WO2023056872A1 - 一种脂质纳米颗粒组合物以及由其制备的药物递送系统 - Google Patents

一种脂质纳米颗粒组合物以及由其制备的药物递送系统 Download PDF

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WO2023056872A1
WO2023056872A1 PCT/CN2022/122221 CN2022122221W WO2023056872A1 WO 2023056872 A1 WO2023056872 A1 WO 2023056872A1 CN 2022122221 W CN2022122221 W CN 2022122221W WO 2023056872 A1 WO2023056872 A1 WO 2023056872A1
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lipid
branched
integer
same
mrna
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French (fr)
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林耀新
乌磊
辛琪
蒋俊
王茜婷
王浩
栗世铀
王利娜
黄晓敏
张欣月
白雪皎
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北京启辰生生物科技有限公司
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the invention belongs to the technical field of biomedical preparations, and in particular relates to a lipid nanoparticle containing a novel lipid compound, and a pharmaceutical composition or drug delivery system prepared therefrom carrying an active ingredient, such as an mRNA vaccine.
  • nucleic acid preparations are being developed for the treatment of various major diseases such as infectious diseases, cancer, rare diseases, etc.
  • nucleic acid preparations include: DNA, antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer (aptamer), ribozyme (ribozyme) etc.
  • ASO antisense nucleic acid
  • siRNA small interfering RNA
  • miRNA microRNA
  • saRNA small activating RNA
  • mRNA messenger RNA
  • aptamer aptamer
  • ribozyme ribozyme
  • mRNA vaccines can induce B cell and T cell immune responses, can cause immune memory effects, deliver more effective antigens, and can express multiple antigens at one time.
  • mRNA only needs to pass through the cell membrane in the cytoplasm to efficiently express antigenic proteins; mRNA has no risk of gene integration into the genome.
  • mRNA is easily degraded after being translated into protein, and its transient expression characteristics not only ensure the safety of mRNA drugs, but also make the dose controllable, avoiding the antigen immune tolerance caused by long-term exposure of vaccine drugs (no specific antigen). state of the reaction).
  • nucleic acid preparations are negatively charged and most of them have relatively large molecular weights, making it difficult to directly enter cells.
  • RNA is unstable and easily degraded by nucleases during the introduction into the body, thereby losing its biological function. Therefore, developing an efficient, safe and universal nucleic acid delivery system is an urgent problem to be solved in the process of nucleic acid drug transformation.
  • nucleic acid delivery methods include chemical modification, bioconjugation technology, nanocarrier technology, lipid-based formulations, exosomes, spherical nucleic acids, DNA nanostructures, stimuli-responsive polymer nanomaterials, etc.
  • the relatively mature nucleic acid delivery carrier is lipid nanoparticle (LNP)
  • siRNA drug Onpattro (patisiran) wrapped in LNP was approved for marketing in 2018, and the mRNA vaccine wrapped in LNP has been officially approved by the FDA in 2021 for the prevention and control of the new crown epidemic.
  • the clinical results show high effectiveness, and there are currently no serious adverse reactions.
  • the main components of such lipid formulations include: cationic/ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-lipid conjugates.
  • the charged head of cationic lipids can bind to negatively charged nucleic acids, and can also bind to phospholipid molecules on the cell membrane, playing a key role in the process of nucleic acid encapsulation and membrane fusion .
  • lipid nanoparticles with ionizable cationic lipids have greater application value.
  • Ionizable cationic lipids consist of three important structural components: a hydrophilic polar head containing an amine group; a hydrophobic lipid chain; and a connecting chain connecting the polar head and the nonpolar tail.
  • commercial ionizable cationic lipids mainly include MC3 series and ALC-0315 and SM-102 for the new crown mRNA vaccine.
  • MC3 has a strong liver targeting, and its application to nucleic acid preparations that may have potential hepatotoxicity will be limited, and MC3 is developed for the delivery of siRNA with a smaller molecular weight, and its loading capacity for nucleic acid preparations with a larger molecular weight Restrictions may apply.
  • the delivery efficiency of ALC-0315 and SM-102 needs to be further improved.
  • nucleic acids such as mRNA molecules
  • mRNA molecules which are structurally unstable, easily degraded by nucleases and difficult to enter cells in biological applications
  • the present invention is based on newly synthesized ionizable cationic lipid molecules to form a lipid-based delivery system with multiple functions .
  • the first aspect of the present invention provides a lipid nanoparticle composition
  • the lipid nanoparticle composition contains lipid nanoparticles
  • the lipid nanoparticles include: ionizable cationic lipid molecules of formula I.
  • the lipid nanoparticle composition further comprises other lipid molecules.
  • the other lipid molecules can be lipid molecules known or routinely used in the art for constructing lipid nanoparticles, including but not limited to neutral lipid molecules, cholesterol-like lipid molecules, PEGylated lipid molecules .
  • the lipid nanoparticle composition further comprises an active ingredient, and the active ingredient may be a small molecule compound, nucleic acid, protein, polypeptide and the like.
  • the active ingredient is located in lipid nanoparticles.
  • the nucleic acid includes but not limited to DNA, antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer (aptamer) and the like.
  • the lipid nanoparticles contain the lipid molecule of formula I accounting for 30-60 mol% of the total lipid molecules, preferably 32-55mol%, more preferably 34-46mol% .
  • the lipid nanoparticles may contain neutral lipid molecules accounting for 5-30mol% of the total lipid molecules, preferably 8-20mol%, more preferably 9-16mol% .
  • the lipid nanoparticles may contain cholesterol lipid molecules accounting for 30-50 mol% of the total lipid molecules, preferably 35-50 mol%, more preferably 37-49 mol% .
  • the lipid nanoparticles may contain PEGylated lipid molecules accounting for 0.4-10 mol% of the total lipid molecules, preferably 0.5-5 mol%, more preferably 1.3-2.7 mol%.
  • the ratio of the total mass of lipid molecules to the mass of nucleic acid in the lipid nanoparticle composition is 5-20:1.
  • the structural formula of the ionizable cationic lipid molecule of formula I is in:
  • Q is a substituted or unsubstituted linear C2-20 alkylene group, one or more C atoms of the alkylene group are optionally replaced by heteroatoms independently selected from O, S and N; or , Q is a substituted or unsubstituted, saturated or unsaturated 4-6 membered ring, and the ring atoms of the 4-6 membered ring optionally contain 1 or more hetero atom; the substituted substituent group is selected from halogen, -OH, straight or branched C1-20 alkyl, straight or branched C1-20 alkoxy, straight or branched C2-20 Alkenyl, straight or branched C2-20 alkynyl, -CH 2 CH(OH)R 5 ,
  • R 1 , R 2 , R 3 , and R 4 may be the same or different, each independently selected from hydrogen, substituted or unsubstituted linear or branched C1-30 alkyl, substituted or unsubstituted linear or branched Chain C2-30 alkenyl, substituted or unsubstituted straight chain or branched C2-30 alkynyl, one or more C atoms of the alkyl, alkenyl or alkynyl are optionally independently selected from The heteroatoms of O, S and N are replaced, or -CH 2 CH(OH)R 5 ; the substituted substituent group is selected from halogen, -OH, linear or branched C1-10 alkyl, linear Or branched C1-10 alkoxy;
  • the condition is that at least one of R 1 , R 2 , R 3 , R 4 is
  • R is selected from hydrogen, substituted or unsubstituted straight chain or branched C1-30 alkyl, substituted or unsubstituted straight chain or branched C2-30 alkenyl, substituted or unsubstituted straight chain or branched Chain C2-30 alkynyl, one or more C atoms of the alkyl, alkenyl or alkynyl are optionally replaced by heteroatoms independently selected from O, S and N; the substituted substituent The group is selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy;
  • R is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, -OH;
  • n is selected from an integer of 1 to 8
  • m is selected from an integer of 0 to 8
  • n and m are independent of each other, and can be the same or different;
  • n and m in each of the groups are independent of each other and may be the same or different.
  • Q is a substituted or unsubstituted linear C2-20 alkylene group, and one or more C atoms of the alkylene group are optionally independently selected from O, S and N heteroatoms replaced;
  • R 8 and R 9 are independently selected from substituted or unsubstituted linear C1-10 alkylene groups, and one or more C atoms of the alkylene groups are optionally independently selected from O, S and N are replaced by heteroatoms;
  • R 7 is hydrogen, halogen, -OH, straight or branched C1-20 alkyl, straight or branched C2-20 alkenyl, straight or branched C2-20 alkyne group, or -CH 2 CH(OH)R 5 , or
  • the substituted substituent group is halogen, -OH, straight-chain or branched C1-10 alkyl, straight-chain or branched C1-10 alkoxy;
  • Q is Wherein: x and y can be the same or different, independently selected from the integers of 1 to 8; the definition of R7 is the same as the above; preferably, x or y are the same or different, and are selected from the integers of 1 to 3, such as 1, 2 or 3; Preferably, R 7 is a straight chain or branched C1-4 alkyl group, such as methyl, ethyl, n-propyl, n-butyl, etc.
  • the saturated or unsaturated 4-6 membered ring is piperazinyl or cyclohexyl.
  • R6 is -OH.
  • n is selected from an integer of 4-8
  • m is selected from an integer of 4-8.
  • the compound of formula I is the following formula A, B, C or D:
  • each n 1 is independent of each other and may be the same or different, each n 1 is selected from an integer of 1 to 8, each m 1 is independent of each other, may be the same or different, and each m 1 is selected from 0 to 8 Integer; preferably, each n 1 is selected from an integer of 4-8, and each m 1 is selected from an integer of 4-8; preferably, each n 1 is the same as each other, and each m 1 is the same as each other.
  • each n 2 is independent of each other and may be the same or different, each n 2 is selected from an integer of 1 to 8, each m 2 is independent of each other, may be the same or different, and each m 2 is selected from 0 to 8 Integer; preferably, each n 2 is selected from an integer of 4 to 8, and each m 2 is selected from an integer of 4 to 8; preferably, each n 2 is the same as each other, and each m 2 is the same as each other.
  • each n 3 is independent of each other and may be the same or different, each n 3 is selected from an integer of 1 to 8, each m 3 is independent of each other, may be the same or different, and each m 3 is selected from 0 to 8 An integer; preferably, each n 3 is selected from an integer of 4 to 8, and each m 3 is selected from an integer of 4 to 8; preferably, each n 3 is the same as each other, and each m 3 is the same as each other.
  • each n 4 is independent of each other and may be the same or different, each n 4 is selected from an integer of 1 to 8, each m 4 is independent of each other, may be the same or different, and each m 4 is selected from 0 to 8 Integer; preferably, each n 4 is selected from an integer of 4 to 8, and each m 4 is selected from an integer of 4 to 8; preferably, each n 4 is identical to each other, and each m 4 is identical to each other.
  • the compound of formula I is selected from the following compounds shown in Table 1:
  • the molar percentage of the lipid molecule of formula I in the lipid of the lipid nanoparticle is 30-60 mol%, for example 32-55 mol%, for example can be 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol% %, 35mol%, 36mol%, 37mol%, 38mol%, 39mol%, 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol%, 46mol%, 47mol%, 48mol%, 49mol%, 50mol%, 51mol%, 52mol%, 53mol%, 54mol%, 55mol%, etc.
  • the neutral lipid molecule is an uncharged lipid molecule or a zwitterionic lipid molecule, such as a phosphatidylcholine compound, or/and a phosphatidylethanolamine compound.
  • the structure of phosphatidylcholine compounds is shown in formula E: E;
  • neutral lipid molecules include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphorylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine ( DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2- Palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidyl
  • the neutral lipid molecule may be selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE ).
  • the neutral lipid molecule may be dimyristoylphosphatidylethanolamine (DMPE).
  • the neutral lipid molecule may be dipalmitoylphosphatidylcholine (DPPC).
  • the mole percentage of neutral lipid molecules in the lipid of lipid nanoparticles is 5-30mol%, such as 8-20mol%, such as 8mol%, 9mol%, 10mol%, 11mol%, 12mol% , 13mol%, 14mol%, 15mol%, 16mol%, 17mol%, 18mol%, 19mol%, 20mol%.
  • cholesterol-like lipid molecules refer to sterols and lipids containing sterol moieties, including but not limited to cholesterol, 5-heptadecylresorcinol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol , brassicasterol, tomatine, tomatine, ursolic acid, ⁇ -tocopherol and its mixture, cholesterol hemisuccinate.
  • the cholesteryl lipid molecule is cholesterol (CHOL).
  • the cholesterol lipid molecule is cholesterol hemisuccinate.
  • the molar percentage of cholesterol-like lipid molecules in the lipids of lipid nanoparticles is 30-50 mol%, for example, it can be 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%. , 37mol%, 38mol%, 39mol%, 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol%, 46mol%, 47mol%, 48mol%, 49mol%, 50mol%, etc.
  • a PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety.
  • the lipid moiety can be derived from diacylglycerol or diacylglycamide, including dialkyl groups having alkyl chain lengths independently comprising about C4 to about C30 saturated or unsaturated carbon atoms. Those with glycerol or dialkylglycerolamide groups, where the chain may contain one or more functional groups, such as amides or esters.
  • the alkyl chain length comprises about C10 to C20.
  • the dialkylglycerol or dialkylglyceramide group may also contain one or more substituted alkyl groups.
  • PEG means any polyethylene glycol or other polyalkylene ether polymer.
  • the PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide.
  • the PEG moiety can be substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups.
  • the PEG moiety comprises a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, e.g., J.
  • the PEG moiety PEG copolymers are not included, for example it could be a PEG monopolymer.
  • the molecular weight of PEG is from about 130 to about 50,000, in a subembodiment, from about 150 to about 30,000, in a subembodiment, from about 150 to about 20,000, in a subembodiment, from about 150 to about 15,000, in a subembodiment, about 150 to about 10,000, in a subembodiment, about 150 to about 6,000, in a subembodiment, about 150 to about 5,000, in a subembodiment, from about 150 to about 4,000, in subembodiments from about 150 to about 3,000, in subembodiments from about 300 to about 3,000, in subembodiments from about 1,000 to about 3,000, and in subembodiments In regimen
  • the PEG is "PEG 2000," which has an average molecular weight of about 2,000 Daltons.
  • n can range from about 30 to about 60.
  • n can range from about 35 to about 55.
  • n can range from about 40 to about 50.
  • n can range from about 42 to about 48. In some embodiments, n can be 45.
  • R can be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R can be unsubstituted C1-C30 alkyl, such as C1-C20 alkyl, C1-C10 alkyl, C1-C6 alkyl. In some embodiments, R can be H, methyl or ethyl.
  • a PEGylated lipid molecule can be expressed as "lipid fraction-PEG-number average molecular weight" or "PEG-lipid fraction” or “PEG-number average molecular weight-lipid fraction", the lipid The main part is diacylglycerol or diacylglycerolamide, selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylglycerol, dilaurylglyceramide, dimyristylglycerolamide, Palmitoylglyceramide, Distearoylglyceramide, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine; PEG The number average molecular weight is from about 130 to about 50,000, such as from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 1
  • the PEGylated lipid molecule may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol ( PEG-DSPE), PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-( Cholesta-5-ene-3[ ⁇ ]-oxy)carboxamido-3',6'-dioxahoctyl]carbamoyl-[ ⁇ ]-methyl-poly(ethylene glycol), PEG -DMB(3,4-Di-tetradecyloxybenzyl-[ ⁇ ]-methyl-poly(ethylene glycol) ether), 1,2-Dimyristo
  • the PEGylated lipid molecule can be DMG-PEG2000. In some embodiments, the PEGylated lipid molecule can be DSG-PEG2000. In one embodiment, the PEGylated lipid molecule can be DSPE-PEG2000. In one embodiment, the PEGylated lipid molecule can be DMA-PEG2000. In one embodiment, the PEGylated lipid molecule can be C-DMA-PEG2000. In one embodiment, the PEGylated lipid molecule can be is DSA-PEG2000. In one embodiment, the PEGylated lipid molecule can be PEG2000-C11. In some embodiments, the PEGylated lipid molecule can be PEG2000-C14.
  • the PEGylated lipid molecule can be PEG2000-C14. In some embodiments, the PEGylated The lipid molecule can be PEG2000-C16. In some embodiments, the PEGylated lipid molecule can be PEG2000-C18.
