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WO2024031051A1 - Lipides pour l'administration d'acides nucléiques - Google Patents

Lipides pour l'administration d'acides nucléiques Download PDF

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Publication number
WO2024031051A1
WO2024031051A1 PCT/US2023/071670 US2023071670W WO2024031051A1 WO 2024031051 A1 WO2024031051 A1 WO 2024031051A1 US 2023071670 W US2023071670 W US 2023071670W WO 2024031051 A1 WO2024031051 A1 WO 2024031051A1
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Prior art keywords
bis
acid
composition
aminopropyl
amino
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Inventor
Arezki Boudif
Neha Nitin PARAYATH
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Life Technologies Corp
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Life Technologies Corp
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Priority to EP23761729.5A priority Critical patent/EP4565572A1/fr
Publication of WO2024031051A1 publication Critical patent/WO2024031051A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
    • C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
    • C07D279/12—1,4-Thiazines; Hydrogenated 1,4-thiazines not condensed with other rings
    • 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

  • Transfection is the process of introducing nucleic acids into eukaryotic cells by non- viral methods. Transfection methods allow the introduction of negatively charged molecules (e.g. phosphate backbones of DNA and RNA) into cells having a negatively charged membrane. Chemicals such as calcium phosphate and DEAE-dextran, or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule.
  • negatively charged molecules e.g. phosphate backbones of DNA and RNA
  • Chemicals such as calcium phosphate and DEAE-dextran, or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule.
  • the DNA- transfection reagent complex easily crosses the cell membrane, especially for lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer of the cell.
  • lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer of the cell.
  • novel ionizable lipids that can be used in vitro and in vivo.
  • Compounds are provided, together with compositions containing these compounds and methods for using these new compounds and compositions for delivery of payloads, (e.g.
  • nucleic acid or small molecule a nucleic acid or small molecule
  • the compounds may be used alone for transfection, or they may be used in combination with additional reagents in transfection compositions.
  • the new compounds may be combined with one or more ionizable lipids and/or neutral lipids, with one or more cell surface ligands, with one or more fusion enhancing agents, and with one or more nuclear localization agents and one or more amphipathic peptides and any combinations thereof.
  • the resulting compositions may be complexed with one or more macromolecules (e.g, nucleic acids, such as DNA or RNA, proteins, ribonucleoproteins, and the like) and used to deliver these macromolecules into cells.
  • macromolecules e.g, nucleic acids, such as DNA or RNA, proteins, ribonucleoproteins, and the like
  • FIG.1 is a graph depicting size (d.nm) and polydispersity index of the lipid-mRNA formulations.
  • FIG.2 is a graph depicting the percent encapsulation efficiency of the lipid-mRNA formulations.
  • FIG.3 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver of mice following intravenous administration of lipid-mRNA formulations.
  • FIG.4 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver by luciferase activity (in bioluminescence flux, photons/second (p/s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.
  • Lipid molecules are provided that are useful for improved methods of delivering payloads such as macromolecules, drugs and/or pharmaceutical agents, nutrients, or the like into eukaryotic cells, and that are particularly effective for delivery of wide variety of cells, tissues and organs, and provide a high efficiency of transfection.
  • lipid molecules are positively charged at about acidic pH, and advantageously can be used to prepare a complex with one or more neutral lipids and additional components such as fusogenic or fusion-enhancing molecules, additional cationic/ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localization agents and endosomal release agents, together with the payload (e.g., macromolecule or pharmaceutical agent, or nutrient, or the like).
  • the payload e.g., macromolecule or pharmaceutical agent, or nutrient, or the like.
  • biodegradable compound of the invention has the structure of compound I where M is
  • G is O or a thiol-group selected from the group consisting of -S-, -SO-, -S(O)2-, and - S(CH2)-
  • X is selected from the group consisting of C and N; each R a , Rb, Rc, Rd, Re, Rf, Rg and Rh are independently selected from the group consisting of H, unbranched C1-C12 alkyl groups, branched C1-C12 alkyl groups, halogenated unbranched C1-C12 alkyl groups, halogenated branched C1-C12 alkyl groups, a C3-C7 membered alkyl ring, a C3-C7 carbocyclic ring formed from two R groups attached to an individual atom and a C3-C7 carbocyclic ring formed from R groups on two adjacent ring carbon atoms;
  • Ri, R2 and R3 are independently selected from the group consisting of H, unbranched Ci- 25 alkyl groups, branched C1.C25 alkyl groups, halogenated unbranched C1-C25 alkyl groups, and halogenated branched C1-C25 alkyl groups; each occurrence of R 4 , R5, R6, and R7 are independently selected from the group consisting of H and a C1-C6 alkyl; w, x, y and z are selected from the group of integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; -(CHa)x-, -(CHb)y-, -(CHc)z- and -(CHd)w- are selected from C1-C12 alkyl, C2-C12 alkene, and the absence of the methylene when w, x, y or z is 0; and a, b, c, and d is 2 for C 1- C 12 alkyl groups and selected
  • Item 2 In some embodiments the compound has the structure of compound I-A, wherein y, z and w are 0, L1, L2 and L3 are a bond [0014]
  • Item 3 In certain embodiments the compound has the structure of compound I-B, wherein X is carbon, x and w are 0, L3 is a bond, R3 is H, and L is a bond [0015]
  • Item 10 Also provided are compounds of compound I, wherein at each occurrence a halogen atom is selected from F, Cl, Br and I [0022]
  • Item 11 In some embodiments of compound I, each of R1, R2 and R3 are independently selected from the group consisting of H, a diene and a triene.
  • Item 12 In some embodiments of compound I, M has a structure selected from the group consisting of
  • each R 1 , R 2 and R 3 of compound I is independently selected from the group consisting of H,
  • Item 14 Also provided are compounds having a structure selected from the group consisting of
  • Item 16 Also provided are compounds having a structure selected from the group consisting of
  • Item 16a Also provided are compounds having a structure selected from the group consisting of
  • Item 17 In some aspects compounds of I-I are provided in which one of L 1 R 1 and L 2 R 2 is an unsaturated fatty acid. [0030] Item 18: In certain embodiments compounds of I-I are provided in which both L1R1, and L 2 R 2 , are an unsaturated fatty acid moiety.
  • Item 19 Also provided are compounds of I-I wherein the unsaturated fatty acid moiety is selected from the group consisting of ⁇ -linolenic acid (C 18:3), stearidonic acid (C 18:4), eicosapentaenoic acid (C 20:5), cervonic acid (C 22:6), linoleic acid (C 18:2), linolelaidic acid (C 18:2), ⁇ -linolenic acid (C 18:3), dihomo- ⁇ -linolenic acid (C 20:3), arachidonic acid (C 20:4), docosatetraenoic acid (C 22:4), palmitoleic acid (C 16:1), vaccenic acid (C 18:1), paullinic acid (C 20:1), oleic acid (C 18:1), elaidic acid (C 18:1), gondoic acid (C 20:1), erucic acid (C 22:
  • Item 20 In some embodiments one of L 1 R 1 , and L 2 R 2 , is a saturated fatty acid moiety.
  • Item 21 In certain embodiments both of L1R1, and L2R2, are saturated fatty acid moiety.
  • Item 22 Also provided are compounds in which the saturated fatty acid moiety is selected from the group consisting of propionic acid (C 3:0), butyric acid (C 4:0), valeric acid (C 5:0), caproic acid (C 6:0), enanthic acid (C 7:0), caprylic acid (C 8:0), pelargonic acid (C 9:0), capric acid (C 10:0), undecylic acid (C 11:0), lauric acid (C 12:0), tridecylic acid (C 13:0), myristic acid (C 14:0), pentadecylic acid (C 15:0), palmitic acid (C 16:0), margaric acid (C 17:0), stearic acid (C 18:0), nonadecylic acid (C 19:0), arachidic acid (C 20:0), heneicosylic acid (C 21:0), behenic acid (C 22:0), tyricosylic acid (C
  • the compound may have has the structure of I-K, where L is a bond and L1R1 and L2R2 form [0036]
  • Item 24 In certain embodiments the compound may be selected from the group consisting of O O [0037]
  • Item 25 Also provided are compounds of structure I-I where the ester moieties -OR1 and –OR 2 may be derived from an alcohol selected from the group consisting of tert-butyl alcohol (C4), tert-amyl alcohol (C 5), 3-methyl-3-pentanol (C 6), 1-hexanol (C 6), 1-heptanol (C 7), 1-octanol (C 8), 1-nonanol (C 9), 1-decanol (C 10), 1-undecanol (C 11), 1-dodecanol (C 12), 1-tridecanol (C 13), 1-tetradecanol (C 14), 1-pentadecanol (C 15), 1-
  • Item 26 In some embodiments, the compound may have the structure of I-L, where L is a bond, and L 1 R 1 and L 2 R 2 form ethers -OR 1 and -OR 2 .
