Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • 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
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
  • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

• LNPs Lipid nanoparticles
• IV intravenously administered LNPs typically accumulate in the liver and are internalized by liver hepatocytes, thereby greatly limiting the scope of their therapeutic applications.
• the present disclosure provides, in some embodiments, a composition comprising a therapeutic agent assembled with a lipid composition.
• the lipid composition may comprise: an ionizable cationinc lipid; a polymer-conjugated lipid comprising one or more hydrocarbon chains that each comprise about 8 to about 20 (e.g., about 8 to about 18, about 8 to about 16, or about 8 to about 14) carbon atoms; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer- conjugated lipid.
• SORT selective organ targeting
• the lipid composition may be characterized by an apparent ionization constant (pKa) from about 6 to about as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay.
• pKa apparent ionization constant
• TMS 2-(p-toluidino)-6-naphthalenesulfonic acid
• the present disclosure provides a composition comprising a therapeutic agent assembled with a lipid composition.
• the lipid composition may comprise: an ionizable cationinc lipid; a polymer-conjugated lipid comprising one or more hydrocarbon chains that each comprise about 8 to about 20 (e.g., about 8 to about 18, about 8 to about 16, or about 8 to about 14) carbon atoms; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer- conjugated lipid.
• SORT selective organ targeting
• the lipid composition may be characterized by an apparent ionization constant (pKa) outside a range of about 6 to about 7 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay.
• the lipid composition is characterized by an apparent ionization constant (pKa) of about 6 or lower as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 3 to about 6 as determined by a 2-(p-toluidino)-6- naphthalenesulfonic acid (TNS) titration assay. [0006] In some embodiments, the present disclosure provides a composition comprising a therapeutic agent assembled with a lipid composition.
• the lipid composition may comprise: an ionizable cationinc lipid; a polymer-conjugated lipid comprising one or more hydrocarbon chains that each comprise about 8 to about 20 (e.g., about 8 to about 18, about 8 to about 16, or about 8 to about 14) carbon atoms; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer- conjugated lipid.
• SORT selective organ targeting
• the lipid composition may be characterized by an apparent ionization constant (pKa) of about 8 or greater as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay.
• the lipid composition is characterized by an apparent ionization constant (pKa) of about 9 or greater as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 8 to about 13 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay.
• pKa apparent ionization constant
• the lipid composition is characterized by an apparent ionization constant (pKa) of about 9 to about 13 as determined by a 2-(p-toluidino)-6- naphthalenesulfonic acid (TNS) titration assay.
• the lipid composition is characterized by a zeta ( ⁇ ) potential of said lipid composition of about -10 millivolts (mV) to about 10 mV as determined by dynamic light scattering (DLS).
• the lipid composition is characterized by a zeta ( ⁇ ) potential of said lipid composition of about 0 millivolt (mV) to about 10 mV as determined by dynamic light scattering (DLS).
• the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid.
• one or more hydrocarbon chains each comprise about 8 to about 18 carbon atoms.
• one or more hydrocarbon chains each comprise about 8 to about 16 carbon atoms.
• one or more hydrocarbon chains each comprise about 8 to about 14 carbon atoms.
• a hydrocarbon chain of the one or more hydrocarbon chains of the polymer-conjugated lipid comprises no more than 3 unsaturated carbon-carbon bonds.
• a hydrocarbon chain of the one or more hydrocarbon chains of the polymer-conjugated lipid comprises no more than 2 unsaturated carbon-carbon bonds.
• the polymer-conjugated lipid comprises a polymer having a molecular weight of about 100 Daltons (Da) to about 100,000 Da. In other embodiments, the polymer- conjugated lipid comprises a polymer having a molecular weight of about 500 Da to about 100,000 Da. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 20%. In other embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 15%. In yet other embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 10%.
• the present disclosure provides cationic ionizable lipids (e.g., ionizable cationic lipids).
• the cationic ionizable lipid comprises a dendron or dendrimer comprising one or more branches, wherein said one or more branches each comprise two or more degradable functional groups.
• said cationic ionizable lipid is a dendron or dendrimer that comprises one or more diacyl groups.
• the ionizable cationic lipid is a dendrimer or dendron of a generation (g) having a structural formula: pharmaceutically acceptable salt thereof.
• the core comprises a structural formula (X Core ): , wherein Q is independently at each occurrence a covalent bond, -O-, -S-, -NR 2 -, or -CR 3a R 3b -, R 2 is independently at each occurrence R 1g or -L 2 -NR 1e R 1f , R 3a and R 3b are each independently at each occurrence hydrogen or an optionally substituted (e.g., C1- C6, such as C1-C3) alkyl, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (if present) are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted (e.g., C1-C12) alkyl, L 0 , L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, (e.g., C1-C12, such as C 1
• each branch of the plurality (N) of branches independently comprises a structural formula (XBranch): wherein * indicates a point of attachment of the branch to the core, g is 1,2 ,3, or 4, Z is 2 (g-1) , G is 0 when g is 1, ⁇ 2 when g ⁇ 1.
• each diacyl group independently comprises a structural formula , wherein * indicates a point of attachment of the diacyl group at the proximal end thereof, ** indicates a point of attachment of the diacyl group at the distal end thereof, Y 3 is independently at each occurrence an optionally substituted (e.g., C 1 -C 12 ): alkylene, an optionally substituted (e.g., C 1 -C 12 ) alkenylene, or an optionally substituted (e.g., C 1 -C 12 ) arenylene, A 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -, wherein R 4 is hydrogen or optionally substituted (e.g., C1-C6) alkyl, m 1 and m 2 are each independently at each occurrence 1, 2, or 3, and R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or an optionally substituted (e.
• * indicates
• each linker group independently comprises a structural formula , wherein ** indicates a point of attachment of the linker to a proximal diacyl group, *** indicates a point of attachment of the linker to a distal diacyl group, and Y1 is independently at each occurrence an optionally substituted (e.g., C1-C12) alkylene, an optionally substituted (e.g., C1-C12) alkenylene, or an optionally substituted (e.g., C1-C12) arenylene.
• each terminating group is independently selected from optionally substituted (e.g., C1-C18, such as C4-C18) alkylthiol, and optionally substituted (e.g., C1-C18, such as C4- C 18 ) alkenylthiol.
• x 1 is 0, 1, 2, or 3.
• R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g are each independently at each occurrence a point of connection to a branch (e.g., as indicated by *), hydrogen, or C 1 -C 12 alkyl (e.g., C 1 -C 8 alkyl, such as C 1 -C 6 alkyl or C 1 -C 3 alkyl), wherein the alkyl moiety is optionally substituted with one or more substituents each independently selected from -OH, C 4 -C 8 (e.g., C 4 -C 6 ) heterocycloalkyl (e.g., piperidinyl (e.g., , N-(C 1 -C 3 alkyl)-piperidinyl (e.g., ), piperazinyl (e.g., ), N-(C 1 -C 3 alkyl)-piperadizinyl R 1d
• R 3a and R 3b are each independently at each occurrence hydrogen.
• the plurality (N) of branches comprises at least 3 (e.g., at least 4, or at least 5) branches.
• each branch of the plurality of branches comprises a structural formula .
• each branch of the plurality of branches comprises a structural formula .
• each branch of the plurality of branches comprises a structural formula .
• each branch of the plurality of branches comprises at least 3 (e.g., at least 4, or at least 5) branches.
• each branch of the plurality of branches comprises a structural formula .
• each branch of the plurality of branches comprises a structural formula .
• the core comprises a structural formula .
• the core comprises a structural formula: (e.g., In some embodiments, the core comprises a structural formula: . In some embodiments, the core comprises a structural formula: , core comprises a structural formula: , such as yet other embodiments, the core comprises a structural formula: wherein Q’ is -NR 2 - or and q 2 are each independently 1
• the core comprises a structural formula: optionally substituted aryl or an optionally substituted (e.g., C3-C12, such as C3-C5) heteroaryl.
• the core comprises has a structural formula .
• the core comprises a structural formula selected from the group consisting of: , , , , , , , , pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.
• a 1 is -O- or -NH-.
• a 2 is -O- or -NH-.
• Y 3 is C1-C12 (e.g., C1-C6, such as C1- C3) alkylene.
• the diacyl group independently at each occurrence comprises a structural formula , such as optionally wherein R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or C 1 -C 3 alkyl.
• L 0 , L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, C1-C6 alkylene (e.g., C1-C3 alkylene), C2-C12 (e.g., C2-C8) alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH2CH2O)1-4-(CH2CH2)-), [(C1-C4) alkylene]-[(C 4 -C 6 ) heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., and [(C 1 -C 4 ) alkylene]-phenylene-[(C1-C4) alkylene] (e.g., In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence selected from C1-C6 alkylene (e.g., C1-C3 alkylene), -(C1- C3 alkylene), -(
• L 0 , L 1 , and L 2 are each independently at each occurrence C 1 -C 6 alkylene (e.g., C 1 -C 3 alkylene). In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence C 2 -C 12 (e.g., C 2 -C 8 ) alkyleneoxide (e.g., -(C 1 -C 3 alkylene-O) 1-4 -(C 1 - C 3 alkylene)).
• L 0 , L 1 , and L 2 are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)- phenylene-(C1-C3 alkylene)-) and [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-).
• each terminating group is independently C1-C18 (e.g., C4-C18) alkenylthiol or C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C 6 -C 12 aryl (e.g., phenyl), C 1 -C 12 (e.g., C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 mono-alkylamino (such as -NHCH 2 CH 2 CH 2 CH 3 ) -C(O)OH, ⁇ C(O)N(C1-C3 alkyl) ⁇ (C1-C6 alkylene) ⁇ (C1-C12 alkylamino (e.g., mono- or di-alkylamino)) , N-heterocyclo
• each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl moiety is optionally substituted with one or more (e.g., one) substituents each independently selected from C6-C12 aryl (e.g., phenyl), C1-C12 (e.g., C1-C8) alkylamino (e.g., C1-C6 mono-alkylamino (such as -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 di-alkylamino (such as , , wherein the C4-C6 N-heterocycloalkyl moiety of any of the preceding substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl.
• substituents each independently selected from C6-C12 aryl (e.g., phenyl), C1-C12 (e.g.,
• each terminating group is independently C1-C18 (e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent -OH.
• each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent selected from C 1 -C 12 (e.g., C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 mono-alkylamino (such as - and C4-C6 N-heterocycloalkyl (e.g., N-pyrrolidinyl ( ), N-piperidinyl ( ), N-azepanyl .
• each terminating group is independently C1-C18 (e.g., C4- C18) alkenylthiol or C1-C18 (e.g., C4-C18) alkylthiol.
• each terminating group is independently C1-C18 (e.g., C4-C18) alkylthiol.
• each terminating group is , [0010]
• the dendrimer or dendron is selected from the group consisting of
• said lipid composition comprises said ionizable cationic lipid at a molar percentage from about 5% to about 30%. In some embodiments, said lipid composition further comprises a phospholipid. In some embodiments, said lipid composition comprises said phospholipid at a molar percentage from about 5% to about 30%. In other embodiments, said lipid composition comprises said phospholipid at a molar percentage from about 8% to about 23%. In some embodiments, said phospholipid is not an ethylphosphocholine. In some embodiments, said lipid composition further comprises a steroid or steroid derivative.
• said lipid composition comprises said steroid or steroid derivative at a molar percentage from about 15% to about 46%.
• said steroid or steroid derivative is cholesterol.
• said SORT lipid is cationic.
• said SORT lipid comprises an ionizable cationic moiety (e.g., a tertiary amine moiety).
• the SORT lipid has a structural formula: .
• L is a bond or a (e.g., biodegradable) linker.
• R1 and R2 are each independently alkyl(C8-C24), alkenyl(C8-C24), or a substituted version of either group.
• R′, R′′ and R′′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6).
• the SORT lipid has a structural formula: [0013]
• R1 and R2 are each independently alkyl(C8-C24), alkenyl(C8-C24), or a substituted version of either group.
• R3, R3′, and R3′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6).
• said SORT lipid comprises a permanent cationic moiety (e.g., a quaternary ammonium ion). In some embodiments, said SORT lipid comprises a counterion to said permanent cationic moiety. In some embodiments, said SORT lipid is an alkylated phosphocholine (e.g., ethylphosphocholine). In some embodiments, said SORT lipid comprises a headgroup having a structural formula: , wherein L is a bond or a (e.g., biodegradable) linker; Z + is positively charged moiety (e.g., a quaternary ammonium ion); and X- is a counterion.
• L is a bond or a (e.g., biodegradable) linker
• Z + is positively charged moiety (e.g., a quaternary ammonium ion)
• X- is a counterion.
• said SORT lipid has a structural formula: .
• R 1 and R 2 are each independently an optionally substituted C 6 -C 24 alkyl, or an optionally substituted C 6 -C 24 alkenyl.
• said SORT lipid has a structural formula: [0015]
• R 1 and R 2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) , or a substituted version of either group.
• R′, R′′ and R′′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6).
• X ⁇ is a monovalent anion.
• L is .
• p and q are each independently 1, 2, or 3.
• R 4 is an optionally substituted C1-C6 alkyl.
• said SORT lipid has a structural formula: [0016]
• R 1 and R 2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) , or a substituted version of either group.
• R 3 , R 3 ′, and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) .
• R 4 is alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) .
• X ⁇ is a monovalent anion.
• said SORT lipid has a structural formula: [0017]
• R1 and R2 are each independently alkyl(C8-C24), alkenyl(C8-C24), or a substituted version of either group.
• R 3 , R 3 ′, and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) .
• X ⁇ is a monovalent anion.
• said SORT lipid has a structural formula: [0018]
• R4 and R4′ are each independently alkyl(C6-C24), alkenyl(C6-C24), or a substituted version of either group.
• R4′′ is alkyl(C ⁇ 24), alkenyl(C ⁇ 24), or a substituted version of either group.
• R4′′′ is alkyl(C1-C8), alkenyl(C2-C8), or a substituted version of either group.
• X 2 is a monovalent anion.
• said lipid composition comprises said SORT lipid at a molar percentage from about 20% to about 65%.
• said SORT lipid is zwitterionic. In some embodiments, said SORT lipid comprises a hydrophobically modified phosphate anion, a sulfonate anion, or a carboxylate anion. In some embodiments, said SORT lipid is anionic. In some embodiments, said SORT lipid has a structural formula: [0019] In some embodiments, R1 and R2 are each independently alkyl(C8-C24), alkenyl(C8-C24), or a substituted version of either group. In some embodiments, R 3 is hydrogen, alkyl (C ⁇ 6) , or substituted alkyl (C ⁇ 6) , or ⁇ Y 1 ⁇ R 4 .
• Y 1 is alkanediyl (C ⁇ 6) or substituted alkanediyl (C ⁇ 6) .
• R 4 is acyloxy (C ⁇ 8-24) or substituted acyloxy (C ⁇ 8-24) .
• said lipid composition is characterized by an average diameter of about 200 nanometers (nm) or less as determined by dynamic light scattering (DLS). In other embodiments, said lipid composition is characterized by an average diameter of about 150 nanometers (nm) or less as determined by dynamic light scattering (DLS). In yet other embodiments, said lipid composition is characterized by an average diameter of about 100 nanometers (nm) or less as determined by dynamic light scattering (DLS).
• said lipid composition is characterized by a polydispersity index (PDI) of about 0.2 or less as determined by dynamic light scattering (DLS). In some embodiments, said lipid composition is characterized by a lipid fusion percentage of at least about 5%, 6%, 7%, 8%, 9%, or 10% as determined by a flurorescence resonance energy transfer (FRET)-based assay.
• said therapeutic agent comprises a compound, a polynucleotide, a polypeptide, a protein, or a combination thereof. In some embodiments, said therapeutic agent comprises a polypeptide or a protein.
• said therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, deoxyribonucle
• said therapeutic agent comprises a polynucleotide; and wherein a molar ratio of nitrogen in said lipid composition to phosphate in said polynucleotide (N/P ratio) is no more than about 20:1. In some embodiments, said N/P ratio is from about 5:1 to about 20:1. In some embodiments, said therapeutic agent comprises two or more polynucleotides that comprises said polynucleotide. In some embodiments, a molar ratio of said therapeutic agent to total lipids of said lipid composition is no more than about 1:1, 1:10, 1:50, or 1:100. In some embodiments, at least about 85% of said therapeutic agent is encapsulated in particles of said lipid compositions.
• said SORT lipid is present in said composition in an amount sufficient to achieve a therapeutic effect at a dose of said therapeutic agent (e.g., at least about 1.1- or 10-fold) lower than that required with a reference lipid composition.
• said therapeutic agent e.g., heterologous polynucleotide
• said therapeutic agent is present in said composition at a dose of no more than about 2 milligram per kilogram (mg/kg, or mpk) body weight.
• said therapeutic agent e.g., heterologous polynucleotide
• the therapeutic agent is present in an aerosol composition at a dose of no more than 1.0, 0.5, 0.1, 0.05, or 0.01 mg/kg body weight.
• said therapeutic agent e.g., heterologous polynucleotide
• said intravenous dosage form at a concentration of no more than about 5 or 2 milligram per milliliter (mg/mL).
• the present disclosure also provides, in some embodiments, a method for targeted delivery of a therapeutic agent to an organ or a cell therein in a subject in need thereof.
• the method may comprise administering to the subject the therapeutic agent assembled with a lipid composition that comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein, upon the administering, a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises a first target protein at a weight or mass ratio of no more than about 20:1, 15:1, or 10:1 to a second target protein that is different from the first target protein, thereby delivering the therapeutic agent to the target organ or the target cell in the subject.
• a lipid composition that comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the io
• the composition is according to any of the compositions described herein.
• the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of said therapeutic agent in said organ or said cell therein in said subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent binding to said plurality of target proteins).
• the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of said therapeutic agent in said organ or said cell therein in said subject as compared to that achieved absent said polymer-conjugated lipid.
• the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of said therapeutic agent in said organ or said cell therein in said subject as compared to that achieved in a reference organ or a reference cell.
• said therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro- ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA),
• siRNA small interfer
• the present disclosure provides a method for targeted delivery of a therapeutic agent to an organ (e.g., liver) or a (e.g., liver) cell therein in a subject in need thereof, the method comprising administering to the subject the therapeutic agent assembled with a lipid composition that comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer- conjugated lipid, wherein, upon the administering, a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises apolipoprotein E (Apo E) and serum albumin, thereby delivering the therapeutic agent to the target organ or the target cell in the subject.
• a lipid composition that comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a
• the Apo E is present at a weight or mass ratio of no more than about 6:1, 5:1, 4:1, or 3:1 to the serum albumin the plurality of target proteins as determined by an incubation assay.
• said plurality of target proteins further comprise complement C1q subcomponent subunit A, immunoglobulin heavy constant mu, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma-2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, or any combination thereof as determined by an incubation assay.
• said SORT lipid comprises an ionizable cationic moiety (e.g., a tertiary amine moiety). In some embodiments, said SORT lipid is an ionizable cationic lipid. In some embodiments, said lipid composition comprises said SORT lipid at a molar percentage from about 5% to about 65%. In some embodiments, said lipid composition is according to any lipid composition provided herein.
• the method provides a (e.g., at least about 2-, 3-, 4-, 5-, or 6-fold) greater amount, expression or activity of said therapeutic agent in said liver or said liver cell in said subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent said binding to said plurality of target proteins).
• a reference lipid composition e.g., absent said binding to said plurality of target proteins.
• the present disclosure provides a method for targeted delivery of a therapeutic agent to a non-liver organ or a non-liver cell therein in a subject in need thereof, the method comprising administering to the subject the therapeutic agent assembled with a lipid composition that comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein, upon the administering, a surface of the lipid composition interacts with apolipoprotein E (Apo E) to a lesser degree than with an exogenous protein that is not Apo E in the subject as determined by an incubation assay, which endogenous protein is not Apor E is selected from beta-2-glycoprotein 1 ( ⁇ 2- GP1) or apolipoprotein H (Apo H), immunoglobulin kappa constant, complement
• a lipid composition that
• said non-liver organ comprises a lung, spleen, bone marrow, or a lymph node.
• said non-liver cell comprises a lung cell, a spleen cell, or a macrophage.
• apolipoprotein E is not the most abundant protein in said plurality of target proteins.
• a surface of said lipid composition interacts with apolipoprotein C (Apo C) to a lesser degree than with apolipoprotein E (Apo E) in said subject as determined by an incubation assay.
• the method provides a lesser amount or activity of said therapeutic agent in liver or a cell therein in said subject as compared to that achieved absent said polymer-conjugated lipid.
• said SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid.
• said lipid composition comprises said SORT lipid at a molar percentage from about 5% to about 65%. In some embodiments, said lipid composition is according to any of the lipid compositions described herein.