  • the molar percentage of PEGylated lipid molecules in the lipid of lipid nanoparticles is 0.4-10 mol%, for example 0.5-5 mol%, for example can be 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8mol%, 0.9mol%, 1.0mol%, 1.1mol%, 1.2mol%, 1.3mol%, 1.4mol%, 1.5mol%, 1.6mol%, 1.7mol%, 1.8mol%, 1.9mol% , 2.0mol%, 2.1mol%, 2.2mol%, 2.3mol%, 2.4mol%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8mol%, 2.9mol%, 3.0mol%, 3.1mol%, 3.2 mol%, 3.3mol%, 3.4mol%, 3.5mol%, 3.6mol%, 3.7mol%, 3.8mol%, 3.9mol%, 4.0mol%, 4.1mol%, 4.2mol%, 4.3mol%,
  • the lipid nanoparticles contain lipid molecules represented by formula C, neutral lipid molecules, cholesterol-like lipid molecules, and PEGylated lipid molecules, wherein:
  • each n 3 is independent of each other and can be the same or different, each n 3 is selected from an integer of 1 to 8, each m 3 is independent of each other, can be the same or different, and each m 3 is selected from an integer of 0 to 8 ; Preferably, each n 3 is selected from an integer of 4 to 8, and each m 3 is selected from an integer of 4 to 8; preferably, each n 3 is the same as each other, and each m 3 is the same as each other.
  • the molar percentage of the ionizable cationic lipid molecule represented by formula C in the lipid in the lipid nanoparticle is 32-55 mol%, preferably 34-46 mol%.
  • the molar percentage of neutral lipid molecules in lipid nanoparticles is 8-20
  • Cholesterol lipid molecules are selected from cholesterol, cholesterol hemisuccinate.
  • the molar percentage of cholesterol-like lipid molecules in the lipid in the lipid nanoparticles is 30-50 mol%, preferably 35-50 mol%, more preferably 37-49 mol%.
  • PEGylated lipid molecules are expressed as "Lipid Fraction-PEG-Number Average Molecular Weight", said lipid moiety is diacylglycerol or diacylglycerolamide selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol Acylglycerol, Distearoylglycerol, Dilaurylglyceramide, Dimyristylglyceramide, Dipalmitoylglyceramide, Distearoylglyceramide, 1,2-Distearoyl-sn-glycerol-3- Phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG is 130-50,000, such as 150-30,000, 150-20,000, 150-15,000, 150-10,000, 150- 6,000, 150 ⁇ 5,000, 150 ⁇ 4,000, 150 ⁇ 3,000, 300 ⁇ 3,000, 1,000 ⁇ 3,000, 1,500 ⁇ 2,500, about 2000
  • said nucleic acid is mRNA.
  • the mRNA may comprise a 5' cap structure, a 5' UTR, an open reading frame (ORF), a 3' UTR and a poly-A tail from the 5' end to the 3' end.
  • the cap structure may be a Cap1 structure, a Cap2 structure or a Cap3 structure.
  • the cap structure is a Cap1 structure.
  • said 5'UTR may comprise the 5'UTR of ⁇ -globin or ⁇ -globin or homologues, fragments thereof.
  • the 5'UTR comprises at least 60%, at least 70%, at least 75%, at least 80% of the 5'UTR nucleotide sequence of ⁇ -globin shown in SEQ ID NO:6 %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homologous to the nucleotide sequence.
  • the 5'UTR comprises the 5'UTR nucleotide sequence of ⁇ -globin shown in SEQ ID NO:6.
  • the 5'UTR further comprises a Kozak sequence.
  • the Kozak sequence is GCCACC.
  • said 3'UTR may comprise the 3'UTR of ⁇ -globin or ⁇ -globin or a homologue, fragment, or combination of fragments thereof.
  • the 3'UTR comprises at least 60%, at least 70%, at least 75%, at least 80%, at least 85% of a fragment of the ⁇ 2-globin 3'UTR shown in SEQ ID NO:7 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence.
  • the 3'UTR comprises two end-to-end fragments that are at least 60%, at least 70%, and at least 75% of the ⁇ 2-globin 3'UTR shown in SEQ ID NO:7 , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to a nucleotide sequence.
  • the 3'UTR comprises two nucleotide sequences shown in SEQ ID NO:7 connected end to end.
  • the length of the poly-A tail can be 50-200 nucleotides, preferably 100-150 nucleotides, such as 110-120 nucleotides, such as about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides.
  • said open reading frame (ORF) is the open reading frame (ORF) of the S protein mutant of coding 2019-nCov, and its nucleic acid sequence is the nucleotide shown in SEQ ID NO:8
  • the sequences are at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homologous Sexual nucleotide sequence.
  • the amino acid sequence of the S protein mutant after translation of the ORF is composed of the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 3 directly connected from the N-terminal to the C-terminal.
  • the nucleotide sequence of the open reading frame (ORF) of the S protein mutant is shown in SEQ ID NO:8.
  • said mRNA comprises at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the nucleotide sequence shown in SEQ ID NO:9, Nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homologous.
  • said mRNA comprises the nucleotide sequence shown in SEQ ID NO:9.
  • the mRNA of the present invention can be prepared using methods known in the art.
  • the nucleic acid sequence encoding mRNA can be artificially synthesized, and the sequence is cloned into a vector to construct a plasmid for in vitro transcription. Transform the constructed plasmid into host bacteria for culture and amplification, and extract the plasmid. The extracted plasmid was digested into a linear molecule using restriction enzymes immediately behind the polyA tail. Using the prepared linearized plasmid molecule as a template, mRNA was prepared by in vitro transcription method.
  • Cap structure analogs can be added during in vitro transcription to directly obtain capped mRNA; capping enzymes and dimethyltransferases can also be used to add cap structures to mRNA after in vitro transcription.
  • the obtained mRNA can be purified by conventional methods in the art, such as chemical precipitation method, magnetic bead method, affinity chromatography and the like.
  • one or more nucleotides in the mRNA may be modified.
  • one or more nucleotides (e.g., all nucleotides) in the mRNA can be independently replaced by naturally occurring nucleotide analogs or artificially synthesized nucleotide analogs, such as selected from pseudouridine (pseudouridine), 2-thiouridine (2-thiouridine), 5-methyluridine (5-methyluridine), 5-methylcytidine (5-methylcytidine), N6-methyladenosine (N6-methyladenosine) , N1-methylpseudouridine (N1-methylpseudouridine), etc.
  • the present invention also provides an mRNA vaccine, which contains lipid nanoparticles, and the lipid nanoparticles contain lipid molecules represented by formula C, neutral lipid molecules, cholesterol-like lipid molecules, PEGylated A lipid molecule and an mRNA encoding a 2019-nCoV S protein mutant, said mRNA comprising at least 60%, at least 70%, at least 75%, at least 80%, at least the nucleotide sequence shown in SEQ ID NO:9 Nucleotide sequences of 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homology; ratio of total mass of lipid molecules to mass of mRNA 5-20:1; where:
  • each n 3 is independent of each other and can be the same or different, each n 3 is selected from an integer of 1 to 8, each m 3 is independent of each other, can be the same or different, and each m 3 is selected from an integer of 0 to 8 ; Preferably, each n 3 is selected from an integer of 4 to 8, and each m 3 is selected from an integer of 4 to 8; preferably, each n 3 is the same as each other, and each m 3 is the same as each other.
  • the lipid molecules represented by formula C account for 34-46mol% of the lipid in the lipid nanoparticles;
  • Neutral lipid molecules account for 9-16mol% of the lipid in the lipid nano
  • Cholesterol lipid molecules are selected from cholesterol, cholesterol hemisuccinate. Cholesterol-like lipid molecules account for 37-49mol% of the lipid in the lipid nanoparticles;
  • PEGylated lipid molecules are expressed as "Lipid Fraction-PEG-Number Average Molecular Weight", said lipid moiety is diacylglycerol or diacylglycerolamide selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol Acylglycerol, Distearoylglycerol, Dilaurylglyceramide, Dimyristylglyceramide, Dipalmitoylglyceramide, Distearoylglyceramide, 1,2-Distearoyl-sn-glycerol-3- Phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; PEG has a number average molecular weight of from about 130 to about 50,000, such as from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, about 150 to about 5,000
  • the molar ratio of lipid molecules represented by formula C, neutral lipid molecules, cholesterol and PEGylated lipid molecules is 35:15:48.5:1.5.
  • the molar ratio of lipid molecules represented by formula C, neutral lipid molecules, cholesterol and PEGylated lipid molecules is 40:10:48.5:1.5.
  • the molar ratio of lipid molecules represented by formula C, neutral lipid molecules, cholesterol and PEGylated lipid molecules is 45:15:38.5:1.5.
  • the lipid molecule of formula C is compound II-37.
  • said neutral lipid molecule is DOPE and/or DSPC.
  • the PEGylated lipid molecule is DMG-PEG2000 and/or DSPE-PEG2000.
  • the present invention also provides a preparation method of the ionizable cationic lipid molecule of formula I.
  • the ionizable lipid compound of the present invention can be synthesized by existing methods in the art, for example, reacting one equivalent or more of an amine with one equivalent or more of an epoxy-terminated compound under suitable conditions form.
  • the synthesis of ionizable lipid compounds is carried out with or without solvents, and the synthesis can be carried out at higher temperatures in the range of 25-100°C.
  • the resulting ionizable lipid compound can optionally be purified.
  • a mixture of ionizable lipid compounds can be purified to obtain a specific ionizable lipid compound.
  • the mixture may be purified to obtain a specific stereoisomer or regioisomer.
  • the epoxides can be purchased commercially or prepared synthetically.
  • the ionizable lipid compound of the present invention can be prepared using the following general preparation methods.
  • reducing agents include, but are not limited to, lithium aluminum hydride, diisobutylaluminum hydride, and the like.
  • solvent used for reaction includes but not limited to ethers (such as diethyl ether, tetrahydrofuran and dioxane etc.), halogenated hydrocarbons (such as chloroform, dichloromethane and dichloroethane etc.), hydrocarbons (such as n-pentane, n-hexane, benzene and toluene, etc.), and mixed solvents formed by two or more of these solvents.
  • oxidizing agent In the presence of an oxidizing agent, the hydroxyl group of compound A2 is oxidized to an aldehyde group to obtain compound A3.
  • oxidizing agents include, but are not limited to, 2-iodylbenzoic acid (IBX), pyridinium chlorochromate (PCC), pyridinium dichlorochromate (PDC), Dess-Martin oxidizer, manganese dioxide, and the like.
  • solvents used in the reaction include but are not limited to halogenated hydrocarbons (such as chloroform, methylene dichloride and ethylene dichloride, etc.), hydrocarbons (such as n-pentane, n-hexane, benzene and toluene, etc.), nitriles (such as acetonitrile, etc. ), and a mixed solvent formed by two or more of these solvents.
  • halogenated hydrocarbons such as chloroform, methylene dichloride and ethylene dichloride, etc.
  • hydrocarbons such as n-pentane, n-hexane, benzene and toluene, etc.
  • nitriles such as acetonitrile, etc.
  • the aldehyde ⁇ -hydrogen of compound A3 is halogenated with a halogenating reagent to obtain an ⁇ -halogenated aldehyde intermediate, and then in the presence of a reducing agent, the aldehyde group of the ⁇ -halogenated aldehyde is reduced to hydroxyl group to obtain compound A4.
  • a reducing agent examples include, but are not limited to, N-chlorosuccinimide (NCS) and N-bromosuccinimide (NBS).
  • reducing agents include, but are not limited to, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.
  • Compound A4 is subjected to an intramolecular nucleophilic substitution reaction in the presence of a base to obtain epoxy compound A5.
  • bases include, but are not limited to, hydroxides or hydrides of alkali metals, such as sodium hydroxide, potassium hydroxide, and sodium hydride.
  • the solvent used in the reaction include, but are not limited to, a mixture of dioxane and water.
  • Compound A5 is subjected to a ring-opening reaction with an amine such as N,N-bis(2-aminoethyl)methylamine to obtain the final compound.
  • an amine such as N,N-bis(2-aminoethyl)methylamine
  • solvents for the reaction include, but are not limited to, ethanol, methanol, isopropanol, tetrahydrofuran, chloroform, hexane, toluene, diethyl ether, and the like.
  • the raw material A1 in the preparation method can be purchased commercially or can be synthesized by conventional methods.
  • the present invention also provides methods of preparing lipid nanoparticle compositions.
  • the preparation method includes: dissolving each lipid molecule in molar ratio with an organic solvent to prepare a mixed lipid solution, using the mixed lipid solution as the organic phase, and using the delivered substance (such as nucleic acid)
  • the aqueous solution is a water phase
  • the organic phase and the water phase are mixed to prepare lipid nanoparticles.
  • Lipid nanoparticles can be prepared using methods including, but not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nanoprecipitation, microfluidics, simple and complex coacervation, and other methods well known to those of ordinary skill in the art .
  • the organic solvent is an alcohol, such as ethanol.
  • the volume ratio of the organic phase to the aqueous phase is (2-4):1.
  • nanoparticles are prepared using a microfluidic platform.
  • the preparation method further includes the step of separating and purifying the lipid nanoparticles.
  • the preparation method further includes the step of freeze-drying the lipid nanoparticles.
  • the ionizable lipid molecular structure of Formula I of the present invention contains two adjacent cis double bonds, so that it has a higher encapsulation efficiency when it is subsequently applied to the delivery system to encapsulate active substances (such as nucleic acids such as mRNA), Better cell transfection rate; in addition, when preparing lipid nanoparticles, the particle size of lipid nanoparticles obtained is more uniform.
  • active substances such as nucleic acids such as mRNA
  • the ionizable lipid compounds of the invention are particularly suitable for the preparation of nanoparticles of solid structure.
  • the S protein mutant encoded by it has high stability, and the lipid nanoparticles of the present invention have high encapsulation efficiency and particle size.
  • the diameter is more uniform and has better cell transfection rate.
  • the mRNA vaccine of the present invention has high mRNA encapsulation rate, drug loading capacity, cell transfection efficiency, efficient and stable translation in vivo, antigen stability, and good immune effect.
  • the particle diameter of the lipid nanoparticles ranges from 1 nm to 1000 nm, such as 10-500 nm, 10-200 nm and the like.
  • the lipid nanoparticle of the present invention can also be modified with targeting molecules, making it a targeting agent capable of targeting specific cells, tissues or organs.
  • Targeting molecules can be located on the particle surface.
  • Targeting molecules can be proteins, peptides, glycoproteins, lipids, small molecules, nucleic acids, etc., examples of which include, but are not limited to, antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, Sialoglycoprotein (asialycoprotein), receptor ligand, sialic acid, aptamer, etc.
  • the targeting molecule can be attached to the cholesterol-like lipid molecule or the PEGylated lipid molecule of the lipid nanoparticle.
  • the lipid nanoparticle composition and vaccine of the present invention may further contain one or more pharmaceutical excipients.
  • pharmaceutical excipient means any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, etc., including but not limited to sugars such as lactose, trehalose, glucose and sucrose; starches such as corn Starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; oils, such as peanut oil, cottonseed oil, safflower oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; surfactants, such as Tween 80 (Tween 80); buffers, such as phosphate buffer, acetate buffer and citrate buffer; coloring agents, sweeteners, flavoring and aroma agents, preservatives
  • sugars
  • the lipid nanoparticle composition and the vaccine are liquid preparations, further containing sucrose, and the concentration of sucrose is 5-20% by mass, preferably 8-10%.
  • the lipid nanoparticle composition and vaccine of the present invention can be administered to human beings orally, rectally, intravenously, intramuscularly, intravaginally, intranasally, intraperitoneally, buccally or in the form of oral or nasal spray and/or or animals.
  • the S protein mutant in the present invention is produced by amino acid mutation of the parent S protein.
  • the parent S protein is the S protein of the 2019-nCoV B1.351 mutant strain, and the S protein of the 2019-nCoV B1.351 mutant strain is similar to the S protein of the 2019-nCoV wild strain.
  • the ratio has the following mutations: L18F, D80A, D215G, L242_244L del, R246I, K417N, E484K, N501Y, D614G, A701V (the site is described by the position of the amino acid sequence shown in SEQ ID NO:1).
  • amino acid positions of the S protein mutant and the parent S protein are described based on the amino acid sequence of the wild-type S protein.
  • the amino acid sequence of the wild-type S protein can be obtained at NCBI GeneID: 43740568, with a total of 1273 amino acids. The sequence is shown below, identified as SEQ ID NO: 1 in the present invention.
