwise specifically in the specification, hydroxylalkyl groups are optionally substituted.
  • Aminoalkyl is an alkyl group comprising at least one amino substituent. Unless stated otherwise specifically in the specification, aminoalkyl groups are optionally substituted.
  • Alkylaminylalkyl is an alkyl group comprising at least one
  • alkylaminyl substituent Unless stated otherwise specifically in the specification, alkylaminylalkyl groups are optionally substituted.
  • Alkoxylalkyl is an alkyl group comprising at least one alkoxy substituent. Unless stated otherwise specifically in the specification, alkoxyalkyl groups are optionally substituted.
  • substituted used herein means any of the above groups (e.g. alkyl, alkylene, alkene, alkenylene, alkyne, alkynylene, alkoxy, alkylaminyl, aryl, arylene, carbocyclic, cycloalkyl, cycloalkylene, heterocyclyl, heterocylene, heteroaryl, heteroaryl ene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl,
  • the substituent is a C 1 -C 12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR ). In other
  • the substituent is a carboxyl group. In other embodiments, the substituent is an amine group(-NR R ).
  • a "polar functional group” is a functional group having at least one polarized bond.
  • Polar functional groups often comprise at least one heteroatom (e.g., O, N or S).
  • Polar functional are also typically hydrophilic and tend to increase the solubility of the compound in water (as compared to solubility of the same compound with a non-polar functional group).
  • Optional or “optionally substituted” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
  • optionally substituted alkyl means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.
  • Prodrug is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention.
  • prodrug refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention.
  • Prodrugs are typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)).
  • prodrugs are provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of a compound of the invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the invention.
  • Prodrugs include compounds of the invention wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the invention is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group,
  • prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds of the invention and the like.
  • Embodiments of the invention disclosed herein are also meant to encompass all pharmaceutically acceptable compounds of the compound of structure (I) or (II) being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number.
  • isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2 H, 3 H, U C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 31 P, 32 P, 35 S, 18 F, 36 C1, 123 I, and 125 I, respectively.
  • radiolabeled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action.
  • Certain isotopically-labelled compounds of structure (I) or (II), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies.
  • the radioactive isotopes tritium, i.e., 3 H, and carbon-14, i.e., 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • substitution with heavier isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
  • Isotopically-labeled compounds of structure (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
  • Embodiments of the invention disclosed herein are also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes.
  • the invention includes compounds produced by a process comprising administering a compound of this invention to a mammal for a period of time sufficient to yield a metabolic product thereof.
  • Such products are typically identified by administering a radiolabeled compound of the invention in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.
  • “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • “Mammal” includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.
  • “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic
  • naphthalene-l,5-disulfonic acid naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ⁇ -toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol,
  • 2-diethylaminoethanol dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, tri ethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine,
  • solvate refers to an aggregate that comprises one or more molecules of a compound of the invention with one or more molecules of solvent.
  • the solvent may be water, in which case the solvate may be a hydrate.
  • the solvent may be an organic solvent.
  • the compounds of the present invention may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms.
  • the compound of the invention may be true solvates, while in other cases, the compound of the invention may merely retain adventitious water or be a mixture of water plus some adventitious solvent.
  • a “pharmaceutical composition” refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans.
  • a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.
  • Effective amount refers to that amount of a compound of the invention which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human.
  • the amount of a lipid nanoparticle of the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • Treating covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes:
  • disease and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.
  • the compounds of the invention may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids.
  • Embodiments of the present invention are meant to include all such possible isomers, as well as their racemic and optically pure forms.
  • Optically active (+) and (-), (R)- and (5)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.
  • Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • stereoisomer refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable.
  • the present invention contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.
  • a “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule.
  • the present invention includes tautomers of any said compounds.
  • the invention provides novel lipid compounds which are capable of combining with other lipid components such as neutral lipids, charged lipids, steroids and/or polymer conjugated-lipids to form lipid nanoparticles with
  • oligonucleotides are lipid nanoparticles shield oligonucleotides from degradation in the serum and provide for effective delivery of oligonucleotides to cells in vitro and in vivo.
  • the compounds have the following structure (I):
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
  • R a is, at each occurrence, independently H, C 1 -C 12 alkyl, C 1 -C 12 hydroxylalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminylalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;
  • R is, at each occurrence, independently either: (a) H or C 1 -C 12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 1 and R 2 have, at each occurrence, the following structure, respectively:
  • a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1 ; c 1 and c 2 are, at each occurrence, independently an integer from 5 to 10; d 1 and d 2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
  • alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
  • X is CH.
  • the sum of a ⁇ ⁇ + c 1 or the sum of a 2 + b 2 + c 2 is an integer from 12 to 26.
  • a 1 and a 2 are independently an integer from 3 to 10.
  • a 1 and a 2 are independently an integer from 4 to 9.
  • b 1 and b 2 are 0. In different embodiments, b 1 and b 2 are 1.
  • c 1 , c 2 , d 1 and d 2 are independently an integer from 6 to 8.
  • c 1 and c 2 are, at each occurrence, independently an integer from 6 to 10, and d 1 and d 2 are, at each occurrence, independently an integer from 6 to 10. In other embodiments of structure (I), c 1 and c 2 are, at each occurrence, independently an integer from 5 to 9, and d 1 and d 2 are, at each occurrence,
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is i . In other embodiments, Z is alkyl.
  • R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • each R is H.
  • at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 1 and R 2 independently have one of the following structures:
  • the compound has one of the following structures:
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
  • R a is, at each occurrence, independently H, C 1 -C 12 alkyl, C 1 -C 12 hydroxylalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminylalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;
  • R is, at each occurrence, independently either: (a) H or C 1 -C 12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 1 and R 2 have, at each occurrence, the following structure, respectively:
  • R' is, at each occurrence, independently H or C1-C12 alkyl; a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1 ;
  • c 1 and c 2 are, at each occurrence, independently an integer from 2 to 12; d 1 and d 2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
  • a , a , c , c , d and d are selected such that the sum of a ⁇ +d 1 is an integer from 18 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
  • G 1 and G 2 are each independently
  • X is CH.
  • the sum of a ⁇ +d 1 is an integer from 20 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 30.
  • the sum of a ⁇ +d 1 is an integer from 20 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 20 to 30.
  • a , a , c 1 , c 2 , d 1 and d 2 are selected such that the sum of a ⁇ +d 1 is an integer from 18 to 28, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 28,
  • a 1 and a 2 are independently an integer from 3 to 10, for example an integer from 4 to 9.
  • b 1 and b 2 are 0.
  • b 1 and b 2 are 1.
  • c 1 , c 2 , d 1 and d 2 are independently an integer from 6 to 8.
  • Z is alkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1.
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is i . In other embodiments, Z is alkyl.
  • R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • each R is H.
  • at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • each R' is H.
  • the sum of a ⁇ +d 1 is an integer from 20 to 25, and the sum of a 2 +c 2 +d 2 is an integer from 20 to 25.
  • R 1 and R 2 independently have one of the following structures:
  • the compound has one of the following structures:
  • n is i . In other of the foregoing embodiments, n is greater than 1.
  • Z is a mono- or polyvalent moiety comprising at least one polar functional group.
  • Z is a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is a polyvalent moiety comprising at least one polar functional group. In more of any of the foregoing embodiments, the polar functional group is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl, heterocyclyl or heteroaryl functional group.
  • Z is hydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl, alkylaminylalkyl, heterocyclyl or heterocyclylalkyl.
  • R 5 and R 6 are independently H or Ci-C 6 alkyl
  • R 7 and R 8 are independently H or Ci-C 6 alkyl or R 7 and R 8 , together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring;
  • x is an integer from 0 to 6.
  • Z has the following structure:
  • R 5 and R 6 are independently H or Ci-C 6 alkyl
  • R 7 and R 8 are independently H or Ci-C 6 alkyl or R 7 and R 8 , together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring;
  • x is an integer from 0 to 6.
  • Z has the following structure:
  • R 5 and R 6 are independently H or Ci-C 6 alkyl
  • R 7 and R 8 are independently H or Ci-C 6 alkyl or R 7 and R 8 , together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring;
  • x is an integer from 0 to 6.
  • Z is hydroxylalkyl, cyanoalkyl or an alkyl substituted with one or more ester or amide groups.
  • Z has one of the following structures:
  • Z-L has one of the following structures:
  • Z-L has one of the following structures:
  • X is CH and Z-L has one of the following structures:
  • the compound has one of the structures set forth in Table 1 below.
  • the invention does not include any of the compounds illustrated in Table 1.
  • any embodiment of the compounds of structure (I) or (II), as set forth above, and any specific substituent and/or variable in the compound structure (I) or (II), as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of compounds of structure (I) or (II) to form embodiments of the inventions not specifically set forth above.
  • compositions comprising any one or more of the compounds of structure (I) or (II) and a therapeutic agent are provided.
  • the compositions comprise any of the compounds of structure (I) or (II) and a therapeutic agent and one or more excipient selected from neutral lipids, steroids and polymer conjugated lipids.
  • excipients and/or carriers are also included in various embodiments of the compositions.
  • the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2: 1 to about 8: 1.
  • compositions further comprise a steroid or steroid analogue.
  • the steroid or steroid analogue is cholesterol.
  • the molar ratio of the compound to cholesterol ranges from about 5: 1 to 1 : 1.
  • the polymer conjugated lipid is a pegylated lipid.
  • some embodiments include a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEGS-DAG) such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(co- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate such as Q-methoxy(polyethoxy)ethyl-N-(2,3- di(
  • the composition comprises a pegylated lipid having the following structure (III):
  • R 3 and R 4 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w has a mean value ranging from 30 to 60.
  • R 3 and R 4 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • the average w is about 45. In other embodiments, the average w ranges from 42 to 55. For example, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, w is about 49.
  • the therapeutic agent comprises a nucleic acid.
  • the nucleic acid is selected from antisense, plasmid DNA, and messenger RNA.
  • the invention is directed to a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing any of the foregoing compositions and administering the composition to the patient.
  • compositions of the present invention comprise a compound of structure (I) or (II) and one or more pharmaceutically acceptable carrier, diluent or excipient.
  • the compound of structure (I) or (II) is present in the composition in an amount which is effective to form a lipid nanoparticle and deliver the therapeutic agent, e.g., for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one skilled in the art.
  • compositions of the invention can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • the pharmaceutical compositions of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques.
  • Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • the composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this invention.
  • a pharmaceutical composition of the invention may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose,
  • microcrystalline cellulose, gum tragacanth or gelatin excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like
  • lubricants such as magnesium stearate or Sterotex
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl salicylate or orange flavoring
  • a coloring agent e.g., pepper
  • the pharmaceutical composition When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • a liquid carrier such as polyethylene glycol or oil.
  • the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions of the invention may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass
  • a liquid pharmaceutical composition of the invention intended for either parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage will be obtained.
  • the pharmaceutical composition of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • the pharmaceutical composition of the invention may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the pharmaceutical composition of the invention may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the pharmaceutical composition of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby assists in the delivery of the compound.
  • Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, or a protein.
  • the pharmaceutical composition of the invention may consist of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.
  • compositions of the invention may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the invention with sterile, distilled water or other carrier so as to form a solution.
  • a surfactant may be added to facilitate the formation of a
  • Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or
  • compositions of the invention are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • compositions of the invention may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents.
  • combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of the composition of the invention and each active agent in its own separate pharmaceutical dosage formulation.
  • a composition of the invention and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
  • the compounds of the invention and one or more additional active agents can be used.
  • combination therapy is understood to include all these regimens.
  • Suitable protecting groups include hydroxy, amino, mercapto and carboxylic acid.
  • Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like.
  • Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like.
  • Suitable protecting groups for mercapto include -C(0)-R" (where R" is alkyl, aryl or arylalkyl), /?-methoxybenzyl, trityl and the like.
  • Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters.
  • Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley.
  • the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.
  • the disclosed compounds can be prepared according to the following General Reaction Scheme I and methods analogous to those disclosed in PCT Pub. No. WO 2013/086322, the full disclosure of which is hereby incorporated by reference in its entirety. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of structure (I) or (II) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed.
  • starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybndge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this invention.
  • Method A provides a method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "G"), wherein R, R 1 , a 1 , a 2 and Z are as defined herein, and PG is an alcohol protecting group such as tetrahydropyran.
  • Compounds of structure A are purchased or prepared according to methods known in the art. Reaction of A with ethyl formate B under Grignard conditions yields alcohol C, which can then be coupled with acid D under standard conditions to yield E. Removal of the protecting group followed by coupling with acid F yields G.
  • Method B provides an alternative method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "P"), wherein R, R 1 , a 1 , a 2 and Z are as defined herein and PG is an alcohol protecting group such as tetrahydropyran.
  • Compounds of structure H are purchased or prepared according to methods known in the art. The hydroxyl group of Compound H is protected using methods known in the art (e.g. pyndinium p-toluenesulfonate, dihydropyran) to yield I.
  • Reaction of I with ethyl formate J under Grignard conditions e.g. with Mg, I 2 ) yields alcohol K.
  • the hydroxyl group of compound K can be oxidized (e.g. with pyridinium chlorochr ornate) and the protecting groups removed (e.g. with pyridinium p-toluenesulfonate) to yield compound L.
  • the free hydroxyl groups of L are then coupled with acid M under standard ester coupling conditions to yield N.
  • the carbonyl of N is then reduced using methods known in the art (e.g. NaBH 4 ) followed by coupling with acid O (e.g. with DMAP, EDC C1) to yield the desired product P.
  • Method C provides another alternative method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "V"), wherein R, R 1 , a 1 , a 2 and Z are as defined herein.
  • Compounds of structure Q are purchased or prepared according to methods known in the art. Compound Q is used to form R under appropriate conditions (e.g. diethyl acetone dicarboxylate, EtONa).
  • Alcohol S is then coupled to R using standard conditions (e.g. DMAP, EDC HC1) to yield T.
  • standard conditions e.g. DMAP, EDC HC1
  • the carbonyl of T is reduced (e.g. with NaBH 4 ) followed by coupling with acid U (e.g. with DMAP, EDC HC1) to yield the desired product V.
  • Lipid nanoparticles were prepared at a total lipid to mRNA weight ratio of approximately 10: 1 to 30: 1. Briefly, the mRNA was diluted to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :5 to 1 :3 (vol/vol) with total flow rates above 15 mL/min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 ⁇ pore sterile filter. Lipid nanoparticle particle size was approximately 55-95 nm diameter, and in some instances
  • Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol at a molar ratio of at a molar ratio of approximately
  • liver tissue was systemically administered by tail vein injection and animals euthanized at specific time points (1, 2, 4, 8 and 24 hrs) post-administration.
  • Liver and spleen were collected in pre-weighed tubes, weights determined, immediately snap frozen in liquid nitrogen and stored at -80°C until processing for analysis.
  • Liver tissues were homogenized with the FastPrep24 instrument (MP Biomedicals) at 2 x 6.0 m/s for 15 seconds. Homogenate was incubated at room temperature for 5 minutes prior to a 1 :4 dilution in GLB and assessed using SteadyGlo Luciferase assay system (Promega). Specifically, 50 ⁇ ⁇ of diluted tissue homogenate was reacted with 50 ⁇ ⁇ of SteadyGlo substrate, shaken for 10 seconds followed by 5 minute incubation and then quantitated using a CentroXS 3 LB 960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was determined by using the BCA protein assay kit (Pierce, Rockford IL).
  • Relative luminescence units were then normalized to total ⁇ g protein assayed.
  • RLU Relative luminescence units
  • a standard curve was generated with QuantiLum Recombinant Luciferase (Promega). Based on the data provided in Figure 1, the four-hour time point was chosen for efficacy evaluation of the lipid formulations (see Example 3).
  • the FLuc mRNA (L-6107) from Trilink Biotechnologies will express a luciferase protein, originally isolated from the firefly, Photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methylcytidine and pseudouridine.
  • the pK a of formulated cationic lipids is correlated with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)).
  • the preferred range of pK a is ⁇ 5 to ⁇ 7.
  • the pK a of each cationic lipid was determined in lipid nanoparticles using an assay based on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid (TNS).
  • Lipid nanoparticles comprising of cationic lipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol%) in PBS at a concentration of 0.4 mM total lipid are prepared using the in-line process as described in Example 1.
  • TNS was prepared as a 100 ⁇ stock solution in distilled water. Vesicles were diluted to 24 ⁇ lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. An aliquot of the TNS solution was added to give a final concentration of 1 ⁇ and following vortex mixing fluorescence intensity was measured at room temperature in a SLM Aminco Series 2 Luminescence
  • Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis was applied to the fluorescence data and the pK a was measured as the pH giving rise to half-maximal fluorescence intensity (see Figure 2).
  • lipid nanoparticles containing the FLuc mRNA (L-6107) using an in line mixing method, as described in Example 1 and in PCT/US 10/22614, which is hereby incorporated by reference in its entirety.
  • Lipid nanoparticles were formulated using the following molar ratio: 50% Cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5%
  • PEG-DMG Cholesterol / 1.5% PEG lipid
  • cationic lipid, DSPC, cholesterol and PEG-lipid are formulated at a molar ratio of approximately
  • Relative activity was determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection as described in Example 1. The activity was compared at a dose of 0.3 and 1.0 mg mRNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration, as described in Example 1.
  • the crude material was added to 5% sodium hydroxide in a 1 : 10 water/methanol solution (150 mL) and heated at 45°C for one hour. The solution was cooled, diluted with water and extracted with hexane. The organic fractions were dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (200 g) column using 0-4% methanol/dichloromethane to afford the desired product (15 g). l,19-Di(tetrahydropyranyloxy)nonadecan-10-one.
  • 1,19-Dihydroxynonadecan-lO-one A solution of 1,19- di(tetrahydropyranyloxy)nonadecan-10-one (3 g) in methanol (100 mL)/water (10 mL) was treated with pyridinium p-toluenesulfonate (1 g) overnight. The solution was filtered to afford the desired product (0.8 g). Dilution of the filtrate with water, followed by extraction using dichloromethane afforded additional desired product (1.0 g). l,19-Di(2'hexyldecanoyloxy)nonadecan-10-one.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Compounds are provided having the following structure (I) or (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R, R1, R2, L, X, L, G1, G2, Z, a1, a2 and n are independently as defined herein for each of structures (I) and (II). Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