  • Item 27 In certain embodiments R 1 and R 2 may be selected from the group consisting of C 1 -C 22 alkyl and C 2 -C 22 alkene groups.
  • Item 27a In some embodiments, the compound may have the structure of any compound in the following table:
  • Item 28 Certain embodiments provide for a composition comprising the compound of any one of items 1-27a and a payload.
  • Item 29 In certain embodiments the composition of item 28, further comprises one or more ionizable lipids.
  • Item 30 In certain embodiments the composition of item 28 or 29, further comprises a helper lipid.
  • Item 31 In certain embodiments the composition of any one of items 28-30, further comprises a stabilizer.
  • Item 32 In certain embodiments of item 31, the stabilizer is selected from the group consisting of a PEGylated lipid, and a surfactant.
  • Item 33 In certain embodiments of item 28, the payload is a nucleic acid, a protein, or a ribonucleoprotein.
  • Item 34 In certain embodiments of item 33, the payload is a nucleic acid.
  • Item 35 In certain embodiments of item 34, the nucleic acid is a ribonucleic acid.
  • Item 36 In certain embodiments of item 35, the ribonucleic acid is selected from antisense RNA, mRNA, o-RNA, miRNA, siRNA, or any combination thereof.
  • Item 37 In certain embodiments of any one of items 28-36, further comprise a delivery-enhancing peptide.
  • Item 38 Certain embodiments provide a method of delivering a payload to a cell, comprises contacting the cell with the composition of any one of items 28-37.
  • Item 39 In certain embodiments of item 38, the cell is a mammalian cell.
  • Item 40 In certain embodiments of item 39, the contacting is performed in vitro, ex- vivo, or in vivo.
  • Item 41 Certain embodiments provide a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing the composition of any one of items 28-37 and administering the composition to the patient.
  • Item 42 Certain embodiments provide a use of a composition according to any of items 28-37 in the manufacture of a medicament.
  • the skilled artisan will recognize that, although the molecules of the invention are shown here for convenience in their neutral (unprotonated) forms, these molecules will exist in a partially or fully protonated form in solutions of appropriate pH, and that the present invention encompasses the molecules in all their protonated, unprotonated, ionized and non-ionized forms without limitation, unless specifically indicated otherwise.
  • GENERAL DEFINITIONS [0057] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.
  • the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.
  • 0.2-5 mg is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
  • Compounds are generally described herein using standard nomenclature. For a recited compound having asymmetric center(s), all of the stereoisomers of the compound and mixtures thereof are encompassed unless otherwise specified. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. Where a recited compound exists in various tautomeric forms, the compound is intended to encompass all tautomeric forms.
  • C1-C6 alkyl refers to an alkyl substituent containing from 1 to 6 carbon atoms.
  • C3-C6 cycloalkyl means a saturated hydrocarbyl ring containing from 3 to 6 carbon ring atoms.
  • a prefix attached to a multiple-component substituent only applies to the first component that immediately follows the prefix.
  • the term "carbocyclylalkyl” contains two components: carbocyclyl and alkyl.
  • C3-C6 carbocyclyl C 1 -C 6 alkyl refers to a C 3 -C 6 carbocyclyl appended to the parent molecular moiety through a C 1 -C 6 alkyl group.
  • the chemical structure is -LS-M-LS''- and M is -N(RB)S(O)-, then the chemical structure is -L S -N(R B )S(O)-L S ''-.
  • a linking element in a depicted structure is a bond, then the element left to the linking element is joined directly to the element right to the linking element via a covalent bond.
  • a chemical structure is depicted as -L S -M-L S ' and M is selected as bond, then the chemical structure will be -L S -L S ''-.
  • the dash(es) indicates the portion of the moiety that has the free valence(s).
  • the moiety may be either substituted or unsubstituted. If a moiety is described as being optionally substituted with up to a particular number of non-hydrogen radicals that moiety may be either unsubstituted, or substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the moiety, whichever is less.
  • any heterocycle with less than three substitutable positions will be optionally substituted by up to only as many non-hydrogen radicals as the heterocycle has substitutable positions.
  • tetrazolyl which has only one substitutable position
  • a primary amino nitrogen will be optionally substituted with up to two non-hydrogen radicals
  • a secondary amino nitrogen will be optionally substituted with up to only one non-hydrogen radical.
  • alkenyl means a straight or branched hydrocarbyl chain containing one or more double bonds. Each carbon-carbon double bond may have either E (cis) or Z (trans) geometry within the alkenyl moiety, relative to groups substituted on the double bond carbons.
  • alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E,Z-, Z,E- and Z,Z-hexadienyl and the like.
  • alkenylene refers to a divalent unsaturated hydrocarbyl chain which may be linear or branched and which has at least one carbon-carbon double bond.
  • alkyl means a straight or branched saturated hydrocarbyl chain.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, iso-amyl, and hexyl.
  • alkylene denotes a divalent saturated hydrocarbyl chain which may be linear or branched. Representative examples of alkylene include, but are not limited to, -CH 2 -, - CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, and -CH 2 CH(CH 3 )CH 2 -.
  • alkynyl means a straight or branched hydrocarbyl chain containing one or more triple bonds.
  • Non-limiting examples of alkynyl include ethynyl, 1-propynyl, 2-propynyl, 3- propynyl, decynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
  • alkynyl alone or in combination with any other term, refers to a straight- chain or branched-chain hydrocarbon radical having one or more triple bonds containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 10 carbon atoms.
  • alkynyl radicals examples include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl and the like.
  • alkoxy refers to an alkyl ether radical, wherein the term “alkyl” is defined above.
  • suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.
  • aryl refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment from 6-15 carbon atoms (i.e. (C 6-15 )aryl), and in another embodiment from 6- 10 carbon atoms (i.e. (C6-10)aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy, (for example methoxy), nitro, halogen, (for example chloro), amino, carboxylate and hydroxy.
  • C 6-15 carbon atoms
  • C6-10aryl 6- 10 carbon atoms
  • aryl radicals include, but are not limited to phenyl, p- tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl and the like.
  • aralkyl alone or in combination, means an alkyl radical as defined above in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl and the like.
  • aralkoxycarbonyl alone or in combination, means a radical of the formula - C(O)-O-aralkyl in which the term “aralkyl” has the significance given above.
  • An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.
  • aryloxy alone or in combination, means a radical of the formula aryl-O- in which the term “aryl” has the significance given above.
  • alkynylene refers to a divalent unsaturated hydrocarbon group which may be linear or branched and which has at least one carbon-carbon triple bonds.
  • alkynylene groups include, by way of example, -C ⁇ C-, -C ⁇ C-CH2-, -C ⁇ C-CH2-CH2-, -CH2- C ⁇ C-CH 2 -, -C ⁇ C-CH(CH 3 )-, and -CH 2 -C ⁇ C-CH(CH 2 CH 3 )-.
  • alkanoyl alone or in combination, means an acyl radical derived from an alkanecarboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4- methylvaleryl, and the like.
  • aryloxyalkanoyl means an acyl radical of the formula aryl-O-alkanoyl wherein aryl and alkanoyl have the significance given above.
  • aralkanoyl means an acyl radical derived from an aryl-substituted alkanecarboxylic acid such as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4- phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4- phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4- methoxyhydrocinnamoyl, and the like.
  • aroyl means an acyl radical derived from an aromatic carboxylic acid.
  • radicals include aromatic carboxylic acids, an optionally substituted benzoic or naphthoic acid such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4- benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6- (benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3- (benzyloxyformamido)-2-naphthoyl, and the like.
  • aminocarbonyl alone or in combination, means an amino-substituted carbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group continuing substituents selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl radicals and the like.
  • aminoalkanoyl means an acyl radical derived from an amino substituted alkanecarboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group containing substituents selected from the group consisting of hydrogen, cycloalkyl, cycloalkylalkyl radicals and the like, examples of which include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.
  • carbocycle or “carbocyclic” or “carbocyclyl” refers to a saturated (e.g., “cycloalkyl"), partially saturated (e.g., “cycloalkenyl” or “cycloalkynyl") or completely unsaturated (e.g., "aryl”) 3- to 8-membered carbon ring system containing zero heteroatom ring atom.
  • Ring atoms or “ring members” are the atoms bound together to form the ring or rings.
  • a carbocyclyl may be, without limitation, a single ring, two fused rings, or bridged or spiro rings.
  • a substituted carbocyclyl may have either cis or trans geometry.
  • carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl, cyclohexadienyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl, cyclohexenyl, phenyl, naphthyl, indanyl, 1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, decalinyl, and norpinanyl.
  • a carbocycle group can be attached to the parent molecular moiety through any substitutable carbon ring atom.