• the present disclosure provides a method for targeted delivery of a therapeutic agent to a lung or a lung cell therein in a subject in need thereof, the method comprising administering to the subject the therapeutic agent assembled with a lipid composition, which lipid composition comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein, upon the administering, a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises vitronectin (Vtn) and clusterin, thereby delivering the therapeutic agent to the lung or the lung cell in the subject.
• a lipid composition comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g.
• the vitronectin is present at a weight or mass ratio of no more than about 6:1 or 5:1 to the clusterin in the plurality of target proteins as determined by an incubation assay.
• said plurality of target proteins further comprise serum paraoxonase/arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H), immunoglobulin (Ig) mu chain C region, alpha-S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen alpha chain, vitamin K-dependent protein Z, alpha-1-antitrypsin 1- 3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1,
• said SORT lipid is a cationic lipid. In some embodiments, said SORT lipid is a permanent cationic lipid. In some embodiments, said SORT lipid is an ionizable cationic lipid. In some embodiments, said lipid composition comprises said SORT lipid at a molar percentage from about 5% to about 65%. In some embodiments, said lipid composition is according to any lipid composition provided herein.
• the method provides a (e.g., at least about 2-, 5-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold) greater amount, expression or activity of said therapeutic agent in said lung or said lung cell in said subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent binding to said plurality of target proteins).
• a reference lipid composition e.g., absent binding to said plurality of target proteins.
• said therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri- miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, deoxyribonucleic acid, deoxy
• the present application provides a method for targeted delivery of a therapeutic agent to spleen, bone marrow, or a lymph node or a cell therein in a subject in need thereof, the method comprising administering to the subject the therapeutic agent assembled with a lipid composition, which lipid composition comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein, upon the administering, a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H) at a weight or mass ratio of no more than about 20:1, 15:1, or 10:1 to a second target protein that is different from the beta-2 glycoprotein 1
• ⁇ 2-GP1 beta
• said cell comprises a spleen cell, or a macrophage.
• said second target protein is selected from: immunoglobulin kappa constant, complement C1q subcomponent subunit A, apolipoprotein E (Apo E), immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, vitronectin, complement C1q subcomponent subunit C, apolipoprotein C-I, immunoglobulin (Ig) gamma-2A chain C region, immunoglobulin (Ig) mu chain C region, serum albumin, serum paraoxonase/arylesterase 1, immunoglobulin heavy constant alpha, and immunoglobulin kappa variable 6-13.
• said SORT lipid is a permanent cationic lipid, or an anionic lipid. In some embodiments, said SORT lipid is a permanent cationic lipid. In some embodiments, said SORT lipid is an anionic lipid. In some embodiments, said lipid composition comprises said SORT lipid at a molar percentage from about 5% to about 65%. In some embodiments, said lipid composition is according to any lipid composition provided herein. In some embodiments, the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of said therapeutic agent in said lugn or said lung cell in said subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent binding to said plurality of target proteins).
• the present disclosure provides a method for targeted delivery of a therapeutic agent to a non-spleen organ or a non-spleen cell therein in a subject in need thereof, the method comprising administering to the subject the therapeutic agent assembled with a lipid composition, which lipid composition comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein, upon the administering, a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises a first target protein at a weight or mass ratio of no more than about 20:1, 15:1, or 10:1 to a second target protein that is different from the first target protein, thereby delivering the therapeutic agent to the non-spleen organ or the non-spleen cell
• a lipid composition comprises
• said non- spleen organ is not spleen, bone marrow, or a lymph node.
• said non-spleen cell is not a spleen cell, or a macrophage.
• beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H) is not the most abundant protein in said plurality of target proteins.
• said plurality of target proteins comprise clusterin.
• said SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid.
• said lipid composition comprises said SORT lipid at a molar percentage from about 5% to about 65%. In some embodiments, said lipid composition is according to any lipid composition provided herein.
• said therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid
• siRNA small interfer
• said polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid.
• said one or more hydrocarbon chains each comprise about 8 to about 20 carbon atoms. In some embodiments, said one or more hydrocarbon chains each comprise about 8 to about 18 carbon atoms. In some embodiments, wherein said one or more hydrocarbon chains each comprise about 8 to about 16 carbon atoms. In some embodiments, said one or more hydrocarbon chains each comprise about 8 to about 14 carbon atoms. In some embodiments, a hydrocarbon chain of said one or more hydrocarbon chains of said polymer-conjugated lipid comprises no more than 3 unsaturated carbon-carbon bonds.
• a hydrocarbon chain of said one or more hydrocarbon chains of said polymer-conjugated lipid comprises no more than 2 unsaturated carbon-carbon bonds.
• said polymer-conjugated lipid comprises a polymer having a molecular weight of about 100 Daltons (Da) to about 100,000 Da.
• said polymer-conjugated lipid comprises a polymer having a molecular weight of about 500 Da to about 100,000 Da.
• said lipid composition comprises said polymer-conjugated lipid at a molar percentage from about 0.5% to about 20%.
• said lipid composition comprises said polymer- conjugated lipid at a molar percentage from about 0.5% to about 15%.
• said lipid composition comprises said polymer-conjugated lipid at a molar percentage from about 0.5% to about 10%.
• said administering comprises intravenously administering.
• a bodily fluid e.g., plasma or serum
• said plurality of target proteins are a plurality of endogenous proteins of said subject.
• FIGs.1A-1E illustrate selective organ targeting (SORT) lipid compositions (e.g., nanoparticles) for tissue-specific mRNA delivery including the unique biodistribution and ionization behavior thereof.
• SORT organ targeting
• a supplemental SORT molecule by adding a supplemental (e.g., 5 th ) SORT molecule to a reference, e.g., four-component lipid composition (15:15:30:3 5A2-SC8:DOPE:Cholesterol:C14-PEG2K mol:mol), the tissue-specific activity of delivered mRNA changes based on the chemical structure of the included SORT molecule.
• a reference e.g., four-component lipid composition (15:15:30:3 5A2-SC8:DOPE:Cholesterol:C14-PEG2K mol:mol
• an ionizable cationic lipid enhances liver- specific mRNA translation (Liver SORT- 15:15:30:3:15.75 5A2-SC8:DOPE:Cholesterol:C14- PEG2K:DODAP), an anionic lipid (18PA) results in spleen-specific mRNA translation (Spleen SORT- 15:15:30:3:275A2-SC8:DOPE:Cholesterol:C14-PEG2K:18PA), and a cationic quaternary ammonium lipid (DOTAP) results in lung-specific mRNA translation (Lung SORT- 15:15:30:3:63 5A2- SC8:DOPE:Cholesterol:C14-PEG2K:DOTAP).
• DODAP ionizable cationic lipid
• FIG.1B illustrates ex vivo fluorescence of Cy5-labelled mRNA in major organs extracted from C57BL/6 mice intravenously injected with example SORT LNPs (as described herein) that incorporate increasing percentages of different SORT molecules (0.5 mg/kg mRNA/body weight, 6h).
• FIG. 1D illustrates representative TNS assay curves for determining the apparent pKa of example SORT LNPs (as described herein) incorporating increasing percentages of ionizable cationic, anionic, or cationic lipid SORT molecules.
• Apparent pKa was defined as the point at which 50% of TNS fluorescence was achieved.
• FIGs.2A-2H illustrate that multiple steps are involved in the mechanism of SORT LNP tissue targeting, including formation of unique protein coronas by way of example(s).
• FIG. 2A illustrates a proposed three-step endogenous targeting mechanism for tissue-specific mRNA delivery by SORT LNPs, by way of example(s) only, where (1) PEG-lipid desorption (2) enables distinct serum proteins to bind SORT LNPs (3) resulting in cellular internalization in the target tissues by receptor-mediated uptake.
• FIG. 2A illustrates a proposed three-step endogenous targeting mechanism for tissue-specific mRNA delivery by SORT LNPs, by way of example(s) only, where (1) PEG-lipid desorption (2) enables distinct serum proteins to bind SORT LNPs (3) resulting in cellular internalization in the target tissues by receptor-mediated uptake.
• FIG. 2B illustrates ex vivo bioluminescence of major organs excised from C57BL/6 mice IV injected with example Liver, Spleen, and Lung SORT LNPs incorporating either sheddable PEG-lipid (C14-PEG2K) or less-sheddable PEG-lipid (C18-PEG2K) (0.1 mg FLuc mRNA/kg body weight, 6h). Total luminescence produced by each organ is reduced when less-sheddable PEG-lipid is used, suggesting PEG-lipid desorption is a key process for efficacious mRNA delivery by the tested SORT LNPs.
• FIG. 1 illustrates ex vivo bioluminescence of major organs excised from C57BL/6 mice IV injected with example Liver, Spleen, and Lung SORT LNPs incorporating either sheddable PEG-lipid (C14-PEG2K) or less-sheddable PEG-lipid (C18-PEG2K) (0.1 mg FLuc mRNA/kg body weight, 6h). Total
• FIG.2C illustrates quantification of total luminescence produced by functional protein translated from FLuc mRNA in target organs of C57BL/6 mice IV injected with example Liver, Spleen, and Lung SORT LNPs incorporating either C14- or C18-PEG2K (0.1 mg FLuc mRNA/kg body weight, 6h).
• FIG.2D illustrates ELISA quantification of serum hEPO in C57BL/6 mice treated with example Liver, Spleen, or Lung SORT LNPs encapsulating hEPO mRNA (0.1 mg hEPO mRNA/kg body weight, 6h). Using a less-sheddable PEG reduces SORT LNP potency.
• FIG.2D illustrates ELISA quantification of serum hEPO in C57BL/6 mice treated with example Liver, Spleen, or Lung SORT LNPs encapsulating hEPO mRNA (0.1 mg hEPO mRNA/kg body weight, 6h). Using a less-
• FIG. 2E illustrates SDS-PAGE of the serum proteins adsorbed to the surface of the reference mDLNP, an example Liver SORT LNP, an example Spleen SORT LNP, and an example Lung SORT LNP. LNPs with different organ-targeting properties bind distinct serum proteins.
• FIG. 2F illustrates average abundance of proteins with distinct biological functions in the protein coronas of the reference mDLNP and an example Liver, Spleen, and Lung SORT LNPs. The choice of SORT molecule leads to large-scale differences in the functional ensemble of serum proteins which bind the LNP.
• FIG.2G illustrates isoelectric point distribution for the most enriched proteins which constitute 80% of the protein corona of the LNPs.
• the chemical structure of SORT molecule affects the number one serum protein that is most highly enriched on the surface of example SORT LNPs. Data are shown as mean ⁇ s.e.m. Statistical significance was determined using an unpaired two-tailed Student’s t test (*, p ⁇ 0.05).
• FIGs. 3A-3D illustrates that distinct serum proteins regulate example SORT LNP uptake and efficacy in vitro.
• example SORT LNPs were pre-incubated with either ApoE, ⁇ 2-GPI, or Vtn prior to treating relevant cell lines to measure cellular uptake (Cy5-mRNA tracking) or functional mRNA delivery (bioluminescence).
• FIGs. 4A-4C illustrate that extrahepatic mRNA delivery occurs via an ApoE-independent mechanism.
• FIG. 4A illustrates ex vivo bioluminescence produced by functional protein translated from FLuc mRNA in major organs excised from wild-type C57BL/6 mice IV injected with the reference mDLNP or an example Liver, Spleen or Lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6h).
• the role of ApoE on the SORT LNP efficacy varies based on the chemical structure of the included SORT molecule
• FIG. 4B illustrates ex vivo bioluminescence produced by functional protein translated from FLuc mRNA in major organs excised from ApoE-/- mice IV injected with reference mDLNP or an example Liver, Spleen or Lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6h).
• FIG. 4C illustrates quantification of total bioluminescence produced by target organs excised from wild-type and ApoE-/- mice treated with reference mDLNP or, an example Liver, Spleen, or Lung SORT LNPs.
• Data are shown as mean ⁇ s.e.m.
• Statistical significance was determined using an unpaired two-tailed Student’s t test (**, p ⁇ 0.01, *, p ⁇ 0.05, ns, p > 0.05).
• Elimination of ApoE from the serum using genetic knockout results in a marked reduction of hepatic mRNA delivery by reference mDLNP and example Liver SORT LNPs.
• example Spleen SORT LNPs have enhanced spleen-targeting when ApoE is depleted from the serum while the efficacy of example Lung SORT LNPs is unaffected by ApoE elimination.
• FIG. 5 illustrate TNS fluorescence curves for example SORT LNPs formulated with various mass fractions of cationic, anionic, ionizable, and zwitterionic lipids. The apparent pKa of an LNP is computed as the pH at which 50% of the TNS fluorescence is measured.
• FIG. 6 illustrates apparent pKa values computed from example SORT LNPs incorporating cationic, anionic, ionizable, and zwitterionic lipids at various mass fractions.
• FIGs. 7A-7B illustrate cellular uptake and functional mRNA delivery to low-density lipoprotein receptor (LDL-R) expressing Hep G2 cells by example SORT LNPs pre-incubated with ApoE.
• FIGs.8A-8B illustrate cellular uptake and functional mRNA delivery to ⁇ v ⁇ 3 expressing U87- MG cells by example SORT LNPs pre-incubated with Vtn.
• FIG.8A-8B illustrate cellular uptake and functional mRNA delivery to ⁇ v ⁇ 3 expressing U87- MG cells by example SORT LNPs pre-incubated with Vtn.
• cleavage sequence means “at least a first cleavage sequence” but includes a plurality of cleavage sequences.
• the operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present application.
• polypeptide peptide
• protein protein are used interchangeably herein to generally refer to polymers of amino acids of any length.
• the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
• N-terminus or “amino terminus”
• C-terminus or “carboxyl terminus”
• N-terminal end sequence as used herein with respect to a polypeptide or polynucleotide sequence of interest, generally means that no other amino acid or nucleotide residues precede the N-terminal end sequence in the polypeptide or polynucleotide sequence of interest at the N-terminal end.
• C-terminal end sequence as used herein with respect to a polypeptide or polynucleotide sequence of interest, generally means that no other amino acid or nucleotide residues follows the C-terminal end sequence in the polypeptide or polynucleotide sequence of interest at the C- terminal end.
• non-naturally occurring and “non-natural” are used interchangeably herein.
• non-naturally occurring or “non-natural,” as used herein with respect to a therapeutic agent or prophylactic agent, generally means that the agent is not biologically derived in mammals (including but not limited to human).
• non-naturally occurring or “non-natural,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally- occurring sequence found in a mammal.
• a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
• “Physiological conditions” refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers are listed in Sambrook et al. (2001).
• Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.
• treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms generally refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
• compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
• a “therapeutic effect” or “therapeutic benefit,” as used herein, generally refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease or an improvement in one or more clinical parameters associated with the underlying disorder in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a polypeptide of the disclosure other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein.
• compositions may be administered to a subject at risk of developing a particular disease, a recurrence of a former disease, condition or symptom of the disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
• therapeutically effective amount and “therapeutically effective dose”, as used herein, generally refer to an amount of a drug or a biologically active protein, either alone or as a part of a polypeptide composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial.
• the term “equivalent molar dose” generally means that the amounts of materials administered to a subject have an equivalent amount of moles, based on the molecular weight of the material used in the dose.
• the term “therapeutically effective and non-toxic dose,” as used herein, generally refers to a tolerable dose of the compositions as defined herein that is high enough to cause depletion of tumor or cancer cells, tumor elimination, tumor shrinkage or stabilization of disease without or essentially without major toxic effects in the subject.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
• the symbol “ ” represents an optional bond, which if present is either single or double.
• the symbol “ ” represents a single bond or a double bond.
• the formula includes And it is understood that no one such ring atom forms part of more than one double bond.
• the covalent bond symbol “ ⁇ ”, when connecting one or two stereogenic atoms does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof.
• the symbol “ ”, when drawn perpendicularly across a bond indicates a point of attachment of the group.
• the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment.
• the symbol “ ” means a single bond where the group attached to the thick end of the wedge is “out of the page.”
• the symbol “ ” means a single bond where the group attached to the thick end of the wedge is “into the page”.
• the symbol “ ” means a single bond where the geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom.
• a bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.
• R may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed.
• R may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise.
• Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals ⁇ CH ⁇ ), so long as a stable structure is formed.
• R may reside on either the 5-membered or the 6-membered ring of the fused ring system.
• the subscript letter “y” immediately following the group “R” enclosed in parentheses represents a numeric variable.
• this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.
• the number of carbon atoms in the group or class is as indicated as follows: “Cn” defines the exact number (n) of carbon atoms in the group/class. “C ⁇ n” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl(C ⁇ 8)” or the class “alkene(C ⁇ 8)” is two.
• alkoxy (C ⁇ 10) designates alkoxy groups having from 1 to 10 carbon atoms.
• Cn-n′ defines both the minimum (n) and maximum number (n′) of carbon atoms in the group.
• alkyl (C2-10) designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning.
• the terms “C5 olefin”, “C5-olefin”, “olefin(C5)”, and “olefinC5” are all synonymous.
• saturated when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below.
• the term when used to modify an atom, it means that the atom is not part of any double or triple bond.
• substituted versions of saturated groups one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon- carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded.
• saturated when used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
• aliphatic when used without the “substituted” modifier signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group.
• the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic).
• Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).
• aromatic when used to modify a compound or a chemical group atom means the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by an interaction of the bonds forming the ring.
• alkyl when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen.
• the groups ⁇ CH3 (Me), ⁇ CH2CH3 (Et), ⁇ CH2CH2CH3 (n-Pr or propyl), ⁇ CH(CH3)2 (i-Pr, i Pr or isopropyl), ⁇ CH2CH2CH2CH3 (n-Bu), ⁇ CH(CH3)CH2CH3 (sec-butyl), ⁇ CH 2 CH(CH 3 ) 2 (isobutyl), ⁇ C(CH 3 ) 3 (tert-butyl, t-butyl, t-Bu or t Bu), and ⁇ CH 2 C(CH 3 ) 3 (neo-pentyl) are non-limiting examples of alkyl groups.
• alkanediyl when used without the “substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
• the groups ⁇ CH2 ⁇ (methylene), ⁇ CH2CH2 ⁇ , ⁇ CH2C(CH3)2CH2 ⁇ , and ⁇ CH2CH2CH2 ⁇ are non-limiting examples of alkanediyl groups.
• An “alkane” refers to the class of compounds having the formula H ⁇ R, wherein R is alkyl as this term is defined above.
• one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH 2 , ⁇ NO 2 , ⁇ CO 2 H, ⁇ CO 2 CH 3 , ⁇ CN, ⁇ SH, ⁇ OCH 3 , ⁇ OCH 2 CH 3 , ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH 2 , ⁇ C(O)NHCH 3 , ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• haloalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e.
• ⁇ F, ⁇ Cl, ⁇ Br, or ⁇ I such that no other atoms aside from carbon, hydrogen and halogen are present.
• the group, ⁇ CH 2Cl is a non-limiting example of a haloalkyl.
• fluoroalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present.
• the groups ⁇ CH2F, ⁇ CF3, and ⁇ CH2CF3 are non-limiting examples of fluoroalkyl groups.
• cycloalkyl when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
• Non-limiting examples include: ⁇ CH(CH 2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy).
• cycloalkanediyl when used without the “substituted” modifier refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
• the group is a non-limiting example of cycloalkanediyl group.
• a “cycloalkane” refers to the class of compounds having the formula H ⁇ R, wherein R is cycloalkyl as this term is defined above.
• alkenyl when used without the “substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
• alkenediyl when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
• alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure.
• alkene and olefin are synonymous and refer to the class of compounds having the formula H ⁇ R, wherein R is alkenyl as this term is defined above.
• terminal alkene and ⁇ -olefin are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.
• one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH2, ⁇ NO2, ⁇ CO2H, ⁇ CO2CH3, ⁇ CN, ⁇ SH, ⁇ OCH3, ⁇ OCH2CH3, ⁇ C(O)CH3, ⁇ NHCH3, ⁇ NHCH2CH3, ⁇ N(CH3)2, ⁇ C(O)NH2, ⁇ C(O)NHCH3, ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• alkynyl when used without the “substituted” modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds.
• alkyne refers to the class of compounds having the formula H ⁇ R, wherein R is alkynyl.
• one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH 2 , ⁇ NO 2 , ⁇ CO 2 H, ⁇ CO 2 CH 3 , ⁇ CN, ⁇ SH, ⁇ OCH 3 , ⁇ OCH 2 CH 3 , ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH 2 , ⁇ C(O)NHCH 3 , ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• aryl when used without the “substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, the carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present.
• Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, ⁇ C 6 H 4 CH 2 CH 3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl.
• the term “arenediyl” when used without the “substituted” modifier refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen.
• the term does not preclude the presence of one or more alkyl, aryl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting).
• arenediyl groups include: , [0090]
• An “arene” refers to the class of compounds having the formula H ⁇ R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.