  • the S protein mutant of the present invention at least comprises an ectodomain, and its ectodomain contains amino acid mutations at the following positions relative to the ectodomain of the parental S protein: F817P, A892P, A899P, A942P, and KV986_987PP and the 682-685 amino acid RRAR mutation It is GSAS; and does not contain the transmembrane domain and cytoplasmic tail of S protein; the domain T4Fibritin Foldon Trimerization Motif that assists in the formation of trimers is directly fused at the C-terminus of the extracellular region.
  • the S protein mutant comprises the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:3 directly connected from N-terminal to C-terminal.
  • alkyl refers to a saturated hydrocarbon group obtained by removal of a single hydrogen atom from a hydrocarbon moiety containing 1 to 30 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl and n-dodecyl.
  • alkenyl denotes a monovalent radical derived from a hydrocarbon moiety having at least one carbon-carbon double bond by removal of a single hydrogen atom.
  • Alkenyl includes, for example, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
  • alkynyl refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by removal of a single hydrogen atom.
  • Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
  • alkoxy means an alkyl group as defined above attached to the parent molecule through an oxygen atom.
  • alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentyloxy, and n-hexyloxy.
  • halo and halogen refer to an atom selected from fluorine, chlorine, bromine and iodine.
  • saturated or unsaturated 4-6 membered ring refers to a ring having 4-6 ring atoms, which may be C, N, S, O, examples of which include but are not limited to 4-6 membered saturated Cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; 4-6 membered aryl, such as phenyl; 4-6 membered heterocyclic group, such as pyrrolidinyl, piperidinyl, piperidine Azinyl, morpholinyl, etc.; 4-6 membered heteroaryl, such as triazolyl, oxazolyl, isoxazolyl, thiazolyl, etc.
  • the saturated or unsaturated 4-6 membered ring is preferably piperazinyl and cyclohexyl.
  • substituted refers to the ability to change one functional group into another, provided that the valences of all atoms are maintained.
  • substituents may be the same or different at each position.
  • nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Nucleobases are referred to herein by the commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The skilled artisan will understand that the T base in the codons disclosed herein is present in DNA, and the T base will be replaced by a U base in the corresponding RNA.
  • a codon-nucleotide sequence disclosed herein in DNA form such as a vector or an in vitro translation (IVT) template, has a T base that is transcribed as a U base in its corresponding transcribed mRNA.
  • codon-optimized DNA sequences comprising T
  • mRNA sequences comprising U
  • equivalent codon maps can be generated by substituting one or more bases with unnatural bases.
  • nucleic acid sequence refers to a contiguous nucleic acid sequence.
  • the sequence may be single- or double-stranded DNA or RNA, such as mRNA.
  • nucleotide sequence encoding refers to a nucleic acid (eg, mRNA or DNA molecule) coding sequence that encodes a polypeptide.
  • the coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in cells of the individual or mammal to which the nucleic acid is administered.
  • the S protein mutants of the present invention are described using the following nomenclature: original amino acid: position: substituted amino acid. According to this nomenclature, for example, substitution of asparagine at position 30 by alanine is represented as: Asn30Ala or N30A; deletion of asparagine at the same position is represented as: Asn30* or N30*; insertion of another amino acid residue, such as Lys Amino acid, expressed as: Asn30AsnLys or N30NK; missing a continuous stretch of amino acid residues, such as missing amino acid residues 242-244, expressed as (242-244)* or ⁇ (242-244) or 242_244del; if combined with other S proteins Compared with the parent, the S protein mutant contains a "deletion" and an insertion at this position, which is expressed as: *36Asp or *36D, indicating that the 36th position is deleted and aspartic acid is inserted at the same time.
  • homology refers to the general relatedness between polymer molecules, for example, between nucleic acid molecules (such as DNA molecules and/or RNA molecules) and/or between polypeptide molecules between.
  • nucleic acid molecules such as DNA molecules and/or RNA molecules
  • homology means the evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestry.
  • homology includes identity and similarity.
  • polymer molecules are considered “homologous” to each other ".
  • the term "homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • identity refers to the overall monomer conservation between polymer molecules, such as between polynucleotide molecules (such as DNA molecules and/or RNA molecules) and/or between polypeptide molecules .
  • the calculation of percent identity between two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences Sequences for optimal alignment and non-identity can be discarded for comparison purposes.Thymine (T) and uracil (U) can be considered equivalent when comparing DNA and RNA.
  • Suitable software programs are available from various sources and are used for the alignment of both protein and nucleotide sequences. For example, Bl2seq, Needle, Stretcher, Water or Matcher etc.
  • coding region and “coding region” refer to the open reading frame (ORF) in a polynucleotide, which when expressed produces a polypeptide or protein.
  • “Operably linked” refers to a functional linkage between two or more molecules, constructs, transcripts, entities, moieties, and the like.
  • domain when referring to a polypeptide refers to a protein having one or more identifiable structural or functional characteristics or properties (e.g., binding ability, serving as a site for protein-protein interaction). ) motif of the polypeptide.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template (e.g., by transcription) from a DNA sequence; (2) processing of an mRNA transcript (e.g., by splicing , editing, 5' capping and/or 3' end processing); (3) translation of mRNA into polypeptide or protein; and (4) post-translational modification of polypeptide or protein.
  • protein mutant or "polypeptide mutant” refers to a molecule whose amino acid sequence differs from a native or reference sequence. Amino acid sequence mutants may have substitutions, deletions, and/or insertions, etc., at certain positions within the amino acid sequence compared to a natural or reference sequence. Typically, a mutant will have at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 80% identity, at least about 90% identity to a native or reference sequence , at least about 95% identity, at least about 99% identity.
  • Figure 1 Analysis of B1.351 mRNA integrity results on the 2100 Bioanalyzer using the RNA 6000nano chip.
  • Figure 2 ELISA method to detect the expression level of S protein in the supernatant after nucleic acid transfection of CHO-K1 cells.
  • Figure 3 3D structure diagram of the S protein mutant.
  • Figure 4 ELISA method to detect the expression level of S protein in the supernatant of cells transfected with the mRNA of the present invention encapsulated in nanoparticles made of II-37.
  • Figure 5 LNP prepared from II-37 and MC3 encapsulates the expression level of S protein in the supernatant of cells transfected with mRNA of the present invention.
  • Figure 6 Statistical graph of intracellular protein expression of mRNA prepared by different in vitro transcription vectors using Firefly Luc as the reporter protein.
  • Fig. 7 Statistical diagram of the production of binding antibodies in vivo after the S protein mutant of the present invention is immunized with BALB/c mice.
  • a is the trimer result of wild-type S protein
  • b is the trimer result of S protein translated from B1.351 mRNA
  • c is the blank control.
  • Figure 8 Statistical graph of the production of binding antibodies and neutralizing antibodies in vivo after the lipid nanoparticles encoding the mRNA of the S protein mutant were immunized in BALB/c mice.
  • d, e, and f are the detection results of binding antibodies after immunization with lipid nanoparticles (LNP) of 5 ⁇ g, 1 ⁇ g, and 0.2 ⁇ g mRNA respectively, and the ordinate in the figure is the concentration ( ⁇ g/ml);
  • g, h, i are the neutralizing antibody detection results after immunization with lipid nanoparticles (LNP) of 5 ⁇ g, 1 ⁇ g, and 0.2 ⁇ g mRNA respectively.
  • the abscissa in the figure is the log-transformed value of the serum dilution factor, and the ordinate is the inhibition rate %.
  • Fig. 10 MTT method to measure the cytotoxicity of II-37-LNP and C14-113-LNP
  • the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
  • the experimental method is a conventional molecular biology method in the field, and can be operated with reference to the molecular biology experiment manual in the field or the instructions of the kit product manual.
  • linolenic alcohol (a2) LiAlH 4 (7.20 g), linolenic acid (50 g, a1) were added to 950 mL THF at 0°C, and the mixture was stirred at 25°C for 2 h.
  • reaction solution was separated through a separatory funnel, washed with saturated brine, dried over sodium sulfate, filtered and concentrated by evaporation, and then the residue was purified by rapid column elution with petroleum ether/ethyl acetate to obtain 29.11 g of the target product 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (a5).
  • Ionizable lipids have two main roles: to bind nucleic acids and to allow the release of nucleic acid molecules in the cell.
  • the pKa of the lipid is an important factor because the lipid needs to be positively charged at low pH to bind nucleic acids, but uncharged at neutral pH so that the formed LNPs do not cause toxicity.
  • the pKa of ionizable lipid II-37 was determined to be 6.81 by TNS dye binding assay.
  • mRNA Take the prepared linearized plasmid molecule as a template, use the in vitro transcription method (the in vitro transcription kit A45975 of Thermo Company) to prepare mRNA, the sequence of the mRNA is as shown in SEQ ID NO: 9, and the mRNA will be referred to as for short below B1.351mRNA, obtained after translation of the mRNA is the S protein mutant of the present invention, and its amino acid sequence is directly connected from the N-terminal to the C-terminal to the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:3.
  • capping enzymes and dimethyltransferases are used to add a cap structure of CAP1 to the mRNA.
  • Quality control of mRNA Use the RNA 6000nano chip to analyze the integrity of the mRNA on the 2100 bioanalyzer. As shown in Figure 1, the transcribed mRNA has a single band and no obvious degradation.
  • the Spike fragment was excised from the commercially available plasmid pCMV3-spike by restriction endonucleases HindIII and EcoRI, and inserted between the HindIII and EcoRI sites of the IVT1 vector of Example 5 to obtain the IVT1-spike plasmid. Point mutations were then performed on the plasmid to obtain the IVT1-spike-D614G plasmid.
  • the spike-D614G mRNA was transcribed in vitro to express the full-length S protein containing the D614G mutation.
  • CHO-K1 cell line was used as the expression system, and the mRNA was transfected with Lipofectamine Messenger MAX Reagent (Invitrogen, Cat#1168-027). After culturing for 48 hours, the cell culture supernatant was collected and used The enzyme-linked immunosorbent assay kit for detecting S protein can detect the expression level of S protein to judge whether mRNA can be translated into protein. The result is shown in Figure 2.
  • spike DNA is the commercialized plasmid pCMV3-spike (purchased from Yiqiao Shenzhou Co., Ltd.), expressing the full-length wild-type S protein;
  • spike-D614G mRNA is the full-length S protein expressed above including the D614G mutation
  • spike B1.351mRNA is the aforementioned B1.351mRNA, which expresses the S protein mutant of the present invention, and the results show that the mRNA of the present invention can highly express the S protein mutant in cells.
  • the S protein mutant After the obtained S protein mutant was purified, its structure was analyzed by cryo-electron microscopy.
  • the 3D structure of the S protein is shown in Figure 3.
  • the S protein mutant is a stable structure of prefusion spike structure. There were 9 differences between the sequence of B1.351 mutant strain and wild strain, 3 of which were in the RBD region.
  • the state of the RBD region of the pre-fusion S protein of the wild strain that has been reported is mainly a structure of 1 OPEN and 2 CLOSE.
  • the structure of the S protein mutant of the present invention is mainly a flexible state of two OPENs and one CLOSE.
  • This structural difference is the structural basis for the enhanced ability of the virus to bind to the receptor ACE2 and the enhanced infectivity, and this structural difference will also lead to significant differences in the immunogenic epitopes of the S protein, so that antibodies induced based on different structures are especially is significantly different from neutralizing antibodies.
  • the organic phase and the aqueous phase are mixed at a volume ratio of 3:1, and a lipid nanoparticle suspension is prepared on a microfluidic platform (eg, PNI Ignite).
  • a lipid nanoparticle suspension is prepared on a microfluidic platform (eg, PNI Ignite).
  • the obtained lipid nanoparticle suspension is centrifugally filtered through a 100KDa ultrafiltration centrifuge tube, purified and concentrated, and the concentrated liquid is divided into packages.
  • the prepared lipid nanoparticles were measured with a laser nanometer particle size analyzer for particle size, PDI, and potential, and the encapsulation efficiency (%) was measured with a UV spectrophotometer in conjunction with a RiboGreen RNA kit.
  • the exemplary results are as follows.
  • lipid nanoparticles with better physical and chemical quality control data can also be obtained.
  • the formula results of II-37:DSPC:CHOL:DMG-PEG2000 are shown in the following table, where tri-009-BJ-LNP-21040601
  • the lipid molar ratio of tri-009-BJ-LNP-21040602 is: 35:15:48.5:1.5; tri-009-BJ-LNP-21040603
  • the lipid molar ratio is: 45:15:38.5:1.5.
  • a part of the samples were transfected into CHO cells according to the method in Example 2, and the protein expression was detected by Elisa to evaluate the cell transfection rate.
  • MC3 is: 4-(N,N-dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptanthic-6,9,28,31-tetraen-19-yl ester.
  • the encapsulation efficiency of lipid nanoparticles prepared by II-37 is as high as 90.5%, which is much higher than that of MC3 lipid nanoparticles, and the particle size is smaller and more uniform, and the potential is higher.
  • the prepared lipid nanoparticles were transfected into cells to understand the expression of the protein.
  • the results are shown in Figure 5.
  • the lipid nanoparticles prepared by II-37 (indicated by C2 in the figure) After the cells were transfected with mRNA, the protein expression in the cells was much higher than that of MC3, indicating that the lipid nanoparticles made of II-37 had a high cell transfection efficiency.
  • sample information Particle size (nm) PDI Zeta potential mRNA-LNP (II-37-LNP) 136.68 0.14 20.07 mRNA-LNP (C14-113-LNP) 152.65 0.12 24.1
  • the transfection efficiency of the prepared LucRNA-LNP HEK293T cells was detected by the luciferin reporter gene method with a multifunctional microplate reader (BioTek, model SLXFATS), and the transfected LucRNA amounts were 0.5 ⁇ g, 1.0 ⁇ g, and 2.0 ⁇ g, respectively.
  • the method for transcribing LucRNA in vitro is as follows: HEK293T cells were plated at a cell density of 2.5 ⁇ 10 5 cells/mL, and transfected when the cell confluence was 30%-50%. 48 h after transfection, the protein expression was detected by a multifunctional microplate reader. The negative control was the cell culture medium without adding LucRNA-LNP.
  • the lipid nanoparticle prepared by the compatibility of the new lipid compound has low cytotoxicity, and its mRNA transfection efficiency is better than that of the structural analog molecule C14-113.
  • Embodiment 5 Efficiency comparison experiment of IVT carrier of the present invention
  • Firefly Luc as a reporter protein
  • different IVT vectors were constructed for in vitro transcription and synthesis of mRNA capable of translating Firefly Luc, and the translation efficiencies of synthesized mRNAs with different sequence characteristics were compared.
  • the coding sequence of Firefly Luc was cloned into the multiple cloning site of the corresponding vector to obtain vectors numbered IVT1, IVT2, IVT3 and IVT4, and then use the AM1344 kit according to the aforementioned vectors in vitro
  • the corresponding Firefly Luc mRNA samples were prepared by transcription.
  • the vectors IVT1 ⁇ IVT4 are all modified on the basis of the commercial vector psp73. The following sequences are inserted at the restriction site XhoI/NdeI of the vector psp73. There is no UTR sequence added to IVT1, and the length of the polyA tail is 64 A; IVT2 The 3'UTR sequence of the 5'UTR shown in SEQ ID NO:6 and GCTCGCTTTCTTGCTGTCCAATTTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAAAACATTTATTTTCATTGC (the 3'UTR sequence of ⁇ globin) was used in the polyA length (the 3'UTR sequence of ⁇ globin), and the length of polyA is 120 NOA shown in ID:6; The 5'UTR shown in SEQ ID NO:7 and the 3'UTR sequence shown in SEQ ID NO:7, the polyA length is 120 A
  • a multiple cloning site containing common restriction sites HindIII and EcoRI was inserted in the middle of the above 5'UTR and 3'UTR sequences, and then the coding sequence of Firefly Luc was cloned into the multiple cloning sites of HindIII and EcoRI. All vectors were constructed by GenScript by gene synthesis.
  • Each Firefly Luc mRNA sample was transfected into CHO cells with Lipofectamine2000 (cat#11668030, purchased from Thermo Fisher) as the transfection reagent, and the Dual-Lumi TM Dual-Luciferase Reporter Gene Detection Kit was used (at#RG088S, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) to detect luciferase.
  • the DNA of Firefly Luc was transformed into psicheck2 plasmid as a positive control (psicheck2 plasmid, cat#60908-6151, purchased from Beijing Tianenze Gene Technology Co., Ltd.).