  • a carbocycle group is a divalent moiety linking two other elements in a depicted chemical structure
  • the carbocycle group can be attached to the two other elements through any two substitutable ring atoms.
  • a carbocycle group is a trivalent moiety linking three other elements in a depicted chemical structure
  • the carbocycle group can be attached to the three other elements through any three substitutable ring atoms, respectively.
  • the carbocycle may be attached at any endocyclic carbon atom which results in a stable structure.
  • Carbocycles in one embodiment have 5-7 carbons.
  • cycloalkyl refers to a saturated carbocyclyl group containing zero heteroatom ring member.
  • Non-limiting examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl and norpinanyl.
  • cycloalkyl alone or in combination, means an alkyl radical which contains from about 3 to about 8 carbon atoms and is cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • cycloalkylalkyl means an alkyl radical as defined above which is substituted by a cycloalkyl radical containing from about 3 to about 8, in one embodiment from about 3 to about 6, carbon atoms.
  • cycloalkylcarbonyl means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, and the like, or from a benz-fused monocyclic cycloalkanecarboxylic acid which is optionally substituted by, for example, alkanoylamino, such as 1,2,3,4-tetrahydro-2- naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.
  • cycloalkylalkoxycarbonyl means an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.
  • carbocyclylalkyl refers to a carbocyclyl group appended to the parent molecular moiety through an alkylene group. For instance, C 3 -C 6 carbocyclylC 1 -C 6 alkyl refers to a C 3 -C 6 carbocyclyl group appended to the parent molecular moiety through C 1 -C 6 alkylene.
  • cycloalkenyl refers to a non-aromatic, partially unsaturated carbocyclyl moiety having zero heteroatom ring member.
  • Representative examples of cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, and octahydronaphthalenyl.
  • halo indicates that the substituent to which the prefix is attached is substituted with one or more independently selected halogen radicals.
  • C 1 - C6haloalkyl means a C1-C6alkyl substituent wherein one or more hydrogen atoms are replaced with independently selected halogen radicals.
  • Non-limiting examples of C1-C6haloalkyl include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and 1,1,1- trifluoroethyl. It should be recognized that if a substituent is substituted by more than one halogen radical, those halogen radicals may be identical or different (unless otherwise stated).
  • heterocycle or “heterocyclo” or “heterocyclyl” refers to a saturated (e.g., “heterocycloalkyl"), partially unsaturated (e.g., “heterocycloalkenyl” or “heterocycloalkynyl”) or completely unsaturated (e.g., “heteroaryl”) ring system where at least one of the ring atoms is a heteroatom (i.e., nitrogen, oxygen or sulfur), with the remaining ring atoms being independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur.
  • a heterocycle may be, without limitation, a single ring, two fused rings, or bridged or spiro rings.
  • a heterocycle group can be linked to the parent molecular moiety via any substitutable carbon or nitrogen atom(s) in the group.
  • a heterocycle group is a divalent moiety that links two other elements in a depicted chemical structure
  • the heterocycle group can be attached to the two other elements through any two substitutable ring atoms.
  • a heterocycle group is a trivalent moiety that links three other elements in a depicted chemical structure
  • the heterocycle group can be attached to the three other elements through any three substitutable ring atoms, respectively.
  • “Het” indicates a heterocycle containing 4-12 carbon atom, where at least one nitrogen atom is present in the ring(s).
  • a heterocyclyl may be, without limitation, a monocycle which contains a single ring.
  • monocycles include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiodiazolyl, oxathiazolyl, oxadiazolyl (including 1,2,3-oxadiazolyl
  • a heterocyclyl may also be, without limitation, a bicycle containing two fused rings, such as, for example, naphthyridinyl (including [1,8]naphthyridinyl, and [1,6]naphthyridinyl), thiazolpyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolizinyl, pyrindinyl, pyranopyrrolyl, 4H-quinolizinyl, purinyl, pyridopyridinyl (including pyrido[3,4-b]-pyridinyl, pyrido[3,2-b]-pyridinyl, and pyrido[4,3-b]- pyridinyl), pyridopyrimidine, and pteridinyl.
  • naphthyridinyl
  • fused-ring heterocycles include benzo-fused heterocyclyls, such as indolyl, isoindolyl, indoleninyl (also known as “pseudoindolyl”), isoindazolyl (also known as “benzpyrazolyl” or indazolyl), benzazinyl (including quinolinyl (also known as “1-benzazinyl”) and isoquinolinyl (also known as “2-benzazinyl”)), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including cinnolinyl (also known as “1,2-benzodiazinyl”) and quinazolinyl (also known as "1,3- benzodiazinyl”)), benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, benzothiadiazolyl, benzimidazolyl, benzothiazolyl
  • a heterocyclyl may also be, without limitation, a spiro ring system, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decanyl.
  • a heterocyclyl may comprise one or more sulfur atoms as ring members; and in some cases, the sulfur atom(s) is oxidized to SO or SO 2 .
  • the nitrogen heteroatom(s) in a heterocyclyl may or may not be quaternized, and may or may not be oxidized to N-oxide. In addition, the nitrogen heteroatom(s) may or may not be N-protected.
  • a heterocycle or carbocycle may be further substituted.
  • substituted refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms with substituents including, but not limited to, -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO 2 , -N 3 , -CN, -NH 2 , protected amino, oxo, thioxo, -NH-C 2 -C 8 -alkenyl, - NH-C2-C8-alkynyl, -NH-C3-C12-cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, - dialkylamino, -diarylamino, -diheteroarylamino, -O-C1-C12-alkyl, -O-C2-C8-alkenyl, alkynyl, -O- C 3
  • N-protecting group or “N-protected” refers to those groups capable of protecting an amino group against undesirable reactions. Commonly used N-protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3 rd ed., John Wiley & Sons, NY (1999)).
  • N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butyl acetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, tri chloroacetyl, phthalyl, o-nitrophenoxy acetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, or 4- nitrobenzoyl; sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl; sulfenyl groups such as phenyl sulfenyl (phenyl-S-) or triphenylmethyl sulfenyl (trityl-S-); sulfinyl groups such as p- methylphenylsulfinyl (p-methylphenyl-S(
  • N- protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
  • halogen means fluorine, chlorine, bromine or iodine.
  • ionizable lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH 4 and a neutral charge at other pHs such as physiological pH 7.
  • lysis agent or “endosomal release agent” as used herein refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the DNA transporter into the cytoplasm of the cell. This term includes but is not limited to viruses, synthetic compounds, lytic peptides, or derivatives thereof.
  • lytic peptide refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both.
  • Examples of lysis agents/endosomal release agents include choroquine, polyamines and polyamidoamines.
  • polycationic nucleic acid binding moiety refers to a moiety containing multiple positive charges at physiological pH that allow the moiety to bind a negatively charged nucleic acid.
  • a polycationic nucleic acid binding moiety may be linked to, for example, a cell surface ligand, a fusion agent, and/or a nuclear localization peptide. The linkage may be covalent.
  • Suitable polycationic nucleic acid binding moieties include polyamines such as PEI, spermine, spermidine, carboxyspermine and polybasic peptides containing, for example, multiple lysine, ornithine, histidine, or arginine residues.
  • polyamines such as PEI, spermine, spermidine, carboxyspermine and polybasic peptides containing, for example, multiple lysine, ornithine, histidine, or arginine residues.
  • the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids.
  • the terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • a “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety.
  • stabilizing agent refers to a compound that mitigates the degradation of the lipid nanoparticles or a subpopulation of the lipid nanoparticles.
  • structural lipid refers to sterols and lipids containing sterol like moieties.
  • surface ligand or "cell surface ligand” refers to a chemical compound or structure which will bind to a surface receptor of a cell.
  • cell surface receptor refers to a specific chemical grouping on the surface of a cell to which the ligand can attach.
  • Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). The receptor facilitates the internalization of the ligand and attached molecules.
  • a cell surface receptor includes but is not limited to a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI)- anchored proteins (e.g., ⁇ -adrenergic receptor, T-cell activating protein, Thy-1 protein, GPI- anchored 5' nucleotidase).
  • GPI glycosylphosphatidylinositol
  • a “receptor” is a molecule to which a ligand binds specifically and with relatively high affinity.
  • a receptor is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx.
  • epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis.
  • surface ligand includes anything which is capable of entering the cell through cytosis (e.g. endocytosis, potocytosis, pinocytosis).
  • ligand refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, RGD-containing cell adhesion peptides and the like.
  • the ligand may also be a plant virus movement protein or peptide derived from such a protein.
  • Suitable peptides and proteins are described, for example, in US Patent No.10,538,784, the contents of which are hereby incorporated by reference in their entirety.
  • a ligand chosen will depend on which receptor is being bound. Since different types of cells have different receptors, this provides one method of targeting nucleic acid to specific cell types, depending on which cell surface ligand is used. Thus, use of a cell surface ligand may depend on the targeted cell type.