• one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH2, ⁇ NO2, ⁇ CO2H, ⁇ CO2CH3, ⁇ CN, ⁇ SH, ⁇ OCH3, ⁇ OCH2CH3, ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH 2 , ⁇ C(O)NHCH 3 , ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• aralkyl when used without the “substituted” modifier refers to the monovalent group ⁇ alkanediyl ⁇ aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above.
• Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
• aralkyl When the term aralkyl is used with the “substituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl group has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH 2 , ⁇ NO 2 , ⁇ CO 2 H, ⁇ CO 2 CH 3 , ⁇ CN, ⁇ SH, ⁇ OCH 3 , ⁇ OCH 2 CH 3 , ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH 2 , ⁇ C(O)NHCH 3 , ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro- 2-phenyl-eth-1-yl.
• the term “heteroaryl” when used without the “substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur.
• Heteroaryl rings may contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen, and sulfur.
• the rings may be fused or unfused.
• the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system.
• heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.
• N-heteroaryl refers to a heteroaryl group with a nitrogen atom as the point of attachment.
• heteroaryl when used without the “substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, the atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused.
• Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system.
• Non-limiting examples of heteroarenediyl groups include: [0093]
• a “heteroarene” refers to the class of compounds having the formula H ⁇ R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.
• one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH2, ⁇ NO2, ⁇ CO2H, ⁇ CO2CH3, ⁇ CN, ⁇ SH, ⁇ OCH3, ⁇ OCH2CH3, ⁇ C(O)CH3, ⁇ NHCH3, ⁇ NHCH2CH3, ⁇ N(CH3)2, ⁇ C(O)NH2, ⁇ C(O)NHCH3, ⁇ C(O)N(CH3)2, ⁇ OC(O)CH3, ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• heterocycloalkyl when used without the “substituted” modifier refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur.
• Heterocycloalkyl rings may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. If more than one ring is present, the rings may be fused or unfused.
• the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic.
• Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl.
• N-heterocycloalkyl refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group.
• heterocycloalkanediyl when used without the “substituted” modifier refers to an divalent cyclic group, with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur.
• the rings may be fused or unfused.
• Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting).
• a covalent bond alkanediyl, or alkenediyl groups (carbon number limitation permitting).
• alkanediyl or alkenediyl groups (carbon number limitation permitting).
• alkyl groups carbon number limitation permitting
• the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic.
• Non-limiting examples of heterocycloalkanediyl groups include: When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH 2 , ⁇ NO 2 , ⁇ CO 2 H, ⁇ CO 2 CH 3 , ⁇ CN, ⁇ SH, ⁇ OCH 3 , ⁇ OCH 2 CH 3 , ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH 2 , ⁇ C(O)NHCH 3 , ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2 OH , or ⁇ S(O) 2 NH 2 .
• acyl when used without the “substituted” modifier refers to the group ⁇ C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as those terms are defined above.
• acyl groups ⁇ CHO, ⁇ C(O)CH3 (acetyl, Ac), ⁇ C(O)CH2CH3, ⁇ C(O)CH2CH2CH3, ⁇ C(O)CH(CH3)2, ⁇ C(O)CH(CH2)2, ⁇ C(O)C6H5, ⁇ C(O)C6H4CH3, ⁇ C(O)CH2C6H5, ⁇ C(O)(imidazolyl) are non-limiting examples of acyl groups.
• a “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group ⁇ C(O)R has been replaced with a sulfur atom, ⁇ C(S)R.
• aldehyde corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a ⁇ CHO group.
• one or more hydrogen atom (including a hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH2, ⁇ NO2, ⁇ CO2H, ⁇ CO2CH3, ⁇ CN, ⁇ SH, ⁇ OCH3, ⁇ OCH2CH3, ⁇ C(O)CH3, ⁇ NHCH3, ⁇ NHCH2CH3, ⁇ N(CH3)2, ⁇ C(O)NH2, ⁇ C(O)NHCH3, ⁇ C(O)N(CH3)2, ⁇ OC(O)CH3, ⁇ NHC(O)CH3, ⁇ S(O)2OH, or ⁇ S(O)2NH
• the groups, ⁇ C(O)CH 2CF3, ⁇ CO 2H (carboxyl), ⁇ CO 2CH3 (methylcarboxyl), ⁇ CO 2 CH 2 CH 3 , ⁇ C(O)NH 2 (carbamoyl), and ⁇ CON(CH 3 ) 2 are non-limiting examples of substituted acyl groups.
• alkoxy when used without the “substituted” modifier refers to the group ⁇ OR, in which R is an alkyl, as that term is defined above.
• Non-limiting examples include: ⁇ OCH3 (methoxy), ⁇ OCH2CH3 (ethoxy), ⁇ OCH2CH2CH3, ⁇ OCH(CH3)2 (isopropoxy), ⁇ OC(CH3)3 (tert-butoxy), ⁇ OCH(CH2)2, ⁇ O ⁇ cyclopentyl, and ⁇ O ⁇ cyclohexyl.
• cycloalkoxy when used without the “substituted” modifier, refers to groups, defined as ⁇ OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively.
• alkoxydiyl refers to the divalent group ⁇ O ⁇ alkanediyl ⁇ , ⁇ O ⁇ alkanediyl ⁇ O ⁇ , or ⁇ alkanediyl ⁇ O ⁇ alkanediyl ⁇ .
• alkylthio and acylthio when used without the “substituted” modifier refers to the group ⁇ SR, in which R is an alkyl and acyl, respectively.
• alcohol corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group.
• ether corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.
• substituted one or more hydrogen atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH 2 , ⁇ NO 2 , ⁇ CO 2 H, ⁇ CO 2 CH 3 , ⁇ CN, ⁇ SH, ⁇ OCH 3 , ⁇ OCH 2 CH 3 , ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH2, ⁇ C(O)NHCH3, ⁇ C(O)N(CH3)2, ⁇ OC(O)CH3, ⁇ NHC(O)CH3, ⁇ S(O)2OH, or ⁇ S(O)2NH2.
• alkylamino when used without the “substituted” modifier refers to the group ⁇ NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: ⁇ NHCH3 and ⁇ NHCH2CH3.
• dialkylamino when used without the “substituted” modifier refers to the group ⁇ NRR′, in which R and R′ can be the same or different alkyl groups, or R and R′ can be taken together to represent an alkanediyl.
• dialkylamino groups include: ⁇ N(CH3)2 and ⁇ N(CH3)(CH2CH3).
• cycloalkylamino when used without the “substituted” modifier, refers to groups, defined as ⁇ NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively.
• a non-limiting example of an arylamino group is ⁇ NHC 6 H 5 .
• alkylaminodiyl refers to the divalent group ⁇ NH ⁇ alkanediyl ⁇ , ⁇ NH ⁇ alkanediyl ⁇ NH ⁇ , or ⁇ alkanediyl ⁇ NH ⁇ alkanediyl ⁇ .
• amido acylamino
• R is acyl, as that term is defined above.
• a non-limiting example of an amido group is ⁇ NHC(O)CH3.
• R is an alkyl
• one or more hydrogen atom attached to a carbon atom has been independently replaced by ⁇ OH, ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ NH 2 , ⁇ NO 2 , ⁇ CO 2 H, ⁇ CO 2 CH 3 , ⁇ CN, ⁇ SH, ⁇ OCH 3 , ⁇ OCH 2 CH 3 , ⁇ C(O)CH 3 , ⁇ NHCH 3 , ⁇ NHCH 2 CH 3 , ⁇ N(CH 3 ) 2 , ⁇ C(O)NH 2 , ⁇ C(O)NHCH 3 , ⁇ C(O)N(CH 3 ) 2 , ⁇ OC(O)CH 3 , ⁇ NHC(O)CH 3 , ⁇ S(O) 2
• the groups ⁇ NHC(O)OCH 3 and ⁇ NHC(O)NHCH 3 are non-limiting examples of substituted amido groups.
• the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
• the term “average molecular weight” refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have different levels of polymerization and thus a different molar mass. The average molecular weight can be used to represent the molecular weight of a plurality of polymer molecules.
• Average molecular weight is typically synonymous with average molar mass.
• the average molecular weight represents either the number average molar mass or weight average molar mass of the formula.
• the average molecular weight is the number average molar mass.
• the average molecular weight may be used to describe a PEG component present in a lipid.
• any forms or tenses of one or more of these verbs are also open-ended.
• any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
• the term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
• IC 50 refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.
• an “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
• the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof.
• the patient or subject is a primate (e.g., non-human primate).
• the patient or subject is a human.
• Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.
• lipid composition generally refers to a composition comprising lipid compound(s), including but not limited to, a lipoplex, a liposome, a lipid particle.
• lipid compositions include suspensions, emulsions, and vesicular compositions.
• the term “detectable” refers to an occurrence of, or a change in, a signal that is directly or indirectly detectable either by observation or by instrumentation.
• a detectable response is an occurrence of a signal wherein the fluorophore is inherently fluorescent and does not produce a change in signal upon binding to a metal ion or biological compound.
• the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters.
• detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density
• a delivery system e.g., a lipid composition
• a therapeutic agent or prophylactic agent such as a desired level of translation, transcription, production, expression, or activity of a protein or gene
• cells e.g., targeted cells
• a lipid composition with a higher potency may achieve a desired therapeutic effect in a greater population of relevant cells, within a shorter response time, or that last a longer period of time.
• pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
• “Pharmaceutically acceptable salts” means salts of compounds of the present application which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity.
• Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid,
• Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
• Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
• Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G.
• pharmaceutically acceptable carrier means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
• prevention includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
• a “repeat unit” is the simplest structural entity of certain materials, for example, frameworks and/or polymers, whether organic, inorganic or metal-organic.
• repeat units are linked together successively along the chain, like the beads of a necklace.
• the repeat unit is ⁇ CH 2 CH 2 ⁇ .
• the subscript “n” denotes the degree of polymerization, that is, the number of repeat units linked together.
• the value for “n” is left undefined or where “n” is absent, it simply designates repetition of the formula within the brackets as well as the polymeric nature of the material.
• the concept of a repeat unit applies equally to where the connectivity between the repeat units extends three dimensionally, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc.
• the repeating unit may also be described as the branching unit, interior layers, or generations.
• the terminating group may also be described as the surface group.
• a “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs.
• “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands.
• “Diastereomers” are stereoisomers of a given compound that are not enantiomers.
• Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer.
• the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds.
• a molecule can have multiple stereocenters, giving it many stereoisomers.
• n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture.
• a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%.
• enantiomers and/or diastereomers can be resolved or separated using techniques known in the art.
• stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures.
• the phrase “substantially free from other stereoisomers” means that the composition contains ⁇ 15%, more preferably ⁇ 10%, even more preferably ⁇ 5%, or most preferably ⁇ 1% of another stereoisomer(s).
• Treatment includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
• the above definitions supersede any conflicting definition in any reference that is incorporated by reference herein.
• lipid composition comprising: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid, e.g., separate from the ionizable cationic lipid.
• SORT selective organ targeting
• the lipid composition comprises an ionizable cationic lipid.
• the cationic ionizable lipids contain one or more groups which is protonated at physiological pH but may deprotonated and has no charge at a pH above 8, 9, 10, 11, or 12.
• the ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH.
• the cationic ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24 alkyl or alkenyl carbon groups.
• the ionizable cationic lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C6-C24 alkyl or alkenyl groups but may have at least 1 or more that 6 tails.
• these cationic ionizable lipids are dendrimers, which are a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core and are characterized by a core, at least one interior branched layer, and a surface branched layer.
• dendrimer as used herein is intended to include, but is not limited to, a molecular architecture with an interior core, interior layers (or “generations”) of repeating units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation.
• a “dendron” is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a larger dendrimer.
• the dendrimer structures have radiating repeating groups from a central core which doubles with each repeating unit for each branch.
• the dendrimers described herein may be described as a small molecule, medium- sized molecules, lipids, or lipid-like material. These terms may be used to described compounds described herein which have a dendron like appearance (e.g. molecules which radiate from a single focal point).
• dendrimers are polymers, dendrimers may be preferable to traditional polymers because they have a controllable structure, a single molecular weight, numerous and controllable surface functionalities, and traditionally adopt a globular conformation after reaching a specific generation.
• Dendrimers can be prepared by sequentially reactions of each repeating unit to produce monodisperse, tree-like and/or generational structure polymeric structures. Individual dendrimers consist of a central core molecule, with a dendritic wedge attached to one or more functional sites on that central core.
• the dendrimeric surface layer can have a variety of functional groups disposed thereon including anionic, cationic, hydrophilic, or lipophilic groups, according to the assembly monomers used during the preparation.
• Modifying the functional groups and/or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can be modulated. Some properties which can be varied include, but are not limited to, solubility, toxicity, immunogenicity and bioattachment capability. Dendrimers are often described by their generation or number of repeating units in the branches. A dendrimer consisting of only the core molecule is referred to as Generation 0, while each consecutive repeating unit along all branches is Generation 1, Generation 2, and so on until the terminating or surface group. In some embodiments, half generations are possible resulting from only the first condensation reaction with the amine and not the second condensation reaction with the thiol.
• Dendrimer synthesis can be of the convergent or divergent type. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process involving attaching one generation to the previous and then changing functional groups for the next stage of reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization would lead to a highly branched molecule that is not monodisperse and is otherwise known as a hyperbranched polymer.
• the dendrimers of G1-G10 generation are specifically contemplated.
• the dendrimers comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein.
• the dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by reducing the spacing units in the branching polymer.
• dendrimers have two major chemical environments: the environment created by the specific surface groups on the termination generation and the interior of the dendritic structure which due to the higher order structure can be shielded from the bulk media and the surface groups. Because of these different chemical environments, dendrimers have found numerous different potential uses including in therapeutic applications. [00125] In some embodiments of the lipid composition of the present disclsoure, the dendrimers or dendrons are assembled using the differential reactivity of the acrylate and methacrylate groups with amines and thiols.
• the dendrimers or dendrons may include secondary or tertiary amines and thioethers formed by the reaction of an acrylate group with a primary or secondary amine and a methacrylate with a mercapto group.
• the repeating units of the dendrimers or dendrons may contain groups which are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diethers, esters, amides, or disulfides groups.
• the core molecule is a monoamine which allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches which each may comprise one or more repeating units.
• the dendrimer or dendron may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom.
• the terminating group is a lipophilic groups such as a long chain alkyl or alkenyl group. In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine ( ⁇ NH2) or a carboxylic acid ( ⁇ CO2H).
• the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.
• the cationic ionizable lipids of the present application may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
• Cationic ionizable lipids may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
• the chiral centers of the cationic ionizable lipids of the present application can have the S or the R configuration. Furthermore, it is contemplated that one or more of the cationic ionizable lipids may be present as constitutional isomers. In some embodiments, the compounds have the same formula but different connectivity to the nitrogen atoms of the core.
• cationic ionizable lipids exist because the starting monomers react first with the primary amines and then statistically with any secondary amines present. Thus, the constitutional isomers may present the fully reacted primary amines and then a mixture of reacted secondary amines.
• Chemical formulas used to represent cationic ionizable lipids of the present application will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups.
• the cationic ionizable lipids of the present application may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
• atoms making up the cationic ionizable lipids of the present application are intended to include all isotopic forms of such atoms.
• Isotopes include those atoms having the same atomic number but different mass numbers.
• isotopes of hydrogen include tritium and deuterium
• isotopes of carbon include 13 C and 14 C.
• the ionizable cationic lipid is a dendrimer or dendron.
• the ionizable cationic lipid comprises an ammonium group which is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH from about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron.
• the ionizable cationic lipid comprises at least two C 6 -C 24 alkyl or alkenyl groups.
• Dendrimers of Formula (I) [00132] In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C8-C24 alkyl groups.
• the ionizable cationic lipid is a dendrimer or dendron further defined by the formula: Core-Repeating Unit-Terminating Group (D-I) wherein the core is linked to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atom with the repeating unit and wherein: the core has the formula: wherein: X1 is amino or alkylamino(C ⁇ 12), dialkylamino(C ⁇ 12), heterocycloalkyl(C ⁇ 12), heteroaryl(C ⁇ 12), or a substituted version thereof; R 1 is amino, hydroxy, or mercapto, or alkylamino (C ⁇ 12) , dialkylamino (C ⁇ 12) , or a substituted version of either of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has the formula: wherein: X 2 is N(R 5 ) y ; R 5 is hydrogen, alkyl (C ⁇ 18) , or substituted alky
• the terminating group is further defined by the formula: wherein: Y 4 is alkanediyl (C ⁇ 18) ; and R10 is hydrogen.
• A1 and A2 are each independently ⁇ O ⁇ or ⁇ NRa ⁇ .
• the core is further defined by the formula: wherein: X2 is N(R5)y; R5 is hydrogen or alkyl(C ⁇ 8), or substituted alkyl(C ⁇ 18); and y is 0, 1, or 2, provided that the sum of y and z is 3; R 2 is amino, hydroxy, or mercapto, or alkylamino (C ⁇ 12) , dialkylamino (C ⁇ 12) , or a substituted version of either of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, 3; provided that the sum of z and y is 3.
• the core is further defined by the formula: wherein: X 3 is ⁇ NR 6 ⁇ , wherein R 6 is hydrogen, alkyl (C ⁇ 8) , or substituted alkyl (C ⁇ 8) , ⁇ O ⁇ , or alkylaminodiyl(C ⁇ 8), alkoxydiyl(C ⁇ 8), arenediyl(C ⁇ 8), heteroarenediyl(C ⁇ 8), heterocycloalkanediyl(C ⁇ 8), or a substituted version of any of these groups; R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino(C ⁇ 12), dialkylamino(C ⁇ 12), or a substituted version of either of these groups; or a group of the , wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3; R c , R d , and
• the terminating group is represented by the formula: wherein: Y 4 is alkanediyl (C ⁇ 18) ; and R10 is hydrogen.
• the core is further defined as: [00137]
• the degradable diacyl is further defined as: [00138]
• the linker is further defined a wherein Y 1 is alkanediyl (C ⁇ 8) or substituted alkanediyl (C ⁇ 8) .
• the dendrimer or dendron of formula (D-I) is selected from the group consisting of: and pharmaceutically acceptable salts thereof.
• Dendrimers or dendrons of Formula (X) [00140]
• the ionizable cationic lipid is a dendrimer or dendron of the formula .
• the ionizable cationic lipid is a dendrimer or dendron of the formula .
• the ionizable cationic lipid is a dendrimer or dendron of a generation (g) having a structural formula: , or a pharmaceutically acceptable salt thereof, wherein: (a) the core comprises a structural formula (X Core ): wherein: Q is independently at each occurrence a covalent bond, -O-, -S-, -NR 2 -, or -CR 3a R 3b -; R 2 is independently at each occurrence R 1g or -L 2 -NR 1e R 1f ; R 3a and R 3b are each independently at each occurrence hydrogen or an optionally substituted (e.g., C1-C6, such as C1-C3) alkyl; R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (if present) are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted
• Q is independently at each occurrence a covalent bond, -O-, - S-, -NR 2 -, or -CR 3a R 3b .
• X Core Q is independently at each occurrence a covalent bond.
• X Core Q is independently at each occurrence an -O-.
• X Core Q is independently at each occurrence a -S-.
• X Core Q is independently at each occurrence a -NR 2 and R 2 is independently at each occurrence R 1g or -L 2 -NR 1e R 1f .
• X Core Q is independently at each occurrence a -CR 3a R 3b R 3a , and R 3a and R 3b are each independently at each occurrence hydrogen or an optionally substituted alkyl (e.g., C1-C6, such as C1-C3).
• R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted alkyl.
• R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g are each independently at each occurrence a point of connection to a branch, hydrogen.
• R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g are each independently at each occurrence a point of connection to a branch an optionally substituted alkyl (e.g., C 1 -C 12 ).
• L 0 , L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L 1 form a heterocycloalkyl (e.g., C4-C6 and containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur) with one of R 1c and R 1d .
• a heterocycloalkyl e.g., C4-C6 and containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur
• L 0 , L 1 , and L 2 are each independently at each occurrence can be a covalent bond. In some embodiments of X Core , L 0 , L 1 , and L 2 are each independently at each occurrence can be a hydrogen. In some embodiments of X Core , L 0 , L 1 , and L 2 are each independently at each occurrence can be an alkylene (e.g., C1-C12, such as C1-C6 or C1-C3).
• alkylene e.g., C1-C12, such as C1-C6 or C1-C3
• L 0 , L 1 , and L 2 are each independently at each occurrence can be a heteroalkylene (e.g., C1-C12, such as C1-C8 or C1-C6). In some embodiments of XCore, L 0 , L 1 , and L 2 are each independently at each occurrence can be a heteroalkylene (e.g., C2-C8 alkyleneoxide, such as oligo(ethyleneoxide)).