Description

LIPIDS AND LIPID NANOPARTICLE FORMULATIONS FOR
DELIVERY OF NUCLEIC ACIDS
BACKGROUND
Technical Field
The present invention generally relates to novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g. oligonucleotides, messenger RNA) both in vitro and in vivo. Description of the Related Art
There are many challenges associated with the delivery of nucleic acids to affect a desired response in a biological system. Nucleic acid based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism in order to realize this potential. Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense
oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme. The therapeutic applications of translatable nucleotide delivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether or not indigenous to the system. The expression products of the nucleic acid can augment existing levels of protein, replace missing or non-functional versions of a protein, or introduce new protein and associated functionality in a cell or organism.
Some nucleic acids, such as miRNA inhibitors, can be used to effect expression of specific cellular products that are regulated by miRNA as would be useful in the treatment of, for example, diseases related to deficiency of protein or enzyme. The therapeutic applications of miRNA inhibition are extremely broad as constructs can be synthesized to inhibit one or more miRNA that would in turn regulate the expression of mRNA products. The inhibition of endogenous miRNA can augment its downstream target endogenous protein expression and restore proper function in a cell or organism as a means to treat disease associated to a specific miRNA or a group of miRNA.
Other nucleic acids can down-regulate intracellular levels of specific mRNA and, as a result, down-regulate the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotide and RNAi are also extremely broad, since oligonucleotide constructs can be synthesized with any nucleotide sequence directed against a target mRNA. Targets may include mRNAs from normal cells, mRNAs associated with disease-states, such as cancer, and mRNAs of infectious agents, such as viruses. To date, antisense oligonucleotide constructs have shown the ability to specifically down-regulate target proteins through degradation of the cognate mRNA in both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.
However, two problems currently face the use of oligonucleotides in therapeutic contexts. First, free RNAs are susceptible to nuclease digestion in plasma. Second, free RNAs have limited ability to gain access to the intracellular compartment where the relevant translation machinery resides. Lipid nanoparticles formed from cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides.
There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles would provide optimal drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. In addition, these lipid-nucleic acid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.
BRIEF SUMMARY
In brief, the present invention provides lipid compounds, including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including for example, all sterols) and/or their analogs, and/or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents. In some instances, the lipid nanoparticles are used to deliver nucleic acids such as antisense and/or messenger RNA. Methods for use of such lipid nanoparticles for treatment of various diseases or conditions, such as those caused by infectious entities and/or insufficiency of a protein, are also provided.
In one embodiment, compounds having the following structure (I) or (II) are provided:
Figure imgf000004_0001
(I) or (II)
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R, R1, R2, L, X, L, G1, G2, Z, a1, a2 and n are independently as defined herein for each of structures (I) and (II).
Pharmaceutical compositions comprising one or more of the foregoing compounds of structure (I) or (II) and a therapeutic agent are also provided. In some embodiments, the pharmaceutical compositions further comprise one or more components selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. Such compositions are useful for formation of lipid nanoparticles for the delivery of the therapeutic agent. In other embodiments, the present invention provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition of lipid nanoparticles comprising the compound of structure (I) or (II) and a therapeutic agent and delivering the composition to the patient.
These and other aspects of the invention will be apparent upon reference to the following detailed description.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
In the figures, identical reference numbers identify similar elements. The sizes and relative positions of elements in the figures are not necessarily drawn to scale and some of these elements are arbitrarily enlarged and positioned to improve figure legibility. Further, the particular shapes of the elements as drawn are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the figures.
Figure 1 shows time course of luciferase expression in mouse liver. Figure 2 illustrates the calculation of pKa for MC3 as a representative example relevant to the disclosed lipids.
DETAILED DESCRIPTION
In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention.
However, one skilled in the art will understand that the invention may be practiced without these details.
The present invention is based, in part, upon the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of an active or therapeutic agent such as a nucleic acid into a cell of a mammal. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo, resulting in a significant increase in the therapeutic index as compared to nucleic acid-lipid nanoparticle compositions previously described.
In particular embodiments, the present invention provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and/or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for expression of protein encoded by mRNA. In other embodiments, these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for down-regulating (e.g., silencing) the protein levels and/or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes. In yet other embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antigen or antibody.
The lipid nanoparticles and compositions of the present invention may be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, both in vitro and in vivo. Accordingly, embodiments of the present invention provide methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel cationic lipids described herein.
As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the lipid nanoparticles and compositions of the present invention may be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or inhibit processes that terminate expression of mRNA (e.g., miRNA inhibitors). Alternatively, the lipid nanoparticles and compositions of the present invention may be used to decrease the expression of target genes and proteins both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of the present invention may also be used for co- delivery of different nucleic acids (e.g. mRNA and plasmid DNA) separately or in combination, such as may be useful to provide an effect requiring colocalization of different nucleic acids (e.g. mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome).
Nucleic acids for use with this invention may be prepared according to any available technique. For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA. In vitro transcription describes a process of template-directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g. including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L. and Conn, G.L., General protocols for preparation of plasmid DNA template and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012)
Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2: 11.6: 11.6.1-11.6.17; Beckert, B. And Masquida, B.,(2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in
Enzymology v. 530, 101-1 14; all of which are incorporated herein by reference).
The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including
unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.). Techniques for the isolation of the mRNA transcripts are well known in the art. Well known procedures include phenol/chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.
Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).
Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain a number of aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self- complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn, can dramatically reduce mRNA translation since protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques to remove these dsRNA contaminants have been developed and are known in the art including but not limited to scaleable HPLC purification (see e.g. Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside- modified, protein-encoding mRNA, Nucl Acid Res, v. 39 el42; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in
Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013). HPLC purified mRNA has been reported to be translated at much greater levels, particularly in primary cells and in vivo.
A significant variety of modifications have been described in the art which are used to alter specific properties of in vitro transcribed mRNA, and improve its utility. These include, but are not limited to modifications to the 5' and 3' termini of the mRNA. Endogenous eukaryotic mRNA typically contain a cap structure on the 5'- end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts. The 5 '-cap contains a 5 '-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional
modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'-hydroxyl group.
Multiple distinct cap structures can be used to generate the 5 '-cap of in vitro transcribed synthetic mRNA. 5 '-capping of synthetic mRNA can be performed co- transcriptionally with chemical cap analogs (i.e. capping during in vitro transcription). For example, the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3'-0-methyl group. However, up to 20% of transcripts remain uncapped during this co- transcriptional process and the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5 '-cap structure that more closely mimics, either structurally or functionally, the endogenous 5 '-cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping. Numerous synthetic 5 '-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see e.g. .Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).
On the 3 '-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3 ' end of the transcript is cleaved to free a 3' hydroxyl to which poly- A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly-A tail has been extensively shown to enhance both translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. I l l, 611- 613).
Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase. The first case allows in vitro transcription of mRNA with poly (A) tails of defined length, depending on the size of the poly (T) tract, but requires additional manipulation of the template. The latter case involves the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 'termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length. 5'- capping and 3 '-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.
In addition to 5' cap and 3' poly adenylation, other modifications of the in vitro transcripts have been reported to provide benefits as related to efficiency of translation and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger potent innate immune responses. The ability to discriminate between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structure and nucleoside modifications since most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus rendering it immunostimulatory which in turn can inhibit effective mRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see e.g. Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013); Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840. The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art. A large variety of nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA (see e.g. US2012/0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.
Other components of mRNA which can be modified to provide benefit in terms of translatability and stability include the 5' and 3' untranslated regions (UTR). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA (see e.g. Pardi, N.,
Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013). In addition to mRNA, other nucleic acid payloads may be used for this invention. For oligonucleotides, methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g. Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J. :
Humana Press, 2005; both of which are incorporated herein by reference).
For plasmid DNA, preparation for use with this invention commonly utilizes but is not limited to expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g. Heilig, J., Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41 :11: 1.7: 1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, S. R. (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; and
US6197553B1). Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents.
Various exemplary embodiments of the cationic lipids of the present invention, lipid nanoparticles and compositions comprising the same, and their use to deliver active (e.g. therapeutic agents), such as nucleic acids, to modulate gene and protein expression, are described in further detail below.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise. Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as,
"comprises" and "comprising" are to be construed in an open and inclusive sense, that is, as "including, but not limited to".
Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.
The phrase "induce expression of a desired protein" refers to the ability of a nucleic acid to increase expression of the desired protein. To examine the extent of protein expression, a test sample (e.g. a sample of cells in culture expressing the desired protein) or a test mammal (e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g. nucleic acid in combination with a lipid of the present invention). Expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g. a sample of cells in culture expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model) that is not contacted with or administered the nucleic acid. When the desired protein is present in a control sample or a control mammal, the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1.0. In particular embodiments, inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1, for example, about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not present in a control sample or a control mammal, inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected. One of ordinary skill in the art will understand appropriate assays to determine the level of protein expression in a sample, for example dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.
The phrase "inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g. a sample of cells in culture expressing the target gene) or a test mammal (e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or a non-human primate (e.g. monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g. a sample of cells in culture expressing the target gene) or a control mammal (e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model) that is not contacted with or administered the nucleic acid. The expression of the target gene in a control sample or a control mammal may be assigned a value of 100%. In particular embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g. an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid. In the case where the expression product is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%), 15%), 10%), 5%), or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.
The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl
phosphonates, 2'-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
"Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
"Gene product," as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide. The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.
A "steroid" is a com ound comprising the following carbon skeleton:
Figure imgf000018_0001
Non-limiting examples of steroids include cholesterol, and the like.
A "cationic lipid" refers to a lipid capable of being positively charged.
Exemplary cationic lipids include one or more amine group(s) which bears the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) critical to the intracellular delivery of nucleic acids.
The term "polymer conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated 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 and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-DMG) and the like.
The term "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. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-s«-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-5«-glycero-3- phosphocholine (DPPC), l,2-Dimyristoyl-s«-glycero-3-phosphocholine (DMPC), 1- Palmitoyl-2-oleoyl-s«-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), phophatidylethanol amines such as l,2-Dioleoyl-s«-glycero-3- phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.
The term "charged lipid" refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g. pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl
dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Choi).
The term "lipid nanoparticle" refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of structure (I) or (II) or other specified cationic lipids. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some
embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) or (II) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub. Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.
As used herein, "lipid encapsulated" refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.
As used herein, the term "aqueous solution" refers to a composition comprising water.
"Serum-stable" in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.
"Systemic delivery," as used herein, refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism.
Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. "Local delivery," as used herein, refers to delivery of an active agent directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.
"Amino" refers to the - H2 radical.
"Cyano" or "nitrile" refers to the -CN radical.
"Hydroxy" or "hydroxyl" refers to the -OH radical.
"Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double (alkenyl) and/or triple bonds (alkynyl)), having, for example, from one to twenty-four carbon atoms (Ci-C24 alkyl), four to twenty carbon atoms (C4-C20 alkyl), six to sixteen carbon atoms (C6-C16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl),one to twelve carbon atoms (Ci-Ci2 alkyl), one to eight carbon atoms (Ci-C8 alkyl) or one to six carbon atoms (Ci-C6 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1 methylethyl (iso propyl), n butyl, n pentyl, 1, 1- dimethylethyl (t butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta-l,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.
"Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (Ci-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene),one to twelve carbon atoms (Ci-Ci2 alkylene), one to eight carbon atoms (Ci- C8 alkylene), one to six carbon atoms (Ci-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, «-butylene, ethenylene, propenylene, «-butenylene, propynylene,
«-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.
"Alkoxy" refers to a radical of the formula -ORa where Ra is an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.
"Alkylaminyl" refers to a radical of the formula -NHRa or - RaRa where each Ra is, independently, an alkyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkylaminyl group is optionally substituted.
"Aryl" refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of this invention, the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. "Arylene" is a divalent aryl radical. Unless stated otherwise specifically in the specification, the term "arylene," "aryl" or the prefix "ar-" (such as in "aralkyl") is meant to include aryl radicals that are optionally substituted.
A "carbocyclic ring" is a ring wherein each ring atom is carbon.
Carbocyclic rings may saturated or unsaturated, including aromatic rings. Unless stated otherwise specifically in the specification, a carbocylic group is optionally substituted. "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl,
7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. "Cycloalkylene" is a divalent cycloalkyl radical. Unless otherwise stated specifically in the specification, cycloalkyl and cycloalkylene groups are optionally substituted.
"Heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered (e.g., 5, 6 or 7-membered) non-aromatic ring radical, wherein at least one ring atom is a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur, and the remaining ring atoms are selected from the group consisting of carbon nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,
1-oxo-thiomorpholinyl, and 1, 1-dioxo-thiomorpholinyl. "Heterocyclene" is a divalent heterocycle radical. Unless stated otherwise specifically in the specification, heterocyclyl and heterocylene groups are optionally substituted.
"Heteroaryl" refers to a 5- to 14-membered ring system radical comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this invention, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[£][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl,
benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl,
dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1 -phenyl- lH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). "Heteroarylene" is a divalent heteroaryl radical. Unless stated otherwise specifically in the specification, heteroaryl and heteroarylene groups are optionally substituted.
"Fused" refers to any ring structure described herein which is fused to an existing ring structure in the compounds of the invention. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.
"Halo" or "halogen" refers to bromo, chloro, fluoro or iodo.
"Hydroxylalkyl" is an alkyl group comprising at least one hydroxyl substituent. Unless stated otherwise specifically in the specification, hydroxylalkyl groups are optionally substituted. "Aminoalkyl" is an alkyl group comprising at least one amino substituent. Unless stated otherwise specifically in the specification, aminoalkyl groups are optionally substituted.
"Alkylaminylalkyl" is an alkyl group comprising at least one
alkylaminyl substituent. Unless stated otherwise specifically in the specification, alkylaminylalkyl groups are optionally substituted.
"Alkoxylalkyl" is an alkyl group comprising at least one alkoxy substituent. Unless stated otherwise specifically in the specification, alkoxyalkyl groups are optionally substituted.
" Alkoxy carbonyl" is radical of formula -C(=0)Ra, wherein Ra is an alkoxy group. Unless stated otherwise specifically in the specification, alkoxycarbonyl groups are optionally substituted.
"Alkylcarbonyloxy" is radical of formula -OC(=0)Ra, wherein Ra is an alkyl group. Unless stated otherwise specifically in the specification, alkylcarbonyloxy groups are optionally substituted.
"Alkylcarbonyloxyalkyl" is radical of formula -RbOC(=0)Ra, wherein Ra is an alkyl group and Rb is alkylene. Unless stated otherwise specifically in the specification, alkylcarbonyloxyalkyl groups are optionally substituted.
"Alkylcarbonyl" is radical of formula -C(=0)Ra, wherein Ra is an alkyl group. Unless stated otherwise specifically in the specification, alkylcarbonyl groups are optionally substituted.
The term "substituted" used herein means any of the above groups (e.g. alkyl, alkylene, alkene, alkenylene, alkyne, alkynylene, alkoxy, alkylaminyl, aryl, arylene, carbocyclic, cycloalkyl, cycloalkylene, heterocyclyl, heterocylene, heteroaryl, heteroaryl ene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl,
alkoxycarbonyl, alkylcarbonyloxyalkyl or alkylcarbonyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to: a halogen atom such as F, CI, Br, or I; oxo groups (=0); hydroxyl groups (-OH); C1-C12 alkyl groups; cycloalkyl groups; -(C=0)OR ; -0(C=0)R ; -C(=0)R ; -OR ; -S(0)xR ; -S-SR ; -C(=0)SR ; -SC(=0)R ; - R R ; - R C(=0)R ; -C(=0) R R ; - R C(=0) R R ; -OC(=0)NR'R'; -NR'C(=0)0R ; -NR'S(0)X R'R ; - R'S(0)XR ; and -S(0)x R R , wherein: R is, at each occurrence, independently H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR ). In other
embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group(-NR R ).
A "polar functional group" is a functional group having at least one polarized bond. Polar functional groups often comprise at least one heteroatom (e.g., O, N or S). Polar functional are also typically hydrophilic and tend to increase the solubility of the compound in water (as compared to solubility of the same compound with a non-polar functional group). Examples of polar functional groups include, but are not limited to groups containing one or more of the following groups: hydroxyl, alkoxy, carbonyl (i.e., C(=0)), ester (-OC(=0)- and -C(=0)0-), cyano, amide (e.g., - RaC(=0)(Ra)- or -C(=0)N(Ra)2-, wherein each Ra is independently alkyl), alkoxy, ether (e.g., RA-0-Ra, wherein each Ra is independently alkyl), amino, alkylamino, heterocyclyl, heteroaryl, S, S(=0) and S(=0)2 groups.
"Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.
"Prodrug" is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention. Thus, the term "prodrug" refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention. Prodrugs are typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of the invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the invention. Prodrugs include compounds of the invention wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the invention is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group,
respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds of the invention and the like.
Embodiments of the invention disclosed herein are also meant to encompass all pharmaceutically acceptable compounds of the compound of structure (I) or (II) being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2H, 3H, UC, 13C, 14C, 13N, 15N, 150, 170, 180, 31P, 32P, 35S, 18F, 36C1, 123I, and 125I, respectively. These radiolabeled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically-labelled compounds of structure (I) or (II), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e., 3H, and carbon-14, i.e., 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Substitution with heavier isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
Substitution with positron emitting isotopes, such as UC, 18F, 150 and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of structure (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
Embodiments of the invention disclosed herein are also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes compounds produced by a process comprising administering a compound of this invention to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound of the invention in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.
"Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. "Mammal" includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.
"Pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
"Pharmaceutically acceptable salt" includes both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid,
naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ^-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol,
2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, tri ethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine,
trimethylamine, dicyclohexylamine, choline and caffeine.
Often crystallizations produce a solvate of the compounds of the invention (i.e., a compound of structure (I) or (II)). As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound of the invention with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compound of the invention may be true solvates, while in other cases, the compound of the invention may merely retain adventitious water or be a mixture of water plus some adventitious solvent.
A "pharmaceutical composition" refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.
"Effective amount" or "therapeutically effective amount" refers to that amount of a compound of the invention which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of a lipid nanoparticle of the invention which constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
"Treating" or "treatment" as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes:
(i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;
(ii) inhibiting the disease or condition, i.e., arresting its development;
(iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or
(iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.
The compounds of the invention (e.g., compounds of structure (I) or (II))„ or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. Embodiments of the present invention are meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (5)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.
A "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any said compounds.
Compounds
In an aspect, the invention provides novel lipid compounds which are capable of combining with other lipid components such as neutral lipids, charged lipids, steroids and/or polymer conjugated-lipids to form lipid nanoparticles with
oligonucleotides. Without wishing to be bound by theory, it is thought that these lipid nanoparticles shield oligonucleotides from degradation in the serum and provide for effective delivery of oligonucleotides to cells in vitro and in vivo.
In one embodiment, the compounds have the following structure (I):
Figure imgf000033_0001
(I)
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
one of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-,
-N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0-, and the other of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, -SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-, -N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0- or a direct bond;
L is, at each occurrence, ~0(C=0)-, wherein ~ represents a covalent bond to X;
X is CRa;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;
R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000034_0001
R1 R2
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1 ; c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,
alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
In some embodiments of structure (I), G1 and G2 are each independently -0(C=0)- or -(C=0)0-.
In other embodiments of structure (I), X is CH.
In different embodiments of structure (I), the sum of a^ ^ + c1 or the sum of a2 + b2 + c2 is an integer from 12 to 26.
In still other embodiments of structure (I), a1 and a2 are independently an integer from 3 to 10. For example, in some embodiments a1 and a2 are independently an integer from 4 to 9.
In various embodiments of structure (I), b1 and b2 are 0. In different embodiments, b1 and b2 are 1.
In more embodiments of structure (I), c1, c2, d1 and d2 are independently an integer from 6 to 8.
In other embodiments of structure (I), c1 and c2 are, at each occurrence, independently an integer from 6 to 10, and d1 and d2 are, at each occurrence, independently an integer from 6 to 10. In other embodiments of structure (I), c1 and c2 are, at each occurrence, independently an integer from 5 to 9, and d1 and d2 are, at each occurrence,
independently an integer from 5 to 9.
In more embodiments of structure (I), Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is i . In other embodiments, Z is alkyl.
In various embodiments of the foregoing, R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond. In certain embodiments, each R is H. In other embodiments at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
In other embodiments of the compound of structure (I), R1 and R2 independently have one of the following structures:
Figure imgf000035_0001
In certain embodiments of structure (I), the compound has one of the following structures:
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
(Π)
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
one of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-,
-N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0-, and the other of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, -SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-, -N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0- or a direct bond;
L is, at each occurrence, ~0(C=0)-, wherein ~ represents a covalent bond to X;
X is CRa;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;
R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000040_0001
R1 R2
R' is, at each occurrence, independently H or C1-C12 alkyl; a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1 ;
c1 and c2 are, at each occurrence, independently an integer from 2 to 12; d1 and d2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
1 2 1 2 1 2 1 1 1 wherein a , a , c , c , d and d are selected such that the sum of a^+d1 is an integer from 18 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
In certain embodiments of structure (II), G1 and G2 are each independently
-0(C=0)- or -(C=0)0-.
In other embodiments of structure (II), X is CH.
In some embodiments of structure (II), the sum of a^^+d1 is an integer from 20 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30. In other
embodiments, the sum of a^^+d1 is an integer from 20 to 30, and the sum of a2+c2+d2 is an integer from 20 to 30. In more embodiments of structure (II), the sum of a1 + b1 +
1 2 2 2 1 2 c or the sum of a + b + c is an integer from 12 to 26. In other embodiments, a , a , c1, c2, d1 and d2 are selected such that the sum of a^^+d1 is an integer from 18 to 28, and the sum of a2+c2+d2 is an integer from 18 to 28,
In still other embodiments of structure (II), a1 and a2 are independently an integer from 3 to 10, for example an integer from 4 to 9. In yet other embodiments of structure (II), b1 and b2 are 0. In different embodiments b1 and b2 are 1.
In certain other embodiments of structure (II), c1, c2, d1 and d2 are independently an integer from 6 to 8.
In different other embodiments of structure (II), Z is alkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1.
In more embodiments of structure (II), Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is i . In other embodiments, Z is alkyl.
In other different embodiments of structure (II), R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond. For example in some embodiments each R is H. In other embodiments at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
In more embodiments, each R' is H.
In certain embodiments of structure (II), the sum of a^+d1 is an integer from 20 to 25, and the sum of a2+c2+d2 is an integer from 20 to 25.
In other embodiments of structure (II), R1 and R2 independently have one of the following structures:
Figure imgf000041_0001
Figure imgf000042_0001
In more embodiments of structure (II), the compound has one of the following structures:
Figure imgf000042_0002
Figure imgf000043_0001
Figure imgf000044_0001
In any of the foregoing embodiments, n is i . In other of the foregoing embodiments, n is greater than 1.
In more of any of the foregoing embodiments, Z is a mono- or polyvalent moiety comprising at least one polar functional group. In some
embodiments, Z is a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is a polyvalent moiety comprising at least one polar functional group. In more of any of the foregoing embodiments, the polar functional group is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl, heterocyclyl or heteroaryl functional group.
In any of the foregoing embodiments, Z is hydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl, alkylaminylalkyl, heterocyclyl or heterocyclylalkyl.
In some other embodiments Z has the following structure:
Figure imgf000045_0001
wherein:
R5 and R6 are independently H or Ci-C6 alkyl;
R7 and R8 are independently H or Ci-C6 alkyl or R7 and R8, together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring; and
x is an integer from 0 to 6.
In still different embodiments, Z has the following structure:
Figure imgf000045_0002
wherein:
R5 and R6 are independently H or Ci-C6 alkyl;
R7 and R8 are independently H or Ci-C6 alkyl or R7 and R8, together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring; and
x is an integer from 0 to 6.
In still different embodiments, Z has the following structure:
O R5
R8 R6 wherein:
R5 and R6 are independently H or Ci-C6 alkyl;
R7 and R8 are independently H or Ci-C6 alkyl or R7 and R8, together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring; and
x is an integer from 0 to 6.
In some other embodiments, Z is hydroxylalkyl, cyanoalkyl or an alkyl substituted with one or more ester or amide groups.
For example, in any of the foregoing embodiments, Z has one of the following structures:
Figure imgf000046_0001
In other embodiments, Z-L has one of the following structures:
Figure imgf000046_0002
Figure imgf000047_0001