  • BALB/c mice were used to evaluate the production of binding antibodies and neutralizing antibodies induced by S protein mutants: 6-week-old female BALB/c mice, the interval between the first immunization and the second immunization was 2 weeks; blood was collected 14 days after immunization.
  • ELISA was used to detect the expression of binding antibody against S protein mutants, and chemiluminescence was used to detect the titer of neutralizing antibodies against S protein mutants.
  • ELISA method to detect binding antibodies by coating commercial S protein on the enzyme plate to capture the binding antibody against the S protein mutant in the plasma of immunized mice, and then detect the absorbance with a biotin-labeled detection antibody.
  • Chemiluminescent detection of neutralizing antibody titers against S protein mutants mouse plasma after immunization and SPIKE lentivirus carrying a luciferase reporter gene (Zhongji Dangkang; trade name: SRAS-CoV-2 pseudovirus (B.1.351 )-LUC; product number: DZPSC-L-0; batch: K05202102) neutralized and then deinfected 293T cells with high expression of ACE-2 (Zhongji Dangkang; product name: "YJ1B09” hACE2-293T cell lines; product number : YJ293T-01; Batch: A23202001), using chemiluminescence (Bright-Lumi II firefly luciferase reporter gene detection kit, brand:
  • mice There were 9 mice in the control group, and each mouse was subcutaneously injected with 2ug protein. Among them, 3 injected proteins were purified trimers of S protein translated from B1.351 mRNA in Example 2, 3 injected proteins were trimers of wild-type S proteins, and 3 injected blank lipids Quality nanoparticles, the lipid formulation of lipid nanoparticles is tri-009-BJ-LNP-21040602 in Example 3.
  • mice in the experimental group were subcutaneously injected with lipid nanoparticles of mRNA; 1-6 were injected with 0.2 ⁇ g of mRNA lipid nanoparticles (LNP), and 7-12 were injected with 1 ⁇ g of mRNA lipid nanoparticles (LNP) , No. 13-18 injection of lipid nanoparticles (LNP) of 5 ⁇ g mRNA, wherein the mRNA is the B1.351mRNA of Example 2, and the formulation of the lipid nanoparticles is tri-009-BJ-LNP-21040602 in Example 3.
  • LNP mRNA lipid nanoparticles
  • both the S protein trimer translated from the B1.351 mRNA of Example 2 and the wild-type S protein trimer can induce binding antibodies against the S protein in mice :
  • a higher concentration of binding antibodies can already be produced.
  • the concentration of the binding antibodies still maintains a high level.
  • the binding antibodies after the second immunization The concentration is about 2.2 ⁇ g/ml, and it can still maintain about 1.6 ⁇ g/ml 8 weeks after the second immunization.

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Abstract

一种新的可电离阳离子脂质分子，以及由该脂质分子和中性脂质分子、胆固醇类脂质分子、PEG化的脂质分子一同组成的脂质纳米颗粒，以及含有所述脂质纳米颗粒的组合物。该脂质纳米颗粒作为活性成分的递送载体，具有颗粒粒径小而均匀、包封率高、细胞转染效率高的优点，尤其适用于核酸分子(例如mRNA)的递送载体。

Description

一种脂质纳米颗粒组合物以及由其制备的药物递送系统
本申请要求2021年10月9日向中国国家知识产权局提交的专利申请号为202111177954.6，发明名称为“一种脂质纳米颗粒组合物以及由其制备的药物递送系统”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物医药制剂技术领域，尤其涉及一种含有新的脂质化合物的脂质纳米颗粒，以及由其制备得到的携带活性成分的药物组合物或药物递送系统，比如mRNA疫苗。
背景技术
不同类型的核酸制剂正被研发用于治疗各种重大疾病如传染性疾病、癌症、罕见病等。该类核酸制剂包括：DNA、反义核酸(ASO)、小干扰RNA(siRNA)、微小RNA(miRNA)、小激活RNA(saRNA)、信使RNA(mRNA)、适配体(aptamer)、核酶(ribozyme)等。其中，mRNA疫苗在安全性、快速制备和免疫原性方面具有颠覆性的优势。基于mRNA修饰和递送工具的发展，一旦获得病毒抗原序列，可在数周内迅速设计和制造具有临床规模的mRNA疫苗，可以实现标准化生产，使其在应对大流行暴发方面非常具有吸引力。且mRNA疫苗不存在减毒疫苗潜在的逆转危险；不存在灭活疫苗的恢复突变问题。在免疫原性上，mRNA疫苗能够诱导B细胞和T细胞免疫应答，能引起免疫记忆效果，传递更多有效抗原，并且能一次表达多个抗原。此外，mRNA只需要穿过细胞膜在细胞质中就可以高效表达抗原蛋白；mRNA没有基因整合到基因组中的风险。再次，mRNA被翻译成蛋白质后易被降解，其瞬时表达的特性不仅确保了mRNA药物的安全性，而且使其剂量可控，避免了疫苗药物长期暴露引起的抗原免疫耐受(对特定抗原无反应的状态)。
但是，一方面核酸制剂带负电而且多数分子量较大很难直接进入细胞内，另一方面，RNA不稳定，在引入体内的过程中极易被核酸酶降解，从而失去生物功能。因此，开发高效安全并且通用的核酸递送系统，是核酸药物转化过程中亟待解决的问题。
目前，核酸的递送方法包括化学修饰、生物偶联技术、纳米载体技术、基于脂质的制剂、外泌体、球形核酸、DNA纳米结构、刺激-响应聚合物纳米材料等。其中，较为成熟的核酸递 送载体是脂质纳米颗粒(LNP)，并且2018年LNP包裹的siRNA药物Onpattro(patisiran)获批上市，2021年LNP包裹的mRNA疫苗已经正式获得FDA审批应用于新冠疫情防控，临床结果显示出较高的有效性，且目前无严重不良反应。
该类脂质制剂的主要成分包括：阳离子/可电离脂质、辅助型脂质、胆固醇和聚乙二醇-脂质结合物。在这四种脂质成分中，阳离子脂质带电荷的头部能够与带负电的核酸结合，同时也能与细胞膜上的磷脂分子结合，在核酸包裹和膜融合过程中都发挥着关键的作用。考虑到永久性的阳离子脂质的潜在毒性，可电离阳离子脂质的脂质纳米颗粒存在更大的应用价值。
可电离的阳离子脂质包括三个重要的结构组成部分：含胺基的亲水极性头部；疏水脂质链；负责连接极性头部和非极性尾部的连接链。目前，商业化的可电离阳离子脂质主要有MC3系列和用于新冠mRNA疫苗的ALC-0315、SM-102。其中，MC3具有很强的肝靶向，对于可能具有潜在肝毒性的核酸制剂其应用将会受限，而且MC3是针对分子量较小的siRNA递送开发的，对于分子量较大的核酸制剂其负载量可能存在限制。ALC-0315和SM-102的递送效率需要进一步提高。
发明内容
为解决活性成分，例如核酸(例如mRNA分子)，生物应用时存在的结构不稳定，易被核酸酶降解以及入胞困难的问题，需要开发新的递送技术。此外，现有递送技术中普遍存在溶酶体逃逸差，递送效率较低的问题，本发明基于新合成的可电离阳离子脂质分子，形成多种功能多样性的以脂质为基础的递送系统。
本发明的第一方面，提供一种脂质纳米颗粒组合物，所述脂质纳米颗粒组合物中含有脂质纳米颗粒，脂质纳米颗粒中包含：式I的可电离阳离子脂质分子。
根据本发明，所述脂质纳米颗粒组合物，进一步包含其他脂质分子。所述其他脂质分子可以是本领域中用于构建脂质纳米颗粒已知或常规使用的脂质分子，包括但不限于中性脂质分子、胆固醇类脂质分子、PEG化的脂质分子。
根据本发明，所述脂质纳米颗粒组合物，进一步包含活性成分，所述活性成分可以是小分子化合物、核酸、蛋白质、多肽等。所述活性成分位于脂质纳米颗粒中。所述核酸包括但不限于DNA、反义核酸(ASO)、小干扰RNA(siRNA)、微小RNA(miRNA)、小激活RNA(saRNA)、信使RNA(mRNA)、适配体(aptamer)等。
根据本发明，所述脂质纳米颗粒组合物中，脂质纳米颗粒中含有占其总体脂质分子30-60mol％的式I的脂质分子，优选32-55mol％，进一步优选34-46mol％。
根据本发明，所述脂质纳米颗粒组合物中，脂质纳米颗粒中可以含有占其总体脂质分子5-30mol％的中性脂质分子，优选8-20mol％，进一步优选9-16mol％。
根据本发明，所述脂质纳米颗粒组合物中，脂质纳米颗粒中可以含有占其总体脂质分子30-50mol％的胆固醇类脂质分子，优选35-50mol％，进一步优选37-49mol％。
根据本发明，所述脂质纳米颗粒组合物中，脂质纳米颗粒中可以含有占其总体脂质分子0.4-10mol％的PEG化的脂质分子，优选0.5-5mol％，进一步优选1.3-2.7mol％。
根据本发明，当活性成分为核酸时，所述脂质纳米颗粒组合物中脂质分子的总质量与核酸质量之比为5-20:1。
根据本发明，式I的可电离阳离子脂质分子的结构式为
Figure PCTCN2022122221-appb-000001
其中：
Q为经取代或未取代的直链C2-20亚烷基，所述亚烷基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；或，Q为经取代或未取代、饱和或不饱和的4-6元环，所述4-6元环的环原子任选含有1个或1个以上独立地选自O、S、N的杂原子；所述取代的取代基团选自卤素、-OH、直链或支链的C1-20烷基、直链或支链的C1-20烷氧基、直链或支链的C2-20烯基、直链或支链的C2-20炔基、-CH 2CH(OH)R 5、
Figure PCTCN2022122221-appb-000002
R 1、R 2、R 3、R 4可以相同或不同，各自独立的选自氢，经取代或未取代的直链或支链C1-30烷基，经取代或未取代的直链或支链C2-30烯基，经取代或未取代的直链或支链C2-30炔基，所述烷基、烯基或炔基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换，或-CH 2CH(OH)R 5；所述取代的取代基团选自卤素、-OH、直链或支链的C1-10烷基、直链或支链的C1-10烷氧基；
条件是，R 1、R 2、R 3、R 4中至少一个为
Figure PCTCN2022122221-appb-000003
R 5选自氢，经取代或未取代的直链或支链C1-30烷基，经取代或未取代的直链或支链C2-30烯基，经取代或未取代的直链或支链C2-30炔基，所述烷基、烯基或炔基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；所述取代的取代基团选自卤素、-OH、直链或支链的C1-10烷基、直链或支链的C1-10烷氧基；
R 6选自氢，C1-3烷基，C1-3烷氧基，-OH；
n选自1～8的整数，m选自0～8的整数，n和m彼此独立，可以相同也可以不同；
当R 1、R 2、R 3、R 4中至少两个为
Figure PCTCN2022122221-appb-000004
时，每个所述基团中的n和m彼此独立，可以相同也可以不同。
在本发明的优选实施方式中，Q为经取代或未取代的直链C2-20亚烷基，所述亚烷基的1 个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；
优选，Q为
Figure PCTCN2022122221-appb-000005
其中，R 8、R 9彼此独立的选自经取代或未取代的直链C1-10亚烷基，所述亚烷基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；R 7为氢，卤素，-OH，直链或支链C1-20烷基，直链或支链C2-20烯基，直链或支链C2-20炔基，或-CH 2CH(OH)R 5，或
Figure PCTCN2022122221-appb-000006
所述取代的取代基团为卤素、-OH、直链或支链的C1-10烷基、直链或支链的C1-10烷氧基；
优选，Q为
Figure PCTCN2022122221-appb-000007
其中：x和y可以相同或者不同，独立地选自1～8的整数；R 7定义和前述相同；优选，x或y相同或不同，选自1～3的整数，例如为1、2或3；优选，R 7为直链或支链C1-4烷基，例如为甲基、乙基、正丙基、正丁基等。
在本发明的一些实施例中，所述饱和或不饱和的4-6元环为哌嗪基或环己基。
在本发明的优选实施方式中，R 6为-OH。
在本发明的优选实施方式中，n选自4～8的整数，m选自4～8的整数。
在本发明的优选实施方式中，所述式I化合物为下式A、B、C或D：
Figure PCTCN2022122221-appb-000008
A，其中每个n 1都彼此独立，可以相同或不同，每个n 1选自1～8的整数，每个m 1都彼此独立，可以相同或不同，每个m 1选自0～8的整数；优选，每个n 1选自4～8的整数，每个m 1选自4～8的整数；优选，每个n 1都彼此相同，每个m 1都彼此相同。
Figure PCTCN2022122221-appb-000009
B，其中每个n 2都彼此独立，可以相同或不同，每个n 2选自1～8的整数，每个m 2都彼此独立，可以相同或不同，每个m 2选自0～8的整数；优选，每个n 2选自4～8的整数，每个m 2选自4～8的整数；优选，每个n 2都彼此相同，每个m 2都彼此相同。
Figure PCTCN2022122221-appb-000010
C，其中每个n 3都彼此独立，可以相同或不同，每个n 3选自1～8的整数，每个m 3都彼此独立，可以相同或不同，每个m 3选自0～8的整数；优选，每个n 3选自4～8的整数，每个m 3选自4～8的整数；优选，每个n 3都彼此相同，每个 m 3都彼此相同。