  • SYNTHESIS OF THE LIPIDS [0115] The synthetic processes of the disclosure can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used.
  • DCM dichloromethane
  • DIEA N,N-Diisopropylethylamine
  • DIPEA N,N-Diisopropylethylamine
  • DMF N,N-dimethylformamide
  • ESBL extended-spectrum ⁇ -lactamase
  • EtOAc ethyl acetate
  • EA ethyl acetate
  • FCC Flash Column Chromatography
  • HATU 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate
  • MeCN acetonitrile
  • NMR nuclear magnetic resonance
  • Preferred protecting groups include, but are not limited to: For a hydroxyl moiety: TBS, benzyl, THP, Ac; For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allyl ester; For amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, benzylideneamine; For diols: Ac (x2) TBS (x2), or when taken together acetonides; For thiols: Ac; For benzimidazoles: SEM, benzyl, PMB, DMB; For aldehydes: di-alkyl acetals such as dimethoxy acetal or diethyl acetyl.
  • the claimed compounds may be produced by (a) reacting propane-1,2,3 triol with acetone in a solvent with a strong acid, typically THF in the presence of p-tolulene sulfonic acid, to form the acetal 1.
  • the free hydroxyl group may be converted (b) to 2 by a variety of methods, such as Mitsunobu coupling in which compound 1 is solubilized in THF or similar solvent with a thio morpholine analog in the presence of triphenyl phosphine.
  • an azodicarboxylate such as diethyl azodicarboxylate (DEAD) is slowly added dropwise resulting in the formation of 2.
  • Deprotection of the hydroxyls may be accomplished by removing the acetal group with trifluoracetic in THF/H2O (c) to form 3.
  • the hydroxyl groups may be subsequently esterified (d) to form 4 in the presence of a carbodiimide and a nucleophilic catalyst such as 4-Dimethylaminopyridine (DMAP).
  • DMAP 4-Dimethylaminopyridine
  • Scheme 2 presents an alternative route to compounds of the invention.
  • a bromo alkyl acid 5 may react with an alcohol 6 to afford ester 7.
  • This step may take place in an organic solvent such as DMF in the presence of a carbodiimide, 4-dimethylaminopyridine and N,N-diisopropyl ethylamine.
  • Compound 7 may react with thio morpholine when heated in DMF in the presence of N-diisopropylethylamine to produce 8.
  • 8 may be produced by incubating 7 and thiol morpholine in acetonitrile, NaI and Na 2 CO 3
  • the thiol group may be oxidized with m-chloroperoxybenzoic or K+H 2 SO 5 - in a methanol/water solution.
  • Scheme 3 shows a third route of synthesis.
  • the thio morpholine compound 10 may be heated with compound 11 in DMF in the presence of N-diisopropylethylamine to form compound 12. Reaction of the acid moiety with a secondary amine results in the formation of the amid 13. The thiol group may be further oxidized to form compound 14.
  • Scheme 4 shows a fourth route of synthesis.
  • the (thio)morpholine compound 15 may be coupled to ester 16 using Mitsunobu coupling conditions to form homoligated compound 17. Transesterification of the diester leads to diester 18. In some instances, diester 18 may be oxidized to form compound 19.
  • the disclosure also includes methods of synthesizing a compound of any of Formulae (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I, (I-J), or (I-K) and intermediate(s) for synthesizing the compound.
  • Other methods of preparing compounds of formula (I) will be apparent to the skilled artisan.
  • COMPOUNDS [0129] Specific examples of compounds of structure (I) include compounds having the structure in Table I below. Table I
  • compositions provided herein encompass complexes in the form of lipid nanoparticles, liposomes (e.g., lipid vesicles) and lipoplexes.
  • liposome encompasses any compartment enclosed by a lipid bilayer.
  • the term liposome includes unilamellar vesicles which are comprised of a single lipid bilayer and generally have a diameter in the range of about 20 to about 400 nm. Liposomes can also be multilamellar having a diameter in the range of approximately 1 ⁇ m to approximately 10 ⁇ m.
  • Multilamellar liposomes may consist of several (anywhere from two to hundreds) unilamellar vesicles forming one inside the other in diminishing size, creating a multilamellar structure of concentric phospholipid spheres separated by layers of water.
  • multilamellar liposomes may consist of many smaller nonconcentric spheres of lipid inside a large liposome.
  • liposomes include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV).
  • the compositions include liposomes which contain any suitable ionizable lipid and neutral lipids, along with the peptide as provided herein.
  • Another aspect of the present application relates to a composition including one or more compounds described herein, and one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and optionally, a payload.
  • the compositions include a compound described herein; one or more structural lipids; one or more stabilizing agents; and optionally, a payload.
  • the compositions include a compound described herein; one or more structural lipids; one or more stabilizing agents; one or more transfection enhancing agents; and optionally, a payload.
  • the compositions include 10 to 80 mol% of one or more of a compound described herein, excluding any payload, if present. Payload [0135]
  • An aspect of the present application relates to a composition including one or more compounds described herein, and a payload.
  • compositions as described herein can further comprise one or more bioactive molecules to be delivered to a cell (i.e., a payload).
  • bioactive molecules can include, e.g., nucleic acids, peptides, active pharmaceutical agents, nutrients, small molecules, or the like.
  • the payloads in the embodiments provided herein are biomolecules, e.g., either nucleic acids (RNA, DNA, etc., as described in more detail herein), peptides, or a combination thereof.
  • the payload can be a therapeutic and/or prophylactic agent.
  • the payload can be used for cosmetic, or nutraceutical applications.
  • the therapeutic and/or prophylactic agents are sometimes referred to as a “therapeutic payload” or “payload” in the present disclosure.
  • the payload can be administered in vivo or in vitro using the compositions provided herein as a delivery vehicle.
  • the payload is a nucleic acid.
  • the compositions include a compound described herein with a charge N and a nucleic acid molecule with a charge P, where the combination of the compound and the nucleic acid contacting the cell comprises an N/P ratio from about 1 to 20.
  • the nucleic acid is an RNA.
  • the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.
  • the RNA is an mRNA.
  • the compositions can include two or more different mRNAs.
  • the RNA encodes an immunogen.
  • the RNA encodes a cancer antigen
  • the nucleic acid is a DNA.
  • the payload includes one or more peptides, and optionally a nucleic acid.
  • the peptide is covalently linked to a nucleic acid.
  • Efficiency of encapsulation [0142] The efficiency of encapsulation of a payload (e.g., macromolecule) describes the amount of payload that is encapsulated or otherwise associated with a lipid composition after preparation, relative to the initial amount provided. In some embodiments, the encapsulation efficiency is desirably high (e.g., close to 100%).
  • the encapsulation efficiency may be measured, for example, by comparing the amount of payload in a solution containing the lipid complex composition before and after breaking up the lipid complex composition with one or more organic solvents or detergents.
  • fluorescence may be used to measure the amount of free payload (e.g., RNA or DNA) in a solution.
  • the encapsulation efficiency of a payload may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.
  • the encapsulation efficiency (EE%) can be measured using a fluorescence plate-based assay employing the RIBOGREENTM reagent. This assay measures the quantity of mRNA in samples with intact lipid complexes (e.g, lipid nanoparticles, or “LNPs”) to determine the quantity of unencapsulated RNA as well as in LNP samples disrupted by triton X-100 to measure the total RNA.
  • LNPs lipid nanoparticles
  • the lipid complexes provided herein include one or more co-lipids, most advantageously neutral co-lipids, although the skilled artisan will recognize that other lipids, including cationic/ionizable lipids, may be used. Some formulations, however, may include just the lipids of formula (I), in combination with a nucleic acid.
  • Lipid nanoparticles LNPs
  • the compositions include lipid nanoparticles (LNPs).
  • the LNP compositions are typically sized on the order of micrometers or small and may include a lipid bilayer.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 1 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 900 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 800 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 700 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 600 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 500 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 400 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 300 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 200 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 100 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 50 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 900 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 800 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 700 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 600 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 500 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 400 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 300 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 200 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 150 ⁇ m.
  • Nucleic Acid [0146]
  • the present lipid compositions include one or more nucleic acid molecules (e.g., DNA or RNA molecules) as the payload.
  • the nucleic acid of the lipid complex composition is an RNA molecule.
  • the RNA payloads can be mRNA, siRNA, shRNA, miRNA, self- replicating RNA (srRNA), self-amplifying RNA (saRNA), stRNA, sgRNA, or combinations thereof.
  • the payload includes more than one mRNA molecule (e.g., at least 2 mRNA molecules, at least 3 mRNA molecules, at least four mRNA molecules, or at least 5 mRNA molecules).
  • the an RNA payload includes at least one sgRNA.