Figure imgf000048_0001
In other embodiments, Z-L has one of the following structures:
Figure imgf000048_0002
In still other embodiments, X is CH and Z-L has one of the following structures:
Figure imgf000048_0003
In various different embodiments, the compound has one of the structures set forth in Table 1 below.
Table 1
Representative Compounds
Figure imgf000048_0004
Figure imgf000049_0001
In other embodiments, the invention does not include any of the compounds illustrated in Table 1.
It is understood that any embodiment of the compounds of structure (I) or (II), as set forth above, and any specific substituent and/or variable in the compound structure (I) or (II), as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of compounds of structure (I) or (II) to form embodiments of the inventions not specifically set forth above. In addition, in the event that a list of substituents and/or variables is listed for any particular R group, L group, G group, Z group, or variables a, n, x, y, or z in a particular embodiment and/or claim, it is understood that each individual substituent and/or variable may be deleted from the particular embodiment and/or claim and that the remaining list of substituents and/or variables will be considered to be within the scope of the invention.
It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compounds.
In some embodiments, compositions comprising any one or more of the compounds of structure (I) or (II) and a therapeutic agent are provided. For example, in some embodiments, the compositions comprise any of the compounds of structure (I) or (II) and a therapeutic agent and one or more excipient selected from neutral lipids, steroids and polymer conjugated lipids. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.
In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2: 1 to about 8: 1.
In various embodiments, the compositions further comprise a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5: 1 to 1 : 1.
In various embodiments, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEGS-DAG) such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(co- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate such as Q-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co- methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the pegylated lipid ranges from about 100: 1 to about 20: 1.
In some embodiments, the composition comprises a pegylated lipid having the following structure (III):
Figure imgf000050_0001
(III)
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
R3 and R4 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
w has a mean value ranging from 30 to 60.
In some embodiments, R3 and R4 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average w is about 45. In other embodiments, the average w ranges from 42 to 55. For example, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, w is about 49.
In some embodiments of the foregoing composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense, plasmid DNA, and messenger RNA.
In other different embodiments, the invention is directed to a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing any of the foregoing compositions and administering the composition to the patient.
For the purposes of administration, the compounds of the present invention (typically in the form of lipid nanoparticles in combination with a therapeutic agent) may be administered as a raw chemical or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present invention comprise a compound of structure (I) or (II) and one or more pharmaceutically acceptable carrier, diluent or excipient. The compound of structure (I) or (II) is present in the composition in an amount which is effective to form a lipid nanoparticle and deliver the therapeutic agent, e.g., for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one skilled in the art.
Administration of the compositions of the invention can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this invention.
A pharmaceutical composition of the invention may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose,
microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil. The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
The liquid pharmaceutical compositions of the invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
A liquid pharmaceutical composition of the invention intended for either parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage will be obtained.
The pharmaceutical composition of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
The pharmaceutical composition of the invention may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The pharmaceutical composition of the invention may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
Alternatively, the active ingredients may be encased in a gelatin capsule.
The pharmaceutical composition of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, or a protein.
The pharmaceutical composition of the invention may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.
One skilled in the art, without undue experimentation may determine preferred aerosols.
The pharmaceutical compositions of the invention may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the invention with sterile, distilled water or other carrier so as to form a solution. A surfactant may be added to facilitate the formation of a
homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or
homogeneous suspension of the compound in the aqueous delivery system.
The compositions of the invention, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
Compositions of the invention may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of the composition of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, a composition of the invention and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Where separate dosage formulations are used, the compounds of the invention and one or more additional active agents can be
administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
Preparation methods for the above compounds and compositions are described herein below and/or known in the art.
It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(0)-R" (where R" is alkyl, aryl or arylalkyl), /?-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.
It will also be appreciated by those skilled in the art, although such protected derivatives of compounds of this invention may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". All prodrugs of compounds of this invention are included within the scope of the invention.
Furthermore, all compounds of the invention which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of the invention can be converted to their free base or acid form by standard techniques.
The disclosed compounds can be prepared according to the following General Reaction Scheme I and methods analogous to those disclosed in PCT Pub. No. WO 2013/086322, the full disclosure of which is hereby incorporated by reference in its entirety. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of structure (I) or (II) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybndge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this invention.
GENERAL REACTION SCHEME 1
Figure imgf000057_0001
General Reaction Scheme 1 ("Method A") provides a method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "G"), wherein R, R1, a1, a2 and Z are as defined herein, and PG is an alcohol protecting group such as tetrahydropyran. Compounds of structure A are purchased or prepared according to methods known in the art. Reaction of A with ethyl formate B under Grignard conditions yields alcohol C, which can then be coupled with acid D under standard conditions to yield E. Removal of the protecting group followed by coupling with acid F yields G. GENERAL REACTI SCHEME 2
Figure imgf000058_0001
General Reaction Scheme 2 ("Method B") provides an alternative method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "P"), wherein R, R1, a1, a2 and Z are as defined herein and PG is an alcohol protecting group such as tetrahydropyran. Compounds of structure H are purchased or prepared according to methods known in the art. The hydroxyl group of Compound H is protected using methods known in the art (e.g. pyndinium p-toluenesulfonate, dihydropyran) to yield I. Reaction of I with ethyl formate J under Grignard conditions (e.g. with Mg, I2) yields alcohol K. The hydroxyl group of compound K can be oxidized (e.g. with pyridinium chlorochr ornate) and the protecting groups removed (e.g. with pyridinium p-toluenesulfonate) to yield compound L. The free hydroxyl groups of L are then coupled with acid M under standard ester coupling conditions to yield N. The carbonyl of N is then reduced using methods known in the art (e.g. NaBH4) followed by coupling with acid O (e.g. with DMAP, EDC C1) to yield the desired product P. GENERAL REACTION SCHEME 3
Figure imgf000059_0001
General Reaction Scheme 3 ("Method C") provides another alternative method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "V"), wherein R, R1, a1, a2 and Z are as defined herein. Compounds of structure Q are purchased or prepared according to methods known in the art. Compound Q is used to form R under appropriate conditions (e.g. diethyl acetone dicarboxylate, EtONa).
Alcohol S is then coupled to R using standard conditions (e.g. DMAP, EDC HC1) to yield T. The carbonyl of T is reduced (e.g. with NaBH4) followed by coupling with acid U (e.g. with DMAP, EDC HC1) to yield the desired product V.
It should be noted that various alternative strategies for preparation of compounds of structure (I) or (II) are available to those of ordinary skill in the art. For example, other compounds of structure (I) or (II) wherein G1 and G2 are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, the General Reaction Schemes above depict preparation of a compound of structure (I) or (II), wherein R1 and R2 as well as a1 and a2 are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein R1 and R2 as well as a1 and a2 are different (i.e. resulting in an asymmetric compound). The use of protecting groups as needed and other modification to the above General Reaction Schemes will be readily apparent to one of ordinary skill in the art. The following examples are provided for purpose of illustration and not limitation.
EXAMPLE 1
LUCIFERASE MRNA IN VIVO EVALUATION USING THE LIPID NANOP ARTICLE COMPOSITIONS
Cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of 50: 10:38.5: 1.5. Lipid nanoparticles (L P) were prepared at a total lipid to mRNA weight ratio of approximately 10: 1 to 30: 1. Briefly, the mRNA was diluted to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :5 to 1 :3 (vol/vol) with total flow rates above 15 mL/min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μπι pore sterile filter. Lipid nanoparticle particle size was approximately 55-95 nm diameter, and in some instances
approximately70-90 nm diameter as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) or by using a Nicomp 370 submicron particle sizer (Santa Barbara, CA).
Alternatively, Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol at a molar ratio of at a molar ratio of approximately
47.5: 10:40.8: 1.7, and the LNPs are prepared as set forth above.
Studies were performed in 6-8 week old female C57BL/6 mice (Charles River) 8-10 week old CD-I (Harlan) mice (Charles River) according to guidelines established by an institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Varying doses of mRNA-lipid nanoparticle were
systemically administered by tail vein injection and animals euthanized at specific time points (1, 2, 4, 8 and 24 hrs) post-administration. Liver and spleen were collected in pre-weighed tubes, weights determined, immediately snap frozen in liquid nitrogen and stored at -80°C until processing for analysis. For liver, approximately 50 mg was dissected for analyses in a 2 mL FastPrep tubes (MP Biomedicals, Solon OH). ¼" ceramic sphere (MP Biomedicals) was added to each tube and 500 μΐ^ of Glo Lysis Buffer - GLB (Promega, Madison WI) equilibrated to room temperature was added to liver tissue. Liver tissues were homogenized with the FastPrep24 instrument (MP Biomedicals) at 2 x 6.0 m/s for 15 seconds. Homogenate was incubated at room temperature for 5 minutes prior to a 1 :4 dilution in GLB and assessed using SteadyGlo Luciferase assay system (Promega). Specifically, 50 μΐ^ of diluted tissue homogenate was reacted with 50 μΐ^ of SteadyGlo substrate, shaken for 10 seconds followed by 5 minute incubation and then quantitated using a CentroXS3 LB 960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was determined by using the BCA protein assay kit (Pierce, Rockford IL). Relative luminescence units (RLU) were then normalized to total μg protein assayed. To convert RLU to ng luciferase a standard curve was generated with QuantiLum Recombinant Luciferase (Promega). Based on the data provided in Figure 1, the four-hour time point was chosen for efficacy evaluation of the lipid formulations (see Example 3).
The FLuc mRNA (L-6107) from Trilink Biotechnologies will express a luciferase protein, originally isolated from the firefly, Photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methylcytidine and pseudouridine.
EXAMPLE 2
DETERMINATION OF PKA OF FORMULATED LIPIDS
As described elsewhere, the pKa of formulated cationic lipids is correlated with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). The preferred range of pKa is ~5 to ~7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid (TNS). Lipid nanoparticles comprising of cationic lipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol%) in PBS at a concentration of 0.4 mM total lipid are prepared using the in-line process as described in Example 1. TNS was prepared as a 100 μΜ stock solution in distilled water. Vesicles were diluted to 24 μΜ lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. An aliquot of the TNS solution was added to give a final concentration of 1 μΜ and following vortex mixing fluorescence intensity was measured at room temperature in a SLM Aminco Series 2 Luminescence
Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis was applied to the fluorescence data and the pKa was measured as the pH giving rise to half-maximal fluorescence intensity (see Figure 2).
EXAMPLE 3
DETERMINATION OF EFFICACY OF LIPID NANOP ARTICLE FORMULATIONS CONTAINING VARIOUS CATIONIC LIPIDS USING AN IN VIVO
LUCIFERASE MRNA EXPRESSION RODENT MODEL
The cationic lipids shown in Table 2 have previously been tested with nucleic acids. For comparative purposes, these lipids were also used to formulate lipid nanoparticles containing the FLuc mRNA (L-6107) using an in line mixing method, as described in Example 1 and in PCT/US 10/22614, which is hereby incorporated by reference in its entirety. Lipid nanoparticles were formulated using the following molar ratio: 50% Cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5%
Cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e.,
l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of 2000). In alternate embodiments, cationic lipid, DSPC, cholesterol and PEG-lipid are formulated at a molar ratio of approximately
47.5: 10:40.8: 1.7. Relative activity was determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection as described in Example 1. The activity was compared at a dose of 0.3 and 1.0 mg mRNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration, as described in Example 1.
Table 2
Comparator Lipids showing activity with mRNA
Figure imgf000063_0001
Representative compounds of the invention shown in Table 3 were formulated using the following molar ratio: 50% cationic lipid / 10%
distearoylphosphatidylcholine (DSPC) / 38.5% Cholesterol / 1.5% PEG lipid ("PEG- DMA" 2-[2-(Q-methoxy(polyethyleneglycol2ooo)ethoxy]-N,N-ditetradecylacetamide). Relative activity was determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection as described in Example 1. The activity was compared relative to activity of MC3 (MC3 activity = 1)
Table 3
Novel Cationic lipids and Relative Activity
Figure imgf000064_0001
EXAMPLE 4
SYNTHESIS OF
10- { [4' -(DIMETHYLAMINO)BUTANOYL]OXY} - 19-[(2-HEXYLDECANOYL)OXY]NONADECYL
2 -HEXYLDEC ANO ATE (COMPOUND 1)
Figure imgf000065_0001
10-{[4'-(dimethylamino)butanoyl]oxy}-19-[(2-hexyldecanoyl)oxy]nonadecyl 2-hexyldecanoate. A solution of 10-[4'-(dimethylamino)butanoyl]oxynonadecan-l,19- diol (0.30 g), 2-hexyldecanoic acid (1.1 g), N-(3-dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride (0.42 g) and 4-dimethylaminopyridine (0.27 g) in dichloromethane (20 mL) was stirred at room temperature overnight. The solution was washed with diluted hydrochloric acid, dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (20 g) column with 0-8% methanol/dichloromethane. The desired product was afforded (0.42 g). EXAMPLE 5
SYNTHESIS OF 1,25- DI-(2'-HEXYLDECYL)13-{[4- (DIMETHYLAMINO BUTANOYL]OXY}PENTACOSANEDIOATE (COMPOUND 2)
Figure imgf000065_0002
13-oxo-pentacosane-l,25-dioic acid. Sodium ethoxide (1.56 g) was dissolved in absolute ethanol (30 mL). Diethylacetone dicarboxylate (4.5 g) was added and the solution heated to reflux. Ethyl 11-bromododecanoate (6.8 g) was slowly added and the solution refluxed for an hour. Sodium ethoxide (1.53 g) was added, followed by ethyl 11-bromododecanoate (18 g). The solution was refluxed overnight. The reaction mixture was cooled, diluted with water, acidified with dilute hydrochloric acid, and extracted with methylene chloride. The organic fraction was washed with water and the solvent removed. The crude product was passed down a silica gel column (80 g) using methanol/methylene chloride to recover unreacted starting materials. The residue containing the product was treated with acetic acid (10 mL) and concentrated hydrochloric acid (20 mL), and then refluxed overnight. The solution was cooled, diluted with water and filtered. The collected precipitate was recrystallized from acetone, affording the desired product as a white powder (2.9 g). l,25-di-(2'-hexyldecyl) 13-oxo-pentacosanedioate. A solution of 13-oxo-pentacosane- 1,25-dioic acid (0.91 g), 4-dimethylaminopyridine (1.1 g), N-(3-dimethylaminopropyl)- N-ethylcarbodiimide hydrochloride (1.0 g) and 2-hexyldecan-l-ol (2.4 g) in
dichloromethane (40 mL) was stirred at room temperature overnight. The solution was washed with diluted hydrochloric acid, dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (20 g) column using 0-4% ethyl acetate/hexane, affording the desired product (1 g). l,25-di-(2'-hexyldecyl) 13-hydroxy-pentacosanedioate. A solution of l,25-di-(2'- hexyldecyl) 13-oxo-pentacosanedioate (1 g) in tetrahydrofuran (10 mL) and methanol (10 mL) was treated with sodium borohydride (0.18 g). The reaction was stirred for 30 minutes and then diluted with water, acidified and extracted with dichloromethane. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent removed to afford the crude product (0.95 g). The crude product was used in the next synthetic step without further purification. 1,25- di-(2'-hexyldecyl)13-{[4-(dimethylamino)butanoyl]oxy}pentacosanedioate (Compound 2). A solution of crude 1,25- di-(2'-hexyldecyl) 13-hydroxy- pentacosanedioate (0.95 g), 4-dimethylaminopyridine (0.42 g), N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.34 g) and N,N- dimethylaminobutyric acid hydrochloride (0.59 g) in dichloromethane (15 mL) was stirred at room temperature for one hour. The solution was washed with diluted hydrochloric acid followed by aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed down a silica gel (20 g) column using 0-6% methanol/dichloromethane to afford the desired product (0.81 g).
EXAMPLE 6
SYNTHESIS OF 10-{ [5-(DIMETHYLAMINO)PENTANOYL]OXY}- 19-[(2- HEX -HEXYLDECANO ATE (COMPOUND 3 )
Figure imgf000067_0001
9-Tetrahydropyranyloxynonan-l-yl bromide. A solution of 9-bromononan-l-ol (25.6 g) and dihydropyran (10.5 g) in dichloromethane (100 mL) was treated with pyridine p- toluenesulfonate (2.8 g) overnight. The solution was diluted with water and extracted with dichloromethane. The organic fractions were dried over anhydrous magnesium sulfate, filtered, and the solvent removed to afford the desired crude product (35 g). The crude product was used in the next synthetic step without further purification. l,19-Di(tetrahydropyranyloxy)nonadecan-10-ol. A solution of 9- tetrahydropyranyloxynonan-l-yl bromide (35 g) in diethyl ether (150 mL) was treated with magnesium (3.0 g). A crystal of iodine was added to initiate the reaction. The solution was refluxed for 4 days, then cooled to room temperature. Ethyl formate (4 mL) was slowly added and the solution refluxed for 2 hours. The solution was allowed to cool, then washed with dilute aqueous sulfuric acid. The organic phase was washed with water, dried over anhydrous magnesium sulfate, filtered, and the solvent removed to yield 25 g of crude product. The crude material was added to 5% sodium hydroxide in a 1 : 10 water/methanol solution (150 mL) and heated at 45°C for one hour. The solution was cooled, diluted with water and extracted with hexane. The organic fractions were dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (200 g) column using 0-4% methanol/dichloromethane to afford the desired product (15 g). l,19-Di(tetrahydropyranyloxy)nonadecan-10-one. A solution of 1,19- di(tetrahydropyranyloxy)nonadecan-10-ol (7.2 g) in dichloromethane (40 mL) was treated with pyridinium chlorochromate (4 g) and stirred overnight. Diethyl ether (200 mL) was added and the solution filtered through a silica gel bed. The solvent was removed and the residue dissolved in hexane, then filtered through a silica gel bed. The solvent was removed and the residue passed down a silica gel (75 g) column using 0- 3% methanol/dichloromethane to afford the desired product (3 g).
1,19-Dihydroxynonadecan-lO-one. A solution of 1,19- di(tetrahydropyranyloxy)nonadecan-10-one (3 g) in methanol (100 mL)/water (10 mL) was treated with pyridinium p-toluenesulfonate (1 g) overnight. The solution was filtered to afford the desired product (0.8 g). Dilution of the filtrate with water, followed by extraction using dichloromethane afforded additional desired product (1.0 g). l,19-Di(2'hexyldecanoyloxy)nonadecan-10-one. A solution of 1, 19- dihydroxynonadecan-10-one (0.87 g), 2-hexyldecanoic acid (2.48 g), 4- dimethylaminopyridine (1.0 g) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.68 g) in dichloromethane (40 mL) was stirred for three days. The solution was diluted with dichloromethane and washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed down a silica gel (50 g) column using dichloromethane to afford the desired product (2.8 g). l,19-Di(2'hexyldecanoyloxy)nonadecan-10-ol. A solution of 1,19- di(2'hexyldecanoyloxy)nonadecan-10-one (1.06 g) in dichloromethane (10 mL) was treated with sodium borohydride (0.15 g). Methanol was added dropwise until the materials dissolved. The solution was stirred for 30 minutes and then partitioned between water and dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (20 g) column using 0-12% ethyl acetate/hexane gradient to afford the desired product (0.40 g).
10-{[5-(dimethylamino)pentanoyl]oxy}-19-[(2-hexyldecanoyl)oxy]nonadecyl 2- hexyldecanoate (Compound 3). A solution of l,19-di(2'hexyldecanoyloxy)nonan-10- ol (0.40 g), 5-N,N-dimethylaminopentanoic acid (0.22 g), 4-dimethylaminopyridine (0.18 g) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.18 g) in dichloromethane (20 mL) was stirred overnight. The solution was diluted with dichloromethane and washed with water. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (20 g) column using 0-3% methanol/dichloromethane to afford the desired product (0.24 g).
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A compound having the following structure (I):
Figure imgf000071_0001
(I)
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
one of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-,
-N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0-, and the other of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, -SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-, -N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0- or a direct bond;
L is, at each occurrence, ~0(C=0)-, wherein ~ represents a covalent bond to X;
X is CRa;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;
R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000072_0001
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1;
c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,
alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
2. The compound of claim 1, wherein G1 and G2 are each independently -0(C=0)- or -(C=0)0-.
3. The compound of claim 1 or 2, wherein X is CH.
4. The compound of any one of claims 1-3, wherein the sum of a1 + b1 + c1 or the sum of a2 + b2 + c2 is an integer from 12 to 26.
5. The compound of any one of claims 1-4, wherein a1 and a2 are independently an integer from 3 to 10.
6. The compound of any one of claims 1-4, wherein a1 and a2 are independently an integer from 4 to 9.
7. The compound of any one of claims 1-6, wherein b1 and b2 are 0.
8 The compound of any one of claims 1-6, wherein b1 and b2 are 1.
9. The compound of any one of claims 1-8, wherein c1, c2, d1 and d2 are independently an integer from 6 to 8.
10. The compound of any one of claims 1-9, wherein R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
11. The compound of any one of claims 1-10, wherein each R is H.
12. The compound of any one of claims 1-10, wherein at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
13. The compound of any one of claims 1-12, wherein R1 and R2 independently have one of the following structures:
Figure imgf000073_0001
14. The compound of any one of claims 1-13, wherein the compound of the following structures:
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
75
15. A compound having the following structure (II):
Figure imgf000077_0001
(Π)
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
one of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-,
-N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0-, and the other of G1 or G2 is, at each occurrence, -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) , -S-S-, -C(=0)S-, -SC(=0)-, -N(Ra)C(=0)-, -C(=0)N(Ra)-, -N(Ra)C(=0)N(Ra)-, -OC(=0)N(Ra)- or -N(Ra)C(=0)0- or a direct bond;
L is, at each occurrence, ~0(C=0)-, wherein ~ represents a covalent bond to X;
X is CRa;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;
R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000078_0001
R1 R2
R' is, at each occurrence, independently H or C1-C12 alkyl; a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1;
c1 and c2 are, at each occurrence, independently an integer from 2 to 12; d1 and d2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
1 2 1 2 1 2 1 1 1 wherein a , a , c , c , d and d are selected such that the sum of a^+d1 is an integer from 18 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
16. The compound of claim 15, wherein G1 and G2 are each independently
-0(C=0)- or -(C=0)0-.
17. The compound of claim 15 or 16, wherein X is CH.
18. The compound of any one of claims 15-17, wherein the sum of a1 + b1 + c1 or the sum of a2 + b2 + c2 is an integer from 12 to 26.
19. The compound of any one of claims 15-18, wherein a1 and a2 are independently an integer from 3 to 10.
20. The compound of any one of claims 15-18, wherein a1 and a2 are independently an integer from 4 to 9.
21. The compound of any one of claims 15-20, wherein b1 and b2 are
0.
22. The compound of any one of claims 15-20, wherein b1 and b2 are
1.
23. The compound of any one of claims 15-22, wherein c1, c2, d1 and d2 are independently an integer from 6 to 8.
24. The compound of any one of claims 15-23, wherein R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
25. The compound of any one of claims 15-24, wherein each R is H.
26. The compound of any one of claims 15-24, wherein at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
27. The compound of any one of claims 15-26, wherein each R' is H.
28. The compound of any one of claims 15-27, wherein the sum of a^c^+d1 is an integer from 20 to 25, and the sum of a2+c2+d2 is an integer from 20 to 25.
Figure imgf000080_0001
30. The compound of any one of claims 15-29, wherein the compound has one of the following structures:
Figure imgf000080_0002
Figure imgf000081_0001
80
Figure imgf000082_0001
81
32. The compound of any one of claims 1-30, wherein n is greater than 1.
33. The compound of any one of claims 1-32, wherein Z is a mono- or polyvalent moiety comprising at least one polar functional group.
34. The compound of claim 33, wherein Z is a monovalent moiety comprising at least one polar functional group.
35. The compound of claim 33, wherein Z is a polyvalent moiety comprising at least one polar functional group.
36. The compound of any one of claims 33-35, wherein the polar functional group is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl, heterocyclyl or heteroaryl functional group.
37. The compound of any one of claims 1-36, wherein Z is hydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl, alkylaminylalkyl, heterocyclyl or heterocyclylalkyl.
38. The compound of any one of claims 1-37, wherein Z, has one of the following structures:
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
= H , Me, Et, iPr . W = H , Me, Et, iPr W = H, Me, Et, iPr
Figure imgf000085_0002
Figure imgf000085_0003
40. A compound selected from a compound in Table 1.
41. A composition comprising the compound of any one of claims 1- 40 and a therapeutic agent.
42. The composition of claim 41, further comprising one or more excipient selected from neutral lipids, steroids and polymer conjugated lipids.
43. The composition of claim 42, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
44. The composition of claim 43, wherein the neutral lipid is DSPC.
45. The composition of any one of claims 42-44, wherein the molar ratio of the compound to the neutral lipid ranges from about 2: 1 to about 8: 1.
46. The composition of any one of claims 42-45, wherein the steroid is cholesterol.
47. The composition of claim 41, wherein the molar ratio of the compound to cholesterol ranges from 5: 1 to 1 : 1.
48. The composition of any one of claims 42-47, wherein the polymer conjugated lipid is pegylated lipid.
49. The composition of claim 48, wherein the molar ratio of the compound to pegylated lipid ranges from about 100: 1 to about 20: 1.
50. The composition of anyone of claims 48 or 49, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG
dialkyoxypropylcarbamate.
51. The composition of any one of claims 48 or 49, wherein the pegylated lipid has the following structure (III):
Figure imgf000086_0001
wherein:
R3 and R4 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
52. The composition of claim 51, wherein R3 and R4 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
53. The composition of any one of claims 51 or 52, wherein the average w is about 45.
54. The composition of any one of claims 41-53, wherein the therapeutic agent comprises a nucleic acid.
55. The composition of claim 54, wherein the nucleic acid is selected from antisense and messenger RNA.
56. A method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing the composition of any one of claims 41-55, and administering the composition to the patient.
PCT/US2016/069491 2015-12-30 2016-12-30 Lipids and lipid nanoparticle formulations for delivery of nucleic acids WO2017117528A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP16829386.8A EP3397613A1 (en) 2015-12-30 2016-12-30 Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US16/067,538 US20190022247A1 (en) 2015-12-30 2016-12-30 Lipids and lipid nanoparticle formulations for delivery of nucleic acids