Figure PCTCN2022122221-appb-000011
D，其中每个n 4都彼此独立，可以相同或不同，每个n 4选自1～8的整数，每个m 4都彼此独立，可以相同或不同，每个m 4选自0～8的整数；优选，每个n 4选自4～8的整数，每个m 4选自4～8的整数；优选，每个n 4都彼此相同，每个m 4都彼此相同。
在本发明的一些具体实施方式中，所述式I化合物选自表1所示的以下化合物：
表1
Figure PCTCN2022122221-appb-000012
Figure PCTCN2022122221-appb-000013
Figure PCTCN2022122221-appb-000014
Figure PCTCN2022122221-appb-000015
Figure PCTCN2022122221-appb-000016
Figure PCTCN2022122221-appb-000017
Figure PCTCN2022122221-appb-000018
Figure PCTCN2022122221-appb-000019
Figure PCTCN2022122221-appb-000020
Figure PCTCN2022122221-appb-000021
根据本发明，式I的脂质分子在脂质纳米颗粒的脂质中的摩尔百分比为30-60mol％，例如32-55mol％，例如可以为30mol％，31mol％，32mol％，33mol％，34mol％，35mol％，36mol％，37mol％，38mol％，39mol％，40mol％，41mol％，42mol％，43mol％，44mol％，45mol％，46mol％，47mol％，48mol％，49mol％，50mol％，51mol％，52mol％，53mol％，54mol％，55mol％等。
根据本发明，所述中性脂质分子是不带电脂质分子或两性离子型脂质分子，例如磷脂酰胆碱类化合物，或/和磷脂酰乙醇胺类化合物。
磷脂酰胆碱类化合物的结构如式E所示：
Figure PCTCN2022122221-appb-000022
E；磷脂酰乙醇胺类化合物的结构如式F所示：
Figure PCTCN2022122221-appb-000023
F，其中Ra、Rb、Rc、Rd独立的选自直链或支链的C1-30烷基，直链或支链的C2-30烯基，优选为直链或支链的C10-30烷基，直链或支链的C10-30烯基，例如CH 3(CH 2) 17CH 2-、CH 3(CH 2) 15CH 2-、CH 3(CH 2) 13CH 2-、CH 3(CH 2) 11CH 2-、CH 3(CH 2) 9CH 2-、CH 3(CH 2) 7CH 2-、CH 3(CH 2) 7-CH＝CH-(CH 2) 7-、CH 3(CH 2) 4CH＝CHCH 2CH＝CH(CH 2) 7-、CH 3(CH 2) 7-CH＝CH-(CH 2) 9-。
中性脂质分子的实例包括但不限于5-十七基苯-1,3-二醇(间苯二酚)、二棕榈酰基磷脂酰胆碱(DPPC)、二硬脂酰基磷脂酰胆碱(DSPC)、磷酸胆碱(DOPC)、二肉豆蔻酰基磷脂酰胆碱(DMPC)、磷脂酰胆碱(PLPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DAPC)、磷脂酰乙醇胺(PE)、卵磷脂酰胆碱(EPC)、二月桂酰基磷脂酰胆碱(DLPC)、二肉豆蔻酰基磷脂酰胆碱(DMPC)、1-肉豆蔻酰基-2-棕榈酰基磷脂酰胆碱(MPPC)、1-棕榈酰基-2-肉豆蔻酰基磷脂酰胆碱(PMPC)、1-棕榈酰基-2-硬脂酰基磷脂酰胆碱(PSPC)、1,2-二花生酰基-sn-甘油-3-磷酸胆碱(DBPC)、1-硬脂酰基-2-棕榈酰基磷脂酰胆碱(SPPC)、1,2-二十碳烯酰基-sn-甘油-3-磷酸胆碱(DEPC)、棕榈酰油酰基磷脂酰胆碱(POPC)、溶血磷脂酰胆碱、二油酰基磷脂酰乙醇胺(DOPE)、二硬脂酰基磷脂酰乙醇胺(DSPE)、二肉豆蔻酰基磷脂酰乙醇胺(DMPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、棕榈酰油酰基磷脂酰乙醇胺(POPE)、溶血磷脂酰乙醇胺以及它们的组合。
在一个实施方案中，中性脂质分子可选自由以下组成的组：二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰乙醇胺(DOPE)和二硬脂酰基磷脂酰乙醇胺(DSPE)。在另一个实施方案中，中性脂质分子可为二肉豆蔻酰基磷脂酰乙醇胺(DMPE)。在另一个实施方案中，中性脂质分子可为二棕榈酰基磷脂酰胆碱(DPPC)。
根据本发明，中性脂质分子在脂质纳米颗粒的脂质中的摩尔百分比为5-30mol％，例如8-20mol％，例如可以为8mol％，9mol％，10mol％，11mol％，12mol％，13mol％，14mol％，15mol％，16mol％，17mol％，18mol％，19mol％，20mol％。
根据本发明，胆固醇类脂质分子是指甾醇以及含有甾醇部分的脂质，包括但不限于胆固醇，5-十七基间苯二酚，粪甾醇，谷甾醇，麦角甾醇，菜油甾醇，豆甾醇，菜子甾醇，番茄 次碱，番茄碱，熊果酸，α-生育酚及其混合物、胆固醇半琥珀酸酯。在一个实施方案中，胆固醇类脂质分子为胆固醇(CHOL)。在一个实施方案中，胆固醇类脂质分子为胆固醇半琥珀酸酯。
根据本发明，胆固醇类脂质分子在脂质纳米颗粒的脂质中的摩尔百分比为30-50mol％，例如可以为30mol％，31mol％，32mol％，33mol％，34mol％，35mol％，36mol％，37mol％，38mol％，39mol％，40mol％，41mol％，42mol％，43mol％，44mol％，45mol％，46mol％，47mol％，48mol％，49mol％，50mol％等。
根据本发明，PEG化的脂质分子包含脂质部分和基于PEG的聚合物部分。在一些实施方案中，脂质部分可衍生自二酰基甘油或二酰基甘油酰胺(diacylglycamide)、包括包含具有独立地包含约C4至约C30饱和或不饱和碳原子的烷基链长度的二烷基甘油或二烷基甘油酰胺基团的那些，其中该链可包含一个或多个官能团，诸如酰胺或酯。在一些实施方案中，烷基链长度包含约C10至C20。二烷基甘油或二烷基甘油酰胺基团可还包含一个或多个取代的烷基。链长可为对称或不对称的。除非另外指明，否则如本文所用，术语“PEG”意指任何聚乙二醇或其他聚亚烷醚聚合物。在一个实施方案中，PEG部分为乙二醇或环氧乙烷的任选地取代的直链或支链聚合物。在某些实施方案中，PEG部分可被例如一个或多个烷基、烷氧基、酰基、羟基或芳基取代。在一个实施方案中，PEG部分包括PEG共聚物，诸如PEG-聚氨酯或PEG-聚丙烯(参见例如J.Milton Harris,Poly(ethylene glycol)chemistry:biotechnical and biomedical applications(1992))；或者，PEG部分不包括PEG共聚物，例如其可为PEG单聚物。在一个实施方案中，PEG的分子量为约130至约50,000，在子实施方案中，为约150至约30,000，在子实施方案中，为约150至约20,000，在子实施方案中，为约150至约15,000，在子实施方案中，为约150至约10,000，在子实施方案中，为约150至约6,000，在子实施方案中，为约150至约5,000，在子实施方案中，为约150至约4,000，在子实施方案中，为约150至约3,000，在子实施方案中，为约300至约3,000，在子实施方案中，为约1,000至约3,000，并且在子实施方案中，为约1,500至约2,500。在某些实施方案中，PEG为“PEG 2000”，其平均分子量为约2,000道尔顿。在本发明的一些实施方式中，PEG在本文中由下式
Figure PCTCN2022122221-appb-000024
表示，对于PEG-2000其中n为45，意指数均聚合度包含约45个亚单位；也可使用本领域中已知的其他PEG实施方案，包括例如其中数均聚合度包含约23个亚单位(n＝23)和/或68个亚单位(n＝68)的那些。在一些实施方案中，n可在约30至约60的范围内。在一些实施方案中，n可在约35至约55的范围内。在一些实施方案中，n可在约40至约50的范围内。在一些实施方案中，n可在约42至约48的范围内。在一些实施方案中，n可为45。在一些实施方案中，R可选自H、取代的烷基和未取代的烷基。在一些实施方案中，R 可为未取代的C1-C30的烷基，例如C1-C20的烷基，C1-C10的烷基，C1-C6的烷基。在一些实施方案中，R可为H，甲基或乙基。
在一些实施方案中，PEG化的脂质分子可表示为“脂质部分-PEG-数均分子量”或者“PEG-脂质部分”或者“PEG-数均分子量-脂质部分”，所述脂质部分是二酰基甘油或二酰基甘油酰胺，选自二月桂酰甘油、二肉豆蔻酰甘油、二棕榈酰甘油、二硬脂酰甘油、二月桂基甘油酰胺、二肉豆蔻基甘油酰胺、二棕榈酰甘油酰胺、二硬脂酰甘油酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺；PEG的数均分子量为约130至约50,000，例如约150至约30,000，约150至约20,000，为约150至约15,000，为约150至约10,000，为约150至约6,000，为约150至约5,000，为约150至约4,000，为约150至约3,000，为约300至约3,000，为约1,000至约3,000，为约1,500至约2,500，例如约2000。
在一些实施方案中，PEG化的脂质分子可选自PEG-二月桂酰甘油、PEG-二肉豆蔻酰甘油(PEG-DMG)、PEG-二棕榈酰甘油、PEG-二硬脂酰甘油(PEG-DSPE)、PEG-二月桂基甘油酰胺、PEG-二肉豆蔻基甘油酰胺、PEG-二棕榈酰甘油酰胺和PEG-二硬脂酰甘油酰胺、PEG-胆固醇(1-[8'-(胆甾-5-烯-3[β]-氧基)甲酰胺基-3',6'-二氧杂辛基]氨基甲酰基-[ω]-甲基-聚(乙二醇)、PEG-DMB(3,4-二-十四氧基苯甲基-[ω]-甲基-聚(乙二醇)醚)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000](DMG-PEG2000)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000](DSPE-PEG2000)、1,2-二硬脂酰基-sn-甘油-甲氧基聚乙二醇(DSG-PEG2000)、聚(乙二醇)-2000-二甲基丙烯酸酯(DMA-PEG2000)和1,2-二硬脂酰氧基丙基-3-胺-N-[甲氧基(聚乙二醇)-2000](DSA-PEG2000)。在一个实施方案中，PEG化的脂质分子可为DMG-PEG2000。在一些实施方案中，PEG化的脂质分子可为DSG-PEG2000。在一个实施方案中，PEG化的脂质分子可为DSPE-PEG2000。在一个实施方案中，PEG化的脂质分子可为DMA-PEG2000。在一个实施方案中，PEG化的脂质分子可为C-DMA-PEG2000。在一个实施方案中，PEG化的脂质分子可为DSA-PEG2000。在一个实施方案中，PEG化的脂质分子可为PEG2000-C11。在一些实施方案中，PEG化的脂质分子可为PEG2000-C14。在一些实施方案中，PEG化的脂质分子可为PEG2000-C16。在一些实施方案中，PEG化的脂质分子可为PEG2000-C18。
根据本发明，PEG化的脂质分子在脂质纳米颗粒的脂质中的摩尔百分比为0.4-10mol％，例如0.5-5mol％，例如可以为0.4mol％，0.5mol％，0.6mol％，0.7mol％，0.8mol％，0.9mol％，1.0mol％，1.1mol％，1.2mol％，1.3mol％，1.4mol％，1.5mol％，1.6mol％，1.7mol％，1.8mol％，1.9mol％，2.0mol％，2.1mol％，2.2mol％，2.3mol％，2.4mol％，2.5mol％，2.6mol％，2.7mol％，2.8mol％，2.9mol％，3.0mol％，3.1mol％，3.2mol％，3.3mol％，3.4mol％，3.5mol％，3.6mol％，3.7mol％，3.8mol％，3.9mol％，4.0mol％，4.1mol％，4.2mol％，4.3mol％，4.4mol％，4.5mol％， 4.6mol％，4.7mol％，4.8mol％，4.9mol％，5.0mol％等。
在本发明的一些实施方式中，所述脂质纳米颗粒中含有式C所示的脂质分子，中性脂质分子、胆固醇类脂质分子、PEG化的脂质分子，其中：
式C
Figure PCTCN2022122221-appb-000025
其中每个n 3都彼此独立，可以相同或不同，每个n 3选自1～8的整数，每个m 3都彼此独立，可以相同或不同，每个m 3选自0～8的整数；优选，每个n 3选自4～8的整数，每个m 3选自4～8的整数；优选，每个n 3都彼此相同，每个m 3都彼此相同。式C所示的可电离阳离子脂质分子占脂质纳米颗粒中脂质的摩尔百分比为32-55mol％，优选为34-46mol％。
中性脂质分子选自式E所示的磷脂酰胆碱类化合物
Figure PCTCN2022122221-appb-000026
E，式F所示的磷脂酰乙醇胺类化合物
Figure PCTCN2022122221-appb-000027
F，其中Ra、Rb、Rc、Rd独立的选自直链或支链的C10-30烷基，直链或支链的C10-30烯基，优选为CH 3(CH 2) 17CH 2-、CH 3(CH 2) 15CH 2-、CH 3(CH 2) 13CH 2-、CH 3(CH 2) 11CH 2-、CH 3(CH 2) 9CH 2-、CH 3(CH 2) 7CH 2-、CH 3(CH 2) 7-CH＝CH-(CH 2) 7-、CH 3(CH 2) 4CH＝CHCH 2CH＝CH(CH 2) 7-、CH 3(CH 2) 7-CH＝CH-(CH 2) 9-。中性脂质分子占脂质纳米颗粒中脂质的摩尔百分比为8-20mol％，优选为9-16mol％；
胆固醇类脂质分子选自胆固醇、胆固醇半琥珀酸酯。胆固醇类脂质分子占脂质纳米颗粒中脂质的摩尔百分比为30-50mol％，优选为35-50mol％，更优选为37-49mol％。
PEG化的脂质分子表示为“脂质部分-PEG-数均分子量”，所述脂质部分是二酰基甘油或二酰基甘油酰胺，选自二月桂酰甘油、二肉豆蔻酰甘油、二棕榈酰甘油、二硬脂酰甘油、二月桂基甘油酰胺、二肉豆蔻基甘油酰胺、二棕榈酰甘油酰胺、二硬脂酰甘油酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺；PEG的数均分子量为130～50,000，例如150～30,000，150～20,000，150～15,000，150～10,000，150～6,000，150～5,000， 150～4,000，150～3,000，300～3,000，1,000～3,000，1,500～2,500，约2000。PEG化的脂质分子占脂质纳米颗粒中脂质的摩尔百分比为0.5-5mol％，优选为1.3-2.7mol％。
在本发明的一个实施方式中，所述核酸是mRNA。
根据本发明，所述mRNA从5’端至3’端可以包含5’帽结构、5’UTR，开放阅读框(ORF)，3’UTR和poly-A尾。
根据本发明，所述帽结构可以是Cap1结构，Cap2结构或Cap3结构。在本发明的一个实施方式中，所述帽结构是Cap1结构。
根据本发明，所述5’UTR可以包含β-珠蛋白或α-珠蛋白的5’UTR或其同源物、片段。在本发明的一些实施方式中，所述5’UTR包含与SEQ ID NO:6所示的β-珠蛋白的5’UTR核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列。在本发明的一个具体实施方式中，所述5’UTR包含SEQ ID NO:6所示的β-珠蛋白的5’UTR核苷酸序列。
在本发明的一些实施方案中，所述5'UTR还包含Kozak序列。在本发明的一个实施方式中，所述Kozak序列为GCCACC。
根据本发明，所述3’UTR可以包含β-珠蛋白或α-珠蛋白的3’UTR或其同源物、片段，或片段的组合。在本发明的一些实施方式中，所述3’UTR包含与SEQ ID NO:7所示α2-珠蛋白3’UTR的片段至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列。在本发明的另一些实施方式中，所述3’UTR包含2个首尾相连的与SEQ ID NO:7所示的α2-珠蛋白3’UTR的片段至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列。在本发明的一个具体实施方式中，所述3’UTR包含2个首尾相连的SEQ ID NO:7所示的核苷酸序列。
根据本发明，所述poly-A尾的长度可以为50-200个核苷酸，优选为100-150个核苷酸，例如110-120个核苷酸，例如约110个核苷酸，约120个核苷酸，约130个核苷酸，约140个核苷酸，约150个核苷酸。