• L 0 , L 1 , and L 2 are each independently at each occurrence can be a [alkylene]-[heterocycloalkyl]-[alkylene] [(e.g., C 1 -C 6 ) alkylene]-[(e.g., C 4 -C 6 ) heterocycloalkyl]-[(e.g., C 1 -C 6 ) alkylene].
• L 0 , L 1 , and L 2 are each independently at each occurrence can be a [alkylene]-(arylene)-[alkylene] [(e.g., C 1 - C6) alkylene]-(arylene)-[(e.g., C1-C6) alkylene].
• L 0 , L 1 , and L 2 are each independently at each occurrence can be a [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6) alkylene]- phenylene-[(e.g., C1-C6) alkylene]).
• L 0 , L 1 , and L 2 are each independently at each occurrence can be a heterocycloalkyl (e.g., C4-C6heterocycloalkyl).
• L 0 , L 1 , and L 2 are each independently at each occurrence can be an arylene (e.g., phenylene).
• part of L 1 form a heterocycloalkyl with one of R 1c and R 1d .
• part of L 1 form a heterocycloalkyl (e.g., C 4 -C 6 heterocycloalkyl) with one of R 1c and R 1d and the heterocycloalkyl can contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.
• a heterocycloalkyl e.g., C 4 -C 6 heterocycloalkyl
• the heterocycloalkyl can contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.
• L 0 , L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, C1-C6 alkylene (e.g., C1-C3 alkylene), C2-C12 (e.g., C2-C8) alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH2CH2O)1-4-(CH2CH2)-), [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., and [(C1-C4) alkylene]-phenylene- [(C 1 -C 4 ) alkylene]
• L 0 , L 1 , and L 2 are each independently at each occurrence selected from C 1 -C 6 alkylene (e.g., C 1 -C 3 alkylene), -(C 1 -
• L 0 , L 1 , and L 2 are each independently at each occurrence C1-C6 alkylene (e.g., C1-C3 alkylene). In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence C2-C12 (e.g., C2-C8) alkyleneoxide (e.g., -(C1-C3 alkylene-O)1-4-(C1- C3 alkylene)).
• L 0 , L 1 , and L 2 are each independently at each occurrence selected from [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 ) heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 alkylene)- phenylene-(C 1 -C 3 alkylene)-) and [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 ) heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 alkylene)-piperazinyl-(C 1 -C 3 alkylene)-).
• x 1 is 0, 1, 2, 3, 4, 5, or 6. In some embodiments of XCore, x 1 is 0. In some embodiments of XCore, x 1 is 1. In some embodiments of XCore, x 1 is 2. In some embodiments of XCore, x 1 is 0, 3. In some embodiments of XCore x 1 is 4. In some embodiments of XCore x 1 is 5. In some embodiments of XCore, x 1 is 6. [00147] In some embodiments of XCore, the core comprises a structural formula: . In some embodiments of XCore , the core comprises a structural formula: .
• the core comprises a structural formula: , In some embodiments of XCore, the core comprises a structural formula: ). In some embodiments of X Core , the core comprises a structural formula: some embodiments of X Core , the core comprises a structural formula: (e.g., . In some embodiments of X Core , the core comprises a structural formula: , , or some embodiments of XCore, the core comprises a structural formula: , wherein Q’ is -NR 2 - or -CR 3a R 3b -; q 1 and q 2 are each independently 1 or 2.
• the core comprises a structural formula: , or some embodiments of XCore, the core comprises a structural formula or , optionally substituted aryl or an optionally substituted (e.g., C3-C12, such as C3-C5) heteroaryl. In some embodiments of XCore, the core comprises has a structural formula . [00148] In some embodiments of XCore, the core comprises a structural formula set forth in Table. 1 and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches. In some embodiments, the example cores of Table. 1 are not limited to the stereoisomers (i.e. enantiomers, diastereomers) listed. Table 1. Example core structures
• the core comprises a structural formula selected from the group , , , and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.
• the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches.
• the plurality (N) of branches comprises at least 3 branches.
• the plurality (N) of branches comprises at least 4 branches.
• the plurality (N) of branches comprises at least 5 branches.
• g is 1, 2, 3, or 4. In some embodiments of X Branch , g is 1.
• each branch of the plurality of branches comprises a structural formula
• Example formulation of the dendrimers or dendrons described herein for generations 1-4 is shown in Table 2.
• the number of diacyl groups, linker groups, and terminating groups can be calculated based on g. Table 2.
• Formulation of Dendrimer or Dendron Groups Based on Generation (g) [00160]
• the diacyl group independently comprises a structural formula , indicates a point of attachment of the diacyl group at the proximal end thereof, and ** indicates a point of attachment of the diacyl group at the distal end thereof.
• Y 3 is independently at each occurrence an optionally substituted; alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the diacyl group of 3 , Y is independently at each occurrence an optionally substituted alkylene (e.g., C1-C12). In some embodiments of the diacyl group , Y 3 is independently at each occurrence an optionally substituted alkenylene (e.g., C 1 -C 12 ). In some embodiments of the diacyl group Y 3 is independently at each occurrence an optionally substituted arenylene (e.g., C1-C12).
• a 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -. In some embodiments of the diacyl group of XBranch, A 1 and A 2 are each independently at each occurrence -O-. In some embodiments of the diacyl group of XBranch, A 1 and A 2 are each independently at each occurrence -S-. In some embodiments of the diacyl group of XBranch, A 1 and A 2 are each independently at each occurrence -NR 4 - and R 4 is hydrogen or optionally substituted alkyl (e.g., C 1 -C 6 ).
• m 1 and m 2 are each independently at each occurrence 1, 2, or 3. In some embodiments of the diacyl group of X h , m 1 and m 2 are each independently at each occurrence 1. In some embodiments of the diacyl group , m 1 and m 2 are each independently at each occurrence 2. In some embodiments of the diacyl group of m 1 and m 2 are each independently at each occurrence 3. In some embodiments of the diacyl group R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or an optionally substituted alkyl.
• diacyl group of X , R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen. In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence an optionally substituted (e.g., C 1 -C 8 ) alkyl. [00163] In some embodiments of the diacyl group, A 1 is -O- or -NH-. In some embodiments of the diacyl group, A 1 is -O-. In some embodiments of the diacyl group, A 2 is -O- or -NH-.
• a 2 is -O-.
• Y 3 is C1-C12 (e.g., C1-C6, such as C1-C3) alkylene.
• the diacyl group independently at each occurrence comprises a structural formula , such as and optionally R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or C 1 -C 3 alkyl.
• linker group independently comprises a structural formula , ** indicates a point of attachment of the linker to a proximal diacyl group, and *** indicates a point of attachment of the linker to a distal diacyl group.
• Y1 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene.
• Y1 is independently at each occurrence an optionally substituted alkylene (e.g., C1-C12).
• Y1 is independently at each occurrence an optionally substituted alkenylene (e.g., C 1 -C 12 ).
• Y 1 is independently at each occurrence an optionally substituted arenylene (e.g., C 1 -C 12 ).
• the terminating group of each terminating group is independently selected from optionally substituted alkylthiol and optionally substituted alkenylthiol.
• each terminating group is an optionally substituted alkylthiol (e.g., C1-C18, such as C4-C18).
• alkylthiol e.g., C1-C18, such as C4-C18
• alkenylthiol e.g., C1-C18, such as C4-C18.
• each terminating group is independently C 1 -C 18 alkenylthiol or C 1 -C 18 alkylthiol, and the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C 6 -C 12 aryl, C 1 - C12 alkylamino, C4-C6 N-heterocycloalkyl , -OH, -C(O)OH, ⁇ C(O)N(C1-C3 alkyl) ⁇ (C1-C6 alkylene) ⁇ (C1-C12 alkylamino), ⁇ C(O)N(C1-C3 alkyl) ⁇ (C1-C6 alkylene) ⁇ (C4-C6 N-heterocycloalkyl), ⁇ C(O) ⁇ (C1-C12 alkylamino), and ⁇ C(O) ⁇ (C4-C6
• each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkenylthiol or C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C6-C12 aryl (e.g., phenyl), C1-C12 (e.g., C1-C8) alkylamino (e.g., C1-C6 mono-alkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 di-alkylamino (such , l - alkylamino)), and ⁇ C(O) ⁇ (C 4 -C 6 N-heterocycloalkyl) , wherein the C 4 -C 6 N-hetero
• each terminating group is independently C1-C18 (e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent -OH.
• each terminating group is independently C1-C18 (e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent selected from C1-C12 (e.g., C1-C8) alkylamino (e.g., C1-C6 mono-alkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 di-alkylamino (such as , , p , each terminating group is independently C1-C18 (e.g., C4-C18) alkenylthiol or C1-C18 (e.g., C4-C18) alkylthiol.
• C1-C18 e.g., C4-C18 alkylthiol
• each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol.
• each terminating group is independently a structural set forth in Table 3.
• the dendrimers or dendrons described herein can comprise a terminating group or pharmaceutically acceptable salt, or thereof selected in Table 3.
• the example terminating group of Table 3 are not limited to the stereoisomers (i.e. enantiomers, diastereomers) listed. Table 3.
• the dendrimer or dendrons of Formula (X) is selected from those set forth in Table 4 and pharmaceutically acceptable salts thereof. Table 4.
• a is 1. In some embodiments of the cationic lipid of formula (D-I’), b is 2. In some embodiments of the cationic lipid of formula (D- I’), m is 1. In some embodiments of the cationic lipid of formula (D-I’), n is 1. In some embodiments of the cationic lipid of formula (D-I’), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or - CH2CH(OH)R 7 .
• R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or .
• R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or .
• R 7 is C 3 -C 18 alkyl (e.g., C 6 -C 12 alkyl).
• the cationic lipid of formula (D-I’) is 13,16,20-tris(2-hydroxydodecyl)- 13,16,20,23-tetraazapentatricontane-11,25-diol: .
• the cationic lipid of formula (D-I’) is (11R,25R)-13,16,20-tris((R)-2- hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: .
• Additional cationic lipids that can be used in the compositions and methods of the present application include those cationic lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210- 217, and International Patent Publication WO 2010/144740, WO 2013/149140, WO 2016/118725, WO 2016/118724, WO 2013/063468, WO 2016/205691, WO 2015/184256, WO 2016/004202, WO 2015/199952, WO 2017/004143, WO 2017/075531, WO 2017/117528, WO 2017/049245, WO 2017/173054 and WO 2015/095340, which are incorporated herein by reference for all purposes.
• Examples of those ionizable cationic lipids include but are not limited to those as shown in Table 5. Table 5: Example ionizable cationic lipids
• the ionizable cationic lipid is present in an amount from about from about 20 to about 23. In some embodiments, the molar percentage is from about 20, 20.5, 21, 21.5, 22, 22.5, to about 23 or any range derivable therein. In other embodiments, the molar percentage is from about 7.5 to about 20. In some embodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 or any range derivable therein. [00178] In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable cationic lipid at a molar percentage from about 5% to about 30%.
• the lipid composition comprises the ionizable cationic lipid at a molar percentage from about 10% to about 25%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable cationic lipid at a molar percentage from about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable cationic lipid at a molar percentage from about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable cationic lipid at a molar percentage from about 20% to about 30%.
• the lipid composition comprises the ionizable cationic lipid at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable cationic lipid at a molar percentage of at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30%.
• the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ.
• the target organ is a lung, a lung tissue or a lung cell.
• the term “preferentially delivered” is used to refer to a composition, upon being delivered, which is delivered to the target organ (e.g., lung), tissue, or cell in at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the amount administered.
• the lipid composition comprises one or more selective organ targeting (SORT) lipid which leads to the selective delivery of the composition to a particular organ.
• SORT lipid may have two or more alkyl or alkenyl chains of C 6 -C 24 .
• the SORT lipid comprises permanently positively charged moiety.
• the permanently positively charged moiety may be positively charged at a physiological pH such that the SORT lipid comprises a positive charge upon delivery of a polynucleotide to a cell.
• the positively charged moiety is quaternary amine or quaternary ammonium ion.
• the SORT lipid comprises, or is otherwise complexed to or interacting with, a counterion.
• the SORT lipid is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group).
• the permanently cationic lipid may contain a group which has a positive charge regardless of the pH.
• One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammonium group.
• the permanently cationic lipid may comprise a structural formula: (S-I), wherein: Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5; provided at least one of Y1, Y2, and Y3 is X2N + R3R4R5; R 1 is C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 alkenyl, C 1 -C 24 substituted alkenyl; X 1 is O or NR a , wherein R a is hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 substituted alkyl; X 2 is C 1 -C 6 alkanediyl or C 1 -C 6 substituted alkanediyl; R 3 , R 4 , and R 5 are each independently C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 al
• the permanently cationic SORT lipid has a structural formula: wherein: R6-R9 are each independently C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, C1-C24 substituted alkenyl; provided at least one of R 6 -R 9 is a group of C 8 -C 24 ; and A 2 is a monovalent anion.
• the SORT lipid is ionizable cationic lipid (i.e., comprising one or more hydrophobic components and an ionizable cationic group).
• the ionizable positively charged moiety may be positively charged at a physiological pH.
• One ionizable cationic group that may be used in the ionizable cationic lipid is a tertiary ammine group.
• the SORT lipid has a structural formula: , wherein: R 1 and R 2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) , or a substituted version of either group; and R 3 and R 3 ′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) .
• the SORT lipid comprises a head group of a particular structure.
• the SORT lipid comprises a headgroup having a structural formula: , wherein L is a linker; Z + is positively charged moiety and X- is a counterion.
• the linker is a biodegradable linker.
• the biodegradable linker may be degradable under physiological pH and temperature.
• the biodegradable linker may be degraded by proteins or enzymes from a subject.
• the positively charged moiety is a quaternary ammonium ion or quaternary amine.
• the SORT lipid has a structural formula: , wherein R 1 and R 2 are each independently an optionally substituted C 6 -C 24 alkyl, or an optionally substituted C 6 -C 24 alkenyl. [00187] In some embodiments of the lipid compositions, the SORT lipid has a structural formula: . [00188] In some embodiments of the lipid compositions, the SORT lipid comprises a Linker (L).
• L is , wherein: p and q are each independently 1, 2, or 3; and R 4 is an optionally substituted C 1 -C 6 alkyl
• the SORT lipid has a structural formula: wherein: R 1 and R 2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) , or a substituted version of either group; R3, R3′, and R3′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6); R4 is alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6); and X ⁇ is a monovalent anion.
• the SORT lipid is a phosphotidylcholine (e.g., 14:0 EPC).
• the phosphatidylcholine compound is further defined as: wherein: R1 and R2 are each independently alkyl(C8-C24), alkenyl(C8-C24), or a substituted version of either group; R3, R3′, and R3′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6); and X ⁇ is a monovalent anion.
• the SORT lipid is a phosphocholine lipid.
• the SORT lipid is an ethylphosphocholine.
• the ethylphosphocholine may be, by way of example, without being limited to, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine.
• the SORT lipid has a structural formula: wherein: R 1 and R 2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) , or a substituted version of either group; R3, R3′, and R3′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6); and X ⁇ is a monovalent anion.
• R 1 and R 2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) , or a substituted version of either group
• R3, R3′, and R3′′ are each independently alkyl(C ⁇ 6) or substituted alkyl(C ⁇ 6)
• X ⁇ is a monovalent anion.
• a SORT lipid of the structural formula of the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).
• the SORT lipid has a structural formula: wherein: R 4 and R 4 ′ are each independently alkyl (C6-C24) , alkenyl (C6-C24) , or a substituted version of either group; R4′′ is alkyl(C ⁇ 24), alkenyl(C ⁇ 24), or a substituted version of either group; R 4 ′′′ is alkyl (C1-C8) , alkenyl (C2-C8) , or a substituted version of either group; and X 2 is a monovalent anion.
• a SORT lipid of the structural formula of the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB) (e.g., bromide salt).
• DDAB dimethyldioctadecylammonium
• the SORT lipid comprises one or more selected from the lipids set forth in Table 6. Table 6.
• Example SORT lipids X- is a counterion (e.g., Cl-, Br-, etc.)
• the lipid composition comprises the SORT lipid at a molar percentage from about 20% to about 65%.
• the lipid composition comprises the SORT lipid at a molar percentage from about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the SORT lipid at a molar percentage from about 30% to about 55%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the SORT lipid at a molar percentage from about 20% to about 50%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the SORT lipid at a molar percentage from about 30% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the SORT lipid at a molar percentage from about 25% to about 60%.
• the lipid composition comprises the SORT lipid at a molar percentage of at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, at least (about) 60%, or at least (about) 65%.
• the lipid composition comprises the SORT lipid at a molar percentage of at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at least (about) 45%, at most (about) 50%, at most (about) 55%, at most (about) 60%, or at most (about) 65%.
• the lipid composition comprises the SORT lipid at a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or of a range between (inclusive) any two of the foregoing values.
• the lipid composition further comprises an additional lipid including but not limited to a steroid or a steroid derivative, a PEG lipid, and a phospholipid.
• Phospholipids or Other Zwitterionic Lipids [00199]
• the lipid composition further comprises a phospholipid.
• the phospholipid may contain one or two long chain (e.g., C6-C24) alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule.
• the small organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine.
• the phospholipid is a phosphatidylcholine.
• the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.
• other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.
• the phospholipid is not an ethylphosphocholine.
• the compositions may further comprise a molar percentage of the phospholipid to the total lipid composition from about 20 to about 23. In some embodiments, the molar percentage is from about 20, 20.5, 21, 21.5, 22, 22.5, to about 23 or any range derivable therein. In other embodiments, the molar percentage is from about 7.5 to about 60. In some embodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 or any range derivable therein. [00202] In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 23%.
• the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 12% to about 18%.
• the lipid composition comprises the phospholipid at a molar percentage of at least (about) 8%, at least (about) 10%, at least (about) 12%, at least (about) 15%, at least (about) 18%, at least (about) 20%, or at least (about) 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage of at most (about) 8%, at most (about) 10%, at most (about) 12%, at most (about) 15%, at most (about) 18%, at most (about) 20%, or at most (about) 23%.
• the lipid composition further comprises a steroid or steroid derivative.
• the steroid or steroid derivative comprises any steroid or steroid derivative.
• the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.
• the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula:
• a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions.
• the steroid or steroid derivative is a sterol wherein the formula is further defined as: .
• the steroid or steroid derivative is a cholestane or cholestane derivative.
• the ring structure is further defined by the formula: described above, a cholestane derivative includes one or more non-alkyl substitution of the above ring system.
• the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both a cholestere and a sterol or a derivative thereof. [00204] In some embodiments of the lipid composition, the compositions may further comprise a molar percentage of the steroid to the total lipid composition from about 40 to about 46. In some embodiments, the molar percentage is from about 40, 41, 42, 43, 44, 45, to about 46 or any range derivable therein.
• the molar percentage of the steroid relative to the total lipid composition is from about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein. [00205] In some embodiments of the lipid composition of the present application, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 15% to about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 40%.
• the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 25% to about 35%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 30% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 30%.
• the lipid composition comprises the steroid or steroid derivative at a molar percentage of at least (about) 15%, of at least (about) 20%, of at least (about) 25%, of at least (about) 30%, of at least (about) 35%, of at least (about) 40%, of at least (about) 45%, or of at least (about) 46%.
• the lipid composition comprises the steroid or steroid derivative at a molar percentage of at most (about) 15%, of at most (about) 20%, of at most (about) 25%, of at most (about) 30%, of at most (about) 35%, of at most (about) 40%, of at most (about) 45%, or of at most (about) 46%.
• Polymer-Conjugated Lipids [00206]
• the lipid composition further comprises a polymer conjugated lipid.
• the polymer conjugated lipid is a PEG lipid.
• the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group.
• the PEG lipid is a compound which contains one or more C6-C24 long chain hydrocarbon groups (e.g., C6-C24 long chain alkyl or alkenyl group or a C6- C 24 fatty acid group) attached to a linker group with a PEG chain.
• the alkyl, alkenyl, or fatty acid group is about 6 carbon atoms to about 24 carbon atoms. In some embodiments, the alkyl, alkenyl, or fatty acid group is at least about 6 carbon atoms.
• the alkyl, alkenyl, or fatty acid group is at most about 24 carbon atoms. In some embodiments, the alkyl, alkenyl, or fatty acid group is about 6 carbon atoms to about 8 carbon atoms, about 6 carbon atoms to about 10 carbon atoms, about 6 carbon atoms to about 12 carbon atoms, about 6 carbon atoms to about 14 carbon atoms, about 6 carbon atoms to about 16 carbon atoms, about 6 carbon atoms to about 20 carbon atoms, about 6 carbon atoms to about 22 carbon atoms, about 6 carbon atoms to about 24 carbon atoms, about 8 carbon atoms to about 10 carbon atoms, about 8 carbon atoms to about 12 carbon atoms, about 8 carbon atoms to about 14 carbon atoms, about 8 carbon atoms to about 16 carbon atoms, about 8 carbon atoms to about 20 carbon atoms, about 8 carbon atoms to about 22 carbon atoms, about 8 carbon atoms to about 8 carbon atom
• the alkyl, alkenyl, or fatty acid group is about 6 carbon atoms, about 8 carbon atoms, about 10 carbon atoms, about 12 carbon atoms, about 14 carbon atoms, about 16 carbon atoms, about 20 carbon atoms, about 22 carbon atoms, or about 24 carbon atoms.