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201562273018P 2015-12-30 2015-12-30
US62/273,018 2015-12-30
US201662302348P 2016-03-02 2016-03-02
US62/302,348 2016-03-02

Publications (1)

Publication Number Publication Date
WO2017117528A1 true WO2017117528A1 (en) 2017-07-06

Family

ID=57868366

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/069491 WO2017117528A1 (en) 2015-12-30 2016-12-30 Lipids and lipid nanoparticle formulations for delivery of nucleic acids

Country Status (3)

Country Link
US (1) US20190022247A1 (en)
EP (1) EP3397613A1 (en)
WO (1) WO2017117528A1 (en)

Cited By (217)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018081480A1 (en) * 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
WO2018191719A1 (en) 2017-04-13 2018-10-18 Acuitas Therapeutics, Inc. Lipid delivery of therapeutic agents to adipose tissue
WO2018213476A1 (en) 2017-05-16 2018-11-22 Translate Bio, Inc. Treatment of cystic fibrosis by delivery of codon-optimized mrna encoding cftr
WO2018236849A1 (en) 2017-06-19 2018-12-27 Translate Bio, Inc. Messenger rna therapy for the treatment of friedreich's ataxia
US10166298B2 (en) 2015-10-28 2019-01-01 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10238754B2 (en) 2011-06-08 2019-03-26 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
WO2019089828A1 (en) 2017-10-31 2019-05-09 Acuitas Therapeutics, Inc. Lamellar lipid nanoparticles
WO2019126593A1 (en) 2017-12-20 2019-06-27 Translate Bio, Inc. Improved composition and methods for treatment of ornithine transcarbamylase deficiency
WO2019131770A1 (en) 2017-12-27 2019-07-04 武田薬品工業株式会社 Nucleic acid-containing lipid nano-particle and use thereof
WO2019222277A1 (en) 2018-05-15 2019-11-21 Translate Bio, Inc. Subcutaneous delivery of messenger rna
WO2019226925A1 (en) 2018-05-24 2019-11-28 Translate Bio, Inc. Thioester cationic lipids
WO2019232208A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Cationic lipids comprising a steroidal moiety
WO2019232097A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Phosphoester cationic lipids
WO2019232103A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Messenger rna vaccines and uses thereof
WO2019232095A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Vitamin cationic lipids
WO2020023533A1 (en) 2018-07-23 2020-01-30 Translate Bio, Inc. Dry power formulations for messenger rna
WO2020047061A1 (en) 2018-08-29 2020-03-05 Translate Bio, Inc. Improved process of preparing mrna-loaded lipid nanoparticles
WO2020056294A1 (en) 2018-09-14 2020-03-19 Translate Bio, Inc. Composition and methods for treatment of methylmalonic acidemia
WO2020061367A1 (en) * 2018-09-19 2020-03-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2020061426A2 (en) 2018-09-21 2020-03-26 Acuitas Therapeutics, Inc. Systems and methods for manufacturing lipid nanoparticles and liposomes
WO2020080475A1 (en) 2018-10-18 2020-04-23 武田薬品工業株式会社 Method for activation/proliferation of t cells
WO2020081933A1 (en) 2018-10-19 2020-04-23 Translate Bio, Inc. Pumpless encapsulation of messenger rna
WO2020097384A1 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. 2,5-dioxopiperazine lipids with intercalated ester, thioester, disulfide and anhydride moieities
WO2020097376A1 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. Multi-peg lipid compounds
WO2020097511A2 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. Messenger rna therapy for treatment of ocular diseases
WO2020097379A2 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. Peg lipidoid compounds
WO2020102172A2 (en) 2018-11-12 2020-05-22 Translate Bio, Inc. Methods for inducing immune tolerance
WO2020106903A1 (en) 2018-11-21 2020-05-28 Translate Bio, Inc. Cationic lipid compounds and compositions thereof for use in the delivery of messenger rna
WO2020146344A1 (en) 2019-01-07 2020-07-16 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
WO2020146805A1 (en) * 2019-01-11 2020-07-16 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
US10723692B2 (en) 2014-06-25 2020-07-28 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2020214946A1 (en) 2019-04-18 2020-10-22 Translate Bio, Inc. Cystine cationic lipids
WO2020219427A1 (en) 2019-04-22 2020-10-29 Translate Bio, Inc. Thioester cationic lipids
WO2020227085A1 (en) 2019-05-03 2020-11-12 Translate Bio, Inc. Di-thioester cationic lipids
WO2020243540A1 (en) 2019-05-31 2020-12-03 Translate Bio, Inc. Macrocyclic lipids
WO2020257611A1 (en) 2019-06-21 2020-12-24 Translate Bio, Inc. Cationic lipids comprising an hydroxy moiety
WO2020257716A1 (en) 2019-06-21 2020-12-24 Translate Bio, Inc. Tricine and citric acid lipids
WO2021007278A1 (en) 2019-07-08 2021-01-14 Translate Bio, Inc. Improved mrna-loaded lipid nanoparticles and processes of making the same
WO2021016430A1 (en) 2019-07-23 2021-01-28 Translate Bio, Inc. Stable compositions of mrna-loaded lipid nanoparticles and processes of making
WO2021016075A1 (en) 2019-07-19 2021-01-28 Flagship Pioneering Innovations Vi, Llc Recombinase compositions and methods of use
WO2021030701A1 (en) 2019-08-14 2021-02-18 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
WO2021046265A1 (en) 2019-09-06 2021-03-11 Generation Bio Co. Lipid nanoparticle compositions comprising closed-ended dna and cleavable lipids and methods of use thereof
WO2021055609A1 (en) 2019-09-20 2021-03-25 Translate Bio, Inc. Mrna encoding engineered cftr
WO2021081058A1 (en) 2019-10-21 2021-04-29 Translate Bio, Inc. Compositions, methods and uses of messenger rna
WO2021102411A1 (en) 2019-11-22 2021-05-27 Generation Bio Co. Ionizable lipids and nanoparticle compositions thereof
WO2021127641A1 (en) 2019-12-20 2021-06-24 Translate Bio, Inc. Improved process of preparing mrna-loaded lipid nanoparticles
WO2021127394A2 (en) 2019-12-20 2021-06-24 Translate Bio, Inc. Rectal delivery of messenger rna
WO2021142245A1 (en) 2020-01-10 2021-07-15 Translate Bio, Inc. Compounds, pharmaceutical compositions and methods for modulating expression of muc5b in lung cells and tissues
WO2021173840A1 (en) 2020-02-25 2021-09-02 Translate Bio, Inc. Improved processes of preparing mrna-loaded lipid nanoparticles
WO2021195529A2 (en) 2020-03-27 2021-09-30 Generation Bio Co. Novel lipids and nanoparticle compositions thereof
WO2021195214A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
WO2021195218A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
WO2021226436A1 (en) 2020-05-07 2021-11-11 Translate Bio, Inc. Optimized nucleotide sequences encoding sars-cov-2 antigens
WO2021226463A1 (en) 2020-05-07 2021-11-11 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
WO2021226468A1 (en) 2020-05-07 2021-11-11 Translate Bio, Inc. Improved compositions for cftr mrna therapy
WO2021231901A1 (en) 2020-05-15 2021-11-18 Translate Bio, Inc. Lipid nanoparticle formulations for mrna delivery
WO2021231697A1 (en) 2020-05-14 2021-11-18 Translate Bio, Inc. Peg lipidoid compounds
WO2021236980A1 (en) 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Coronavirus antigen compositions and their uses
WO2021236930A1 (en) 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2021243301A2 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc. Trem compositions and methods relating thereto
WO2021243290A1 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods relating thereto
WO2021257595A1 (en) 2020-06-15 2021-12-23 Research Institute At Nationwide Children's Hospital Adeno-associated virus vector delivery for muscular dystrophies
WO2022006527A1 (en) 2020-07-02 2022-01-06 Maritime Therapeutics, Inc. Compositions and methods for reverse gene therapy
WO2022023284A1 (en) 2020-07-27 2022-02-03 Anjarium Biosciences Ag Compositions of dna molecules, methods of making therefor, and methods of use thereof
WO2022051629A1 (en) 2020-09-03 2022-03-10 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
CN114213347A (en) * 2022-02-22 2022-03-22 中国科学院基础医学与肿瘤研究所(筹) Bivalent ionizable lipid compound, composition and application thereof
CN114213346A (en) * 2022-02-22 2022-03-22 中国科学院基础医学与肿瘤研究所(筹) Bivalent ionizable lipid compound, composition and application thereof
WO2022076562A1 (en) 2020-10-06 2022-04-14 Translate Bio, Inc. Improved process and formulation of lipid nanoparticles
WO2022081548A1 (en) 2020-10-12 2022-04-21 Translate Bio, Inc. Improved process of preparing ice-based lipid nanoparticles
WO2022081544A1 (en) 2020-10-12 2022-04-21 Translate Bio, Inc. Improved process of preparing mrna-loaded lipid nanoparticles
WO2022099194A1 (en) 2020-11-09 2022-05-12 Translate Bio, Inc. Improved compositions for delivery of codon-optimized mrna
WO2022115547A1 (en) 2020-11-25 2022-06-02 Translate Bio, Inc. Stable liquid lipid nanoparticle formulations
US11357856B2 (en) 2017-04-13 2022-06-14 Acuitas Therapeutics, Inc. Lipids for delivery of active agents
WO2022140702A1 (en) 2020-12-23 2022-06-30 Flagship Pioneering, Inc. Compositions of modified trems and uses thereof
WO2022155404A1 (en) 2021-01-14 2022-07-21 Translate Bio, Inc. Methods and compositions for delivering mrna coded antibodies
WO2022169508A1 (en) 2021-02-08 2022-08-11 The Board Of Regents Of The University Of Texas System Unsaturated dendrimers compositions,related formulations, and methods of use thereof
EP4046629A1 (en) 2021-02-19 2022-08-24 ModernaTX, Inc. Lipid nanoparticle compositions and methods of formulating the same
WO2022204549A1 (en) 2021-03-25 2022-09-29 Translate Bio, Inc. Optimized nucleotide sequences encoding the extracellular domain of human ace2 protein or a portion thereof
WO2022212784A1 (en) 2021-03-31 2022-10-06 Flagship Pioneering Innovations V, Inc. Thanotransmission polypeptides and their use in treating cancer
WO2022225918A1 (en) 2021-04-19 2022-10-27 Translate Bio, Inc. Improved compositions for delivery of mrna
WO2022223556A1 (en) 2021-04-20 2022-10-27 Anjarium Biosciences Ag Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof
WO2022232289A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing therapeutic antibodies and uses thereof
WO2022232286A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing anti-coronavirus antibodies and uses thereof
US11524932B2 (en) 2017-08-17 2022-12-13 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US11542225B2 (en) 2017-08-17 2023-01-03 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2023278754A1 (en) 2021-07-01 2023-01-05 Translate Bio, Inc. Compositions for delivery of mrna
WO2023009547A1 (en) 2021-07-26 2023-02-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and uses thereof
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
WO2023023055A1 (en) 2021-08-16 2023-02-23 Renagade Therapeutics Management Inc. Compositions and methods for optimizing tropism of delivery systems for rna
US11597698B2 (en) 2019-09-19 2023-03-07 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023044006A1 (en) 2021-09-17 2023-03-23 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
WO2023044343A1 (en) 2021-09-14 2023-03-23 Renagade Therapeutics Management Inc. Acyclic lipids and methods of use thereof
WO2023044333A1 (en) 2021-09-14 2023-03-23 Renagade Therapeutics Management Inc. Cyclic lipids and methods of use thereof
WO2023069498A1 (en) 2021-10-22 2023-04-27 Senda Biosciences, Inc. Mrna vaccine composition
WO2023069397A1 (en) 2021-10-18 2023-04-27 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
US11639329B2 (en) 2017-08-16 2023-05-02 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
WO2023081526A1 (en) 2021-11-08 2023-05-11 Orna Therapeutics, Inc. Lipid nanoparticle compositions for delivering circular polynucleotides
WO2023086893A1 (en) 2021-11-10 2023-05-19 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
WO2023091787A1 (en) 2021-11-22 2023-05-25 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023091490A1 (en) 2021-11-16 2023-05-25 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023097003A2 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and their uses
WO2023096963A1 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Varicella-zoster virus immunogen compositions and their uses
WO2023096990A1 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovation Vi, Llc Coronavirus immunogen compositions and their uses
WO2023096858A1 (en) 2021-11-23 2023-06-01 Senda Biosciences, Inc. A bacteria-derived lipid composition and use thereof
WO2023115013A1 (en) 2021-12-17 2023-06-22 Flagship Pioneering Innovations Vi, Llc Methods for enrichment of circular rna under denaturing conditions
WO2023122789A1 (en) 2021-12-23 2023-06-29 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
WO2023122752A1 (en) 2021-12-23 2023-06-29 Renagade Therapeutics Management Inc. Constrained lipids and methods of use thereof
WO2023122080A1 (en) 2021-12-20 2023-06-29 Senda Biosciences, Inc. Compositions comprising mrna and lipid reconstructed plant messenger packs
WO2023122745A1 (en) 2021-12-22 2023-06-29 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2023135273A2 (en) 2022-01-14 2023-07-20 Anjarium Biosciences Ag Compositions of dna molecules encoding factor viii, methods of making thereof, and methods of use thereof
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023177904A1 (en) 2022-03-18 2023-09-21 Modernatx, Inc. Sterile filtration of lipid nanoparticles and filtration analysis thereof for biological applications
WO2023177655A1 (en) 2022-03-14 2023-09-21 Generation Bio Co. Heterologous prime boost vaccine compositions and methods of use
WO2023183616A1 (en) 2022-03-25 2023-09-28 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023196931A1 (en) 2022-04-07 2023-10-12 Renagade Therapeutics Management Inc. Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents
WO2023196634A2 (en) 2022-04-08 2023-10-12 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2023220083A1 (en) 2022-05-09 2023-11-16 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods of use for treating proliferative disorders
WO2023220729A2 (en) 2022-05-13 2023-11-16 Flagship Pioneering Innovations Vii, Llc Double stranded dna compositions and related methods
US11820728B2 (en) 2017-04-28 2023-11-21 Acuitas Therapeutics, Inc. Carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2023239756A1 (en) 2022-06-07 2023-12-14 Generation Bio Co. Lipid nanoparticle compositions and uses thereof
WO2023250112A1 (en) 2022-06-22 2023-12-28 Flagship Pioneering Innovations Vi, Llc Compositions of modified trems and uses thereof
WO2024030856A2 (en) 2022-08-01 2024-02-08 Flagship Pioneering Innovations Vii, Llc Immunomodulatory proteins and related methods
WO2024035952A1 (en) 2022-08-12 2024-02-15 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
WO2024044147A1 (en) 2022-08-23 2024-02-29 Modernatx, Inc. Methods for purification of ionizable lipids
WO2024049979A2 (en) 2022-08-31 2024-03-07 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine
WO2024077191A1 (en) 2022-10-05 2024-04-11 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
WO2024097664A1 (en) 2022-10-31 2024-05-10 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2024102730A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and nanoparticle compositions for delivering polynucleotides
WO2024102799A1 (en) 2022-11-08 2024-05-16 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
WO2024102762A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024102434A1 (en) 2022-11-10 2024-05-16 Senda Biosciences, Inc. Rna compositions comprising lipid nanoparticles or lipid reconstructed natural messenger packs
WO2024102677A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Circular rna compositions
WO2024112652A1 (en) 2022-11-21 2024-05-30 Translate Bio, Inc. Compositions of dry powder formulations of messenger rna and methods of use thereof
WO2024119039A2 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticles and uses thereof
WO2024119074A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticle compositions for cell targeting
WO2024119051A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same
WO2024119103A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers
WO2024121814A1 (en) 2022-12-09 2024-06-13 Takeda Pharmaceutical Company Limited Modified immunomodulators
WO2024126809A1 (en) 2022-12-15 2024-06-20 Sanofi Mrna encoding influenza virus-like particle
WO2024129988A1 (en) 2022-12-14 2024-06-20 Flagship Pioneering Innovations Vii, Llc Compositions and methods for delivery of therapeutic agents to bone
WO2024133515A1 (en) 2022-12-20 2024-06-27 Sanofi Rhinovirus mrna vaccine
WO2024151673A2 (en) 2023-01-09 2024-07-18 President And Fellows Of Harvard College Recombinant nucleic acid molecules and their use in wound healing
WO2024151685A1 (en) 2023-01-09 2024-07-18 Beth Israel Deaconess Medical Center, Inc. Recombinant nucleic acid molecules and their use in wound healing
WO2024151687A1 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
WO2024151583A2 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2024156291A1 (en) * 2023-01-29 2024-08-02 Geneditbio Limited Cationic lipids and lipid nanoparticles
WO2024159172A1 (en) 2023-01-27 2024-08-02 Senda Biosciences, Inc. A modified lipid composition and uses thereof
WO2024167885A1 (en) 2023-02-06 2024-08-15 Flagship Pioneering Innovations Vii, Llc Immunomodulatory compositions and related methods
US12065396B2 (en) 2017-08-17 2024-08-20 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2024173836A2 (en) 2023-02-17 2024-08-22 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified cytosine
WO2024173828A1 (en) 2023-02-17 2024-08-22 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified uracil
WO2024173307A2 (en) 2023-02-13 2024-08-22 Flagship Pioneering Innovation Vii, Llc Cleavable linker-containing ionizable lipids and lipid carriers for therapeutic compositions
US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024192422A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2024192420A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Compositions comprising polyribonucleotides and uses thereof
WO2024205657A2 (en) 2023-03-29 2024-10-03 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024200826A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell inhibiting molecule and use thereof
WO2024200823A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell enhancing molecule and use thereof
WO2024210160A1 (en) 2023-04-07 2024-10-10 Takeda Pharmaceutical Company Limited Conjugation complex
WO2024216128A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Trems for use in correction of missense mutations
WO2024216191A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Modified trems, compositions, and related methods thereof
US12121610B2 (en) 2021-03-22 2024-10-22 Recode Therapeutics, Inc. Compositions and methods for targeted delivery to cells
WO2024218166A1 (en) 2023-04-17 2024-10-24 Sanofi Reconstitutable dry powder formulations and methods of use thereof
WO2024220712A2 (en) 2023-04-19 2024-10-24 Sail Biomedicines, Inc. Vaccine compositions
WO2024220625A1 (en) 2023-04-19 2024-10-24 Sail Biomedicines, Inc. Delivery of polynucleotides from lipid nanoparticles comprising rna and ionizable lipids
WO2024220746A2 (en) 2023-04-21 2024-10-24 Flagship Pioneering Innovations Vii, Llc Rnai agents targeting fatty acid synthase and related methods
WO2024220752A2 (en) 2023-04-19 2024-10-24 Sail Biomedicines, Inc. Rna therapeutic compositions
US12129223B2 (en) 2021-12-16 2024-10-29 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US12133923B2 (en) 2022-12-08 2024-11-05 Recode Therapeutics, Inc. Lipid nanoparticle compositions and uses thereof
WO2024230934A1 (en) 2023-05-11 2024-11-14 CureVac SE Therapeutic nucleic acid for the treatment of ophthalmic diseases
WO2024233308A2 (en) 2023-05-05 2024-11-14 Orna Therapeutics, Inc. Circular rna compositions and methods
WO2024236504A1 (en) 2023-05-15 2024-11-21 Takeda Pharmaceutical Company Limited Sequences and methods for delivery of dna and rna
WO2024236361A1 (en) 2023-05-15 2024-11-21 Takeda Pharmaceutical Company Limited Compositions and methods for delivery of nucleic acids to cells
US12151995B2 (en) 2015-09-17 2024-11-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2024258829A1 (en) 2023-06-12 2024-12-19 Flagship Pioneering Innovations Vii, Llc Sars-cov-2 vaccine compositions and related methods
WO2025006684A1 (en) 2023-06-28 2025-01-02 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
WO2025024486A2 (en) 2023-07-25 2025-01-30 Flagship Pioneering Innovations Vii, Llc Cas endonucleases and related methods
WO2025027116A1 (en) 2023-08-01 2025-02-06 Institut Curie Nanoparticles comprising nucleic acid sequences encoding cyclic gmp-amp synthase
WO2025042786A1 (en) 2023-08-18 2025-02-27 Flagship Pioneering Innovations Vi, Llc Compositions comprising circular polyribonucleotides and uses thereof
WO2025049690A1 (en) 2023-08-29 2025-03-06 Orna Therapeutics, Inc. Circular polyethylene glycol lipids
WO2025054236A2 (en) 2023-09-06 2025-03-13 Flagship Pioneering Innovations Vii, Llc Sars-cov-2 vaccine compositions and related methods
WO2025051994A1 (en) 2023-09-07 2025-03-13 Coave Therapeutics Ionizable lipid nanoparticles
WO2025052180A2 (en) 2023-09-07 2025-03-13 Axelyf ehf. Lipids and lipid nanoparticles
US12257317B2 (en) 2017-03-17 2025-03-25 Newcastle University Adeno-associated virus vector delivery of a fragment of micro-dystrophin to treat muscular dystrophy
US12257318B2 (en) 2021-03-23 2025-03-25 Recode Therapeutics, Inc. Polynucleotide compositions, related formulations, and methods of use thereof
WO2025064475A2 (en) 2023-09-18 2025-03-27 Flagship Pioneering Innovations Vii, Llc Ionizable lipidoid compositions and therapeutic uses thereof
WO2025072331A1 (en) 2023-09-26 2025-04-03 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025081042A1 (en) 2023-10-12 2025-04-17 Renagade Therapeutics Management Inc. Nickase-retron template-based precision editing system and methods of use
WO2025096807A2 (en) 2023-10-31 2025-05-08 Flagship Pioneering Innovations Vii, Llc Novel therapeutic dna forms
WO2025101501A1 (en) 2023-11-07 2025-05-15 Orna Therapeutics, Inc. Circular rna compositions
WO2025106930A1 (en) 2023-11-17 2025-05-22 Sail Biomedicines, Inc. Circular polyribonucleotides encoding glucagon-like peptide 2 (glp-2) and uses thereof
WO2025106915A1 (en) 2023-11-17 2025-05-22 Sail Biomedicines, Inc. Circular polyribonucleotides encoding glucagon-like peptide 1 (glp-1) and uses thereof
WO2025106670A1 (en) 2023-11-14 2025-05-22 Flagship Pioneering Innovations Vii, Llc Ionizable lipidoid compositions and therapeutic uses thereof
WO2025106806A1 (en) 2023-11-17 2025-05-22 Acuitas Therapeutics, Inc. Pegylated lipids
WO2025111526A1 (en) 2023-11-22 2025-05-30 Flagship Pioneering Innovations Vii, Llc Methods and compositions for treating non-alcoholic fatty liver disease
US12318441B2 (en) 2022-12-29 2025-06-03 Rinuagene Biotechnology Co., Ltd. Polynucleotide molecules used for the prevention or treatment of HPV infection related diseases
WO2025117877A2 (en) 2023-12-01 2025-06-05 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025114520A1 (en) 2023-12-01 2025-06-05 Coave Therapeutics Ionizable lipid nanoparticles
US12324859B2 (en) 2017-03-15 2025-06-10 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2025128696A1 (en) 2023-12-12 2025-06-19 Acuitas Therapeutics, Inc. Cationic lipid compounds for use in lipid nanoparticles
WO2025126071A1 (en) 2023-12-14 2025-06-19 Pfizer Inc. Rna molecules
WO2025132122A1 (en) 2023-12-13 2025-06-26 Berlin Institute Of Health Methods of delivering therapeutics using lipid nanoparticles
WO2025137646A1 (en) 2023-12-22 2025-06-26 Recode Therapeutics, Inc. Gene editing methods and compositions for treating cystic fibrosis
WO2025133115A1 (en) 2023-12-21 2025-06-26 Ose Immunotherapeutics Lipid-based nanoparticles comprising il-35
WO2025140421A1 (en) * 2023-12-29 2025-07-03 北京剂泰医药科技有限公司 Ionizable lipid compound and use thereof in delivering nucleic acid vaccine
EP4484408A4 (en) * 2022-03-25 2025-07-09 Shenzhen Neocurna Biotechnology Corp CATIONIC lipid compound, method of preparation and use thereof, and mRNA delivery system