在本发明的一个实施方案中，所述开放阅读框(ORF)是编码2019-nCov的S蛋白突变体的开放阅读框(ORF)，其核酸序列是与SEQ ID NO:8所示核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列。该ORF翻译后的S蛋白突变体的氨基酸序列是由从N端向C端直接连接的SEQ ID NO:2所示氨基酸序列和SEQ ID NO:3所示氨基酸序列组成。在本发明的一个具体实施方式中，所述S蛋白突变体的开放阅读框(ORF)的核苷酸序列如SEQ ID NO:8所示。
在本发明的一个实施方案中，所述mRNA包含与SEQ ID NO:9所示的核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列。在本发明的一个具体实施方式中，所述mRNA包含SEQ ID NO:9所示的核苷酸序列。
可采用本领域已知的方法制备本发明的mRNA。在本发明的一些实施方式中，可以人工合成编码mRNA的核酸序列，将该序列克隆到载体中，构建体外转录用质粒。将构建的质粒转化到宿主菌中培养扩增，提取质粒。将提取的质粒使用紧邻着polyA尾后面的限制性内切酶酶切消化成线性分子。以制备的线性化质粒分子为模板，使用体外转录法制备mRNA。可以在体外转录过程中添加帽结构类似物，直接得到具有帽结构的mRNA；也可以在体外转录结束后，使用加帽酶和二甲基转移酶给mRNA添加上帽结构。可采用本领域常规的方法纯化得到的mRNA，例如化学沉淀法、磁珠法、亲和层析法等。
根据本发明，所述mRNA中的一个或多个核苷酸可以是经修饰的。例如，所述mRNA中的一个或多个核苷酸(例如所有核苷酸)可以各自独立替换为天然存在的核苷酸类似物或人工合成的核苷酸类似物，例如选自假尿苷(pseudouridine)、2-硫尿苷(2-thiouridine)、5-甲基尿苷(5-methyluridine)、5-甲基胞苷(5-methylcytidine)、N6-甲基腺苷(N6-methyladenosine)、N1-甲基假尿苷(N1-methylpseudouridine)等。
本发明还提供一种mRNA疫苗，所述疫苗中含有脂质纳米颗粒，所述脂质纳米颗粒中含有式C所示的脂质分子，中性脂质分子、胆固醇类脂质分子、PEG化的脂质分子和编码2019-nCoV S蛋白突变体的mRNA，所述mRNA包含与SEQ ID NO:9所示的核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列；脂质分子的总质量与mRNA的质量比为5-20:1；其中：
式C
Figure PCTCN2022122221-appb-000028
其中每个n 3都彼此独立，可以相同或不同，每个n 3选自1～8的整数，每个m 3都彼此独立，可以相同或不同，每个m 3选自0～8的整数；优选，每个n 3选自4～8的整数，每个m 3选自4～8的整数；优选，每个n 3都彼此相同，每个m 3都彼此相同。式C所示的脂质分子占脂质纳米颗粒中脂质的摩尔百分比为34-46mol％；
中性脂质分子选自式E所示的磷脂酰胆碱类化合物
Figure PCTCN2022122221-appb-000029
E，式F所示 的磷脂酰乙醇胺类化合物
Figure PCTCN2022122221-appb-000030
F，其中Ra、Rb、Rc、Rd独立的选自直链或支链的C10-30烷基，直链或支链的C10-30烯基，优选为CH 3(CH 2) 17CH 2-、CH 3(CH 2) 15CH 2-、CH 3(CH 2) 13CH 2-、CH 3(CH 2) 11CH 2-、CH 3(CH 2) 9CH 2-、CH 3(CH 2) 7CH 2-、CH 3(CH 2) 7-CH＝CH-(CH 2) 7-、CH 3(CH 2) 4CH＝CHCH 2CH＝CH(CH 2) 7-、CH 3(CH 2) 7-CH＝CH-(CH 2) 9-。中性脂质分子占脂质纳米颗粒中脂质的摩尔百分比为9-16mol％；
胆固醇类脂质分子选自胆固醇、胆固醇半琥珀酸酯。胆固醇类脂质分子占脂质纳米颗粒中脂质的摩尔百分比为37-49mol％；
PEG化的脂质分子表示为“脂质部分-PEG-数均分子量”，所述脂质部分是二酰基甘油或二酰基甘油酰胺，选自二月桂酰甘油、二肉豆蔻酰甘油、二棕榈酰甘油、二硬脂酰甘油、二月桂基甘油酰胺、二肉豆蔻基甘油酰胺、二棕榈酰甘油酰胺、二硬脂酰甘油酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺；PEG的数均分子量为约130至约50,000，例如约150至约30,000，约150至约20,000，为约150至约15,000，为约150至约10,000，为约150至约6,000，为约150至约5,000，为约150至约4,000，为约150至约3,000，为约300至约3,000，为约1,000至约3,000，为约1,500至约2,500，例如约2000。PEG化的脂质分子占脂质纳米颗粒中脂质的摩尔百分比为1.3-2.7mol％。
在本发明的一些实施方式中，式C所示的脂质分子、中性脂质分子、胆固醇和PEG化的脂质分子摩尔比为35:15:48.5:1.5。
在本发明的一些实施方式中，式C所示的脂质分子、中性脂质分子、胆固醇和PEG化的脂质分子摩尔比为40:10:48.5:1.5。
在本发明的一些实施方式中，式C所示的脂质分子、中性脂质分子、胆固醇和PEG化的脂质分子摩尔比为45:15:38.5:1.5。
在本发明的一个实施方式中，式C脂质分子为化合物II-37。
在本发明的一个实施方式中，所述中性脂质分子是DOPE和/或DSPC。
在本发明的一个实施方式中，所述PEG化的脂质分子是DMG-PEG2000和/或DSPE-PEG2000。
本发明还提供式I的可电离阳离子脂质分子的制备方法。
本发明的可电离脂质化合物可以采用本领域已有的方法进行合成，例如，使一当量或一当量以上的胺与一当量或一当量以上的环氧基封端化合物在合适的条件下反应形成。可电离脂质化合物的合成是在有或无溶剂的情况下进行，并且所述合成可在25-100℃范围内的较高 温度下进行。可任选纯化制得的可电离脂质化合物。举例来说，可纯化可电离脂质化合物的混合物，得到特定的可电离脂质化合物。或可纯化混合物，得到特定立体异构体或区域异构体。所述环氧化物可以商业购买，或者合成制备。
在本发明的一些实施方式中，本发明的可电离脂质化合物可以采用如下的一般制备方法进行制备。
Figure PCTCN2022122221-appb-000031
Figure PCTCN2022122221-appb-000032
式A、B、C或D
步骤1：还原
在还原剂的存在下，将化合物A1的羧基还原成羟基以获得化合物A2。还原剂的实例包括但不限于氢化铝锂、二异丁基氢化铝等。反应所用溶剂的实例包括但不限于醚类(如乙醚、四氢呋喃和二氧六环等)，卤代烃(如氯仿、二氯甲烷和二氯乙烷等)，烃类(如正戊烷、正己烷、苯和甲苯等)，及这些溶剂的两种或以上形成的混合溶剂。
步骤2：氧化
在氧化剂的存在下，将化合物A2的羟基氧化成醛基以获得化合物A3。氧化剂的实例包括但不限于2-碘酰基苯甲酸(IBX)、氯铬酸吡啶(PCC)、二氯铬酸吡啶盐(PDC)、戴斯-马丁氧化剂、二氧化锰等。反应所用溶剂的实例包括但不限于卤代烃(如氯仿、二氯甲烷和二氯乙烷等)，烃类(如正戊烷、正己烷、苯和甲苯等)，腈类(例如乙腈等)，及这些溶剂的两种或以上形成的混合溶剂。
步骤3：卤代-还原
首先在酸性条件下，将化合物A3的醛α-氢与卤代试剂发生卤代反应以获得α-卤代醛中间体，然后在还原剂存在下，将α-卤代醛的醛基还原成羟基以获得化合物A4。提供酸性条件的实例包括但不限于DL-脯氨酸。卤代试剂的实例包括但不限于N-氯代丁二酰亚胺(NCS)和N-溴代丁二酰亚胺(NBS)。还原剂的实例包括但不限于硼氢化钠、氰基硼氢化钠和三乙酰氧基硼氢化钠。
步骤4：环氧化
将化合物A4在碱的存在下进行分子内的亲核取代反应以获得环氧化合物A5。碱的实例包括但不限于碱金属的氢氧化物或氢化物，例如氢氧化钠、氢氧化钾和氢化钠。反应所用溶剂的实例包括但不限于二氧六环和水的混合物。
步骤5：开环反应
使化合物A5与胺(例如N,N-二(2-氨基乙基)甲胺)发生开环反应以获得最终化合物。反 应用溶剂的实例包括但不限于乙醇、甲醇、异丙醇、四氢呋喃、三氯甲烷、己烷、甲苯、乙醚等。
所述制备方法中的原料A1可以商购也可以采用常规方法合成。
本发明还提供制备脂质纳米颗粒组合物的方法。
根据本发明，所述制备方法，包括：将各脂质分子按摩尔比用有机溶剂溶解制成混合脂质的溶液，以混合脂质的溶液为有机相，以被递送物(例如核酸)的水溶液为水相，混合有机相和水相，制备脂质纳米颗粒。可以采用包括但不限于喷雾干燥、单一和双重乳液溶剂蒸发、溶剂萃取、相分离、纳米沉淀、微流控、简单和复杂凝聚以及所属领域的普通技术人员熟知的其它方法来制备脂质纳米颗粒。
在一些实施方式中，所述有机溶剂是醇，例如乙醇。
在一些实施方式中，所述有机相和水相的体积比为(2～4):1。
在一些实施方案中，采用微流控平台制备纳米颗粒。
根据本发明，所述制备方法还进一步包括，分离和纯化得到所述脂质纳米颗粒的步骤。
根据本发明，所述制备方法还进一步包括，冻干所述脂质纳米颗粒的步骤。
本发明式I的可电离脂质分子结构中含有两个相邻的顺式双键，使其在后续应用于递送系统包裹活性物质(例如核酸如mRNA)时，具有较高的包封率，较好的细胞转染率；此外，在制备脂质纳米颗粒时，得到的脂质纳米颗粒粒径更均匀。本发明的可电离脂质化合物尤其适合于制备实心结构的纳米颗粒。
此外，对于本发明的mRNA疫苗，由于本发明的mRNA具有高的翻译效率和稳定性，其所编码的S蛋白突变体具有高的稳定性，加之本发明脂质纳米颗粒包封率高、粒径更均匀，具有较好的细胞转染率，本发明的mRNA疫苗具有高的mRNA包封率、载药量，细胞转染效率，高效和稳定的体内翻译，抗原稳定性，免疫效果好。
本发明中脂质纳米颗粒的粒径范围在1nm到1000nm范围，例如10～500nm，10～200nm等。
本发明的脂质纳米颗粒还可修饰上靶向分子，使其成为靶向剂而能靶向特定细胞、组织或器官。靶向分子可位于颗粒表面上。靶向分子可为蛋白质、肽、糖蛋白、脂质、小分子、核酸等，其实例包括(但不限于)抗体、抗体片段、低密度脂蛋白(LDL)、转铁蛋白(transferrin)、脱唾液酸糖蛋白(asialycoprotein)、受体配体、唾液酸、适体等。靶向分子可以连接在脂质纳米颗粒的胆固醇类脂质分子或者PEG化的脂质分子上。
本发明的脂质纳米颗粒组合物和疫苗中，可以进一步含有一种或一种以上医药赋形剂。术语“医药赋形剂”的意思是任何类型的无毒、惰性固体、半固体或液体填充剂、稀释剂等，包括但不限于糖，诸如乳糖、海藻糖、葡萄糖和蔗糖；淀粉，诸如玉米淀粉和马铃薯淀粉；纤维素和其衍生物，诸如羧甲基纤维素钠、乙基纤维素和乙酸纤维素；明胶；滑石；油，诸 如花生油、棉籽油、红花油、橄榄油、玉米油和大豆油；二醇，诸如丙二醇；酯，诸如油酸乙酯和月桂酸乙酯；表面活性剂，诸如吐温80(Tween 80)；缓冲剂，诸如磷酸盐缓冲溶液、乙酸盐缓冲液和柠檬酸盐缓冲液；着色剂、甜味剂、调味剂和芳香剂、防腐剂和抗氧化剂等。
在本发明的一个实施方式中，所述脂质纳米颗粒组合物和疫苗是液体制剂，进一步含有蔗糖，蔗糖的质量百分比浓度为5-20％，优选为8-10％。
本发明的脂质纳米颗粒组合物和疫苗，可经口、经直肠、静脉内、肌注、阴道内、鼻内、腹膜内、经颊或以口服或鼻用喷雾等形式投予人类和/或动物。
本发明中的S蛋白突变体是亲本S蛋白发生氨基酸突变产生的。在本发明的一个实施方式中，所述亲本S蛋白是2019-nCoV B1.351突变毒株的S蛋白，2019-nCoV B1.351突变毒株的S蛋白与2019-nCoV野生株的S蛋白相比具有以下突变：L18F，D80A，D215G，L242_244L del，R246I，K417N，E484K，N501Y，D614G，A701V(所述位点以SEQ ID NO:1所示氨基酸序列的位置来定位描述)。
在本发明中，S蛋白突变体和亲本S蛋白的氨基酸位置均以野生型S蛋白的氨基酸序列为描述依据，野生型S蛋白的氨基酸序列可以在NCBI GeneID:43740568获得，共有1273个氨基酸，其序列如下所示，在本发明中标示为SEQ ID NO:1。
本发明所述S蛋白突变体至少包含胞外域，其胞外域相对于亲本S蛋白的胞外域包含如下位置的氨基酸突变：F817P、A892P、A899P、A942P、和KV986_987PP以及将682-685位氨基酸RRAR突变为GSAS；以及不包含S蛋白的跨膜域和胞质尾；在胞外区的C端直接融合辅助形成三聚体的结构域T4Fibritin Foldon Trimerization Motif。所述S蛋白突变体包含从N端向C端直接连接的SEQ ID NO:2的氨基酸序列和SEQ ID NO:3的氨基酸序列。
本发明上述序列的列表：
Figure PCTCN2022122221-appb-000033
Figure PCTCN2022122221-appb-000034
Figure PCTCN2022122221-appb-000035
Figure PCTCN2022122221-appb-000036
Figure PCTCN2022122221-appb-000037
Figure PCTCN2022122221-appb-000038
Figure PCTCN2022122221-appb-000039
Figure PCTCN2022122221-appb-000040
术语说明：
术语“烷基”是指从含有1到30个碳原子的烃部分通过去除单个氢原子得到的饱和烃基。烷基的实例包括(但不限于)甲基、乙基、丙基、异丙基、正丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基、正己基、正庚基、正辛基、正癸基、正十一烷基和正十二烷基。
术语“烯基”表示从具有至少一个碳-碳双键的烃部分通过去除单个氢原子得到的单价基团。烯基包括例如例如乙烯基、丙烯基、丁烯基、1-甲基-2-丁烯-1-基等。
术语“炔基”是指从具有至少一个碳-碳三键的烃通过去除单个氢原子得到的单价基团。代表性炔基包括乙炔基、2-丙炔基(炔丙基)、1-丙炔基等。
术语“烷氧基”是指如前文所定义的烷基通过氧原子连接于母体分子。烷氧基的实例包括(但不限于)甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、叔丁氧基、新戊氧基和正己氧基。
术语“卤基”和“卤素”是指选自氟、氯、溴和碘的原子。
术语“饱和或不饱和的4-6元环”是指具有4-6个环原子的环，所述环原子可以是C、N、S、O，其实例包括但不限于4-6元饱和的环烷基，例如环丙基、环丁基、环戊基、环己基；4-6元芳基，例如苯基；4-6元杂环基，例如吡咯烷基、哌啶基、哌嗪基、吗啉基等；4-6元杂芳基，例如三唑基、唑基、异唑基、噻唑基等。在本发明的一些实施例中，所述饱和或不饱和的4-6元环优选为哌嗪基、环己基。
术语“经取代”(无论前面是否存在术语“任选地”)和“取代基”是指将一个官能团变为另一个官能团的能力，条件是维持所有原子的价数。当任何特定结构中的一个以上位置可经一个以上选自指定群组的取代基取代时，所述取代基可在每一位置相同或不同。
“和/或”将被视为具有或不具有另一个的两个指定特征或组件中的每一个的具体公开。因此，在诸如“A和/或B”之类的短语中使用的术语“和/或”旨在包括“A和B”、“A或B”、“A”(单独)和“B”(单独)。同样地，在诸如“A、B和/或C”的短语中使用的术语“和/或”旨在涵盖以下方面中的每一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；和C(单独)。
“包括”和“包含”具有相同的含义，旨在是开放的并且允许但不要求包括额外的元件 或步骤。当在本文中使用术语“包括”或“包含”时，因此也包括和公开了术语“由......组成”和/或“基本上由……组成”。
在本说明书和权利要求书中，核苷酸通过其通常接受的单字母代码来指代。除非另有说明，否则核苷酸序列以5'至3'方向从左向右书写。核碱基在本文中由IUPAC-IUB生物化学命名委员会推荐的通常已知的单字母符号表示。技术人员将理解，本文公开的密码子中的T碱基存在于DNA中，而T碱基在相应RNA中将被U碱基取代。例如，本文公开的DNA形式的密码子-核苷酸序列，例如载体或体外翻译(IVT)模板，其T碱基在其相应的转录mRNA中转录为U碱基。在这一方面，密码子优化的DNA序列(包含T)和它们相应的mRNA序列(包含U)都被认为是本公开的密码子优化的核苷酸序列。本领域技术人员还将理解，可以通过用非天然碱基替换一个或多个碱基来产生等同的密码子图谱。
术语“核酸序列”、“核苷酸序列”或“多核苷酸序列”可互换使用，并且是指连续的核酸序列。序列可以是单链或双链的DNA或RNA，例如mRNA。