• the long chain hydrocarbon group or groups may comprise one or more unsaturated carbon bonds (e.g., double or triple carbon-carbon bonds). In some embodiments, the long chain hydrocarbon groups may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more unsaturated carbon bonds. In some embodiments, the hydrocarbon groups may comprise no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer unsaturated carbon bonds.
• a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified 1,2- diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols.
• the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000.
• the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000.
• the molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000.
• the polymer-conjugated lipid has a molecular weight from about 500 to about 100,000 Daltons (Da).
• the polymer-conjugated lipid has a a molecular weight of about 100, 200, 300, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, or more Da. In some embodiments, the polymer-conjugated lipid has a molecular weight of more than about 100, 200, 300, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, or more. In some embodiments, the polymer-conjugted lipid has a molecular weight of no more than 100,000, 50,000, 20,000, 10,000, 5,000, 2,000, 1,000, 500, 300, 200, 100, or less.
• the polymer-conjugated lipid has a molecular weight of about 100 Da to about 100,000 Da. In some embodiments, the polymer-conjugated lipid has a molecular weight of at least about 100 Da. In some embodiments, the polymer-conjugated lipid has a molecular weight of at most about 100,000 Da.
• the polymer-conjugated lipid has a molecular weight of about 100 Da to about 200 Da, about 100 Da to about 300 Da, about 100 Da to about 500 Da, about 100 Da to about 1,000 Da, about 100 Da to about 2,000 Da, about 100 Da to about 5,000 Da, about 100 Da to about 10,000 Da, about 100 Da to about 20,000 Da, about 100 Da to about 50,000 Da, about 100 Da to about 100,000 Da, about 200 Da to about 300 Da, about 200 Da to about 500 Da, about 200 Da to about 1,000 Da, about 200 Da to about 2,000 Da, about 200 Da to about 5,000 Da, about 200 Da to about 10,000 Da, about 200 Da to about 20,000 Da, about 200 Da to about 50,000 Da, about 200 Da to about 100,000 Da, about 300 Da to about 500 Da, about 300 Da to about 1,000 Da, about 300 Da to about 2,000 Da, about 300 Da to about 5,000 Da, about 300 Da to about 10,000 Da, about 300 Da to about 20,000 Da, about 300 Da to about 50,000 Da, about 200 Da to about 100,000 Da, about 300 Da to about 500 Da,
• the polymer- conjugated lipid has a molecular weight of about 100 Da, about 200 Da, about 300 Da, about 500 Da, about 1,000 Da, about 2,000 Da, about 5,000 Da, about 10,000 Da, about 20,000 Da, about 50,000 Da, or about 100,000 Da.
• lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010/141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.
• the PEG lipid has a structural formula: , wherein: R12 and R13 are each independently alkyl(C ⁇ 24), alkenyl(C ⁇ 24), or a substituted version of either of these groups; Re is hydrogen, alkyl(C ⁇ 8), or substituted alkyl(C ⁇ 8); and x is 1-250. In some embodiments, Re is alkyl(C ⁇ 8) such as methyl. R12 and R13 are each independently alkyl (C ⁇ 4-20) . In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250.
• the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.
• the PEG lipid has a structural formula: , wherein: n1 is an integer between 1 and 100 and n2 and n3 are each independently selected from an integer between 1 and 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein.
• n1 is from about 30 to about 50.
• n2 is from 5 to 23.
• n 2 is 11 to about 17.
• n 3 is from 5 to 23.
• n 3 is 11 to about 17.
• the compositions may further comprise a molar percentage of the PEG lipid to the total lipid composition from about 4.0 to about 4.6.
• the molar percentage is from about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, to about 4.6 or any range derivable therein.
• the molar percentage is from about 1.5 to about 4.0.
• the molar percentage is from about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, to about 4.0 or any range derivable therein.
• the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 1% to about 8%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer- conjugated lipid at a molar percentage from about 2% to about 7%.
• the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 3% to about 5%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 5% to about 10%.
• the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10%.
• the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5%, or at most (about) 10%.
• a pharmaceutical composition comprising a therapeutic agent (or prophylactic agent) assembled to a lipid composition as described herein.
• the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof.
• the compound, the polynucleotide, the polypeptide, or a combination thereof is exogenous or heterologous to the cell or the subject being treated by the pharmaceutical compositions described herein.
• the therapeutic agent (or prophylactic agent) comprises a compound described herein.
• the therapeutic agent comprises a polynucleotide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polypeptide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof. [00213] In some embodiments, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) for treating a lung disease such as asthma, COPD, or lung cancer.
• the therapeutic agent comprises a steroid such as prednisone, hydrocortisone, prednisolone, methylprednisolone, or dexamethasone.
• the therapeutic agent comprises Abraxane, Afatinib Dimaleate, Afinitor, Afinitor Disperz, Alecensa, Alectinib, Alimta, Alunbrig, Atezolizumab, Avastin, Bevacizumab, Brigatinib, Capmatinib Hydrochloride, Carboplatin, Ceritinib, Crizotinib, Cyramza, Dabrafenib Mesylate, Dacomitinib, Docetaxel, Doxorubicin Hydrochloride, Durvalumab, Entrectinib, Erlotinib Hydrochloride, Everolimus, Gavreto, Gefitinib, Gilotrif, Gem
• therapeutic agents comprising compounds include small molecule selected from 7-Methoxypteridine, 7 Methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acetretin, acrivastine, adenine, adenosine, alatrofloxacin, albendazole, albuterol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital, allopurinol, all-transretinoic acid (ATRA), aloxiprin, alprazolam, alprenolol, altretamine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone HCl
• the therapeutic agent (or prophylactic agent) assembled to the lipid composition comprises one or more polynucleotides.
• the present application is not limited in scope to any particular source, sequence, or type of polynucleotide; however, as one of ordinary skill in the art could readily identify related homologs in various other sources of the polynucleotide including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).
• non-human species e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species.
• the polynucleotide used in the present application can comprises a sequence based upon a naturally-occurring sequence. Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the naturally-occurring sequence.
• the polynucleotide comprises nucleic acid sequence that is a complementary sequence to a naturally occurring sequence, or complementary to 75%, 80%, 85%, 90%, 95% and 100%.
• the polynucleotide used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the polynucleotide would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as "mini-genes.” At a minimum, these and other nucleic acids of the present application may be used as molecular weight standards in, for example, gel electrophoresis.
• cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
• mRNA messenger RNA
• the polynucleotide comprises one or more segments comprising a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a
• siRNA small interfering
• the polynucleotide encodes at least one of the therapeutic agent (or prophylactic agent) described herein.
• the polynucleotide encodes at least one guide polynucleotide, such as guide RNA (gRNA) or guide DNA (gDNA), for complexing with a guide RNA guided nuclease described herein.
• the polynucleotide encodes at least one guide polynucleotide guided heterologous nuclease.
• the nuclease may be an endonuclease.
• Non-limiting example of the guide polynucleotide guided heterologous endonuclease may be selected from CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA- binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argona
• the therapeutic (or prophylactic) agent is a transfer ribonucleic acid (tRNA) that introduces an amino acid into a growing peptide chain of a protein of a target gene.
• tRNA transfer ribonucleic acid
• Some embodiments of the therapeutic agent (or prophylactic agent) provided herein comprise a heterologous polypeptide comprising an actuator moiety.
• the actuator moiety can be configured to complex with a target polynucleotide corresponding to a target gene.
• administration of the therapeutic agent (or prophylactic agent) results in a modified expression or activity of the target gene.
• the modified expression or activity of the target gene can be detectable, for example, in at least about 1% (e.g., at least about 2%, 5%, 10%, 15%, or 20%) cells (e.g., lung cells, such as lung basal cells) of the subject.
• the therapeutic agent or prophylactic agent
• the actuator moiety may be configured to complex with a target polynucleotide corresponding to a target gene.
• the heterologous polynucleotide may encode a guide polynucleotide configured to direct the actuator moiety to the target polynucleotide.
• the actuator moiety may comprise a heterologous endonuclease or a fragment thereof (e.g., directed by a guide polynucleotide to specifically bind the target polynucleotide).
• the heterologous endonuclease may be (1) part of a ribonucleoprotein (RNP) and (2) complexed with the guide polynucleotide.
• the heterologous endonuclease may be part of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein complex.
• the heterologous endonuclease may be a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonuclease.
• CRISPR CRISPR-associated
• the heterologous endonuclease may comprise a deactivated endonuclease.
• the deactivated endonuclease may be fused to a regulatory moiety.
• the regulatory moiety may comprise a transcription activator, a transcription repressor, an epigenetic modifier, or a fragment thereof.
• the polynucleotide encodes at least one guide polynucleotide (such as guide RNA (gRNA) or guide DNA (gDNA)) guided heterologous endonuclease.
• gRNA guide RNA
• gDNA guide DNA
• the polynucleotide encodes at least one guide polynucleotide and at least one heterologous endonuclease, where the guide polynucleotide can be complexed with and guides the at least one heterologous endonuclease to cleave a genetic locus of any one of the genes described herein.
• the polynucleotide encodes at least one guide polynucleotide guided heterologous endonuclease such as Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, Cas14, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas10, Cas10d, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (Cas9, Ca
• Cas13 can include, but are not limited to, Cas13a, Cas13b, Cas13c, and Cas 13d (e.g., CasRx).
• the heterologous endonuclease comprises a deactivated endonuclease, optionally fused to a regulatory moiety, such as an epigenetic modifier to remodel the epigenome that mediates the expression of the selected genes of interest.
• the epigenetic modifier can include methyltransferase, demethylase, dismutase, an alkylating enzyme, depurinase, oxidase, photolyase, integrase, transposase, recombinase, polymerase, ligase, helicase, glycosylase, acetyltransferase, deacetylase, kinase, phosphatase, ubiquitin-activating enzymes, ubiquitin- conjugating enzymes, ubiquitin ligase, deubiquitinating enzyme, adenylate-forming enzyme, AMPylator, de-AMPylator, SUMOylating enzyme, deSUMOylating enzyme, ribosylase, deribosylase, N-myristoyltransferase, chromotine remodeling enzyme, protease, oxidoreductase, transferase, hydrolase, ly
• the epigenetic modifier can comprise one or more selected from the group consisting of p300, TET1, LSD1, HDAC1, HDAC8, HDAC4, HDAC11, HDT1, SIRT3, HST2, CobB, SIRT5, SIR2A, SIRT6, NUE, vSET, SUV39H1, DIM5, KYP, SUVR4, Set4, Set1, SETD8, and TgSET8.
• the polynucleotide encodes a guide polynucleotide (such as guide RNA (gRNA) or guide DNA (gDNA)) that is at least partially complementary to the genomic region of a gene, where upon binding of the guide polynucleotide to the gene the guide polynucleotide recruits the guide polynucleotide guided nuclease to cleave and genetically modified the region.
• a guide polynucleotide such as guide RNA (gRNA) or guide DNA (gDNA)
• genes that may be modified by the guide polynucleotide guided nuclease include CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.
• the polynucleotide comprises or encodes at least one mRNA that, upon expression of the mRNA, restores the function of a defective gene in a subject being treated by the pharmaceutical composition described herein.
• the polynucleotide comprises or encodes an mRNA that expresses a wild type CFTR protein, which may be used to rescue a subject who is afflicted with inborn mutation in CFTR protein.
• mRNA that can be expressed from the polynucleotide includes mRNA that encodes DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.
• the polynucleotides of the present application comprise at least one chemical modifications of the one or more nucleotides.
• the chemical modification increases specificity of the guide polynucleotide (such as guide RNA (gRNA) or guide DNA (gDNA)) binding to a complementary genomic locus (e.g., the genomic locus of any one of the genes described herein).
• the at least one chemical modification increases resistance to nuclease digestion, when then polynucleotide is administered to a subject in need thereof.
• the at least one chemical modification decreases immunogenicity, when then polynucleotide is administered to a subject in need thereof.
• the at least one chemical modification stabilizes scaffold such as a tRNA scaffold.
• Such chemical modification may have desirable properties, such as enhanced resistance to nuclease digestion or increased binding affinity with a target genomic locus relative to a polynucleotide without the at least one chemical modification.
• the at least one chemical modification comprises modification to sugar moiety.
• modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2' and/or 5' positions.
• sugar substituents suitable for the 2'-position include, but are not limited to: 2'- F, 2'-OCH3 ("OMe” or “O-methyl”), and 2'-O(CH2)2OCH3 ("MOE”).
• sugar substituents at the 5'-position include, but are not limited to: 5'-methyl (R or S); 5'-vinyl, and 5'- methoxy.
• substituted sugars comprise more than one non-bridging sugar substituent, for example, T-F-5'-methyl sugar moieties.
• Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides.
• These 2'- substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
• R m and R n is, independently, H, an amino protecting group or substituted or unsubstituted C 1 -C 10 alkyl.
• a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'- substituent group selected from F, O--CH 3 , and OCH 2 CH 2 OCH 3 .
• modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
• the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms.
• Examples of such 4' to 2' sugar substituents include, but are not limited to: --[C(Ra)(Rb)]n--, --[C(Ra)(Rb)]n--O--, --C(RaRb)--N(R)--O-- or, -- C(RaRb)--O--N(R)--; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)--O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2--O-2' (ENA); 4'-CH(CH3)--O-2' (cEt) and 4'-CH(CH2OCH3)--O-2', and analogs thereof; 4'-C(CH3)(CH3)--O- 2' and analogs thereof; 4'-CH2--N(OCH3)-2' and analogs thereof; 4'-CH2--O-N(CH3)-2'; 4'-CH2--CH2-
• Bicyclic nucleosides include, but are not limited to, (A) ⁇ -L-Methyleneoxy (4'-CH2--O-2') BNA, (B) ⁇ -D-Methyleneoxy (4'-CH 2 --O-2') BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4'-(CH 2 ) 2 --O-2') BNA, (D) Aminooxy (4'-CH 2 --O--N(R)-2') BNA, (E) Oxyamino (4'- CH2--N(R)--O-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH3)--O-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2--S), (G) methylene-thio (4'-CH2--S), (G) methylene-thio (4'-CH2--S)
• bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
• a nucleoside comprising a 4'-2' methylene-oxy bridge may be in the .alpha.-L configuration or in the .beta.-D configuration.
• ⁇ -L-methyleneoxy (4'-CH2--O-2') bicyclic nucleosides have been incorporated into antisense polynucleotides that showed antisense activity.
• substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).
• modified sugar moieties are sugar surrogates.
• the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom.
• such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above.
• sugar surrogates comprise a 4'-sulfur atom and a substitution at the 2'-position and/or the 5' position.
• carbocyclic bicyclic nucleosides having a 4'-2' bridge have been described.
• sugar surrogates comprise rings having other than 5-atoms.
• a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted.
• Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA), and fluoro HNA (F-HNA).
• HNA hexitol nucleic acid
• ANA anitol nucleic acid
• MNA manitol nucleic acid
• F-HNA fluoro HNA
• THP nucleosides of Formula VII are provided wherein one of R1 and R2 is F.
• R1 is fluoro and R2 is H
• R1 is methoxy and R2 is H
• R1 is methoxyethoxy and R2 is H.
• Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds.
• modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modifications are selected such that the resulting polynucleotide possesses desirable characteristics.
• polynucleotide comprises one or more RNA- like nucleosides.
• polynucleotide comprises one or more DNA-like nucleotides.
• nucleosides of the present application comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present application comprise one or more modified nucleobases.
• modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
• nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)- one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)- one), carbazole cytidine ( 2 H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
• tricyclic pyrimidines such as phenoxa
• Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone.
• the present application provides poylnucleotide comprising linked nucleosides.
• nucleosides may be linked together using any internucleoside linkage.
• the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
• Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiester (--O--- C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)2--O--); and N,N'- dimethylhydrazine (--CH2--N(CH3)--N(CH3)--).
• Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide.
• internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
• Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
• the polynucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), ⁇ or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
• Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
• Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
• one additional modification of the ligand conjugated polynucleotides of the present application involves chemically linking to the oligonucleotide one or more additional non- ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
• Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
• lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e
• the polynucleotides described herein comprise or encode at least one tRNA described herein.
• the tRNA expressed from the polynucleotide restores the function of at least one defective tRNA in a subject who is being treated by the pharmaceutical composition described herein.
• the at least one tRNA expressed by the polynucleotide described herein may include tRNA that encodes alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucin, lysine, methionine, phenylaniline, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, or valine.
• the at least one tRNA expressed by the polynucleotide described herein may include tRNA that encodes arginine, tryptophan, glutamic acid, glutamine, serine, tyrosine, lysine, leucine, glycine, or cysteine.
• the tRNA encoded by the polynucleotide described herein may restore the expression of any one of the genes described herein.
• the tRNA encoded by the polynucleotide described herein may restore the expression of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.
• the therapeutic agent (or prophylactic agent) assembled to the lipid composition comprises one or more one or more polypeptides.
• Some polypeptide may include enzymes such as any one of the nuclease enzymes described herein.
• the nuclease enzyme may include from CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR- associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR- associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR- associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (
• the nuclease enzyme may include Cas proteins such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.
• Cas proteins such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas
• the Cas protein may be complexed with a guide polynucleotide described herein to be form a CRISPR ribonucleoprotein (RNP).
• RNP CRISPR ribonucleoprotein
• the nuclease in the compositions described herein may be Cas9 (e.g., from S. pyogenes or S. pneumonia).
• the CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence of any one of the genes described herein.
• the CRISPR enzyme may be directed and cleaved a genomic locus of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.
• the CRISPR enzyme may be mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
• an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
• a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
• guide sequence(s) e.g., two guide sequences
• This combination allows both strands to be nicked and used to induce NHEJ or HDR.
• the present disclosure provides polypeptide containing one or more therapeutic proteins.
• the therapeutic proteins that may be included in the composition include a wide range of molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anti-coagulants, blood factors, bone morphogenic proteins, immunoglobulins, and enzymes.
• EPO Erythropoietin
• G-CSF Granulocyte colony-stimulating factor
• Alpha-galactosidase A Alpha-L-iduronidase
• Thyrotropin ⁇ N-acetylgalactosamine-4-sulfatase
• Dornase alfa Tissue plasminogen activator (TPA) Activase, Glucocerebrosidase, Interferon (IF) ⁇ -1a, Interferon ⁇ -1b, Interferon ⁇ , Interferon ⁇ , TNF- ⁇ , IL-1 through IL-36, Human growth hormone (rHGH), Human insulin (BHI), Human chorionic gonadotropin ⁇ , Darbepoetin ⁇ , Follicle-stimulating hormone (FSH), and Factor VIII.
• EPO Erythropoietin
• G-CSF Granulocyte colony-stimulating factor
• Alpha-galactosidase A
• the polypeptide comprises a peptide sequence that is at least partially identical to any of the therapeutic agent (or prophylactic agent) comprising a peptide sequence.
• the polypeptide may comprise a peptide sequence that is at least partially identical to an antibody (e.g., a monoclonal antibody) for treating a lung disease such as lung cancer.
• the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject being treated by the pharmaceutical composition described herein.
• the polynucleotide comprises a peptide or protein that restores function of cystic fibrosis transmembrane conductance regulator (CFTR) protein, which may be used to rescue a subject who is afflicted with inborn error leading to the expression of the mutated CFTR protein.
• CFTR cystic fibrosis transmembrane conductance regulator
• the rescue may include administering to a subject in need thereof a polypeptide comprising a peptide or protein of wild type Dynein axonemal heavy chain 5, Dynein axonemal heavy chain 11,Bone morphogenetic protein receptor type 2,Fumarylacetoacetate hydrolase, Phenylalanine hydroxylase, Alpha-L-iduronidase, Collagen type IV alpha 3 chain, Collagen type IV alpha 4 chain, Collagen type IV alpha 5 chain, Polycystin 1, Polycystin 2, Fibrocystin (or polyductin), Solute carrier family 3 member 1,Solute carrier family 7 member 9,Paired box gene 9,Myosin VIIA, Cadherin related 23, Usherin, Clarin 1, Gap junction beta-2 protein, Gap junction beta-6 protein, Rhodopsin, dystrophia myotonica protein kinase, Dystrophin, Sodium voltage-gated channel alpha subunit 1, Sodium voltage-gated channel beta subunit 1, Coagulation
• the pharmaceutical composition of the present application comprises a plurality of payloads assembled with (e.g., encapsulated within) a lipid composition.