Families Citing this family (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180303925A1 (en) 2015-04-27 2018-10-25 The Trustees Of The University Of Pennsylvania Nucleoside-Modified RNA For Inducing an Adaptive Immune Response
US11241490B2 (en) 2017-01-11 2022-02-08 The Trustees Of The University Of Pennsylvania Nucleoside-modified RNA for inducing an immune response against zika virus
AU2018258575A1 (en) 2017-04-27 2019-11-07 The Johns Hopkins University Hepatitis C virus gene sequences and methods of use therefor
EP3820890A1 (en) 2018-07-13 2021-05-19 Genmab A/S Trogocytosis-mediated therapy using cd38 antibodies
EP3820900A1 (en) 2018-07-13 2021-05-19 Genmab A/S Variants of cd38 antibody and uses thereof
AU2020295012A1 (en) 2019-06-18 2022-02-17 Janssen Sciences Ireland Unlimited Company Combination of hepatitis B virus (HBV) vaccines and anti-PD-1 or anti-PD-L1 antibody
US20220233685A1 (en) 2019-06-18 2022-07-28 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and anti-pd-1 antibody
US20220305117A1 (en) 2019-06-18 2022-09-29 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and hbv-targeting rnai
WO2020255010A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of recombinant interleukin 12 construct and hepatitis b virus (hbv) vaccines
US20250041386A1 (en) 2019-06-18 2025-02-06 Janssen Sciences Ireland Unlimited Company Recombinant Interleukin 12 Construct and Uses Thereof
AU2020295807A1 (en) 2019-06-20 2022-02-17 Janssen Sciences Ireland Unlimited Company Lipid nanoparticle or liposome delivery of hepatitis B virus (HBV) vaccines
WO2021061815A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4α) GENE EXPRESSION
JP2022548320A (en) 2019-09-23 2022-11-17 オメガ セラピューティクス， インコーポレイテッド Compositions and methods for modulating apolipoprotein B (APOB) gene expression
CN116096886A (en) 2020-03-11 2023-05-09 欧米茄治疗公司 Compositions and methods for modulating fork-box P3 (FOXP 3) gene expression
US12194157B2 (en) 2020-04-09 2025-01-14 Finncure Oy Carrier for targeted delivery to a host
EP4132478A1 (en) 2020-04-09 2023-02-15 Finncure Oy Mimetic nanoparticles for preventing the spreading and lowering the infection rate of novel coronaviruses
PH12023550030A1 (en) 2020-07-08 2024-03-11 Janssen Sciences Ireland Unlimited Co Rna replicon vaccines against hbv
TW202245809A (en) 2020-12-18 2022-12-01 美商詹森藥物公司 Combination therapy for treating hepatitis b virus infection
JP2024501022A (en) 2020-12-28 2024-01-10 アークトゥルス セラピューティクス， インコーポレイテッド Transcription activator-like effector nuclease (TALEN) targeting HBV
US20240124396A1 (en) * 2021-02-07 2024-04-18 Shenzhen Shenxin Biotechnology Co., Ltd. Amino lipid compound, preparation method therefor, and use thereof
EP4367242A2 (en) 2021-07-07 2024-05-15 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
CA3229889A1 (en) 2021-09-03 2023-03-09 Glaxosmithkline Biologicals Sa Substitution of nucleotide bases in self-amplifying messenger ribonucleic acids
EP4399227A1 (en) 2021-09-06 2024-07-17 Genmab A/S Antibodies capable of binding to cd27, variants thereof and uses thereof
TW202330610A (en) 2021-10-08 2023-08-01 丹麥商珍美寶股份有限公司 Antibodies binding to cd30 and cd3
WO2023064469A1 (en) 2021-10-13 2023-04-20 Modernatx, Inc. Compositions of mrna-encoded il15 fusion proteins and methods of use thereof
EP4508211A2 (en) 2022-04-13 2025-02-19 Caribou Biosciences, Inc. Therapeutic applications of crispr type v systems
CN116969851A (en) * 2022-04-29 2023-10-31 北京剂泰医药科技有限公司 Ionizable lipid compounds
WO2023218420A1 (en) 2022-05-13 2023-11-16 Janssen Pharmaceuticals, Inc. Mrna compositions for inducing latent hiv-1 reversal
WO2023233290A1 (en) 2022-05-31 2023-12-07 Janssen Sciences Ireland Unlimited Company Rnai agents targeting pd-l1
EP4539876A2 (en) 2022-06-18 2025-04-23 GlaxoSmithKline Biologicals S.A. Recombinant rna molecules comprising untranslated regions or segments encoding spike protein from the omicron strain of severe acute respiratory coronavirus-2
WO2024133160A1 (en) 2022-12-19 2024-06-27 Glaxosmithkline Biologicals Sa Hepatitis b compositions
WO2024178305A1 (en) 2023-02-24 2024-08-29 Modernatx, Inc. Compositions of mrna-encoded il-15 fusion proteins and methods of use thereof for treating cancer
TW202509071A (en) 2023-05-12 2025-03-01 丹麥商珍美寶股份有限公司 Antibodies capable of binding to ox40, variants thereof and uses thereof
WO2024243438A2 (en) 2023-05-23 2024-11-28 Omega Therapeutics, Inc. Compositions and methods for reducing cxcl9, cxcl10, and cxcl11 gene expression
WO2025007109A1 (en) * 2023-06-30 2025-01-02 RVAC Medicines (US), Inc. Ionizable lipids and lipid nanoparticle compositions for delivery of nucleic acids
TW202509077A (en) 2023-06-30 2025-03-01 丹麥商珍美寶股份有限公司 Antibodies binding to fibroblast activation protein alpha and death receptor 4
WO2025038371A1 (en) * 2023-08-11 2025-02-20 Purdue Research Foundation Salt-loaded solid lipid nanoparticles loaded with an active agent
WO2025129128A1 (en) 2023-12-15 2025-06-19 Vivasor, Inc. Compositions and methods for non-viral delivery of therapeutic compounds
WO2025144938A1 (en) 2023-12-26 2025-07-03 Emmune, Inc. Systems for nucleic acid transfer

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197553B1 (en) 1994-07-15 2001-03-06 Merck & Co., Inc. Method for large scale plasmid purification
US20120251618A1 (en) 2011-03-31 2012-10-04 modeRNA Therapeutics Delivery and formulation of engineered nucleic acids
WO2013086322A1 (en) * 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Branched alkyl and cycloalkyl terminated biodegradable lipids for the delivery of active agents
WO2013086354A1 (en) * 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
WO2014089239A1 (en) * 2012-12-07 2014-06-12 Alnylam Pharmaceuticals, Inc. Improved nucleic acid lipid particle formulations

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018081480A1 (en) * 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197553B1 (en) 1994-07-15 2001-03-06 Merck & Co., Inc. Method for large scale plasmid purification
US20120251618A1 (en) 2011-03-31 2012-10-04 modeRNA Therapeutics Delivery and formulation of engineered nucleic acids
WO2013086322A1 (en) * 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Branched alkyl and cycloalkyl terminated biodegradable lipids for the delivery of active agents
WO2013086354A1 (en) * 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
WO2014089239A1 (en) * 2012-12-07 2014-06-12 Alnylam Pharmaceuticals, Inc. Improved nucleic acid lipid particle formulations

Non-Patent Citations (33)