  • the nucleic acid of the lipid complex composition molecule includes an sgRNA molecule and an mRNA molecule.
  • the lipid composition includes a gene editing reagent (or a gene editing composition), and the gene editing reagent includes a gene editing protein, an RNA or DNA (e.g., encoding a gene editing protein), a donor nucleic acid, a ribonucleoprotein (RNP), or any combination thereof.
  • the gene editing protein includes a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a MegaTal, a Cre recombinase, a Hin Recombinase, or a Flp recombinase.
  • the RNA molecule includes sgRNA, a crRNA, and/or a tracrRNA.
  • the lipid complex composition includes an RNP and an sgRNA, and optionally a donor nucleic acid.
  • the RNP can include a Cas protein and a sgRNA, a crRNA or a tracrRNA, and optionally a donor nucleic acid.
  • the payload can include a gene editing protein covalently or non-covalently bound to a donor nucleic acid (e.g., a donor DNA or a donor RNA).
  • the payload includes a nucleic acid that encodes a gene editing protein (e.g., a DNA or an RNA encoding a ZFN, TALEN, Cas protein, Cre recombinase, etc).
  • a gene editing protein e.g., a DNA or an RNA encoding a ZFN, TALEN, Cas protein, Cre recombinase, etc.
  • the lipid complex composition includes an RNA encoding a gene editing protein and an sgRNA.
  • the lipid complex composition can include an RNA encoding a Cas protein and a sgRNA, a crRNA or a tracrRNA, and optionally a donor nucleic acid.
  • the payload includes a nucleic acid that encodes a therapeutic protein, such as an antibody, growth factor, cytokine, enzyme, or the like.
  • the nucleic acid of the lipid complex compositions is a single- stranded molecule.
  • the nucleic acid of the lipid complex is double stranded.
  • the lipid may include donor DNA.
  • the payload may be a plasmid DNA or linear DNA.
  • the payload may be doggiebone DNA, or “dbDNA.”
  • the payload can be an oligonucleotide, such as an antisense oligonucleotide.
  • the gene editing composition induces single-strand or double- strand breaks in DNA within the cells.
  • the gene editing reagent (or gene editing composition) further comprises a repair template polynucleotide.
  • the repair template comprises (a) a first flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on one side of the single or double strand break and a second flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on the other side of the single or double strand break; or (b) a first flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single or double strand break and a second flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side
  • Non-limiting descriptions relating to gene editing (including repair templates) using the CRISPR-Cas system are discussed in Ran et al. (2013) Nat Protoc.2013 Nov; 8(11): 2281-2308, the entire content of which is incorporated herein by reference. Embodiments involving repair templates are not limited to those comprising the CRISPR-Cas system.
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.
  • the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2 and in the NCBI database as under accession number Q99ZW2.1.
  • UniProt database accession numbers A0A0G4DEU5 and CDJ55032 provide another example of a Cas9 protein amino acid sequence.
  • Another non-limiting example is a Streptococcus thermophilus Cas9 protein, the amino acid sequence of which may be found in the UniProt database under accession number Q03JI6.1.
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme is Cas9, and may be Cas9 from S.
  • the CRISPR enzyme directs 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. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated to 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 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).
  • Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • nickases may be used for genome editing via homologous recombination.
  • 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.
  • the nucleic acid of the lipid composition encodes for an immunogen. In some examples the nucleic acid of the lipid composition encodes for a hemagglutinin (HA) protein or fragment thereof. In some examples the nucleic acid of the lipid composition encodes for ovalbumin or fragment thereof.
  • the lipid composition induces an immune response in a subject to the nucleic acid-encoded protein of the lipid composition.
  • the amount of nucleic acid e.g., DNA, mRNA, self-amplifying RNA, dbDNA or the like
  • the amount of nucleic acid in a lipid nanoparticle formulation may depend on the size, sequence, and other characteristics of the nucleic acid.
  • the amount of nucleic acid in a lipid nanoparticle formulation may also depend on the size, composition, desired target, and other characteristics of lipid nanoparticle formulation.
  • the relative amounts of payload, e.g., mRNA or other macromolecule and other elements (e.g., lipids) may also vary.
  • the wt/wt ratio of the lipid component to a nucleic acid, such as an mRNA, in a nanoparticle composition may be from about 5:1 to about 50:1, such as 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, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1.
  • the wt/wt ratio of the lipid component to a nucleic acid, such as an mRNA may be from about 10:1 to about 40:1.
  • the amount of nucleic acid in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible (UV-vis) spectroscopy).
  • the lipid nanoparticle formulations can comprise a nucleic acid in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
  • the one or more nucleic acids e.g. mRNAs
  • lipids, and amounts thereof may be selected to provide a specific N:P ratio.
  • the N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in a nucleic acid (e.g., an mRNA). In general, a lower N:P ratio is preferred.
  • the one or more mRNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio may be from about 2:1 to about 5:1.
  • the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. See, for example, US Patent No.11,318,213 and U.S, Published Application Nos.2020/0163878 each of which is incorporated by reference. [0159] In some embodiments of the nucleic acid-containing lipid composition, nucleic acid is complexed to the exterior of the lipid complex (e.g., liposomes, lipid nanoparticles).
  • the lipid complex e.g., liposomes, lipid nanoparticles.
  • the compositions have from about 20% to about 50% of the nucleic acid complexed to the exterior of the lipid complex. In other embodiments, the compositions have about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the nucleic acid complexed to the exterior of the lipid complex. Exterior complexation of a nucleic acid can be measured by methods know in the art, such as in Blakney et al. (2019) Gene Therapy 26:363-372. [0160] In some embodiments of the nucleic acid-containing lipid composition, nucleic acid is complexed on the interior of the lipid complex (e.g., liposomes, lipid nanoparticles).
  • the compositions have an encapsulation efficiency from about 75% to about 95%, or from about 85% to about 90%. In some examples, the encapsulation efficiency is from about 75% to about 100%. In some examples, the encapsulation efficiency is from about 75% to about 95%. In some examples, the encapsulation efficiency is from about 75% to about 90%. In some examples, the encapsulation efficiency is from about 75% to about 85%. In some examples, the encapsulation efficiency is from about 75% to about 80%. In some examples, the encapsulation efficiency is from about 80% to about 95%. In some examples, the encapsulation efficiency is from about 80% to about 90%. In some examples, the encapsulation efficiency is from about 80% to about 85%.
  • Exosomes exosome lipids
  • the lipid compositions provided herein can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes.
  • exosome refers to the small membrane vesicles secreted by most cells that contain cell specific payloads of proteins, lipids and, genetic material and other biomolecules that are transported to other cells in different location of the tissue. Exosomes can be considered liposomal particles. Exosomes or lipid mixtures obtained therefrom, can be used in combination with other transfection agents or helper lipid mixtures.
  • Exosomes are also referred to as microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes and oncosomes.
  • compositions can include, for example, a lipid of Formula (I) and one or more exosomes (and optionally a payload); a lipid of Formula (I), and one or more exosomes, and one or more neutral lipids (and optionally a payload); a lipid of Formula (I), and one or more exosomes, one or more neutral lipids, and one or more stabilizing agents (and optionally a payload); a lipid of Formula (I), and one or more exosomes, and one or more neutral lipids, optionally one or more stabilizing agents, and optionally one or more cell penetrating peptides (and optionally a payload).
  • compositions include, for example; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes (and optionally a payload); a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, and one or more neutral lipids (and optionally a payload); a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, one or more neutral lipids, and one or more stabilizing agents (and optionally a payload); a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, and one or more neutral lipids, optionally one or more stabilizing agents, and optionally one or more cell penetrating peptides (and optionally a payload).
  • compositions may include a lipid of Formula (I), and one or more biological materials derived or purified from viruses, such a viral lipids, viral proteins or a viral nucleic acid fragment, optionally with one or more stabilizing agents.
  • viruses such as a viral lipids, viral proteins or a viral nucleic acid fragment
  • stabilizing agents such as a stabilizing agents for stabilizing the lipid compositions.
  • the lipid compositions provided herein can include one or more cationinc/ionizable lipids, in addition to the lipid of Formula (I).
  • some lipid compositions include a cationic/ionizable lipid selected from the group consisting of DOTMA, DOTAP, DMRIE, DC- Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, DHDMS, HDMS TMDOS,, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N- 1-(2,3-diapalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3- diaoleoyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1
  • the ionizable lipid includes one or more cationic lipid selected from GeneIn TM , LipofectAmine TM 2000, LipofectAmine TM , Lipofectin®, DMRIE-C, CellFectin® (Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega, Madison, WI), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega), Transfectin TM (BioRad, Hercules, CA), SilentFect TM (Bio-Rad), Effectene® (Qiagen, Valencia, CA), DC-chol (Avanti Polar Lipids), GenePorter® (Gene Therapy Systems, San Diego, CA), DharmaFect 1® (D
  • the lipid of Formula (I), or the combination of the lipid of Formula (I) with one or more cationic/ionizable lipids is present at about 5-80 mol% of the lipid composition.