• the plurality of payloads assembled with the lipid composition may be configured for gene-editing or gene-expression modification.
• the plurality of payloads assembled with the lipid composition may comprise a polynucleotide encoding an actuator moiety (e.g., comprising a heterologous endonuclease such as Cas) or a polynucleotide encoding the actuator moiety.
• the plurality of payloads assembled with the lipid composition may further comprise one or more (e.g., one or two) guide polynucleotides.
• the plurality of payloads assembled with the lipid composition may further comprise one or more donor or template polynucleotides.
• the plurality of payloads assembled with the lipid composition may comprise a ribonucleoprotein (RNP).
• RNP ribonucleoprotein
• the therapeutic agent or prophylactic agent
• N/P ratio a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide
• the therapeutic agent is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is no less than (about) 20:1, no less than (about) 15:1, no less than (about) 10:1, or no less than (about) 5:1.
• the therapeutic agent (or prophylactic agent) is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 5:1 to about 20:1.
• the therapeutic agent is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 10:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 15:1 to about 20:1.
• the therapeutic agent is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 5:1 to about 10:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 5:1 to about 15:1.
• the therapeutic agent is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is from about 15:1 to about 20:1.
• a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 1:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 1:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 50:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 1:1 to about 1:20.
• a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 20:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 50:1 to about 1:70. In some embodiments of the pharmaceutical composition of the present application, a molar ratio of the therapeutic agent to total lipids of the lipid composition is from about 70:1 to about 1:100.
• a molar ratio of the therapeutic agent to total lipids of the lipid composition is no more than (about) 1:1, no more than (about) 1:5, no more than (about) 1:10, no more than (about) 1:15, no more than (about) 1:20, no more than (about) 1:25, no more than (about) 1:30, no more than (about) 1:35, no more than (about) 1:40, no more than (about) 1:45, no more than (about) 1:50, no more than (about) 1:60, no more than (about) 1:70, no more than (about) 1:80, no more than (about) 1:90, or more than (about) 1:100.
• a molar ratio of the therapeutic agent to total lipids of the lipid composition is no less than (about) 1:1, no less than (about) 1:5, no less than (about) 1:10, no less than (about) 1:15, no less than (about) 1:20, no less than (about) 1:25, no less than (about) 1:30, no less than (about) 1:35, no less than (about) 1:40, no less than (about) 1:45, no less than (about) 1:50, no less than (about) 1:60, no less than (about) 1:70, no less than (about) 1:80, no less than (about) 1:90, or less than (about) 1:100.
• the lipid composition comprises a plurality of particles characterized by one or more characteristics of the following: (1) a (e.g., average) size of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of no more than about 0.2; and (3) a zeta potential of -10 millivolts (mV) to 10 mV.
• the lipid composition comprises a plurality of particles with a (e.g., average) size from about 50 nanometers (nm) to about 100 nanometers (nm).
• the lipid composition comprises a plurality of particles with a (e.g., average) size from about 70 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a (e.g., average) size from about 50 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a (e.g., average) size from about 60 nanometers (nm) to about 80 nanometers (nm).
• the lipid composition comprises a plurality of particles with a (e.g., average) size of at most about 100 nanometers (nm), at most about 90 nanometers (nm), at most about 85 nanometers (nm), at most about 80 nanometers (nm), at most about 75 nanometers (nm), at most about 70 nanometers (nm), at most about 65 nanometers (nm), at most about 60 nanometers (nm), at most about 55 nanometers (nm), or at most about 50 nanometers (nm).
• a (e.g., average) size of at most about 100 nanometers (nm), at most about 90 nanometers (nm), at most about 85 nanometers (nm), at most about 80 nanometers (nm), at most about 75 nanometers (nm), at most about 70 nanometers (nm), at most about 65 nanometers (nm), at most about 60 nanometers (nm), at most about 55 nanometers (nm), or at most about 50 nano
• the lipid composition comprises a plurality of particles with a (e.g., average) size of at least about 100 nanometers (nm), at least about 90 nanometers (nm), at least about 85 nanometers (nm), at least about 80 nanometers (nm), at least about 75 nanometers (nm), at least about 70 nanometers (nm), at least about 65 nanometers (nm), at least about 60 nanometers (nm), at least about 55 nanometers (nm), or at least about 50 nanometers (nm).
• the lipid composition comprises a plurality of particles with a polydispersity index (PDI) from about 0.05 to about 0.5. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a polydispersity index (PDI) from about 0.1 to about 0.5.
• PDI polydispersity index
• the lipid composition comprises a plurality of particles with a polydispersity index (PDI) from about 0.1to about 0.3. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a polydispersity index (PDI) from about 0.2 to about 0.5. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a polydispersity index (PDI) of no more than about 0.5, no more than about 0.4, no more than about 0.3, no more than about 0.2, no more than about 0.1, or no more than about 0.05.
• PDI polydispersity index
• the lipid composition comprises a plurality of particles with a negative zeta potential of -5 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -10 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -15 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -20 millivolts (mV) or less.
• the lipid composition comprises a plurality of particles with a negative zeta potential of -30 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles with a zeta potential of 0 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles with a zeta potential of 5 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles with a zeta potential of 10 millivolts (mV) or less.
• the lipid composition comprises a plurality of particles with a negative zeta potential of 15 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of 20 millivolts (mV) or less. [00261] In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -5 millivolts (mV) or more.
• the lipid composition comprises a plurality of particles with a negative zeta potential of -10 millivolts (mV) or more In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -15 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -20 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of -30 millivolts (mV) or more.
• the lipid composition comprises a plurality of particles with a zeta potential of 0 millivolts (mV) or more. In some embodiments, the lipid composition comprises a plurality of particles with a zeta potential of 5 millivolts (mV) or more. In some embodiments, the lipid composition comprises a plurality of particles with a zeta potential of 10 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles with a negative zeta potential of 15 millivolts (mV) or more.
• the lipid composition comprises a plurality of particles with a negative zeta potential of 20 millivolts (mV) or more.
• the lipid composition has an apparent ionization constant (pKa) outside a range of 6 to 7.
• the lipid composition has an apparent pKa of about 8 or higher, about 9 or higher, about 10 or higher, about 11 or higher, about 12 or higher, or about 13 or higher.
• the lipid composition has an apparent pKa of about 8 to about 13.
• the lipid composition has an apparent pKa of about 8 to about 10. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 9 to about 11. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 10 to about 13. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 8 to about 12. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 10 to about 12.
• the SORT lipid in the pharmaceutical composition effects a delivery of the therapeutic agent characterized by one or more of the following: (a) a greater therapeutic effect in a cell of the subject compared to that achieved with a reference lipid composition; (b) a therapeutic effect in a greater plurality of cells of the subject compared to that achieved with a reference lipid composition; (c) a therapeutic effect in a first plurality of cells of a first cell type and in a greater second plurality of cells of a second cell type; and (d) a greater therapeutic effect in a first cell of a first cell type of the subject compared to that in a second cell of a second cell type of the subject.
• the first cell type is different from the second cell type.
• the cell is a lung cell.
• the lung cell is a lung airway cell.
• Example lung airway cells that can be targeted by the delivery of the present disclosure includes but is not limited to basal cell.
• the therapeutic effect is characterized by a therapeutically effective amount of the therapeutic agent, for example, in a lung, a lung cell, a plurality of lung cells, or a lung cell type of the subject.
• the therapeutic effect is characterized by an activity of the therapeutic agent, for example, in a lung, a lung cell, a plurality of lung cells, or a lung cell type of the subject. In some embodiments, the therapeutic effect is characterized by an effect of the therapeutic agent, for example, in a lung, a lung cell, a plurality of lung cells, or a lung cell type of the subject. In some embodiments, the greater therapeutic effect is characterized by a greater therapeutic amount of the therapeutic agent. In some embodiments, the greater therapeutic effect is characterized by a greater activity of the therapeutic agent. In some embodiments, the greater therapeutic effect is characterized by a greater effect of the therapeutic agent.
• the SORT lipid in the pharmaceutical composition effects delivery of the therapeutic agent to the cell of the subject characterized by a greater therapeutic effect compared to that achieved with a reference lipid composition.
• the reference lipid composition does not comprise the SORT lipid.
• the reference lipid composition does not comprise the amount of the SORT lipid.
• the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23- tetraazapentatricontane-11,25-diol (“LF92”), a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold therapeutic effect compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold therapeutic effect compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold therapeutic effect compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold therapeutic effect compared to that achieved with a reference lipid composition.
• the SORT lipid achieves at least about 1.1- fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold therapeutic effect compared to that achieved with a reference lipid composition.
• the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold therapeutic effect compared to that achieved with a reference lipid composition in basal cell. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold therapeutic effect compared to that achieved with a reference lipid composition in basal cell. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold therapeutic effect compared to that achieved with a reference lipid composition in basal cell. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold therapeutic effect compared to that achieved with a reference lipid composition in basal cell.
• the SORT lipid achieves at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold therapeutic effect compared to that achieved with a reference lipid composition in basal cell.
• the SORT lipid in the pharmaceutical composition effects delivery of the therapeutic agent to cells of the subject characterized by a therapeutic effect in a greater plurality of cells compared to that achieved with a reference lipid composition.
• the reference lipid composition does not comprise the SORT lipid.
• the reference lipid composition does not comprise the amount of the SORT lipid.
• the reference lipid comprises 13,16,20-tris(2- hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol (“LF92”), a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid in the pharmaceutical composition achieves therapeutic effect in about 1.1-fold to about 20-fold cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves therapeutic effect in about 1.1-fold to about 10-fold cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves therapeutic effect in about 5-fold to about 10-fold cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves therapeutic effect in about 10-fold to about 20-fold cells compared to that achieved with a reference lipid composition.
• the SORT lipid achieves therapeutic effect in at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12- fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold cells compared to that achieved with a reference lipid composition.
• the SORT lipid in the pharmaceutical composition achieves therapeutic effect in about 1.1-fold to about 20-fold cells compared to that achieved with a reference lipid composition in basal cell. In some embodiments, the SORT lipid achieves therapeutic effect in about 1.1-fold to about 10-fold more cells compared to that achieved with a reference lipid composition in basal cell. In some embodiments, the SORT lipid achieves therapeutic effect in about 5-fold to about 10-fold more cells compared to that achieved with a reference lipid composition in basal cell. In some embodiments, the SORT lipid achieves therapeutic effect in about 10-fold to about 20-fold more cells compared to that achieved with a reference lipid composition in basal cell.
• the SORT lipid achieves therapeutic effect in about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold more cells compared to that achieved with a reference lipid composition in basal cell.
• the SORT lipid in the pharmaceutical composition effects delivery of the therapeutic agent to cells of the subject characterized by a therapeutic effect in a first plurality of cells of a first cell type and in a greater therapeutic effect in a second plurality of cells of a second cell type.
• the first cell type is different from the second cell type.
• the first cell type is a lung cell.
• the first cell type is a lung airway cell.
• Example lung airway cells that can be targeted by the delivery of the present application includes but is not limited to basal cell.
• the second cell type is a lung cell. In some embodiments, the second cell type is a lung airway cell. [00275] In some embodiments of the pharmaceutical composition of the present disclosure, the SORT lipid in the pharmaceutical composition achieves therapeutic effect in about 1.1-fold to about 20-fold greater second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves therapeutic effect in about 1.1-fold to about 10-fold greater second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type.
• the SORT lipid achieves therapeutic effect in about 5-fold to about 10-fold greater second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves therapeutic effect in about 10-fold to about 20-fold greater second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type.
• the SORT lipid achieves therapeutic effect in at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type.
• the SORT lipid in the pharmaceutical composition effects delivery of the therapeutic agent to cells of the subject characterized by a greater therapeutic effect in a first cell of a first cell type compared to that in a second cell of a second cell type.
• the first cell type is different from the second cell type.
• the first cell type is a lung cell.
• the first cell type is a lung airway cell. Examples of lung airway cells that can be targeted by the delivery of the present application include but is not limited to basal cells.
• the second cell type is a lung cell.
• the second cell type is a lung airway cell.
• the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold therapeutic effect in first cell of the first cell type compared to that achieved in the second cell of the second cell type. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold therapeutic effect in first cell of the first cell type compared to that achieved in the second cell of the second cell type. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold therapeutic effect in first cell of the first cell type compared to that achieved in the second cell of the second cell type.
• the SORT lipid achieves about 10-fold to about 20-fold therapeutic effect in first cell of the first cell type compared to that achieved in the second cell of the second cell type. In some embodiments of the method, the SORT lipid achieves at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11- fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20- fold therapeutic effect in first cell of the first cell type compared to that achieved in the second cell of the second cell type.
• compositions that comprise components that allow for an improved efficacy or outcome based on the delivery of the polynucleotide.
• the compositions described elsewhere herein may be more effective at delivery to a particular cell, cell type, organ, or bodily region as compared to a reference composition or compound.
• the compositions described elsewhere herein may be more effective at generating increase expression of a corresponding polypeptide of a delivered polynucleotide.
• the compositions described elsewhere herein may be more effective at generating a larger number of cells that express a corresponding polypeptide of a delivered polynucleotide.
• compositions described elsewhere herein may result in an increase uptake of the polynucleotide as compared to a reference polynucleotide.
• the increased uptake may be result of improved stability of polynucleotide or an improved targeting of the composition to a particular cell type or organ.
• the SORT lipid is present in an amount in the lipid composition to effect a greater expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol (“LF92”), a phospholipid, cholesterol, and a PEG-lipid.
• a reference lipid composition comprising 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol (“LF92”), a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect at least a 1.1 fold greater expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect at least a 2-fold greater expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid. In some embodiments, the SORT lipid is present in an amount in the lipid composition to effect at least a 5 fold greater expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect at least a 10- fold greater expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in a greater plurality of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in at least a 1.1-fold greater plurality of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in at least a 2-fold greater plurality of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in at least a 5- fold greater plurality of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an expression or activity of the polynucleotide (or corresponding polypeptide of the polynucleotide) in at least a 10-fold greater plurality of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an uptake of the polynucleotide in a greater plurality of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• the SORT lipid is present in an amount in the lipid composition to effect an uptake of the polynucleotide in a greater amount to a cell compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.
• Protein corona binding [00283]
• a surface of a pharmaceutical composition as described hererin binds to one or more target proteins comprising a protein corona.
• the surface of the pharmaceutical composition may bind a first target protein.
• the surface of the pharmacetutical composition may bind a first and a second target protein.
• a surface of a pharmaceutical composition as disclosed herein may comprise a protein corona.
• the protein corona may comprise one or more (e.g., serum or blood) proteins.
• the one or more protiens may comprise an apolipoprotein, a complement protein, an immune protein, a coagulation protein, or any other protein.
• the one or more proteins comprise target proteins.
• the one or more target protiens may comprise an apolipoprotein, a complement protein, an immune protein, a coagulation protein, or any other protein.
• the one or more target proteins comprise alpha-2-HS-glycoprotein, aomplement C1q subcomponent subunit C, alpha-1-antitrypsin 1- 3, Ig alpha chain C region, Ig mu chain C region (fragment), serine protease inhibitor A3K, apolipoprotein C-I, serum albumin, immunoglobulin heavy variable 1-34 (fragmnent), vitamin K- dependent protein Z, immunoglobulin kappa variable 6-13, Ig gamma-2B chain C region, histone H, beta-2-glycoprotein 1, Ig heavy chain V region X44, protein Z-dependent protease inhibitor, immunoglobulin heavy constant alpha (fragment), C-reactive protein, mannose-binding protein C, immunoglobulin kappa variable 1-110 (fragmnet), beta-casein, immunoglobulin heavy constant mu, serum paraoxonase/arylesterase, glycosylphosphatidylinositol specific phosphose,
• coagulation factor V Ig kappa chain V-II region 26-10, Ig gamma- 1 chain C region secreted form (fragmnet), Immunoglobulin heavy constant gamma 3 (fragment), platelet factor 4, apolipoprotein A-I, lipopolysaccharide-binding protein, immunoglobulin heavy variable 5-9 (fragment), Ig kappa chain V-V region HP 124E,.histidine-rich glycoprotein, Ig heavy chain V-III region J606, Ig kappa chain V-III region PC 2880/PC 1229, Ig kappa chain V-V region HP 124E1, band 3 anion transport protein, immunoglobulin kappa variable 17-121 (fragment), apolipoprotein N, plasminogen, immunoglobulin kappa chain variable 8-30 (fragmnet), complement C1s-A subcomponent, hemoglobin subunit beta-2, immunoglobulin kappa variable 1-135 (fragmnent
• immunoglobulin heavy variable 7-1 fragment
• immunoglobulin kappa variable 4-57 fragment
• immunoglobulin heavy variable V1-5 Ig heavy chain V region 914, histone H2B
• immunoglobulin heavy constant gamma 3 fragment
• apolipoprotein E fibrinogen alpha chain
• complement C1q subcomponent subunit A immunoglobulin heavy variable 5-9 (fragment)
• immunoglobulin kappa constant apolipoprotein C-III
• immunoglobulin heavy constant gamma 2B fragment
• prothrombin complement C1q subcomponent subunit, carboxypeptidase N catalytic chain, vitronectin
• immunoglobulin kappa variable 12-46 fragment
• Ig gamma-2A chain C region membrane-bound form
• immunoglobulin kappa variable 12-44 fragment
• the pharmaceutical composition may bind a first and a second target protein at a certain weight or mass ratio.
• the weight or mass ratio may range from 1:1 to 20:1 or more.
• the weight or mass ration may be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1.9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or more.
• the weight or mass ratio may be 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1 or less.
• the compositon of a protein corona may determine an organ and/or cell type tropism (e.g., targeting) of a pharmaceutical composition as described herein.
• the organ and/or cell type targeting may be determined by the presence or absence of a certain target protein, the weight or mass ratio between one target protein and another target protein or proteins, or some combination thereof.
• the presence of absence of a protein and/or a weight or mass ratio between one protein or another or among a group of proteins may be determined by an incubation assay.
• the incubation assay may comprise incubating a lipid composition in a serum or plasma sample from an organism (e.g., mouse).
• Protein bound to the lipid composition may be isolated and/or purified and quantified by any suitable technique known in the art such as Bradford or other colorimetric assays, UV-vis spectroscopy, biuret assays, and fluorescent assays. Proteins isolated from such samples may be futher characterized or identified by mass spectrometry, gel electrophoresis (e.g, native gel, SDS PAGE gel), or other suitable techniques known in the art.
• the targeting of a composition comprising a given protein corona may be determined by measuring (directly, indirecrly, and/or relatively) the amount of the composition or portion thereof (e.g., payload, therapeutic agent) delivered to one or more organs or cell types.
• measuring the targeting of the composition comprising the protein corona may comprise measuring the amount, expression, or activity of a therapeutic payload contained in the composition in a target organ or cell type versus a reference organ or cell type.
• the amount, expression, or activity of a therapeutic agent deliverd by the composition comprising the protein corona is at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold or higher.
• the amount, expression, or activity of a therapeutic agent delivered by the composition comprising the protein corona is less than about 20-fold, about 19-fold, about 18-fold, about 17-fold, about 16-fold, about 15-fold, about 14-fold, about 13-fold, about 12-fold, about 11-fold, about 10-fold, about 9-fold, about 8-fold, about 7-fold, about 6-fold, about 5-fold, about 4-fold, about 3-fold, about 2-fold, or less.
• Assays to determine the targeting of a composition comprising a protein corona may comprise any suitable quantification procedure or functional assay known in the art.
• such assays may comprise quantification of luminescence of a fluorescent payload or transcription/translation product thereof, immunofluorescence assays targeting a payload or translation product thereof, and the like.
• the protein corona comprises apolipoprotein E (Apo E) and serum albumin.
• the composition comprising the protein corona may target a liver or liver cell.
• the Apo E is present a weight or mass ratio or no more than about 6:1, about 5:1, about 4:1, or about 3:1 to the serum albumin.
• the protein corona further comprises complement C1q subcomponent subunit A, immunoglobulin heavy constant mu, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma-2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, clusterin, another protein, or any combination thereof as determined by an incubation assay.
• the protein corona comprises at least one, at least two, or at least three protein(s) listed in Table 10 (e.g., those not listed in or listed different from Table 9).
• the protein corona comprises Apo E in a lesser amount than an endogenous protein which is not Apo E, as determined by an incubation assay.
• the endogenous protein is beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H), immunoglobulin kappa constant, complement C1q subcomponent subunit A, vitronectin, and serum paraoxonase/arylesterase 1, clusterin another protein, or combinations thererof.
• the protein corona further comprises apolipoprotein C (Apo C). In such cases, the protein corona may comprise less Apo C than Apo E, as determined by an incubation assay.