* Cited by examiner, † Cited by third party
Title
"Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", December 2000, WILEY
"Bioreversible Carriers in Drug Design", 1987, AMERICAN PHARMACEUTICAL ASSOCIATION AND PERGAMON PRESS
"Methods in Molecular Biology", 2005, HUMANA PRESS, article "Oligonucleotide synthesis: methods and applications"
"Oligonucleotide synthesis: a practical approach", 1984, IRL PRESS
"Remington: The Science and Practice of Pharmacy", 2000, PHILADELPHIA COLLEGE OF PHARMACY AND SCIENCE
BATZER ET AL., NUCLEIC ACID RES., vol. 19, 1991, pages 5081
BECKERT, B.; MASQUIDA, B.: "Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology", vol. 703, 2010, HUMANA PRESS
BERNSTEIN, P.; ROSS, J.: "Poly (A), poly (A) binding protein and the regulation of mRNA stability", TRENDS BIO SCI, vol. 14, 1989, pages 373 - 377, XP023669459, DOI: doi:10.1016/0968-0004(89)90011-X
BOWMAN, J.C. ET AL: "RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods", vol. 941, 2012, HUMANA PRESS
BRUNELLE, J.L.; GREEN, R.: "Methods in Enzymology", vol. 530, 2013, article "Five - In vitro transcription from plasmid or PCR-amplified DNA", pages: 101 - 114
BUNDGARD, H.: "Design of Prodrugs", 1985, ELSEVIER, pages: 7 - 9,21-24
DREYFUS, M.; REGNIER, P.: "The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria", CELL, vol. 111, 2002, pages 611 - 613
GREEN, T.W.; P.G.M. WUTZ: "Protective Groups in Organic Synthesis", 1999, WILEY
GRUDZIEN-NOGALSKA, E.; KOWALSKA, J.; SU, W.; KUHN, A.N.; SLEPENKOV, S.V.; DARYNKIEWICZ, E.; SAHIN, U.; JEMIELITY, J.; RHOADS, R.E.: "Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology", vol. 969, 2013
GUHANIYOGI, J.; BREWER, G.: "Regulation of mRNA stability in mammalian cells", GENE, vol. 265, 2001, pages 11 - 23, XP004230718, DOI: doi:10.1016/S0378-1119(01)00350-X
HAFEZ, I.M. ET AL., GENE THER, vol. 8, 2001, pages 1188 - 1196
HEILIG, J. ET AL: "Large-Scale Preparation of Plasmid DNA", CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vol. 41, no. II, 2001, pages 1.7.1 - 1.7.16, XP002005961, DOI: doi:10.1002/0471142727.mb0107s41
HIGUCHI, T. ET AL., A.C.S. SYMPOSIUM SERIES, vol. 14
JAYARAMAN ET AL., ANGEWANDTE CHEMIE, INTERNATIONAL EDITION, vol. 51, no. 34, 2012, pages 8529 - 8533
KAMAKAKA, R. T.; KRAUS, W. L.: "In Vitro Transcription", CURRENT PROTOCOLS IN CELL BIOLOGY, vol. 2, no. 11.6, 2001, pages 11.6.1 - 11.6.17
KARIKO, K. ET AL: "Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA", NUCL ACID RES, vol. 39, 2011, pages E142
KARIKO, K.; MURAMATSU, H.; WELSH, F.A.; LUDWIG, J.; KATO, H.; AKIRA, S.; WEISSMAN, D.: "Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability", MOL THER, vol. 16, 2008, pages 1833 - 1840
KARIKO, K.; WEISSMAN, D.: "Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development", CURR OPIN DRUG DISCOV DEVEL, vol. 10, 2007, pages 523 - 532, XP009154595
LINPINSEL, J.L.; CONN, G.L.: "RNA in vitro transcription and RNA purification", vol. 941, 2012, HUMANA PRESS, article "General protocols for preparation of plasmid DNA template and Bowman"
LOSICK, R.: "In vitro transcription", ANN REV BIOCHEM, vol. 41, 1972, pages 409 - 446
LUKAVSKY, P.J.; PUGLISI, J.D.: "Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides", RNA, vol. 10, 2004, pages 889 - 893
OHTSUKA ET AL., J. BIOL. CHEM., vol. 260, 1985, pages 2605 - 2608
PARDI, N.; MURAMATSU, H.; WEISSMAN, D.; KARIKO, K.: "In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology", vol. 969, 2013
ROSSOLINI ET AL., MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98
ROZKOV, A. ET AL: "Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture", BIOTECHNOL. BIOENG., vol. 99, 2008, pages 557 - 566
SEMPLE ET AL., NATURE BIOTECHNOLOGY, vol. 28, 2010, pages 172 - 176
SEMPLE, S.C. ET AL., ADV. DRUG DELIV REV, vol. 32, 1998, pages 3 - 17
WEISSMAN, D.; PARDI, N.; MURAMATSU, H.; KARIKO, K.: "HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology", vol. 969, 2013