“编码…的核苷酸序列”是指编码多肽的核酸(例如，mRNA或DNA分子)编码序列。编码序列可以进一步包括与调控元件可操作地连接的起始和终止信号，所述调控元件包括能够指导在施用核酸的个体或哺乳动物的细胞中的表达的启动子和多腺苷酸化信号。
在本说明书和权利要求书中，使用氨基酸残基的常规单字母或三字母代码。除非另有说明，氨基酸序列以氨基至羧基取向从左至右书写。
“约”：在整个说明书和权利要求中与数值结合使用的术语“约”表示本领域技术人员熟悉和可接受的准确度区间。通常，这种精确度的区间为±10％。
为了便于参照，本发明的S蛋白突变体使用如下命名规则描述：原始氨基酸:位置:取代氨基酸。根据该命名规则，例如在第30位天冬酰胺被丙氨酸取代表示为：Asn30Ala或N30A；在相同位置缺失天冬酰胺表示为：Asn30*或N30*；插入另一氨基酸残基，例如赖氨酸，表示为：Asn30AsnLys或N30NK；缺失连续的一段氨基酸残基，例如缺失氨基酸残基242-244，表示为(242-244)*或Δ(242-244)或242_244del；如果与其它S蛋白亲本相比，S蛋白突变体含有“缺失”且在该位置含有插入，则表示为：*36Asp或*36D，表示在第36位缺失同时插入天冬氨酸。当在给定位置可插入一个或多个可选择的氨基酸残基时，表示为：N30A,E，或N30A或N30E。
同源性：如本文所用，术语“同源性”是指聚合物分子之间的总体相关性，例如，在核酸分子(例如DNA分子和/或RNA分子)之间和/或在多肽分子之间。通常，术语“同源性”意味着两个分子之间的进化关系。因此，两个同源的分子将具有共同的进化祖先。在本公开的背景下，术语同源性包括同一性和相似性。
在一些实施方案中，如果分子中至少25％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％，95％，96％，97％，98％，99％或100％的单体 是相同的(完全相同的单体)或相似(保守置换)，聚合物分子被认为是彼此“同源的”。术语“同源的”必然是指至少两个序列(多核苷酸或多肽序列)之间的比较。
同一性：如本文所用，术语“同一性”是指聚合物分子之间，例如多核苷酸分子(例如DNA分子和/或RNA分子)之间和/或多肽分子之间的整体单体保守性。例如，可以通过以进行最佳比较目的比对两个序列来进行两个多核苷酸序列的百分同一性的计算(例如，可以在第一和第二核酸序列之一或两个中引入空位用于最佳比对和非相同的序列可以对于比较目的放弃。当比较DNA和RNA时，胸腺嘧啶(T)和尿嘧啶(U)可被认为是等同的。
合适的软件程序可从各种来源获得，并用于蛋白质和核苷酸序列两者的比对。例如，Bl2seq，Needle，Stretcher，Water或Matcher等。
术语“编码区”和“编码区域”是指多核苷酸中的开放阅读框(ORF)，其在表达时产生多肽或蛋白质。
“可操作地连接”是指两个或更多个分子，构建体，转录物，实体，部分等之间的功能性连接。
结构域：如本文所用，当提及多肽时，术语“结构域”是指具有一个或多个可识别的结构或功能特征或性质(例如，结合能力，用作蛋白质-蛋白质相互作用的位点)的多肽的基序。
表达：如本文所用，核酸序列的“表达”是指一个或多个以下事件：(1)从DNA序列产生mRNA模板(例如，通过转录)；(2)mRNA转录物的加工(例如，通过剪接，编辑，5'帽形成和/或3'末端加工)；(3)将mRNA翻译成多肽或蛋白质；和(4)多肽或蛋白质的翻译后修饰。
术语“蛋白突变体”或“多肽突变体”是指其氨基酸序列与天然或参考序列不同的分子。与天然或参考序列相比，氨基酸序列突变体可在氨基酸序列内的某些位置具有置换、缺失和/或插入等。通常，突变体与天然或参考序列将具有至少约50％的同一性，至少约60％的同一性，至少约70％的同一性，至少约80％的同一性，至少约90％的同一性，至少约95％的同一性，至少约99％的同一性。
附图说明
图1：使用RNA 6000nano chip在2100生物分析仪上分析B1.351mRNA完整性结果。
图2：ELISA法检测核酸转染CHO-K1细胞后上清中S蛋白表达水平。
图3：S蛋白突变体的3D结构图。
图4：ELISA法检测用II-37制成的纳米颗粒包封本发明mRNA转染细胞后上清中S蛋白表达水平。
图5：II-37和MC3制备的LNP包封本发明mRNA转染细胞后上清中S蛋白表达水平。
图6：以Firefly Luc为报告蛋白，不同的体外转录载体制备的mRNA在细胞内蛋白表达量 的统计图。
图7：本发明S蛋白突变体免疫BALB/c鼠后体内结合抗体的产生情况统计图。图中a为野生型S蛋白三聚体结果，b为B1.351mRNA翻译出的S蛋白的三聚体结果，c为空白对照。
图8：编码所述S蛋白突变体的mRNA的脂质纳米颗粒免疫BALB/c鼠后，体内结合抗体和中和抗体的产生情况统计图。图中d、e、f分别为5μg、1μg、0.2μg mRNA的脂质纳米颗粒(LNP)免疫后的结合抗体检测结果，图中纵坐标为浓度(μg/ml)；图中g、h、i分别为5μg、1μg、0.2μg mRNA的脂质纳米颗粒(LNP)免疫后的中和抗体检测结果，图中横坐标为血清稀释倍数的log转换值，纵坐标为抑制率％。
图9可电离脂质II-37和C14-113分别形成的包裹mRNA的LNP的细胞转染效率对比
图10MTT法测定II-37-LNP和C14-113-LNP的细胞毒性
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。所述实验方法是本领域常规的分子生物学方法，可以参照本领域的分子生物学实验手册或试剂盒产品说明书的指引进行操作。
实施例1脂质II-37的合成
Figure PCTCN2022122221-appb-000041
亚麻油醇(a2)的合成：在0℃下，向950mL四氢呋喃中加入LiAlH 4(7.20g)、亚麻油酸(50g,a1)，之后混合物在25℃下搅拌2h。薄层色谱法(TLC)显示反应完成后，向反应液依次加入水(7.2mL)，NaOH水溶液(7.2mL，质量分数为15％)和水(21.6mL)淬灭，并加入适量Na 2SO 4搅拌15分钟后通过布氏漏斗过滤并用乙酸乙酯洗涤滤饼，收集滤液并蒸发浓缩，得到目标产物亚麻油醇(a2)47.4g。
1H NMR(400MHz,CDCl 3):δ5.27-5.44(m,4H),3.63(t,J＝6.63Hz,2H),2.77(t,J＝6.44Hz,2H),1.97-2.12(m,4H),1.57-1.63(m,1H),1.20-1.46(m,18H),0.83-0.95(m,3H)
(9Z,12Z)-十八碳-9,12-二烯醛(a3)的合成：在室温下，向170mL乙腈中加入亚麻油醇(25.0g，a2)和2-碘酰基苯甲酸(39.4g)，之后混合物在85℃下搅拌4h。反应液通过布氏漏斗过滤并用二氯甲烷洗涤滤饼，收集滤液并蒸发浓缩，得到目标产物(9Z,12Z)-十八碳-9,12-二烯醛(a3)24.0g。
1H NMR(400MHz,CDCl 3):δ9.76(t,J＝1.76Hz,1H),5.25-5.43(m,4H),2.76(t,J＝6.17Hz,2H),2.41(td,J＝7.33,1.87Hz,2H),2.04(q,J＝6.84Hz,4H),1.56-1.68(m,2H),1.22-1.36(m,14H),0.88(t,J＝6.73Hz,3H)
(9Z,12Z)-2-氯代-十八碳-9,12-二烯-1-醇(a4)的合成：在0℃下，向246mL乙腈中加入(9Z,12Z)-十八碳-9,12-二烯醛(43.0g，a3)，DL-脯氨酸(5.62g)和N-氯代丁二酰亚胺，然后在0℃下搅拌2h。反应完成后，用无水乙醇(246mL)稀释反应液，再加入硼氢化钠(8.8g)，之后在0℃下搅拌4h。向反应混合物中加水(120mL)淬灭，并用甲基叔丁基醚萃取，合并有机相后用饱和食盐水洗涤，硫酸钠干燥之后过滤、蒸发浓缩，得到目标产物(9Z,12Z)-2-氯代-十八碳-9,12-二烯-1-醇(a4,46g)，直接用于下一步。
1H NMR(400MHz,CDCl 3):δ5.25-5.51(m,4H),3.97-4.07(m,1H),3.79(dd,J＝12.01,3.63Hz,1H),3.59-3.70(m,1H),2.67-2.90(m,2H),1.96-2.15(m,5H),1.64-1.82(m,1H),1.20-1.49(m,15H),0.89(br t,J＝6.75Hz,3H)
2-[(7Z,10Z)-十六碳烷-7,10-二烯]环氧乙烷(a5)的合成：在室温下，向450mL1,4-二氧六环中加入(9Z,12Z)-2-氯代-十八碳-9,12-二烯-1-醇(45g，a4)和氢氧化钠水溶液(120g氢氧化钠溶于585mL水)，滴加完毕后混合物在35℃下搅拌2h。TLC显示反应完成后，反应液通过分液漏斗分离，并用饱和食盐水洗涤，硫酸钠干燥后过滤并蒸发浓缩，然后通过快速过柱法用石油醚/乙酸乙酯洗脱来纯化残余物，得到目标产物2-[(7Z,10Z)-十六碳烷-7,10-二烯]环氧乙烷(a5)29.11g。
1H NMR(400MHz,CDCl 3):δ5.27-5.46(m,4H),2.87-2.98(m,1H),2.70-2.85(m,3H),2.46(dd,J＝5.00,2.75Hz,1H),1.94-2.21(m,4H),1.24-1.58(m,17H),0.78-1.00(m,3H)
II-37的合成：在室温下，向10mL乙醇中加入2-[(7Z,10Z)-十六碳烷-7,10-二烯]环氧乙烷(5g)和N,N-二(2-氨基乙基)甲胺(739mg)，之后混合物在90℃下搅拌36h。反应液蒸发浓缩，然后通过快速过柱法用二氯甲烷/甲醇洗脱来纯化残余物，得到粗产物II-37(4g)。再次通过快速过柱法用二氯甲烷/甲醇纯化目标产物，得到II-37(2.2g)。
1H NMR(400MHz,CDCl 3):δ5.27-5.44(m,12H),3.48-3.79(m,3H),2.63-3.00(m,12H),2.16-2.61(m,12H),2.05(q,J＝6.80Hz,12H),1.18-1.57(m,51H),0.89(t,J＝6.88Hz,9H)
ESI-MS：m/z 910.8[M+H] +,911.8[M+2H] +,912.8[M+3H] +
可电离脂质有两个主要作用：结合核酸和允许核酸分子在细胞中释放。脂质的pKa是一个重要因素，因为脂质需要在低pH值下带正电荷才能与核酸结合，但在中性pH值下不带电荷， 才能使形成的LNP不会引起毒性。通过TNS染料结合试验测定可电离脂质Ⅱ-37的pKa在6.81。
实施例2 B1.351mRNA的制备及其翻译
1.人工合成能够编码前述SEQ ID No.8所示mRNA的核酸序列，将该序列克隆到pUC57-kana载体的T7启动子后面，所述载体在之前经过改造，已含有能够编码SEQ ID NO:6、Kozak序列、2个首尾相连的SEQ ID NO:7、polyA尾的序列。编码前述SEQ ID NO:8所示mRNA的核酸序列克隆在Kozak序列和2个首尾相连SEQ ID NO:7之间的多克隆位点上，构建体外转录用质粒。
2.将构建的质粒转化到大肠杆菌Dh5a中，培养扩增，提取质粒。
3.将提取的质粒使用紧邻着polyA尾后面的限制性内切酶SpeI酶切消化成线性分子。
4.以制备的线性化质粒分子为模板，使用体外转录法(Thermo公司的体外转录试剂盒A45975)制备mRNA，所述mRNA的序列如SEQ ID NO:9所示，以下将所述mRNA简称为B1.351mRNA，由该mRNA翻译后获得的是本发明S蛋白突变体，其氨基酸序列从N端向C端为直接连接的SEQ ID NO:2的氨基酸序列和SEQ ID NO:3的氨基酸序列。在体外转录结束后，使用加帽酶和二甲基转移酶给mRNA添加上CAP1的帽结构。
5.mRNA的纯化：得到的mRNA原液使用亲和层析法纯化。
6.mRNA的质控：将制备出来的mRNA，使用RNA 6000nano chip在2100生物分析仪上分析mRNA完整性，结果如图1所示，转录mRNA条带单一，无明显降解。
另外，通过限制性内切酶HindIII和EcoRI从商品化的质粒pCMV3-spike上切下Spike片段，插入到实施例5的IVT1载体的HindIII和EcoRI位点之间，得到IVT1-spike质粒。再对该质粒进行点突变，得到IVT1-spike-D614G质粒，以该质粒为模板，体外转录得到spike-D614G mRNA，表达包含D614G突变的全长S蛋白。
7.B1.351mRNA细胞水平表达检测：以CHO-K1细胞系为表达体系，使用Lipofectamine Messenger MAX Reagent(Invitrogen,Cat#1168-027)将mRNA转染，培养48h后，收集细胞培养上清，采用检测S蛋白的酶联免疫法检测试剂盒，检测S蛋白表达水平，以评判mRNA可否翻译成蛋白。结果如图2所示。图2中，“spike DNA”为商品化的质粒pCMV3-spike(采购自义翘神州公司)，表达全长野生型S蛋白；“spike-D614G mRNA”为前述表达包含D614G突变的全长S蛋白的mRNA，“spike B1.351mRNA”为前述B1.351mRNA，表达的是本发明所述的S蛋白突变体，结果表明本发明的mRNA可以在细胞内高表达出S蛋白突变体。
将得到的S蛋白突变体纯化后，采用冷冻电镜进行结构解析，该S蛋白的3D结构如图3所示，S蛋白突变体为预融合(prefusion spike structure)的稳定结构。B1.351突变毒株的序列和野生毒株的序列有9个突变位点的差异，其中3个在RBD区域。已经报道的野生毒株的预融合S 蛋白的RBD区域状态主要为1个OPEN、2个CLOSE的结构。本发明的S蛋白突变体的结构主要为2个OPEN和1个CLOSE的柔性状态。这种结构差异，是病毒与受体ACE2结合能力增强和传染性增强的结构基础，并且该结构差异，也会导致S蛋白的免疫原性表位的显著差异，从而基于不同结构诱导的抗体尤其是中和抗体的显著不同。
实施例3 包含核酸的脂质纳米颗粒组合物的制备
准确称取化合物II-37、DOPE、CHOL、DSPE-PEG2000、DSPC、DMG-PEG2000等，将每种脂质于适当容器中，无水乙醇充分溶解备用。
将各脂质按下表中的摩尔比例混合均匀，作为有机相，将核酸(mRNA或DNA)配置成水溶液(以纯水为溶剂)作为水相pH＝4。
有机相与水相体积比例为3:1混合，在微流控平台(如，PNI Ignite)上制备得到脂质纳米颗粒混悬液。将得到的脂质纳米颗粒悬浮液经100KDa超滤离心管离心过滤，纯化浓缩，将浓缩后的液体进行分装。
将制备所得脂质纳米颗粒用激光纳米粒度仪测粒径、PDI、电位，用紫外分光光度计结合RiboGreen RNA试剂盒测包封率(％)，示例性结果如下。
Figure PCTCN2022122221-appb-000042
Figure PCTCN2022122221-appb-000043
Figure PCTCN2022122221-appb-000044
在优化制备工艺后，还能获得理化质控数据更好的脂质纳米颗粒，以II-37:DSPC:CHOL:DMG-PEG2000的配方结果示例如下表，其中tri-009-BJ-LNP-21040601的脂质摩尔配比为:40:10:48.5:1.5；tri-009-BJ-LNP-21040602的脂质摩尔配比为:35:15:48.5:1.5；tri-009-BJ-LNP-21040603的脂质摩尔配比为:45:15:38.5:1.5。将其中一部分样本按照实施例2的方式转染细胞CHO，并通过Elisa检测蛋白表达量以评估细胞转染率。
样品编号 PDI 直径(nm) EE％ Zeta电位(mV)
tri-009-BJ-LNP-21040601 0.1429 140.36±27.54 100.00 24.91
tri-009-BJ-LNP-21040602 0.2335 120.48±21.39 100.00 27.58
tri-009-BJ-LNP-21040603 0.1885 134.20±22.20 100.00 28.04
细胞转染结果见图4，图4中，“tri-009-BJ-LNP-21040601”、“tri-009-BJ-LNP-21040602”、“tri-009-BJ-LNP-21040603”是上述对应配方包载实施例2的B1.351mRNA，“lipoMax-spike mRNA”是用lipoMax TM包载实施例2的B1.351mRNA，“Nagative control”为不含mRNA的空白脂质纳米颗粒。从图4中可以看到基于II-37所获得的包载核酸的脂质纳米颗粒转染细胞48小时后，通过ELISA检测均可检测到有抗原蛋白表达，其细胞转染效率与商品化的lipoMax TM相当甚至更好。
实施例4
1、II-37和商业化可电离阳离子脂质分子MC3的效果对比
MC3为：4-(N,N-二甲基氨基)丁酸(6Z,9Z,28Z,31Z)-庚三十碳-6,9,28,31-四稀-19-基脂。