• the protein corona comprises vitronectin and clusterin. In such cases, the composition comprising the protein corona may target a lung or a lung cell. In some embodiments, the vitronectin is present at a weight or mass ratio of no more than about 6:1 or about 5:1 to the clusterin, as determined by an incubation assay.
• the protein corona further comprises serum paraoxonase/arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H), immunoglobulin (Ig) mu chain C region, alpha- S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen alpha chain, vitamin K-dependent protein Z, alpha-1-antitrypsin 1-3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1, coagulation factor X, apolipoprotein A- I, immunoglobulin heavy constant alpha, immunoglobulin (Ig) gamma-2A chain C region, beta-globin, complement C1
• the protein corona comprises at least one, at least two, or at least three protein(s) listed in Table 12 (e.g., those not listed in or listed different from Table 9).
• the protein corona comprises beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H) and a second target protein that is different from beta-2 glycoprotein 1 or Apo H.
• the beta-2 glycoprotein 1 or Apo H may be present at a weight or mass ratio of no more than about 20:1, 15:1, of 10: 1 to the second target protein.
• the pharmaceutical compostion may target the spleen, bone marrow, or a lymph node or a cell therein.
• the cell is a spleen cell or a macrophase.
• the second target protein comprises immunoglobulin kappa constant, complement C1q subcomponent subunit A, apolipoprotein E (Apo E), immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, vitronectin, complement C1q subcomponent subunit C, apolipoprotein C-I, immunoglobulin (Ig) gamma-2A chain C region, immunoglobulin (Ig) mu chain C region, serum albumin, serum paraoxonase/arylesterase 1, immunoglobulin heavy constant alpha, clusterin, and immunoglobulin kappa variable 6-13, another protein, or a combination thereof.
• the protein corona comprises at least one, at least two, or at least three protein(s) listed in Table 11 (e.g., those not listed in or listed different from Table 9). In some embodiments, the protein corona does not comprise beta-2 glycoprotein 1 or Apo H. In such cases, the composition may target an organ that is not a spleen bone marrow, or lymph node or a cell therein. The composition may not target a spleen cell or macrophage. METHODS FOR TARGETED DELIVERY [00293] In some embodiments, provided herein is a method for targeted delivery of a therapeutic agent to an organ or a cell therein in a subject in need thereof.
• the method may comprise administering to the subject the therapeutic agent assembled with a lipid composition such as those described herein.
• the lipid composition comprises: an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid.
• the administering comprises intravenously administering.
• a bodily fluid (e.g., plasma or serum) of the subject comprises the plurality of target proteins.
• the plurality of target proteins is a plurality of endogenous proteins of the subject.
• a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises a first target protein at a weight or mass ratio of no more than about 20:1, 15:1, or 10:1 to a second target protein that is different from the first target protein, thereby delivering the therapeutic agent to the target organ or the target cell in the subject.
• the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of the therapeutic agent in the organ or the cell therein in the subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent binding to the plurality of target proteins).
• the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of the therapeutic agent in the organ or the cell therein in the subject as compared to that achieved absent the polymer-conjugated lipid. In some embodiments, in the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of the therapeutic agent in the organ or the cell therein in the subject as compared to that achieved in a reference organ or a reference cell. [00295] In some embodiments, the method described herein is for targeted delivery of a therapeutic agent to liver or a liver cell therein in a subject in need thereof.
• a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises apolipoprotein E (Apo E) and serum albumin, thereby delivering the therapeutic agent to the liver or the liver cell in the subject.
• the Apo E is present at a weight or mass ratio of no more than about 6:1, 5:1, 4:1, or 3:1 to the serum albumin in the plurality of target proteins as determined by an incubation assay.
• the plurality of target proteins further comprise complement C1q subcomponent subunit A, immunoglobulin heavy constant mu, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma- 2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, or any combination thereof as determined by an incubation assay.
• the plurality of target proteins comprises at least one, at least two, or at least three protein(s) listed in Table 10 (e.g., those not listed in or listed different from Table 9).
• the SORT lipid comprises an ionizable cationic moiety (e.g., a tertiary amine moiety).
• the SORT lipid is an ionizable cationic lipid.
• the lipid composition comprises the SORT lipid at a molar percentage from about 5% to about 65%.
• the method provides a (e.g., at least about 2-, 3-, 4-, 5-, or 6-fold) greater amount, expression or activity of the therapeutic agent in the liver or the liver cell in the subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent the binding to the plurality of target proteins).
• a corresponding reference lipid composition e.g., absent the binding to the plurality of target proteins.
• a surface of the SORT lipid composition interacts with apolipoprotein E (Apo E) to a lesser degree than with an endogenous protein that is not Apo E in the subject as determined by an incubation assay, which endogenous protein that is not Apo E is selected from beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H), immunoglobulin kappa constant, complement C1q subcomponent subunit A, vitronectin, and serum paraoxonase/arylesterase 1, thereby delivering the therapeutic agent to the non-liver organ or the non-liver cell in the subject.
• ⁇ 2-GP1 beta-2 glycoprotein 1
• Apo H apolipoprotein H
• the non-liver organ comprises a lung, spleen, bone marrow, or a lymph node.
• non-liver cell comprises a lung cell, a spleen cell, or a macrophage.
• apolipoprotein E is not the most abundant protein in the plurality of target proteins.
• a surface of the lipid composition interacts with apolipoprotein C (Apo C) to a lesser degree than with apolipoprotein E (Apo E) in the subject as determined by an incubation assay.
• the method provides a lesser amount or activity of the therapeutic agent in liver or a cell therein in the subject as compared to that achieved absent the polymer-conjugated lipid.
• the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid.
• the lipid composition comprises the SORT lipid at a molar percentage from about 5% to about 65%.
• the method described herein is for targeted delivery of a therapeutic agent to a lung or a lung cell therein in a subject in need thereof.
• a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises vitronectin (Vtn) and clusterin, thereby delivering the therapeutic agent to the lung or the lung cell in the subject.
• the vitronectin is present at a weight or mass ratio of no more than about 6:1, or 5:1 to the clusterin in the plurality of target proteins as determined by an incubation assay.
• the plurality of target proteins further comprise serum paraoxonase/arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H), immunoglobulin (Ig) mu chain C region, alpha-S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen alpha chain, vitamin K-dependent protein Z, alpha-1-antitrypsin 1-3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1, coagulation factor X, apolipoprotein A-I, immunoglobulin heavy constant alpha, immunoglobulin (Ig) gamma-2A chain C region, beta-globin, complement C
• the plurality of target proteins comprises comprises at least one, at least two, or at least three protein(s) listed in Table 12 (e.g., those not listed in or listed different from Table 9).
• the SORT lipid is a cationic lipid.
• the SORT lipid is a permanent cationic lipid.
• the SORT lipid is an ionizable cationic lipid.
• the lipid composition comprises the SORT lipid at a molar percentage from about 5% to about 65%.
• the method provides a (e.g., at least about 2-, 5-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold) greater amount, expression or activity of the therapeutic agent in the lung or the lung cell in the subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent binding to the plurality of target proteins).
• a corresponding reference lipid composition e.g., absent binding to the plurality of target proteins.
• the method described herein is for targeted delivery of a therapeutic agent to spleen, bone marrow, or a lymph node or a cell therein in a subject in need thereof.
• a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H) at a weight or mass ratio of no more than about 20:1, 15:1, or 10:1 to a second target protein that is different from the beta-2 glycoprotein 1 ( ⁇ 2-GP1) or apolipoprotein H (Apo H), thereby delivering the therapeutic agent to the spleen, bone marrow, or a lymph node or the cell in the subject.
• the cell comprises a spleen cell, or a macrophage.
• the second target protein is selected from: immunoglobulin kappa constant, complement C1q subcomponent subunit A, apolipoprotein E (Apo E), immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, vitronectin, complement C1q subcomponent subunit C, apolipoprotein C-I, immunoglobulin (Ig) gamma-2A chain C region, immunoglobulin (Ig) mu chain C region, serum albumin, serum paraoxonase/arylesterase 1, immunoglobulin heavy constant alpha, and immunoglobulin kappa variable 6-13.
• the SORT lipid is a permanent cationic lipid, or an anionic lipid.
• the plurality of target proteins comprises comprises at least one, at least two, or at least three protein(s) listed in Table 11 (e.g., those not listed in or listed different from Table 9).
• the SORT lipid is a permanent cationic lipid.
• the SORT lipid is an anionic lipid.
• the lipid composition comprises the SORT lipid at a molar percentage from about 5% to about 65%.
• the method provides a (e.g., at least about 2-fold) greater amount, expression or activity of the therapeutic agent in the lugn or the lung cell in the subject as compared to that achieved with a corresponding reference lipid composition (e.g., absent binding to the plurality of target proteins).
• a corresponding reference lipid composition e.g., absent binding to the plurality of target proteins.
• a surface of the lipid composition binds to a plurality of target proteins as determined by an incubation assay, which plurality of target proteins comprises a first target protein at a weight or mass ratio of no more than about 20:1, 15:1, or 10:1 to a second target protein that is different from the first target protein, thereby delivering the therapeutic agent to the non-spleen organ or the non-spleen cell in the subject.
• the non-spleen organ is not spleen, bone marrow, or a lymph node.
• the non-spleen cell is not a spleen cell, or a macrophage.
• beta-2 glycoprotein 1 ⁇ 2-GP1
• apolipoprotein H apolipoprotein H
• the plurality of target proteins comprises clusterin.
• the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid.
• the lipid composition comprises the SORT lipid at a molar percentage from about 5% to about 65%.
• the therapeutic agent may comprise a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribon
• siRNA small interfering ribonucleic
• the therapeutic agents provided herein may be present in intravenous compositions.
• the therapeutic agents provided herein may be present in aerosol compositions.
• the lipid composition may be formulated as an aerosol.
• the compositions provided herein may be formulated as an aerosol dosage form.
• the compositions provided herein are formulated as intravenous dosage forms.
• the lipid composition may be formulated as a nebulizer.
• the compositions described herein are dispensed via a nebulizer.
• the lipid composition may be dispensed as an aerosol.
• the compositions described herein may be stored at or below -70°C. [00302] In some embodiments, the compositions described herein may be formulated as a dispersion. In some embodiments, the the concentration of the dispersion is about 0.5 mg/mL to about 5 mg/mL. In some embodiments, the concentration of the dispersion is about 0.5 mg/mL to about 1 mg/mL. In some embodiments, the concentration of the dispersion is about 0.5 mg/mL to about 2 mg/mL. In some embodiments, the concentration of the dispersion is about 0.5 mg/mL to about 3 mg/mL. In some embodments, the concentration of the dispersion is about 2 mg/mL to about 3 mg/mL.
• the concentration of the dispersion is about 2 mg/mL to about 4 mg/mL. In some embodiments, the concentration of the dispersion is no more than 5 mg/mL. In some embodiments, the concentration of the dispersion is 1 mg/mL. In some embodiments, the compositions described herein are dispersed at pH 7.5. [00303] In some embodiments, the compositions provided herein are administered to a human. In some embodiments, the compositions provided herein are administered to an adult. In other embodiments, the compositions provided herein are administered to a child. In some embodiments, the compositions provided herein are administered to a patient with a body mass index of 18 to 35 kg/m 2 .
• the compositions provided herein are administered to a patient with a total body weight of ⁇ 50 kg.
• the compositions described herein are administered intravenously.
• the compositions described herein are delivered via inhalation.
• the compositions described herein may comprise administration by nebulization.
• the compositions described herein may comprise administration to a lung by nebulization.
• the compositions described herein are administered at least once a week.
• the compositions described herein are administered at least twice a week.
• the compositions are administered in any suitable dose.
• the dose refers to the amount of the composition.
• the dose refers to the amount of the therapeutic agent.
• the administered dose is about 1 mg to about 30 mg.
• the administered dose is about 1 mg to about 2.5 mg, about 1 mg to about 5 mg, about 1 mg to about 7.5 mg, about 1 mg to about 10 mg, about 1 mg to about 15 mg, about 1 mg to about 20 mg, about 1 mg to about 25 mg, about 1 mg to about 30 mg, about 2.5 mg to about 5 mg, about 2.5 mg to about 7.5 mg, about 2.5 mg to about 10 mg, about 2.5 mg to about 15 mg, about 2.5 mg to about 20 mg, about 2.5 mg to about 25 mg, about 2.5 mg to about 30 mg, about 5 mg to about 7.5 mg, about 5 mg to about 10 mg, about 5 mg to about 15 mg, about 5 mg to about 20 mg, about 5 mg to about 25 mg, about 5 mg to about 30 mg, about 7.5 mg to about 10 mg, about 7.5 mg to about 15 mg, about 7.5 mg to about 20 mg, about 7.5 mg to about 25 mg, about 5 mg to about 30 mg, about 7.5 mg to about 10
• the administered dose is about 1 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, or about 30 mg. In some embodiments, the administered dose is at least about 1 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg. In some embodiments, the administered dose is at most about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, or about 30 mg. In some embodiments, the administered dose is about 2.5 mg. In some embodiments, the administered dose is about 5.0 mg. In some embodiments, the admistered dose is about 10.0 mg.
• the administered dose is about 20.0 mg.
• the dose may be determined in reference to body weight. In any of these compositions or methods, any suitable dose may be used. In some embodiments, the dose is about 0.01 mg/kg body weight to about 1 mg/kg body weight.
• the dose is about 0.01 mg/kg body weight to about 0.05 mg/kg body weight, about 0.01 mg/kg body weight to about 0.1 mg/kg body weight, about 0.01 mg/kg body weight to about 0.5 mg/kg body weight, about 0.01 mg/kg body weight to about 0.8 mg/kg body weight, about 0.01 mg/kg body weight to about 1 mg/kg body weight, about 0.05 mg/kg body weight to about 0.1 mg/kg body weight, about 0.05 mg/kg body weight to about 0.5 mg/kg body weight, about 0.05 mg/kg body weight to about 0.8 mg/kg body weight, about 0.05 mg/kg body weight to about 1 mg/kg body weight, about 0.1 mg/kg body weight to about 0.5 mg/kg body weight, about 0.1 mg/kg body weight to about 0.8 mg/kg body weight, about 0.1 mg/kg body weight to about 1 mg/kg body weight, about 0.5 mg/kg body weight, about 0.1 mg/kg body weight to about 0.8 mg/kg body weight, about 0.1 mg/kg body weight to about 1
• the dose is about 0.01 mg/kg body weight, about 0.05 mg/kg body weight, about 0.1 mg/kg body weight, about 0.5 mg/kg body weight, about 0.8 mg/kg body weight, or about 1 mg/kg body weight. In some embodiments, the dose is at least about 0.01 mg/kg body weight, about 0.05 mg/kg body weight, about 0.1 mg/kg body weight, about 0.5 mg/kg body weight, or about 0.8 mg/kg body weight. In some embodiments, the dose is at most about 0.05 mg/kg body weight, about 0.1 mg/kg body weight, about 0.5 mg/kg body weight, about 0.8 mg/kg body weight, or about 1 mg/kg body weight.
• a dose comprises no more than about 1.0, 0.5, 0.1, 0.05, or 0.01 mg/kg body weight.
• the dose may be determined in reference to lung weight. In any of these compositions or methods, any suitable dose may be used. In some embodiments, the administered dose is about 0.002 mg/g lung weight to about 0.03 mg/g lung weight.
• the administered dose is about 0.002 mg/g lung weight to about 0.005 mg/g lung weight, about 0.002 mg/g lung weight to about 0.008 mg/g lung weight, about 0.002 mg/g lung weight to about 0.01 mg/g lung weight, about 0.002 mg/g lung weight to about 0.015 mg/g lung weight, about 0.002 mg/g lung weight to about 0.02 mg/g lung weight, about 0.002 mg/g lung weight to about 0.025 mg/g lung weight, about 0.002 mg/g lung weight to about 0.03 mg/g lung weight, about 0.005 mg/g lung weight to about 0.008 mg/g lung weight, about 0.005 mg/g lung weight to about 0.01 mg/g lung weight, about 0.005 mg/g lung weight to about 0.015 mg/g lung weight, about 0.005 mg/g lung weight to about 0.02 mg/g lung weight, about 0.005 mg/g lung weight to about 0.025 mg/g lung weight, about 0.005 mg/g lung weight to about 0.005 mg
• the administered dose is about 0.002 mg/g lung weight, about 0.005 mg/g lung weight, about 0.008 mg/g lung weight, about 0.01 mg/g lung weight, about 0.015 mg/g lung weight, about 0.02 mg/g lung weight, about 0.025 mg/g lung weight, or about 0.03 mg/g lung weight.
• LNPs Lipid nanoparticles
• LNPs are composed of 4 components: an ionizable cationic lipid, zwitterionic phospholipid, cholesterol, and lipid poly(ethylene glycol) (PEG).
• PEG lipid poly(ethylene glycol)
• these LNPs result in only general delivery of nucleic acids, rather than organ or tissue targeted delivery.
• LNPs typically delivery RNAs only to the liver. Therefore, new formulations of LNPs were sought in an effort to provide targeted nucleic acid delivery.
• the four canonical types of lipids were mixed in a 15:15:30:3 molar ratio, with or without the addition of a permanently cationic lipid.
• LNPs were prepared by mixing a dendrimer or dendron lipid (ionizable cationic), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanently cationic).
• DOPE dendrimer or dendron lipid
• DOPE dendrimer or dendron lipid
• DOTAP permanently cationic
• DODAP permanently cationic
• DOTAP can be substituted for DODAP to generate an LNP comprising DODAP.
• a dendrimer or dendron lipid, DOPE, Cholesterol and DMG-PEG were dissolved in ethanol at desired molar ratios.
• the mRNA was dissolved in citrate buffer (10 mM, pH 4.0).
• mRNA was then diluted into the lipids solution to achieve a weight ratio of 40:1 (total lipids: mRNA) by rapidly mixing the mRNA into the lipid solution at a volume ratio of 3:1 (mRNA: lipids, v/v). This solution was then incubated for 10 min at room temperature.
• mRNA was dissolved in 1 ⁇ PBS or citrate buffer (10 mM, pH 4.0), and mixed rapidly into ethanol containing 5A2-SC8, DOPE, Cholesterol, DMG-PEG and DOTAP, fixing the weight ratio of 40:1 (total lipids:mRNA) and volume ratio of 3:1 (mRNA:lipids).
• Formulations are named X% DOTAP Y (or X%DODAP Y) where X represents the DOTAP (or DODAP) molar percentage in total lipids, and Y represents the type of dendrimer or dendron lipid.
• formulation may be named Y X%DOTAP or Y X%DODAP where X represents the DOTAP (or DODAP) molar percentage in total lipids, and Y represents the type of dendrimer or dendron lipid.
• Example 2 SORT LNP Stability [00311] Lipid (LNP) compositions are tested for stability.
• Lipid compositions as described herein such as those comprising a dendrimer or dendron (e.g., 5A2-SC8), as an ionizable cationic lipid, and a selective organ-targeting (SORT) lipid (e.g., DODAP), e.g., at a molar percentage in total lipids from 20% to 50%, are generated using either a microfluidic mixing method or a cross/tee mixing method. Size, polydispersity index (PDI) and zeta-potential of different LNP formulations are characterized by dynamic light scattering (DLS) (3 separate times for each formulation).
• a dendrimer or dendron e.g., 5A2-SC8
• SORT selective organ-targeting
• DODAP selective organ-targeting
• Size, polydispersity index (PDI) and zeta-potential of different LNP formulations are characterized by dynamic light scattering (DLS) (3 separate times for each formulation).
• Encapsulation efficiency of the LNPs is tested using a Ribogreen RNA assay (Zhao et al., 2016). Briefly, mRNA is encapsulated with > 90% (e.g., > 95%) efficiency in LNPs when the mRNA is dissolved in acidic buffer (e.g., 10 mM citrate, pH 4). The characteristics are observed over 28 days for the tested LNPs, e.g., over the course of 28 days. [00313] In addition, stability of the lipid compositions as described herein (LNPs) in solution and resulting mRNA expression are observed in mice. Briefly, mice are injected intravenously with less than 1 mg/kg and observed in vivo.
• Luciferin is added 5 hours after injection and visualized.
• SORT e.g., lung-SORT
• LNP generated tissue specific radiance in the lungs remain highly detectable even after 14 day with a slight decay in signal by the 21 st and 28 th day. Images of organs of the tested mice at specific time periods after treated with example SORT LNP are taken.
• Example 3A SORT molecules alter LNP biodistribution [00314] Messenger RNA therapeutics must overcome multiple barriers for intracellular delivery as they do not readily diffuse through anionic cellular membranes and are susceptible to degradation by RNases in the blood.
• LNPs will encapsulate and protect mRNA from enzymatic degradation, enable accumulation in the target organ, facilitate receptor-mediated endocytosis into cells, and release mRNA from endosomes into the cytosol to undergo translation into a functional protein.