Cited By (271)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11951179B2 (en) 2011-06-08 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US11730825B2 (en) 2011-06-08 2023-08-22 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10888626B2 (en) 2011-06-08 2021-01-12 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11547764B2 (en) 2011-06-08 2023-01-10 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US11052159B2 (en) 2011-06-08 2021-07-06 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US12121592B2 (en) 2011-06-08 2024-10-22 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10238754B2 (en) 2011-06-08 2019-03-26 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US11291734B2 (en) 2011-06-08 2022-04-05 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11338044B2 (en) 2011-06-08 2022-05-24 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10507249B2 (en) 2011-06-08 2019-12-17 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10350303B1 (en) 2011-06-08 2019-07-16 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10413618B2 (en) 2011-06-08 2019-09-17 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11185595B2 (en) 2011-06-08 2021-11-30 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11951181B2 (en) 2011-06-08 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11951180B2 (en) 2011-06-08 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US11634379B2 (en) 2014-06-25 2023-04-25 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10723692B2 (en) 2014-06-25 2020-07-28 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11168051B2 (en) 2015-06-29 2021-11-09 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US12151995B2 (en) 2015-09-17 2024-11-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11648324B2 (en) 2015-10-28 2023-05-16 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10166298B2 (en) 2015-10-28 2019-01-01 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11040112B2 (en) 2015-10-28 2021-06-22 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11712481B2 (en) 2015-10-28 2023-08-01 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
WO2018081480A1 (en) * 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
US12144895B2 (en) 2016-11-08 2024-11-19 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US12324859B2 (en) 2017-03-15 2025-06-10 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US12257317B2 (en) 2017-03-17 2025-03-25 Newcastle University Adeno-associated virus vector delivery of a fragment of micro-dystrophin to treat muscular dystrophy
US11357856B2 (en) 2017-04-13 2022-06-14 Acuitas Therapeutics, Inc. Lipids for delivery of active agents
WO2018191719A1 (en) 2017-04-13 2018-10-18 Acuitas Therapeutics, Inc. Lipid delivery of therapeutic agents to adipose tissue
US11820728B2 (en) 2017-04-28 2023-11-21 Acuitas Therapeutics, Inc. Carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2018213476A1 (en) 2017-05-16 2018-11-22 Translate Bio, Inc. Treatment of cystic fibrosis by delivery of codon-optimized mrna encoding cftr
US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2018236849A1 (en) 2017-06-19 2018-12-27 Translate Bio, Inc. Messenger rna therapy for the treatment of friedreich's ataxia
US11639329B2 (en) 2017-08-16 2023-05-02 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US11524932B2 (en) 2017-08-17 2022-12-13 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US12065396B2 (en) 2017-08-17 2024-08-20 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US11542225B2 (en) 2017-08-17 2023-01-03 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2019089828A1 (en) 2017-10-31 2019-05-09 Acuitas Therapeutics, Inc. Lamellar lipid nanoparticles
WO2019126593A1 (en) 2017-12-20 2019-06-27 Translate Bio, Inc. Improved composition and methods for treatment of ornithine transcarbamylase deficiency
WO2019131770A1 (en) 2017-12-27 2019-07-04 武田薬品工業株式会社 Nucleic acid-containing lipid nano-particle and use thereof
WO2019222277A1 (en) 2018-05-15 2019-11-21 Translate Bio, Inc. Subcutaneous delivery of messenger rna
US12076439B2 (en) 2018-05-24 2024-09-03 Translate Bio, Inc. Thioester cationic lipids
WO2019226925A1 (en) 2018-05-24 2019-11-28 Translate Bio, Inc. Thioester cationic lipids
CN112437767B (en) * 2018-05-24 2023-10-27 川斯勒佰尔公司 thioester cationic lipids
CN112437767A (en) * 2018-05-24 2021-03-02 川斯勒佰尔公司 Thioester cationic lipids
CN117430538A (en) * 2018-05-24 2024-01-23 川斯勒佰尔公司 Thioester cationic lipids
AU2019275068B2 (en) * 2018-05-24 2024-09-26 Translate Bio, Inc. Thioester cationic lipids
WO2019232208A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Cationic lipids comprising a steroidal moiety
WO2019232095A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Vitamin cationic lipids
WO2019232103A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Messenger rna vaccines and uses thereof
WO2019232097A1 (en) 2018-05-30 2019-12-05 Translate Bio, Inc. Phosphoester cationic lipids
EP4442831A2 (en) 2018-05-30 2024-10-09 Translate Bio, Inc. Cationic lipids comprising a steroidal moiety
WO2020023533A1 (en) 2018-07-23 2020-01-30 Translate Bio, Inc. Dry power formulations for messenger rna
AU2019333042B2 (en) * 2018-08-29 2025-06-19 Translate Bio, Inc. Improved process of preparing mRNA-loaded lipid nanoparticles
WO2020047061A1 (en) 2018-08-29 2020-03-05 Translate Bio, Inc. Improved process of preparing mrna-loaded lipid nanoparticles
US12064515B2 (en) 2018-08-29 2024-08-20 Translate Bio, Inc. Process of preparing mRNA-loaded lipid nanoparticles
US11357726B2 (en) 2018-08-29 2022-06-14 Translate Bio, Inc. Process of preparing mRNA-loaded lipid nanoparticles
WO2020056294A1 (en) 2018-09-14 2020-03-19 Translate Bio, Inc. Composition and methods for treatment of methylmalonic acidemia
EP3853202A1 (en) * 2018-09-19 2021-07-28 ModernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12263248B2 (en) 2018-09-19 2025-04-01 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2020061367A1 (en) * 2018-09-19 2020-03-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
JP2024038014A (en) * 2018-09-19 2024-03-19 モデルナティエックス インコーポレイテッド Compounds and compositions for intracellular delivery of therapeutic agents
JP7410135B2 (en) 2018-09-19 2024-01-09 モデルナティエックス インコーポレイテッド Compounds and compositions for intracellular delivery of therapeutic agents
JP2022501360A (en) * 2018-09-19 2022-01-06 モデルナティエックス インコーポレイテッドModernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2020061426A2 (en) 2018-09-21 2020-03-26 Acuitas Therapeutics, Inc. Systems and methods for manufacturing lipid nanoparticles and liposomes
US12329857B2 (en) 2018-09-21 2025-06-17 Acuitas Therapeutics, Inc. Systems and methods for manufacturing lipid nanoparticles and liposomes
WO2020080475A1 (en) 2018-10-18 2020-04-23 武田薬品工業株式会社 Method for activation/proliferation of t cells
WO2020081933A1 (en) 2018-10-19 2020-04-23 Translate Bio, Inc. Pumpless encapsulation of messenger rna
WO2020097384A1 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. 2,5-dioxopiperazine lipids with intercalated ester, thioester, disulfide and anhydride moieities
WO2020097379A2 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. Peg lipidoid compounds
WO2020097379A3 (en) * 2018-11-09 2020-07-23 Translate Bio, Inc. Peg lipidoid compounds
WO2020097511A2 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. Messenger rna therapy for treatment of ocular diseases
US12280147B2 (en) 2018-11-09 2025-04-22 Translate Bio, Inc. PEG lipidoid compounds
WO2020097376A1 (en) 2018-11-09 2020-05-14 Translate Bio, Inc. Multi-peg lipid compounds
WO2020102172A2 (en) 2018-11-12 2020-05-22 Translate Bio, Inc. Methods for inducing immune tolerance
WO2020106903A1 (en) 2018-11-21 2020-05-28 Translate Bio, Inc. Cationic lipid compounds and compositions thereof for use in the delivery of messenger rna
WO2020146344A1 (en) 2019-01-07 2020-07-16 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
US12151996B2 (en) 2019-01-11 2024-11-26 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
IL284535B2 (en) * 2019-01-11 2025-03-01 Acuitas Therapeutics Inc Lipids for lipid nanoparticle delivery of active agents
CN113474328A (en) * 2019-01-11 2021-10-01 爱康泰生治疗公司 Lipids for lipid nanoparticle delivery of active agents
WO2020146805A1 (en) * 2019-01-11 2020-07-16 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
JP2022517783A (en) * 2019-01-11 2022-03-10 アクイタス セラピューティクス インコーポレイテッド Lipids for Activator Lipid Nanoparticle Delivery Lipids
JP7523449B2 (en) 2019-01-11 2024-07-26 アクイタス セラピューティクス インコーポレイテッド Lipids for lipid nanoparticle delivery of active agents
IL284535B1 (en) * 2019-01-11 2024-11-01 Acuitas Therapeutics Inc Lipids for lipid nanoparticle delivery of active substances
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
EP4450487A3 (en) * 2019-01-11 2024-12-25 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
WO2020214946A1 (en) 2019-04-18 2020-10-22 Translate Bio, Inc. Cystine cationic lipids
WO2020219427A1 (en) 2019-04-22 2020-10-29 Translate Bio, Inc. Thioester cationic lipids
WO2020227085A1 (en) 2019-05-03 2020-11-12 Translate Bio, Inc. Di-thioester cationic lipids
EP3962902A1 (en) * 2019-05-03 2022-03-09 Translate Bio, Inc. Di-thioester cationic lipids
WO2020243540A1 (en) 2019-05-31 2020-12-03 Translate Bio, Inc. Macrocyclic lipids
WO2020257716A1 (en) 2019-06-21 2020-12-24 Translate Bio, Inc. Tricine and citric acid lipids
WO2020257611A1 (en) 2019-06-21 2020-12-24 Translate Bio, Inc. Cationic lipids comprising an hydroxy moiety
WO2021007278A1 (en) 2019-07-08 2021-01-14 Translate Bio, Inc. Improved mrna-loaded lipid nanoparticles and processes of making the same
EP4467653A2 (en) 2019-07-08 2024-11-27 Translate Bio, Inc. Improved mrna-loaded lipid nanoparticles and processes of making the same
WO2021016075A1 (en) 2019-07-19 2021-01-28 Flagship Pioneering Innovations Vi, Llc Recombinase compositions and methods of use
WO2021016430A1 (en) 2019-07-23 2021-01-28 Translate Bio, Inc. Stable compositions of mrna-loaded lipid nanoparticles and processes of making
WO2021030701A1 (en) 2019-08-14 2021-02-18 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
DE112020003843T5 (en) 2019-08-14 2022-05-19 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
EP4013385B1 (en) 2019-08-14 2024-07-03 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
EP4454640A2 (en) 2019-08-14 2024-10-30 Acuitas Therapeutics Inc. Improved lipid nanoparticles for delivery of nucleic acids
WO2021046265A1 (en) 2019-09-06 2021-03-11 Generation Bio Co. Lipid nanoparticle compositions comprising closed-ended dna and cleavable lipids and methods of use thereof
US12312293B2 (en) 2019-09-19 2025-05-27 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11597698B2 (en) 2019-09-19 2023-03-07 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
WO2021055609A1 (en) 2019-09-20 2021-03-25 Translate Bio, Inc. Mrna encoding engineered cftr
WO2021081058A1 (en) 2019-10-21 2021-04-29 Translate Bio, Inc. Compositions, methods and uses of messenger rna
WO2021102411A1 (en) 2019-11-22 2021-05-27 Generation Bio Co. Ionizable lipids and nanoparticle compositions thereof
WO2021127641A1 (en) 2019-12-20 2021-06-24 Translate Bio, Inc. Improved process of preparing mrna-loaded lipid nanoparticles
WO2021127394A2 (en) 2019-12-20 2021-06-24 Translate Bio, Inc. Rectal delivery of messenger rna
WO2021142245A1 (en) 2020-01-10 2021-07-15 Translate Bio, Inc. Compounds, pharmaceutical compositions and methods for modulating expression of muc5b in lung cells and tissues
WO2021173840A1 (en) 2020-02-25 2021-09-02 Translate Bio, Inc. Improved processes of preparing mrna-loaded lipid nanoparticles
WO2021195214A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
WO2021195218A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
WO2021195529A2 (en) 2020-03-27 2021-09-30 Generation Bio Co. Novel lipids and nanoparticle compositions thereof
WO2021226436A1 (en) 2020-05-07 2021-11-11 Translate Bio, Inc. Optimized nucleotide sequences encoding sars-cov-2 antigens
WO2021226463A1 (en) 2020-05-07 2021-11-11 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
WO2021226468A1 (en) 2020-05-07 2021-11-11 Translate Bio, Inc. Improved compositions for cftr mrna therapy
WO2021231697A1 (en) 2020-05-14 2021-11-18 Translate Bio, Inc. Peg lipidoid compounds
WO2021231901A1 (en) 2020-05-15 2021-11-18 Translate Bio, Inc. Lipid nanoparticle formulations for mrna delivery
WO2021236980A1 (en) 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Coronavirus antigen compositions and their uses
WO2021236930A1 (en) 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2021243301A2 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc. Trem compositions and methods relating thereto
WO2021243290A1 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods relating thereto
WO2021257595A1 (en) 2020-06-15 2021-12-23 Research Institute At Nationwide Children's Hospital Adeno-associated virus vector delivery for muscular dystrophies
WO2022006527A1 (en) 2020-07-02 2022-01-06 Maritime Therapeutics, Inc. Compositions and methods for reverse gene therapy
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
WO2022023284A1 (en) 2020-07-27 2022-02-03 Anjarium Biosciences Ag Compositions of dna molecules, methods of making therefor, and methods of use thereof
WO2022051629A1 (en) 2020-09-03 2022-03-10 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2022076562A1 (en) 2020-10-06 2022-04-14 Translate Bio, Inc. Improved process and formulation of lipid nanoparticles
WO2022081548A1 (en) 2020-10-12 2022-04-21 Translate Bio, Inc. Improved process of preparing ice-based lipid nanoparticles
WO2022081544A1 (en) 2020-10-12 2022-04-21 Translate Bio, Inc. Improved process of preparing mrna-loaded lipid nanoparticles
WO2022099194A1 (en) 2020-11-09 2022-05-12 Translate Bio, Inc. Improved compositions for delivery of codon-optimized mrna
WO2022115547A1 (en) 2020-11-25 2022-06-02 Translate Bio, Inc. Stable liquid lipid nanoparticle formulations
WO2022140702A1 (en) 2020-12-23 2022-06-30 Flagship Pioneering, Inc. Compositions of modified trems and uses thereof
WO2022155404A1 (en) 2021-01-14 2022-07-21 Translate Bio, Inc. Methods and compositions for delivering mrna coded antibodies
WO2022169508A1 (en) 2021-02-08 2022-08-11 The Board Of Regents Of The University Of Texas System Unsaturated dendrimers compositions,related formulations, and methods of use thereof
EP4046629A1 (en) 2021-02-19 2022-08-24 ModernaTX, Inc. Lipid nanoparticle compositions and methods of formulating the same
US12121610B2 (en) 2021-03-22 2024-10-22 Recode Therapeutics, Inc. Compositions and methods for targeted delivery to cells
US12257318B2 (en) 2021-03-23 2025-03-25 Recode Therapeutics, Inc. Polynucleotide compositions, related formulations, and methods of use thereof
WO2022204549A1 (en) 2021-03-25 2022-09-29 Translate Bio, Inc. Optimized nucleotide sequences encoding the extracellular domain of human ace2 protein or a portion thereof
WO2022212784A1 (en) 2021-03-31 2022-10-06 Flagship Pioneering Innovations V, Inc. Thanotransmission polypeptides and their use in treating cancer