  • some lipid compositions include less than 50 mol% Formula (I), or combination of Formula (I) and one or more additional cationic/ionizable lipids.
  • Other lipid compositions include more than 50 mol% Formula (I), or combination of Formula (I) and one or more additional cationic/ionizable lipids.
  • some lipid compositions include a lipid of Formula (I), or a combination of Formula (I) and one or more cationic/ionizable lipids at 15-80 mol%, a sterol at 20-60 mol%, a stabilizing agent at 0.5-5 mol%, and a phospholipid at 1-40 mol% of the lipid composition.
  • An exemplary lipid composition can include about 20-60 mol % Formula (I) lipid or combination of Formula (I) lipid and one or more additional cationic/ionizable lipids, about 5-25 mol % phospholipid, about 25-55 mol% sterol or sterol derivative; and about 0.5-15 mol% stabilizing agent.
  • Another exemplary lipid composition includes a about 50 mol % lipid of Formula (I) or combination of lipid of Formula (I) and one or more cationic/ionizable lipids, about 1.5 mol% stabilizing agent, about 38.5 mol% sterol or sterol derivative, and about 10 mol% phospholipid.
  • Another exemplary lipid composition comprises about 55% lipid of Formula (I) or combination of lipid of Formula (I) and one or more cationic/ionizable lipids, about 2.5 mol % stabilizing agent, about 32.5 mol % sterol or sterol derivative, and about 10 mol % phospholipid.
  • the lipid compositions include one or more neutral co-lipids, although the skilled artisan will recognize that other co- lipids may be used.
  • the neutral lipid may be, for example, selected from the group consisting of a sterol or sterol derivative, a phospholipid, or a combination thereof.
  • the neutral lipid can be present at about 5-60 mol% of the overall lipid formulation. In some embodiments, neutral lipid(s) are present from about 15-50 mol%, e.g., 25-40 mol %.
  • the amount of the neutral lipid in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % of the overall lipid formulation.
  • the lipid composition includes a neutral lipid, and the neutral lipid includes cholesterol.
  • the neutral lipid includes sterols, or lipids containing sterol moieties (“sterol derivatives”).
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • exemplary sterols and lipids containing sterol moieties useful in the lipid composition formulations provided herein include, but are not limited to cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • Some lipid composition formulations provided herein include a sterol or sterol derivative.
  • the sterols or sterol derivatives can be present at about 5-60 mol% of the overall lipid composition formulation.
  • the sterol or sterol derivatives are present from about 15-50 mol%, e.g., 25-40 mol %.
  • the amount of the sterol (such as cholesterol) or sterol derivative in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % of the overall lipid formulation.
  • compositions provided herein do not include a sterol or sterol derivative.
  • the compositions include a structural lipid in a concentration of 14-50 mol% of the composition, excluding any payload, if present.
  • the structural lipid is selected from the group consisting of: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha- tocopherol, hopanoids, phytosterols, steroids, and any combination thereof.
  • the neutral lipid includes a phospholipid.
  • the neutral lipid is a phospholipid such as dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • the neutral lipid includes N-Palmitoyl phosphatidylethanolamine.
  • the neutral lipid includes N-Oleoyl-DPPE.
  • the neutral lipid includes diphytanoylphosphatidylethanolamine (DPhPE).
  • the neutral lipid includes Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3- phosphoethanolamine).
  • the neutral lipid includes Lyso-PC (1-acyl-3- hydroxy-sn-glycero-3-phosphocholine).
  • the neutral lipid includes distearoylphosphatidylcholine (DSPC). In some embodiments, the neutral lipid includes dioleoylphosphatidylcholine (DOPC). In some embodiments, the neutral lipid includes dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the neutral lipid includes palmitoyloleoylphosphatidylcholine (POPC). In some embodiments, the neutral lipid includes palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal).
  • POPE palmitoyloleoyl-phosphatidylethanolamine
  • DOPE-mal dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate
  • the neutral lipid includes dipalmitoyl phosphatidyl ethanolamine (DPPE). In some embodiments, the neutral lipid includes dimyristoylphosphoethanolamine (DMPE). In some embodiments, the neutral lipid includes distearoyl-phosphatidylethanolamine (DSPE). In some embodiments, the neutral lipid includes 16-O-monomethyl PE. In some embodiments, the neutral lipid includes 16-O-dimethyl PE. In some embodiments, the neutral lipid includes 18-1-trans PE. In some embodiments, the neutral lipid includes 1-stearoyl-2-oleoyl-phosphatidyethanol amine (SOPE).
  • SOPE 1-stearoyl-2-oleoyl-phosphatidyethanol amine
  • the neutral lipid includes 1,2-dioleoyl-sn-glycero-3-phophoethanolamine (trans DOPE). In some embodiments, the neutral lipid includes any combinations thereof.
  • Exemplary phospholipids useful in the compositions disclosed herein include, but are not limited to, dioleoylphosphoethanolamine (DOPE), diphytanolphosphatidylethanolamine (DPhPE), Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3- hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE),
  • DOPE dio
  • the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 0.5 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 1 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 2 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 3 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 4 mol %.
  • the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 5 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 10 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 12 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 15 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 16 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 17 mol %.
  • the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 18 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 19 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 20 mol %.
  • PEG-modified lipids [0179]
  • the lipid compositions provided herein can also include a stabilizing agent, such as a stabilizing lipid. Stabilizing lipids can be neutral lipids, or they can be charged.
  • Stabilizing lipids that can advantageously be used in the formulations provided herein include, but are not limited to, polyethylene glycol (PEG)-modified lipids.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG- modified 1,2-diacyloxypropan-3-amines.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG- modified 1,2-diacyloxypropan-3-amines.
  • PEGylated lipids PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • PEGylated lipids include DSPE-PEG 2000 (distearoyl- poly(ethylene glycol)), PEG2000-DSPE-PEG500, 1,2-distearyloxypropyl-3-amine-PEG2000, hexadecylcarbamoylmethyl hexadecanoate-PEG2000, cholesteryl hemisuccinate-PEG2000, photocleavable cholesteryl-PEG 2000 , cholesterol-hyperbranched polyglycerol, poly(2-methyl-2- oxazoline) (PMOZ)- or poly(2-ethyl-2-oxazoline) (PEOZ)-DSPE, poly(hydroxyethyl-l- asparagine)-succinyldioc
  • the lipid compositions do not include a PEGylated lipid.
  • Other stabilizing agents useful in the compositions disclosed herein include, e.g., polyglycol lipids, oxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co-polymers, and amphiphilic branched polymers.
  • the stabilizing agent e.g., PEGylated lipid or other stabilizing agent
  • the stabilizing agent is present at about 0.1 - 5 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol %, 3 mol%, 3.5 mol %, 4 mol %, 4.5 mol%, 5 mol%, or any value in between, of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol% to about 5 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol% to about 4 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol% to about 3 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 0.5 mol% to about 2 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 0.5 mol% to about 1 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 1 mol% to about 5 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 1 mol% to about 4 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 1 mol% to about 3 mol% of the lipid composition.
  • the stabilizing agent is present at about 1 mol% to about 2 mol% of the lipid composition.
  • the compositions include a stabilizing agent in a concentration of 0.1-10 mol% of the composition, excluding any payload, if present.
  • stabilizing agent is selected from the group consisting of: a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof.
  • the stabilizing agent is a PEG-modified lipid.
  • Exemplary PEG-modified lipids include, but are not limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-ceramide conjugate, a PEG-modified dialkylamine, a PEG-modified 1,2-diacyloxypropan-3-amine, and any combination thereof.
  • the PEG-modified lipid is selected from PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and any combination thereof.
  • the stabilizing agent comprises one or more phospholipids selected from the group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleo
  • the formulations may also include one or more lipids derived from viral capsids, e.g., from enveloped viruses.
  • Polyamine Components / Polymers may also include one or more polyamine transfection agent or polymer, such as dense star dendrimers, PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine, polyethylenimine conjugates, polylysine, polyarginine, polyornithine, histone, and any combination thereof.
  • the polymer is a linear or branched PEI.
  • transfection Enhancing Agents such as a fusion agent (such as an endosomal release agent), a cell surface ligand and/or a nuclear localization agent such as a nuclear receptor ligand peptide
  • transfection enhancing agents include, but are not limited to, reovirus-related fusogenic peptides (see, e.g., WO07/130073, which is hereby incorporated by reference in its entirety), enveloped and nonenveloped virus particles or inactivated virus, particles or proteins form these viruses, insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein
  • compositions described herein may include one or more transfection enhancing agents (e.g., a polycationic nucleic acid binding moiety).