• SORT LNPs we showed that the chemical identity and amount of the incorporated SORT molecule systematically alters tissue-specific protein expression following mRNA delivery.
• Inclusion of ionizable cationic lipids enhanced liver targeting, anionic lipids resulted in retargeting of delivery to the spleen, and permanently cationic lipids bearing a quaternary ammonium headgroup retargeted delivery to the lungs (FIG. 1A).
• Example 3B SORT molecules alter apparent LNP pK a
• TMS 6-(p-toluidino)-2-naphthalenesulfonic acid
• mRNA formulations 60 ⁇ M total lipids
• TNS probe 2 ⁇ M
• 10 mM HEPES 10 mM MES (4-morpholineethanesulfonic acid)
• 10 mM ammonium acetate 130 mM NaCl
• the mean fluorescence intensity of each well black bottom 96- well plate
• ⁇ Ex excitation wavelength
• ⁇ Em emission wavelength
• the apparent pKa is defined by the pH at half-maximum fluorescence.
• this method was useful for estimating LNP global/apparent pKa for most all LNPs, it could not be used for example SORT LNPs containing >40% permanently cationic lipid because these LNPs are always charged. Therefore, the relative pK a was instead estimated compared to base LNP formulation (no added SORT lipid) when 50% fluorescence normalized to the lowest fluorescence measurement was produced. This alternative calculation did not change the pK a for most LNPs but did allow estimation of the pK a of example SORT LNPs with >40% cationic lipid that agreed with experimental results for tissue selective RNA delivery. Representative results of the assay are shown in FIG.1D. Full results of the assay for the 67 different example SORT LNPs are shown in FIG.6. Table 7. Molecular composition of example SORT LNPs studied in the TNS assay.
• FIG. 1E shows a plot of relative pKa with respect to tissue-specific activity.
• spleen targeting 2 LNPs had a lower pKa, by way of example(s).
• siRNA delivery to liver hepatocytes strongly depends on LNP pKa, with a pKa of 6.2-6.4 being optimal for gene silencing; LNPs with a pKa outside of that narrow range were unable to functionally deliver siRNA to the liver.
• SORT LNPs 6(p-toluidino)-2- naphthalenesulfonic acid (TNS) assay
• LNPs incorporate a PEG-lipid on the surface to promote colloidal stability. Since PEG-lipid molecules are non-covalently incorporated into the LNP, they desorb at a rate inversely proportional to the length of their corresponding lipid anchor. Further, PEG-lipid on an LNP surface is known to impair serum or plasma protein adsorption.
• An mPEG with a shorter alkyl tail is expected to be less-sheddable from the LNP compared to an mPEG with a longer alkyl tail.
• the effects of increasing the PEG-lipid anchor length on the delivery of luciferase mRNA was measured by injecting C57BL/6 mice IV at a dosage of 0.1 mg/kg mRNA and imaging organ luminescence ex vivo at a timepoint of 6 hours post-injection. Imaging results are shown in FIG.2A. Average fluorescence of the liver, lung, and spleen were measured using Living Image Software (Perkin Elmer) by drawing regions of interest around each organ. The relative fluorescence for each organ was calculated as: . As shown in FIG.
• FIG.2C shows quantification of luciferase mRNA (as determined by total luminescence) in target organs of treated mice.
• human erythropoietin (hEPO) mRNA was delivered to target tissues in vivo using example Liver, Spleen, or Lung SORT LNPs incorporating either shorter (e.g., C14-PEG2K) or longer (e.g, C18-PEG2K) alkyl chain PEG-lipid. Since hEPO is a secreted protein, delivery efficacy was quantified by measuring hEPO levels in the serum or plasma of treated mice.
• Serum or plasma hEPO concentration was lower in mice treated with C18-PEG2K SORT LNPs compared to those treated with C14-PEG2K SORT LNPs, as shown in FIG.2D.
• SORT LNPs which incorporate a PEG-lipid with a longer hydrophobic anchor were less effective for mRNA delivery to target organs and further suggested that PEG-lipid desorption is a necessary process for effective mRNA delivery to target tissues by LNPs.
• LNPs low-density lipoprotein receptor
• example SORT LNPs might operate by a similar mechanism, where (1) PEG-lipid desorbs from the LNP to expose underlying SORT molecules, (2) distinct serum proteins recognize the exposed SORT molecules and adsorb to the LNP surface, and (3) these surface-adsorbed proteins interact with cognate receptors which mediate uptake of the LNPs by cells in target tissues (FIG. 2A).
• PEG-lipid desorbs from the LNP to expose underlying SORT molecules
• distinct serum proteins recognize the exposed SORT molecules and adsorb to the LNP surface
• these surface-adsorbed proteins interact with cognate receptors which mediate uptake of the LNPs by cells in target tissues (FIG. 2A).
• LNPs incorporate a PEG-lipid on the surface to promote colloidal stability.
• PEG-lipid molecules are non-covalently incorporated into the LNP, they spontaneously desorb at a rate inversely proportional to the length of the PEG-lipid’s hydrophobic anchor.
• PEG-lipid on the LNP surface can impair serum protein adsorption, resulting in reduced cellular targeting and delivery efficacy. Shedding of PEG-lipid would be expected to expose the underlying SORT molecules for recognition by serum proteins, promoting their binding of the example SORT LNP in the blood.
• hEPO human erythropoietin
• example SORT LNPs which incorporate a PEG-lipid with a longer hydrophobic anchor are less effective for mRNA delivery to target organs.
• Example 5 SORT molecule choice influences LNP-protein interactions in the serum [00326]
• the the plasma proteins which bind a conventional four-component LNP formulation (mDLNP), and three different example SORT LNP formulations as described herein Liver SORT, comprising an ionizable cationic lipid (e.g., DODAP); Lung SORT, comprising a cationic lipid (e.g., DOTAP); Spleen SORT, comprising an anionic lipid (e.g., 18PA)) were isolated using differential centrifugation following ex vivo incubation with mouse plasma. Details of the SORT formulations are described in Table 8.
• mouse plasma was added to a solution of each LNP (prepared as in Example 1 and diluted with 1X PBS 1 g/L concentration of lipid) at a 1:1 volume ratio and incubated for 15 minutes at 37 °C.
• a 0.7 M sucrose solution was prepared by dissolving solid sucrose in MilliQ water.
• the LNP/Plasma mixture was loaded onto a 0.7 M sucrose cushion of equal volume to the mixture and centrifuged at 15,300 g and 4 °C for 1 hour. The supernatant was removed, and the pellet was washed with 1X PBS. Next the pellet was centrifuged at 15,300 g and 4 °C for 5 minutes, and the supernatant was removed.
• an ionizable cationic lipid e.g., DODAP
• inclusion of an ionizable cationic lipid did not greatly alter the pI distribution of the major proteins which bound the LNP.
• the inclusion of a lipid with an anionic headgroup e.g., 18PA
• the inclusion of a lipid with a cationic head group e.g., DOTAP
• the enrichment of proteins with a pI below physiological pH as shown in the bottom panel of FIG.2G).
• LNPs were further characterized based on the 5 most abundant proteins in their respective coronas.
• FIG.2H shows that the most highly enriched protein in each corona was found to depend on the identity of SORT molecule.
• the top left and right panel of FIG.2H shows ApoE was found to be the most highly enriched serum or plasma protein for mDLNP (reference) and Lung SORT LNPs, on average composing 13.9% of mDLNP’s protein corona and 13.3% for Lung SORT.
• Spleen SORT LNPs were most highly enriched in ⁇ 2-glycoprotein I ( ⁇ 2-GPI), at an average abundance of 20.1% as shown in the bottom left of FIG.
• FIG. 2H thus demonstrates that different SORT molecules may result in unique protein corona signatures.
• Average abundance, isoelectronic point, and physiological function of the proteins which constitute 80% of the protein corona for each LNP formulation are listed in Tables 9-12. Table 9. Average abundance, isoelectric point, and physiological function of the proteins which constitute 80% of reference mDLNP’s protein corona
• Table 10 Average abundance, isoelectric point, and physiological function of the proteins which constitute 80% of example Liver SORT’s protein corona.
• Table 11 Average abundance, isoelectric point, and physiological function of the proteins which constitute 80% of example Spleen SORT’s protein corona.
• the second step of the hypothesized endogenous targeting mechanism for example SORT LNPs involves the adsorption of distinct proteins to the LNP surface to form unique protein coronas (FIG. 2A). Following PEG-lipid desorption, serum proteins readily adsorb to the surface of intravenously-administered LNPs, forming an interfacial layer known as the “protein corona” that defines their biological identity. Adsorption of ApoE has been shown to drive liver targeting of conventional four-component LNPs through binding of the LDL-R highly expressed on hepatocytes.
• a key band is highly enriched for Spleen SORT at a Mw of 54 kDa while a band near 65 kDa is highly enriched for example Lung SORT (FIG. 2E).
• adding a SORT molecule to mDLNP alters the composition of the protein corona in accordance with the SORT molecule’s chemical structure.
• Unbiased mass spectrometry proteomics enabled identification and quantification of which proteins bind example SORT LNPs in the plasma.
• a protein’s isoelectric point (pI) is defined as the pH at which the protein molecule bears no net charge, providing an estimate of a protein’s charge state in the physiological milieu.
• DODAP an ionizable cationic lipid
• 18PA a lipid with an anionic headgroup
• DOTAP a lipid with a cationic quaternary ammonium headgroup
• Liver SORT most avidly bound ApoE at an average abundance of 13.3%, representing a 55-fold enrichment compared to native mouse plasma (FIG.2H).
• Spleen SORT was most highly enriched in ⁇ 2-glycoprotein I ( ⁇ 2- GPI), at an average abundance of 20.1% (125-fold higher than native mouse plasma) (FIG.2H), while Lung SORT was most highly enriched in vitronectin (Vtn), at an average abundance of 12.2% (108- fold higher than native mouse plasma) (FIG.2H).
• example SORT LNPs for extrahepatic mRNA most avidly bind proteins distinct from ApoE compared to mDLNP.
• example SORT LNPs bind low abundance serum proteins to form unique protein coronas with quantitatively distinct composition, based on the nature of the incorporated SORT molecule, which may impact tissue-specific mRNA delivery.
• Example 6 Key serum proteins define SORT LNP cellular targeting [00334] As illustrated in FIG.3A, SORT LNPs were incubated with either ApoE, ⁇ 2-GPI, or Vtn, the most highly enriched serum or plasma proteins to associate with SORT LNPs as described in Example 5 hereinabove, and the effects of these proteins on LNP uptake and mRNA delivery efficiency in vitro were investigated. SORT LNPs encapsulating Cy5-mRNA were incubated with ApoE, and cellular uptake in HuH-7 and Hep G2 cells, two cell lines which highly express ApoE’s receptor, LDL-R, was measured.
• cells e.g., HuH-7, Hep G2, A-498, U-87 MG cells
• cells were seeded into 12 well plates at a density of 1 ⁇ 105 cells per well and incubated at 37 °C overnight while undifferentiated THP-1 cells were seeded into 12 well plates at a density of 2 ⁇ 105 cells per well and treated with 100 nM of Phorbol 12-myristate 13-acetate overnight (to enable differentiation into macrophages). All cells were cultured in complete medium, as recommended by the suppliers. Then, the old media was refreshed and cells treated with 250 ng of Cyanine 5 FLuc mRNA encapsulated in SORT LNPs.
• LNPs were either uncoated, pre-incubated with 1.0 g protein/g total lipid of ApoE, 1,0 g protein/g total lipid ⁇ 2-GPI, or pre- incubated with 0.25 g protein/g total lipid of Vtn.
• 1.0 g protein/g total lipid of ApoE 1,0 g protein/g total lipid ⁇ 2-GPI
• pre- incubated with 0.25 g protein/g total lipid of Vtn were washed two times with 1X PBS and stained with Hoechst 33342 (0.1 mg mL ⁇ 1 ) for 10 min at 37 °C. Cells were washed twice more with PBS, then imaged by fluorescence microscopy (Keyence BZ-X800). Images were captured using a 10x magnification. All image settings were kept consistent for a single experiment.
• Cy5 fluorescence intensity was quantified using ImageJ software version 1.53c (NIH). Each treatment group was performed in duplicates and five images were taken for each well. [00336] Intracellular accumulation of Cy5-mRNA was increased in HuH-7 and Hep G2 cells by a factor of 2.4, as shown in the top row of each of FIGs.3B-3C, and the 6.5, respectively, when Liver SORT LNPs were incubated with ApoE.
• THP-1 macrophages a cell line known to interact with ⁇ 2-GPI-bound particles containing anionic phospholipids, was incubated with ⁇ 2-GPI enhanced Cy5-mRNA SORT LNPs and showed increased update by a factor of 2.1 as shown by representative middle row of each of FIGs.3B-3C and second panel of FIG.3D.
• A-498 and U-87 MG cells, which highly express Vtn’s receptor ⁇ v ⁇ 3 integrin were examined.
• Luciferase activity was enhanced in HuH-7 and Hep G2 cells by ApoE-coated Liver SORT LNPs whereas luciferase activity in these cells was not improved by Spleen or Lung SORT LNPs pre-incubated with ApoE), as shown in the top panel of FIG. 3D, suggesting that Spleen and Lung SORT LNPs do not efficiently bind ApoE and enter LDL-R expressing cells.
• ApoE exclusively enhanced functional mRNA delivery by Liver SORT LNPs in LDL-R expressing cells.
• example Spleen and Lung SORT LNPs do not efficiently bind ApoE and enter LDL-R expressing cells.
• ApoE exclusively enhanced functional mRNA delivery by example Liver SORT LNPs in LDL-R expressing cells.
• incubating example Spleen SORT LNPs with ⁇ 2-GPI exclusively enhanced functional mRNA delivery to THP-1 macrophages (FIG.3D), while incubating Lung SORT LNPs with Vtn exclusively enhanced functional mRNA delivery to A-498 and U-87 MG cells (FIG.3D, FIG.8).
• example Lung SORT LNPs incubating example Lung SORT LNPs at a ratio of 1.0 g vitronectin/g total lipid resulted in diminished luciferase activity, suggesting that excess (free in media) vitronectin may be inhibiting ⁇ v ⁇ 3 integrin to limit mRNA delivery.
• these studies reveal that unique interactions between specific example SORT molecules and individual serum proteins selectively promote example SORT LNP targeting to distinct cell types by enhancing cellular uptake.
• Example 7 Extrahepatic mRNA delivery by SORT LNPs occurs by an ApoE-independent mechanism
• IP intraperitoneal
• mDLNPs By eliminating ApoE from the serum or plasma, mDLNPs exhibited significantly reduced mRNA delivery to the liver, with an average reduction of luciferase activity by 78%, compared to WT C57BL/6 mice, as shown in the top row of FIGs.4A-4B.
• Liver SORT LNPs which also bind ApoE selectively from the serum or plasma, maintaiend this dependence on ApoE for mRNA delivery to the liver: knockout of ApoE significantly impaired liver targeting, with the second row of FIGs.4A-4B showing an average reduction in luciferase activity by 87% compared to WT C57BL/6 mice.
• ApoE is indispensable for efficacious targeting of the liver by both mDLNPs and Liver SORT LNPs.
• mRNA delivery by Spleen SORT to the spleen was enhanced by a factor of 2.2 in ApoE-/- mice (third row of FIGs. 4A-4B), suggesting that ApoE plays an antagonistic role in efficacious mRNA delivery to the spleen.
• Tissue-specific mRNA delivery by Lung SORT LNPs as shown and measured in the bottom row of FIGs. 4A-4B, was not significantly different in ApoE-/- mice compared to WT mice, indicating that this serum or plasma protein is not functionally important for modulating lung targeting.
• Vtn could bind its cognate receptor, ⁇ v ⁇ 3 integrin, which is highly expressed by the pulmonary endothelium but not by liver cells or other vascular beds providing a plausible explanation for why Vtn promotes lung-specificity.
• ⁇ 2-GPI can bind phosphatidyl serine, an anionic lipid exposed by senescent red blood cells to promote their filtration from the circulation in the s[leen, suggesting how ⁇ 2-GPI could be implicated in Spleen SORT LNP targeting.
• clusterin highly enriched in Lung SORT, could play a role in evading the mononuclear phagocyte system to promote lung targeting.
• the reduced binding of ApoE to Spleen and Lung SORT LNPs could promote extrahepatic mRNA delivery, possibly due to displacement of ApoE by apolipoprotein C, which is present in the protein coronas of Spleen and Lung SORT LNPs but not Liver SORT.
• Adsorption of ApoE to the surface of conventional four-component LNPs is a crucial process necessary for highly efficacious RNA delivery to liver hepatocytes.
• mDLNP which is a four-component LNP for mRNA delivery to the liver, avidly binds to ApoE in the serum.
• the addition of a SORT molecule to mDLNP alters which serum proteins adsorb to the LNP surface.
• the set of proteins which bind the LNP might play a role in defining the organ-targeting properties of example SORT LNPs via an endogenous mechanism, whereby surface-adsorbed proteins interact with cognate receptors expressed by cells in the target organ.
• Lung and Spleen SORT LNPs include molecules (DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and 18PA (1,2-dioleoyl-sn-glycero- 3-phosphate), respectively) that are not common to conventional LNPs, we reasoned that differences in organ targeting could be driven by differences in protein corona composition. Genetically modified mice can be used to deplete key proteins from the serum, preventing their adsorption to the LNP surface and possibly impairing expected organ targeting.
• DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
• 18PA 1,2-dioleoyl-sn-glycero- 3-phosphate
• mDLNP By eliminating ApoE from the serum, mDLNP exhibited significantly reduced mRNA delivery to the liver, with an average reduction of luciferase activity by 78%, compared to WT C57BL/6 mice (FIGs.4A-4C).
• Example liver SORT LNPs which also bind ApoE selectively from the serum, maintain this dependence on ApoE for mRNA delivery to the liver: knockout of ApoE significantly impaired liver targeting, with an average reduction in luciferase activity by 87% compared to WT C57BL/6 mice (FIGs.4A-4C).
• ApoE is indispensable for efficacious targeting of the liver by both mDLNP reference and example Liver SORT LNPs.
• mRNA delivery by example Spleen SORT to the spleen was enhanced by a factor of 2.2 in ApoE-/- mice, suggesting that ApoE plays an antagonistic role in efficacious mRNA delivery to the spleen (FIGs. 4A-4C).
• Tissue- specific mRNA delivery by example Lung SORT LNPs was not significantly different in ApoE-/- mice compared to WT mice, indicating that this serum protein is not functionally important for modulating lung targeting (FIGs. 4A-4C). Together, these results indicate that mRNA delivery is no longer an ApoE-dependent process upon inclusion of either an anionic lipid or cationic lipid to mDLNP.
• example Spleen SORT and Lung SORT LNPs can enable extrahepatic mRNA delivery via an ApoE- independent mechanism. It is plausible that other serum proteins, such as those identified in the proteomics study, may be responsible for endogenous targeting to the spleen and lungs in vivo.
• Other serum proteins such as those identified in the proteomics study, may be responsible for endogenous targeting to the spleen and lungs in vivo.
• Clinical applications of genetic medicines are limited by the availability of efficacious carriers for the intracellular delivery of nucleic acid biomolecules to target tissues. Since intravenously administered LNPs have been limited to targeting liver hepatocytes to date, there is great need to develop drug delivery systems capable of extrahepatic targeting.
• we identified mechanistic factors which underly the tissue-targeting properties of example SORT LNPs for extrahepatic mRNA delivery.
• the proposed mechanism may have similarity with that of lipoproteins, which can be considered as a natural class of nanoparticles for physiological cholesterol transport through the blood.
• Acquisition of ApoE in the serum results in uptake of lipoproteins by liver hepatocytes through receptor- mediated endocytosis by LDL-R.
• This physiological function of ApoE is leveraged by DLin-MC3- DMA LNPs for siRNA delivery to the liver; by binding ApoE in the serum, these nanoparticles can endogenously target LDL-R highly expressed by liver hepatocytes.
• the hypothesized mechanism we provide evidence for herein builds upon and extends the scope of this endogenous targeting concept. Importantly.
• example SORT LNPs include classes of “out of the box” supplemental molecules that can tune an LNP’s molecular composition to promote the binding of distinct protein species to the LNP, which are not typically observed in the protein corona, and enable mRNA delivery to cells and organs beyond liver hepatocytes. Because example SORT LNPs include additional classes of molecules that have not been included in LNPs before, the SORT platform expands the toolbox of molecules available to control the protein corona without loss of efficacy.
• ⁇ 2-GPI can bind phosphatidyl serine, an anionic lipid exposed by senescent red blood cells to promote their filtration from the circulation in the spleen, suggesting how ⁇ 2-GPI could be implicated in Spleen SORT LNP targeting.

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