WO2022225918A1 (en) 2021-04-19 2022-10-27 Translate Bio, Inc. Improved compositions for delivery of mrna
WO2022223556A1 (en) 2021-04-20 2022-10-27 Anjarium Biosciences Ag Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof
WO2022232289A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing therapeutic antibodies and uses thereof
WO2022232286A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing anti-coronavirus antibodies and uses thereof
WO2023278754A1 (en) 2021-07-01 2023-01-05 Translate Bio, Inc. Compositions for delivery of mrna
WO2023009547A1 (en) 2021-07-26 2023-02-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and uses thereof
WO2023023055A1 (en) 2021-08-16 2023-02-23 Renagade Therapeutics Management Inc. Compositions and methods for optimizing tropism of delivery systems for rna
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023044333A1 (en) 2021-09-14 2023-03-23 Renagade Therapeutics Management Inc. Cyclic lipids and methods of use thereof
WO2023044343A1 (en) 2021-09-14 2023-03-23 Renagade Therapeutics Management Inc. Acyclic lipids and methods of use thereof
WO2023044006A1 (en) 2021-09-17 2023-03-23 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
EP4464783A2 (en) 2021-09-17 2024-11-20 Flagship Pioneering Innovations VI, LLC Compositions and methods for producing circular polyribonucleotides
WO2023069397A1 (en) 2021-10-18 2023-04-27 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2023069498A1 (en) 2021-10-22 2023-04-27 Senda Biosciences, Inc. Mrna vaccine composition
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
WO2023081526A1 (en) 2021-11-08 2023-05-11 Orna Therapeutics, Inc. Lipid nanoparticle compositions for delivering circular polynucleotides
WO2023086893A1 (en) 2021-11-10 2023-05-19 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
WO2023091490A1 (en) 2021-11-16 2023-05-25 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023091787A1 (en) 2021-11-22 2023-05-25 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023096858A1 (en) 2021-11-23 2023-06-01 Senda Biosciences, Inc. A bacteria-derived lipid composition and use thereof
WO2023097003A2 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and their uses
WO2023096963A1 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Varicella-zoster virus immunogen compositions and their uses
WO2023096990A1 (en) 2021-11-24 2023-06-01 Flagship Pioneering Innovation Vi, Llc Coronavirus immunogen compositions and their uses
US12129223B2 (en) 2021-12-16 2024-10-29 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2023115013A1 (en) 2021-12-17 2023-06-22 Flagship Pioneering Innovations Vi, Llc Methods for enrichment of circular rna under denaturing conditions
WO2023122080A1 (en) 2021-12-20 2023-06-29 Senda Biosciences, Inc. Compositions comprising mrna and lipid reconstructed plant messenger packs
WO2023122745A1 (en) 2021-12-22 2023-06-29 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2023122789A1 (en) 2021-12-23 2023-06-29 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
WO2023122752A1 (en) 2021-12-23 2023-06-29 Renagade Therapeutics Management Inc. Constrained lipids and methods of use thereof
WO2023135273A2 (en) 2022-01-14 2023-07-20 Anjarium Biosciences Ag Compositions of dna molecules encoding factor viii, methods of making thereof, and methods of use thereof
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
CN114213346A (en) * 2022-02-22 2022-03-22 中国科学院基础医学与肿瘤研究所(筹) Bivalent ionizable lipid compound, composition and application thereof
CN114213347A (en) * 2022-02-22 2022-03-22 中国科学院基础医学与肿瘤研究所(筹) Bivalent ionizable lipid compound, composition and application thereof
WO2023177655A1 (en) 2022-03-14 2023-09-21 Generation Bio Co. Heterologous prime boost vaccine compositions and methods of use
WO2023177904A1 (en) 2022-03-18 2023-09-21 Modernatx, Inc. Sterile filtration of lipid nanoparticles and filtration analysis thereof for biological applications
EP4484408A4 (en) * 2022-03-25 2025-07-09 Shenzhen Neocurna Biotechnology Corp CATIONIC lipid compound, method of preparation and use thereof, and mRNA delivery system
WO2023183616A1 (en) 2022-03-25 2023-09-28 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2023196931A1 (en) 2022-04-07 2023-10-12 Renagade Therapeutics Management Inc. Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents
WO2023196634A2 (en) 2022-04-08 2023-10-12 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2023220083A1 (en) 2022-05-09 2023-11-16 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods of use for treating proliferative disorders
WO2023220729A2 (en) 2022-05-13 2023-11-16 Flagship Pioneering Innovations Vii, Llc Double stranded dna compositions and related methods
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2023239756A1 (en) 2022-06-07 2023-12-14 Generation Bio Co. Lipid nanoparticle compositions and uses thereof
WO2023250112A1 (en) 2022-06-22 2023-12-28 Flagship Pioneering Innovations Vi, Llc Compositions of modified trems and uses thereof
WO2024030856A2 (en) 2022-08-01 2024-02-08 Flagship Pioneering Innovations Vii, Llc Immunomodulatory proteins and related methods
WO2024035952A1 (en) 2022-08-12 2024-02-15 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
WO2024044147A1 (en) 2022-08-23 2024-02-29 Modernatx, Inc. Methods for purification of ionizable lipids
WO2024049979A2 (en) 2022-08-31 2024-03-07 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
WO2024077191A1 (en) 2022-10-05 2024-04-11 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine
WO2024097664A1 (en) 2022-10-31 2024-05-10 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2024102677A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Circular rna compositions
WO2024102762A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024102730A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and nanoparticle compositions for delivering polynucleotides
WO2024102799A1 (en) 2022-11-08 2024-05-16 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
WO2024102434A1 (en) 2022-11-10 2024-05-16 Senda Biosciences, Inc. Rna compositions comprising lipid nanoparticles or lipid reconstructed natural messenger packs
WO2024112652A1 (en) 2022-11-21 2024-05-30 Translate Bio, Inc. Compositions of dry powder formulations of messenger rna and methods of use thereof
WO2024119074A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticle compositions for cell targeting
WO2024119039A2 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticles and uses thereof
WO2024119103A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers
WO2024119051A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same
US12133923B2 (en) 2022-12-08 2024-11-05 Recode Therapeutics, Inc. Lipid nanoparticle compositions and uses thereof
US12337068B2 (en) 2022-12-08 2025-06-24 Recode Therapeutics, Inc. Lipid nanoparticle compositions and uses thereof
WO2024121814A1 (en) 2022-12-09 2024-06-13 Takeda Pharmaceutical Company Limited Modified immunomodulators
WO2024129988A1 (en) 2022-12-14 2024-06-20 Flagship Pioneering Innovations Vii, Llc Compositions and methods for delivery of therapeutic agents to bone
WO2024126809A1 (en) 2022-12-15 2024-06-20 Sanofi Mrna encoding influenza virus-like particle
WO2024133515A1 (en) 2022-12-20 2024-06-27 Sanofi Rhinovirus mrna vaccine
US12318441B2 (en) 2022-12-29 2025-06-03 Rinuagene Biotechnology Co., Ltd. Polynucleotide molecules used for the prevention or treatment of HPV infection related diseases
WO2024151685A1 (en) 2023-01-09 2024-07-18 Beth Israel Deaconess Medical Center, Inc. Recombinant nucleic acid molecules and their use in wound healing
WO2024151583A2 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2024151673A2 (en) 2023-01-09 2024-07-18 President And Fellows Of Harvard College Recombinant nucleic acid molecules and their use in wound healing
WO2024151687A1 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
WO2024159172A1 (en) 2023-01-27 2024-08-02 Senda Biosciences, Inc. A modified lipid composition and uses thereof
WO2024156291A1 (en) * 2023-01-29 2024-08-02 Geneditbio Limited Cationic lipids and lipid nanoparticles
WO2024167885A1 (en) 2023-02-06 2024-08-15 Flagship Pioneering Innovations Vii, Llc Immunomodulatory compositions and related methods
WO2024173307A2 (en) 2023-02-13 2024-08-22 Flagship Pioneering Innovation Vii, Llc Cleavable linker-containing ionizable lipids and lipid carriers for therapeutic compositions
WO2024173828A1 (en) 2023-02-17 2024-08-22 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified uracil
WO2024173836A2 (en) 2023-02-17 2024-08-22 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified cytosine
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024192420A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Compositions comprising polyribonucleotides and uses thereof
WO2024192422A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2024205657A2 (en) 2023-03-29 2024-10-03 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024200820A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Method of synthesis of targeted lipid nanoparticle and uses thereof
WO2024200823A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell enhancing molecule and use thereof
WO2024200826A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell inhibiting molecule and use thereof
WO2024210160A1 (en) 2023-04-07 2024-10-10 Takeda Pharmaceutical Company Limited Conjugation complex
WO2024216128A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Trems for use in correction of missense mutations
WO2024216191A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Modified trems, compositions, and related methods thereof
WO2024218166A1 (en) 2023-04-17 2024-10-24 Sanofi Reconstitutable dry powder formulations and methods of use thereof
WO2024220712A2 (en) 2023-04-19 2024-10-24 Sail Biomedicines, Inc. Vaccine compositions
WO2024220625A1 (en) 2023-04-19 2024-10-24 Sail Biomedicines, Inc. Delivery of polynucleotides from lipid nanoparticles comprising rna and ionizable lipids
WO2024220752A2 (en) 2023-04-19 2024-10-24 Sail Biomedicines, Inc. Rna therapeutic compositions
WO2024220746A2 (en) 2023-04-21 2024-10-24 Flagship Pioneering Innovations Vii, Llc Rnai agents targeting fatty acid synthase and related methods
WO2024233308A2 (en) 2023-05-05 2024-11-14 Orna Therapeutics, Inc. Circular rna compositions and methods
WO2024230934A1 (en) 2023-05-11 2024-11-14 CureVac SE Therapeutic nucleic acid for the treatment of ophthalmic diseases
WO2024236361A1 (en) 2023-05-15 2024-11-21 Takeda Pharmaceutical Company Limited Compositions and methods for delivery of nucleic acids to cells
WO2024236504A1 (en) 2023-05-15 2024-11-21 Takeda Pharmaceutical Company Limited Sequences and methods for delivery of dna and rna
WO2024258829A1 (en) 2023-06-12 2024-12-19 Flagship Pioneering Innovations Vii, Llc Sars-cov-2 vaccine compositions and related methods
WO2025006684A1 (en) 2023-06-28 2025-01-02 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
WO2025024486A2 (en) 2023-07-25 2025-01-30 Flagship Pioneering Innovations Vii, Llc Cas endonucleases and related methods
WO2025027116A1 (en) 2023-08-01 2025-02-06 Institut Curie Nanoparticles comprising nucleic acid sequences encoding cyclic gmp-amp synthase
WO2025042786A1 (en) 2023-08-18 2025-02-27 Flagship Pioneering Innovations Vi, Llc Compositions comprising circular polyribonucleotides and uses thereof
WO2025049690A1 (en) 2023-08-29 2025-03-06 Orna Therapeutics, Inc. Circular polyethylene glycol lipids
WO2025054236A2 (en) 2023-09-06 2025-03-13 Flagship Pioneering Innovations Vii, Llc Sars-cov-2 vaccine compositions and related methods
WO2025051994A1 (en) 2023-09-07 2025-03-13 Coave Therapeutics Ionizable lipid nanoparticles
WO2025052180A2 (en) 2023-09-07 2025-03-13 Axelyf ehf. Lipids and lipid nanoparticles
WO2025064475A2 (en) 2023-09-18 2025-03-27 Flagship Pioneering Innovations Vii, Llc Ionizable lipidoid compositions and therapeutic uses thereof
WO2025072331A1 (en) 2023-09-26 2025-04-03 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025081042A1 (en) 2023-10-12 2025-04-17 Renagade Therapeutics Management Inc. Nickase-retron template-based precision editing system and methods of use
WO2025096807A2 (en) 2023-10-31 2025-05-08 Flagship Pioneering Innovations Vii, Llc Novel therapeutic dna forms
WO2025101501A1 (en) 2023-11-07 2025-05-15 Orna Therapeutics, Inc. Circular rna compositions
WO2025106670A1 (en) 2023-11-14 2025-05-22 Flagship Pioneering Innovations Vii, Llc Ionizable lipidoid compositions and therapeutic uses thereof
WO2025106930A1 (en) 2023-11-17 2025-05-22 Sail Biomedicines, Inc. Circular polyribonucleotides encoding glucagon-like peptide 2 (glp-2) and uses thereof
WO2025106806A1 (en) 2023-11-17 2025-05-22 Acuitas Therapeutics, Inc. Pegylated lipids
WO2025106915A1 (en) 2023-11-17 2025-05-22 Sail Biomedicines, Inc. Circular polyribonucleotides encoding glucagon-like peptide 1 (glp-1) and uses thereof
WO2025111526A1 (en) 2023-11-22 2025-05-30 Flagship Pioneering Innovations Vii, Llc Methods and compositions for treating non-alcoholic fatty liver disease
WO2025114520A1 (en) 2023-12-01 2025-06-05 Coave Therapeutics Ionizable lipid nanoparticles
WO2025117877A2 (en) 2023-12-01 2025-06-05 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025128696A1 (en) 2023-12-12 2025-06-19 Acuitas Therapeutics, Inc. Cationic lipid compounds for use in lipid nanoparticles
WO2025132122A1 (en) 2023-12-13 2025-06-26 Berlin Institute Of Health Methods of delivering therapeutics using lipid nanoparticles
WO2025126071A1 (en) 2023-12-14 2025-06-19 Pfizer Inc. Rna molecules
WO2025133115A1 (en) 2023-12-21 2025-06-26 Ose Immunotherapeutics Lipid-based nanoparticles comprising il-35
WO2025137646A1 (en) 2023-12-22 2025-06-26 Recode Therapeutics, Inc. Gene editing methods and compositions for treating cystic fibrosis
WO2025140421A1 (en) * 2023-12-29 2025-07-03 北京剂泰医药科技有限公司 Ionizable lipid compound and use thereof in delivering nucleic acid vaccine

Also Published As

Publication number Publication date
EP3397613A1 (en) 2018-11-07
US20190022247A1 (en) 2019-01-24

Similar Documents

Publication Publication Date Title
AU2022200794B2 (en) Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
AU2021201646B2 (en) Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US12151996B2 (en) Lipids for lipid nanoparticle delivery of active agents
WO2017117528A1 (en) Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11524932B2 (en) Lipids for use in lipid nanoparticle formulations
AU2016343803B2 (en) Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
AU2025202533A1 (en) Lipids for lipid nanoparticle delivery of active agent
WO2019036008A1 (en) Lipids for use in lipid nanoparticle formulations
WO2018191657A1 (en) Lipids for delivery of active agents
WO2024259322A1 (en) Lipids for use in lipid nanoparticles
HK1248232B (en) Lipids and lipid nanoparticle formulations for delivery of nucleic acids

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16829386

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2016829386

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2016829386

Country of ref document: EP

Effective date: 20180730