  • the transfection enhancing agent selected from an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, and any combination thereof.
  • the one or more transfection enhancing agents includes an amphipathic peptide.
  • the lipid nanoparticle compositions provided herein can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes.
  • Other cell penetrating peptides useful in the compositions provided herein include those provided in Table II (See WO 2023/018990, which is hereby incorporated by reference in its entirety), below: TABLE II
  • compositions described herein can be used to transfect cells in vitro or ex vivo.
  • WO07/130073 at pages 54-60 describes "before” and “after” protocols for transfection where the components of a transfection complex are mixed in differing orders prior to addition to a cell culture.
  • a liposomal preparation of the lipid, with or without colipid is prepared, and is then mixed with a payload/macromolecule, such as a nucleic acid molecule, to form a transfection complex.
  • the complex is then added to a cell culture and transfection is monitored using well known methods. Additional components such as cell surface ligands, fusion agents, nuclear localization agents and the like may be added to the nucleic acid prior to admixture with the liposome, or may be added to the liposome prior to addition of nucleic acid.
  • the contacting of the cell occurs in vitro. In some embodiments, the contacting of the cell occurs ex vivo. In some embodiments, the contacting of the cell occurs in vivo.
  • Cells which can be transfected according to these methods include, but are not limited to, virtually any eukaryotic cell including primary cells, cells in culture, a passaged cell culture or a cell line, and cells in cultured tissue.
  • Suitable cells include human cell lines and animal cell lines.
  • the cell may be a fibroblast, an immune cell (e.g., a T cell, and NK cell) a pluripotent cell (e.g., a pluripotent stem cell, an iPSC, a neural stem cell, a mesenchymal stem cell, a progenitor cell, or the like).
  • the cells can be attached cells or cells in suspension (suspension cells).
  • the cells can also be an organoid or a spheroid.
  • the cells are suspension CHO-S cells and suspension 293-F cells.
  • cells that may be used include, without limitation, 293, 293-S, CHO, CHO K-1, Cos, 3T3, Hela, primary fibroblasts, A549, Be2C, SW480, CHOK1, Griptite 293, HepG2, Jurkat, LNCap, MCF-7, NIH-3T3, PC12, C6, Caco-2, COS-7, HL60, HT-1080, IMR-90, K-562, SK-BR3, PHP1, HUVEC, MJ90, NHFF, NDFF and primary neurons.
  • the cell is a mammalian cell.
  • the lipid nanoparticle formulations are preferably administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • a bolus injection see Stadler, et al., U.S. Pat. No.5,286,634, which is incorporated herein by reference.
  • Intracellular nucleic acid delivery has also been discussed in Straubringer, et al., Methods in Enzymology, Academic Press, New York.101:512-527 (1983); Mannino et al., Biotechniques 6:682-690 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst.6:239-271 (1989), and Behr, Acc. Chem. Res.26:274-278 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, Rahman et al., U.S. Pat. No.3,993,754; Sears, U.S. Pat.
  • the administration is systemic.
  • the administration is selected from: subcutaneous administration, intramuscular administration, intranasal administration, intra-tumoral administration, administration to the brain, administration to the spinal cord, administration to the eye, administration to the lymph node of a subject, and any combination thereof.
  • a method for producing a protein which includes contacting a cell with a lipid-nucleic acid complex as described above, where the nucleic acid encodes the protein.
  • protein production is in vitro, e.g., bioproduction.
  • cells are incubated to produce the protein and the protein is collected.
  • Cells which can be used for protein production are described above.
  • any composition which includes a lipid of Formula (I) or (II) can be used for transfection of cells.
  • compositions are further discussed herein, and include, but are not limited to compositions comprising lipids of Formula (I) or (II), a co-lipid and an optional transfection enhancing agent such as a fusogenic peptide or protein.
  • the methods provided herein include methods of producing therapeutic or prophylactic proteins, either in vivo or in vitro.
  • the compositions provided herein can be used to deliver mRNA encoding immunogens (e.g, for vaccines), mRNA encoding therapeutic proteins (e.g, growth factors, enzymes, cytokines, or the like), or the like.
  • RNAi double stranded RNA molecule
  • siRNA siRNA molecule designed to inhibit expression of the protein.
  • Methods of designing such RNA molecules are well known in the art.
  • Lipids of Formula (I) are particularly suitable for deliver of RNAi molecules in this fashion. The cells are incubated and the phenotypic consequence of inhibiting production of the selected protein is observed.
  • An aspect of the present application relates to a method of delivering a payload to a cell.
  • the method includes providing a composition described herein; providing a cell; and contacting the cell with the composition.
  • Another aspect of the present application relates to a method for delivering a composition to a subject.
  • the method includes administering the composition described herein to the subject.
  • METHODS OF MANUFACTURE AND METHODS OF USE OF LIPID FORMULATIONS [0200]
  • the lipids described above may be formulated by various methods to be used in transfection.
  • One of the simplest methods for formulation is reverse evaporation, as described in U.S. Pat. No.9,259,475, which is hereby incorporated by reference in its entirety.
  • Other methods for formulation that can be used are sonication and microfluidization.
  • the lipids are formulated as lipid nanoparticles using microfluidic mixing as described, for example, in Roces et al., Pharmaceutics, 12:1095 (2020).
  • Suitable microfluidic mixing devices are commercially available from, for example, Precision Nanosystems (Vancouver, BC).
  • microfluidic mixing combines two fluid streams, one containing the nucleic acid(s) and one containing the lipid of Formula (I) and other components, such as the peptide, ligand and other lipid components as described below.
  • lipid nanoparticle compositions including an RNA
  • solutions of the RNA at concentrations of 0.1 mg/ml in deionized water are diluted in 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution.
  • Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange. Formulations may be dialyzed against phosphate buffered saline (PBS), pH 7.4, using a desired molecular weight cutoff, e.g.10 kD. The resulting nanoparticle suspension may be filtered through a 0.2 ⁇ m sterile filters (Sarstedt, Numbrecht, Germany) into glass vials and sealed.
  • Methods of determining particle size in nanoparticles formulations are well-known in the art.
  • a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the nanoparticle compositions.
  • UV-visible spectroscopy can be used to determine the concentration of nucleic acid (e.g., mRNA) in nanoparticle compositions.
  • a quantity of the composition is diluted in a suitable solvent and the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a spectrophotometer.
  • the concentration of therapeutic and/or prophylactic in the nanoparticle composition can be calculated based on the extinction coefficient of the therapeutic and/or prophylactic used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.
  • a QUANT-ITTM RIBOGREEN ® RNA assay can be used to evaluate the encapsulation of an RNA by the nanoparticle composition using methods provided by the manufacturer.
  • the fluorescence intensity generated after addition of the RIBOGREEN reagent can be measured using a fluorescence plate reader at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm.
  • the fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).
  • REAGENT KITS [0204]
  • the present invention also provides packaging and kits comprising pharmaceutical compositions for use in the methods of the present invention.
  • a lipid composition in the kit is suitable for delivery (e.g., local injection) to a subject.
  • the kit can comprise one or more containers selected from the group consisting of a bottle, a vial, an ampoule, a blister pack, and a syringe.
  • the kit can further include one or more of instructions for use in treating and/or preventing a disease, condition or disorder of the present invention, one or more syringes, one or more applicators, or a sterile solution suitable for reconstituting a pharmaceutical composition of the present invention.
  • the kit comprising a lipid composition provides that the ionizable lipid and neutral lipid are in a separate container from the peptide.
  • the kit comprising a lipid composition provides that the ionizable lipid and neutral lipid are in the same container as the peptide.
  • Components of the transfection compositions described above can be provided in a reagent kit.
  • the kits contain the lipid of Formula (I) above, together with additional components, such as a neutral lipid, a cationic lipid, cell surface ligands, fusion agents, and/or nuclear localization agents and the like.
  • the kit components may be separate or may be premixed in any manner.
  • the lipid of formula (I) may be admixed with one or more neutral lipid. Additional components may also be present in the same container or may be present in one or more separate containers.
  • kits typically include vessels, such as vials and/or tubes, which are packaged together, for example in a cardboard box.
  • the kits can be shipped from a supplier to a customer.
  • a kit that includes a vial that includes a liposomal formulation as described above and, optionally, a transfection agent and a transfection enhancing peptide.
  • the kit can also include, for example, a separate vessel that includes a transfection enhancing agent, such as a transfection enhancing peptide, for example Plus ReagentTM (Invitrogen Corp., Carlsbad, CA).
  • the kit can also include in separate containers, cells, cell culture medium, and a reporter nucleic acid sequence, such as a plasmid that expresses a reporter gene.
  • the culture medium can be reduced-serum medium and/or protein expression medium.
  • kits containing a compound of Formula (I) or (Ia) and additional reagents such as a cationic lipid, a neutral lipid, an amphipathic peptide, an amphipathic peptide comprising a polycationic nucleic acid binding moiety, a cell surface ligand, a cell surface ligand comprising a polycationic nucleic acid binding moiety, a fusion agent, a fusion agent comprising a polycationic nucleic acid binding moiety, a nuclear localization peptide or protein, and a nuclear localization peptide or protein comprising a polycationic nucleic acid binding moiety.
  • additional reagents such as a cationic lipid, a neutral lipid, an amphipathic peptide, an amphipathic peptide comprising a polycationic nucleic acid binding moiety, a cell surface ligand, a cell surface ligand comprising a polycationic nucleic acid binding moiety, a fusion agent,
  • kits may contain one, some, or all of these additional reagents, in any possible combination.
  • the additional reagents include a cationic lipid, an amphipathic peptide and a cell surface ligand that contains a polycationic nucleic acid binding moiety.
  • the cell surface ligand is a peptide or protein
  • the polycationic nucleic acid binding moiety is a polybasic amino acid sequence.
  • a kit comprises individual portions of, or a mixture of, cationic lipid, such as a lipid of Formula (I) or (Ia) and peptide, protein or fragment thereof or modified peptide, protein or fragment thereof.
  • kits comprises individual portions of, or a mixture of, polycationic polymers and peptide, protein or fragments thereof or modified peptide, protein or fragments thereof.
  • Cationic lipid transfection kits can optionally include neutral lipid as well as other transfection-enhancing agents or other additives, and the relative amounts of components in the kit may be adjusted to facilitate preparation of transfection compositions.
  • Kit components can include appropriate medium or solvents for other kit components.
  • An aspect of the present application relates to a kit including one or more compounds described herein and one or more of a structural lipid, an ionizable lipid, and a stabilizing agent.
  • the kit includes one or more compounds described herein, one or more of a structural lipid, one or more stabilizing agent and optionally a payload. In some embodiments, the kit includes one or more compounds described herein, one or more of a structural lipid, one or more stabilizing agent, one or more fusion agent, and optionally a payload.
  • Payloads that can be delivered by the methods of this invention include nucleic acids, proteins, ribonucloeproteins, and the like, including DNA and RNA (including RNAi/siRNA) of any size from any source comprising natural bases or non-natural bases, and include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those which catalyze reactions (ribozymes), and those which function in diagnostic assays (e.g., diagnostic nucleic acids).
  • DNA and RNA including RNAi/siRNA of any size from any source comprising natural bases or non-natural bases
  • RNAi/siRNA include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those which catalyze reactions (ribozymes
  • Therapeutic nucleic acids include those nucleic acids that encode or can express therapeutically useful proteins, peptides or polypeptides in cells, those which inhibit undesired expression of nucleic acids in cells, and those which inhibit undesired enzymatic activity or activate desired enzymes in cells.
  • the payload comprises an RNA molecule.
  • the compositions can be used to deliver RNA payloads such as mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self- amplifying RNA, stRNA, sgRNA, or combinations thereof.
  • the RNA molecule comprises more than one RNA molecule, e.g., more than one mRNA.
  • compositions and methods provided herein can also be readily adapted in view of the disclosure herein to introduce biologically active macromolecules other than nucleic acids including, among others, polyamines, polyamine acids, polypeptides and proteins into eukaryotic cells.
  • Other materials useful for example as therapeutic agents, diagnostic materials, research reagents, which can be bound to the peptides and modified peptides and introduced into eukaryotic cells by the methods of this invention.
  • Yet other payloads include small molecules, nutrients, and the like.
  • the compositions provided herein can be delivered to cells via in vivo administration.
  • the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally).
  • parenterally e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally.
  • the pharmaceutical compositions are administered intravenously, intrathecally, or intraperitoneally by a bolus injection.
  • Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal administration.
  • Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets in vitro and in vivo.
  • applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides.
  • therapy is provided for genetic diseases by supplying deficient or absent gene products.
  • Methods of the present invention may be practiced in vitro, ex vivo, or in vivo.
  • the compositions of the present invention can also be used for delivery of payloads to cells in vivo, using methods which are known to those of skill in the art.
  • the compositions of the invention can be used for delivery of a payload to a sample of patient cells that are ex vivo, then are returned to the patient.
  • the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally).
  • parenterally e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally.
  • the compositions provided herein are administered intravenously, intrathecally, or intraperitoneally by a bolus injection.
  • Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.
  • compositions provided herein are preferably administered to biological samples that have been removed from the organism, then the cells are washed and restored to the organism.
  • the organism may be a mammal, and in particular may be a mammal (e.g., a primate), such as a human. This process is used for cell reprogramming, genetic restoration, immunotherapy, for example.
  • PHARMACEUTICAL COMPOSITIONS [0216]
  • nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions.
  • Pharmaceutical compositions can include one or more nanoparticle compositions.
  • a pharmaceutical composition can include one or more nanoparticle compositions including one or more different therapeutic and/or prophylactic agents.
  • compositions can further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein.
  • pharmaceutically acceptable excipients or accessory ingredients such as those described herein.
  • General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams &Wilkins, Baltimore, Md., 2006.
  • Conventional excipients and accessory ingredients can be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient can be incompatible with one or more components of a nanoparticle composition.
  • Triphenyl phosphine (2.183g, 8.32 mmol, 1.1 eq.) was dissolved in 30 mL of dry THF under inert atmosphere and the temperature was lowered to -78 o C using a methanol/dry ice bath. Diethyl azo dicarboxylate (DEAD) (1.449g, 1.1 eq.) was added dropwise and the mixture stirred for 30 minutes. Then the protected glycerol (1a) (1.0, 7.56 mmol, 1.1 eq.) was added dropwise. The mixture was allowed to stir for 60 minutes at -78 o C. Finally, thiomorpholine (0.78g, 1 eq.) was added dropwise as solution in dry THF (5mL).
  • DEAD Diethyl azo dicarboxylate
  • Di-isopropyl ethyl amine (DIPEA) (2.82 mL, 7 eq.) and myristoyl chloride (2.34g, 4 eq.) were added and the mixture stirred at room temperature and inert atmosphere overnight (about 17h).
  • the reaction mixture was then diluted with dichloromethane, washed twice with water (30mL), dried over anhydrous sodium sulfate, filtered off, and the solvent evaporated under reduced pressure to afford 3.25g of crude product.
  • the crude product was taken up in a mixture of hexane-dichloromethane 9:1 (20mL) resulting in the precipitation of a white solid which was filtered off.
  • compositions including Thiomorpholines and helper lipids were made and complexed with mRNA. As shown in Table 1, the formulations examined varied in molar ratios of thiomorpholines and helper lipids.
  • Lipid nano particle (LNP) formulations were screened and assessed by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload- encoded protein.
  • All the LNP formulations contained one of the thiomorpholines and DOPE, Cholesterol, and DMG-PEG. Some formulations had peptide SEQ ID NO: 47. All the lipids were weighed and solubilized in ethanol at the desired molar ratio. This lipid mix and firefly luciferase (fLuc) mRNA were complexed into LNPs using a microfluidic device.
  • Bioluminescence imaging was quantified in vivo (whole body) and ex vivo (organ) using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.
  • All the LNP formulations with thiomorpholines had particle size >120nm, polydispersity index ⁇ 0.3, (Fig.1) and % encapsulation efficiency (%EE) > 70% except formulation ID 12 (Fig.2).
  • Intravenous administration of the LNP formulations with thiomorpholines resulted in mRNA delivery and luciferase expression distributed between the liver (Fig.3) and the spleen (Fig.4) of the injected mice.

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Abstract

L'invention concerne des lipides ionisables, des compositions comprenant les lipides ionisables, et des méthodes de fabrication et d'utilisation associées. Les lipides ionisables de l'invention peuvent être préparés sous la forme de compositions lipidiques pour l'administration de macromolécules, telles que des acides nucléiques, in vitro, ex vivo, ou in vivo.
PCT/US2023/071670 2022-08-05 2023-08-04 Lipides pour l'administration d'acides nucléiques Ceased WO2024031051A1 (fr)

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CN118480173A (zh) * 2024-05-11 2024-08-13 深圳市信必递生物科技有限公司 一种阳离子聚酯及其制备方法和应用
CN120590288A (zh) * 2025-08-07 2025-09-05 北京悦康科创医药科技股份有限公司 多胺基、多羟基可电离阳离子脂质、包含其的组合物及用途

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118480173A (zh) * 2024-05-11 2024-08-13 深圳市信必递生物科技有限公司 一种阳离子聚酯及其制备方法和应用
CN120590288A (zh) * 2025-08-07 2025-09-05 北京悦康科创医药科技股份有限公司 多胺基、多羟基可电离阳离子脂质、包含其的组合物及用途

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