按照实施例3所述的方法，分别使用II37和MC3制备脂质纳米颗粒，具体摩尔配比为：II-37:DSPC:CHOL:DMG-PEG2000＝45:15:38.5:1.5；MC3:DSPC:CHOL:DMG-PEG2000＝45:15:38.5:1.5；包载实施例2中B1.351mRNA。
制备得到的脂质纳米颗粒理化质控数据如下表所示：
样本信息 粒径(nm) PDI Zeta电位 包封率
mRNA-LNP(II-37) 154.58±27.75 0.1068 22.07 90.5
mRNA-LNP(MC3) 234.08±40.11 0.1259 2.44 40.7
由上表可见，在相同的制备工艺下，II-37制备的脂质纳米颗粒包封率高达90.5％，远高于MC3的脂质纳米颗粒，并且粒径更小更均一，电位更高。
采用实施例2相同的转染方式，将制备的脂质纳米颗粒转染细胞，了解蛋白的表达情况，结果如图5所示，II-37(图中以C2表示)制备的脂质纳米颗粒携带mRNA转染细胞之后，细胞中蛋白表达量远远高于MC3的，说明II-37制成的脂质纳米颗粒的细胞转染效率很高。
2、II-37和其结构类似物分子C14-113的对比
C14-113的结构式为：
Figure PCTCN2022122221-appb-000045
按照实施例3所述的方法，分别使用II37和C14-113制备脂质纳米颗粒，具体摩尔配比为：II-37:DSPC:CHOL:DMG-PEG2000＝45:15:38.5:1.5；C14-113:DSPC:CHOL:DMG-PEG2000＝45:15:38.5:1.5；N/P比为10:1。
制备得到的脂质纳米颗粒理化质控数据如下表所示：
样本信息 粒径(nm) PDI Zeta电位
mRNA-LNP(II-37-LNP) 136.68 0.14 20.07
mRNA-LNP(C14-113-LNP) 152.65 0.12 24.1
用多功能酶标仪(BioTek，型号为SLXFATS)荧光素报告基因法来检测制备的LucRNA-LNP HEK293T细胞的转染效率，转染的LucRNA量分别为0.5μg、1.0μg、2.0μg。体外转录LucRNA的方法如下：HEK293T细胞铺板，细胞密度为2.5×10 5个细胞/mL，当细胞融合度为30％-50％进行转染。转染48h后使用多功能酶标仪检测蛋白表达量。阴性对照为不添加LucRNA-LNP的细胞培养基。结果如图9所示，在转染的mRNA量相同的情况下，II-37(图中以II-37-LNP表示)制备的脂质纳米颗粒携带mRNA转染细胞之后，细胞中蛋白表达量远远高于C14-113的，说明II-37制成的脂质纳米颗粒的细胞转染效率很高。
此外，采用MTT法测定II-37-LNP和C14-113-LNP的细胞毒性，考察载体剂量、作用时间等因素对正常细胞(293T)细胞增殖的影响。结果如图10所示，II-37(图中以II-37-LNP表示)制备的脂质纳米颗粒携带mRNA转染细胞48小时之后，在较高的剂量(2μg/mL)时依然保持较好细胞活性，说明II-37制成的脂质纳米颗粒的细胞毒性很低。
从结果可以看出，由新型脂质化合物配伍制备的脂质纳米颗粒细胞毒性低，且在mRNA转染效率优于结构类似物分子C14-113。
实施例5 本发明IVT载体的效率对比实验
本实施例以Firefly Luc为报告蛋白，构建了不同的IVT载体用于在体外转录合成能够翻译Firefly Luc的mRNA，对比合成的不同序列特征的mRNA的翻译效率。
采用本领域常规的质粒载体构建技术，将Firefly Luc的编码序列克隆到相应载体的多克隆位点上，得到编号分别为IVT1，IVT2，IVT3和IVT4的载体，之后使用AM1344试剂盒依据前述载体体外转录制备得到对应的Firefly Luc mRNA样品。
载体IVT1～IVT4均为在商业化载体psp73的基础上改造而来，以下序列在载体psp73酶切位点XhoI/NdeI处插入，其中IVT1中没有添加UTR序列，polyA尾长度为64个A；IVT2中使用了SEQ ID NO:6所示的5’UTR和GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC的3’UTR序列(β珠蛋白的3’UTR序列)，polyA长度为120个A；IVT3中使用了SEQ ID NO:6所示的5’UTR和SEQ ID NO:7所示的3’UTR序列，polyA长度为120个A；IVT4中使用了SEQ ID NO:6所示的5’UTR和2个串联重复的SEQ ID NO:7所示的3’UTR序列，polyA长度为120个A。在上述5’UTR和3’UTR的序列中间插入包含常见酶切位点HindIII和EcoRI的多克隆位点，再将Firefly Luc的编码序列克隆到HindIII和EcoRI的多克隆位点中。所有载体均为金斯瑞公司使用基因合成的方法构建得到。
将每种Firefly Luc mRNA样品以Lipofectamine2000(cat#11668030，购自赛默飞世尔公司)为转染试剂，转染到CHO细胞中，使用Dual-Lumi TM双萤光素酶报告基因检测试剂盒(at#RG088S，购自上海碧云天生物技术有限公司)检测荧光素酶。将Firefly Luc的DNA转入psicheck2质粒作为阳性对照(psicheck2质粒，cat#60908-6151，购自北京天恩泽基因科技有限公司)。具体如下：第一天，将CHO细胞种到96孔板，每孔1.5×10 4个细胞，使用F12K+10％FBS培养过夜；第二天，转染前将培养基换成无血清的F12K培养基，使用Lipofectamine2000，将mRNA或DNA转染到CHO细胞中；每孔使用的核酸量为100ng，脂质体用量为0.3μl，每孔总体积为100μl，过夜培养；第三天，将无血清培养基换成完全培养基(F12K+10％FBS)，继续培养24小时；第四天(转染后48小时)，检测Firefly Luc荧光值。
结果如图6所示。图中“DNA”是阳性对照(携带有Firefly Luc的DNA的psicheck2质粒)，“IVT1-Luc”、“IVT2-Luc”、“IVT3-Luc”、“IVT4-Luc”分别代表由IVT1，IVT2，IVT3和IVT4的载体体外转录得到的对应Firefly Luc mRNA，“Nagative control”为阴性对照。由图6可见，在相同mRNA的转染量下，IVT4-Luc的蛋白表达量远远高于其他三个mRNA，为其2-3倍，说明IVT4-Luc的稳定性好且翻译效率高。
实施例6 S蛋白突变体免疫原性的测定
使用BALB/c鼠评价S蛋白突变体诱导结合抗体和中和抗体的产生：6周龄雌性BALB/c鼠，初次免疫及二次免疫间隔2周；免疫14天后采血。ELISA法检测针对S蛋白突变体的结合抗体的表达，化学发光检测针对S蛋白突变体的中和抗体滴度。
ELISA方法检测结合抗体：通过在酶标板上包被商业化S蛋白捕获免疫小鼠血浆中针对S蛋白突变体的结合抗体，再用生物素标记的检测抗体进行吸光度检测。化学发光检测针对S蛋白突变体的中和抗体滴度：免疫后小鼠血浆与携带荧光素酶报告基因的SPIKE慢病毒(中吉当康；商品名称：SRAS-CoV-2假病毒(B.1.351)-LUC；商品编号：DZPSC-L-0；批次：K05202102)中和后去感染高表达ACE-2的293T细胞(中吉当康；商品名称：“YJ1B09”hACE2-293T cell lines；商品编号：YJ293T-01；批次：A23202001)，用化学发光(Bright-Lumi II萤火虫萤光素酶报告基因检测试剂盒，品牌：碧云天；商品编号：RG052M)评定血浆的中和抗体滴度。
对照组共9只小鼠，每只小鼠皮下注射2ug蛋白。其中，3只注射的蛋白为实施例2的B1.351mRNA翻译出的S蛋白的三聚体纯化物，3只注射的蛋白为野生型的S蛋白的三聚体，3只注射的是空白脂质纳米颗粒，脂质纳米颗粒的脂质配方是实施例3中tri-009-BJ-LNP-21040602。
实验组共18只小鼠，皮下注射mRNA的脂质纳米颗粒；其中1-6号注射0.2μg mRNA的脂质纳米颗粒(LNP)，7-12号注射1μg mRNA的脂质纳米颗粒(LNP)，13-18号注射5μg mRNA的脂质纳米颗粒(LNP)，其中的mRNA是实施例2的B1.351mRNA，脂质纳米颗粒的配方是实施例3中tri-009-BJ-LNP-21040602。
初次免疫及二次免疫后小鼠结合抗体表达水平以及中和抗体水平如图7-8所示。
由图7中的a、b、c可见，实施例2的B1.351mRNA翻译出的S蛋白三聚体及野生型的S蛋白三聚体在小鼠体内均可以诱导出抗S蛋白的结合抗体：在实验小鼠中第二次免疫时已经能产生较高浓度的结合抗体，在二次免疫后8周，所述结合抗体的浓度仍保持较高水平，经计算，二次免疫后结合抗体浓度在2.2μg/ml左右，二次免疫后8周，仍能维持1.6μg/ml左右。
由图8中的d、e、f可见，二次免疫LNP包裹的mRNA制剂后的小鼠，即使是低剂量(0.2μg)注射组也可在小鼠体内诱导出抗S蛋白的结合抗体，经计算，结合抗体水平约为0.1-0.3μg/ml。由图8中的g、h、i可见，二次免疫LNP包裹的mRNA制剂后的小鼠体内诱导出了较好的中和抗体，中和抗体的GMT值分别为78.69，21.9和72.19。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种脂质纳米颗粒组合物，其含有脂质纳米颗粒，所述脂质纳米颗粒中包含式I的脂质分子，
    Figure PCTCN2022122221-appb-100001
    其中：
    Q为经取代或未取代的直链C2-20亚烷基，所述亚烷基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；或，Q为经取代或未取代、饱和或不饱和的4-6元环，所述4-6元环的环原子任选含有1个或1个以上独立地选自O、S、N的杂原子；所述取代的取代基团选自卤素、-OH、直链或支链的C1-20烷基、直链或支链的C1-20烷氧基、直链或支链的C2-20烯基、直链或支链的C2-20炔基、-CH 2CH(OH)R 5、
    Figure PCTCN2022122221-appb-100002
    R 1、R 2、R 3、R 4可以相同或不同，各自独立的选自氢，经取代或未取代的直链或支链C1-30烷基，经取代或未取代的直链或支链C2-30烯基，经取代或未取代的直链或支链C2-30炔基，所述烷基、烯基或炔基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换，或-CH 2CH(OH)R 5；所述取代的取代基团选自卤素、-OH、直链或支链的C1-10烷基、直链或支链的C1-10烷氧基；
    条件是，R 1、R 2、R 3、R 4中至少一个为
    Figure PCTCN2022122221-appb-100003
    R 5选自氢，经取代或未取代的直链或支链C1-30烷基，经取代或未取代的直链或支链C2-30烯基，经取代或未取代的直链或支链C2-30炔基，所述烷基、烯基或炔基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；所述取代的取代基团选自卤素、-OH、直链或支链的C1-10烷基、直链或支链的C1-10烷氧基；
    R 6选自氢，C1-3烷基，C1-3烷氧基，-OH；
    n选自1～8的整数，m选自0～8的整数，n和m彼此独立，可以相同也可以不同；
    当R 1、R 2、R 3、R 4中至少两个为
    Figure PCTCN2022122221-appb-100004
    时，每个所述基团中的n和m彼此独立，可以相同也可以不同。
  2. 如权利要求1所述的脂质纳米颗粒组合物，其中式I中的Q为经取代或未取代的直链C2-20亚烷基，所述亚烷基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；
    优选，Q为
    Figure PCTCN2022122221-appb-100005
    其中，R 8、R 9彼此独立的选自经取代或未取代的直链C1-10亚烷基，所述亚烷基的1个或1个以上C原子任选被独立地选自O、S和N的杂原子所替换；R 7为氢，卤素，-OH，直链或支链C1-20烷基，直链或支链C2-20烯基，直链或支链C2-20炔基，或-CH 2CH(OH)R 5，或
    Figure PCTCN2022122221-appb-100006
    所述取代的取代基团为卤素、-OH、直链或支链的C1-10烷基、直链或支链的C1-10烷氧基；
    更优选，Q为
    Figure PCTCN2022122221-appb-100007
    其中：x和y可以相同或者不同，独立地选自1～8的整数；R 7定义和前述相同；优选，x或y相同或不同，选自1～3的整数，例如为1、2或3；优选，R 7为直链或支链C1-4烷基，例如为甲基、乙基、正丙基、正丁基等。
  3. 如权利要求1或2所述的脂质纳米颗粒组合物，其中式I中所述饱和或不饱和的4-6元环为哌嗪基或环己基；
    优选，R 6为-OH；
    优选，n选自4～8的整数，m选自4～8的整数。
  4. 如权利要求1-3任一项所述的脂质纳米颗粒组合物，所述式I化合物为下式A、B、C或D：
    Figure PCTCN2022122221-appb-100008
    其中每个n 1都彼此独立，可以相同或不同，每个n 1选自1～8的整数，每个m 1都彼此独立，可以相同或不同，每个m 1选自0～8的整数；优选，每个n 1选自4～8的整数，每个m 1选自4～8的整数；优选，每个n 1都彼此相同，每个m 1都彼此相同；
    Figure PCTCN2022122221-appb-100009
    其中每个n 2都彼此独立，可以相同或不同，每个n 2选自1～8的整数，每个m 2都彼此独立，可以相同或不同，每个m 2选自0～8的整数；优选，每个n 2选自4～8的整数，每个m 2选自4～8的整数；优选，每个n 2都彼此相同，每个m 2都彼此相同；
    Figure PCTCN2022122221-appb-100010
    其中每个n 3都彼此独立，可以相同或不同，每个n 3选自1～8的整数，每个m 3都彼此独立，可以相同或不同，每个m 3选自0～8的整 数；优选，每个n 3选自4～8的整数，每个m 3选自4～8的整数；优选，每个n 3都彼此相同，每个m 3都彼此相同；
    Figure PCTCN2022122221-appb-100011
    其中每个n 4都彼此独立，可以相同或不同，每个n 4选自1～8的整数，每个m 4都彼此独立，可以相同或不同，每个m 4选自0～8的整数；优选，每个n 4选自4～8的整数，每个m 4选自4～8的整数；优选，每个n 4都彼此相同，每个m 4都彼此相同。
  5. 如权利要求1-4任一项所述的脂质纳米颗粒组合物，脂质纳米颗粒中含有占其总体脂质分子30-60mol％的式I的脂质分子，优选32-55mol％，进一步优选34-46mol％；
    优选，所述脂质纳米颗粒中含有占其总体脂质分子5-30mol％的中性脂质分子，优选8-20mol％，进一步优选9-16mol％；
    优选，所述脂质纳米颗粒中可以含有占其总体脂质分子30-50mol％的胆固醇类脂质分子，优选35-50mol％，进一步优选37-49mol％；
    优选，所述脂质纳米颗粒中可以含有占其总体脂质分子0.4-10mol％的PEG化的脂质分子，优选0.5-5mol％，进一步优选1.3-2.7mol％；
    优选，所述脂质纳米颗粒组合物，进一步包含活性成分，所述活性成分位于脂质纳米颗粒中；优选，所述活性成分是核酸；优选，所述活性成分是mRNA。
  6. 如权利要求5所述的脂质纳米颗粒组合物，所述中性脂质分子选自式E所示磷脂酰胆碱类化合物
    Figure PCTCN2022122221-appb-100012
    式F所示磷脂酰乙醇胺类化合物
    Figure PCTCN2022122221-appb-100013
    其中Ra、Rb、Rc、Rd独立的选自直链或支链的C1-30烷基，直链或支链的C2-30烯基，优选为直链或支链的C10-30烷基，直链或支链的C10-30烯基，更优 选为CH 3(CH 2) 17CH 2-、CH 3(CH 2) 15CH 2-、CH 3(CH 2) 13CH 2-、CH 3(CH 2) 11CH 2-、CH 3(CH 2) 9CH 2-、CH 3(CH 2) 7CH 2-、CH 3(CH 2) 7-CH＝CH-(CH 2) 7-、CH 3(CH 2) 4CH＝CHCH 2CH＝CH(CH 2) 7-、CH 3(CH 2) 7-CH＝CH-(CH 2) 9-；例如DOPE和/或DSPC；
    胆固醇类脂质分子选自胆固醇、粪甾醇，谷甾醇，麦角甾醇，菜油甾醇，豆甾醇，菜子甾醇，番茄次碱，番茄碱，熊果酸，α-生育酚及其混合物、5-十七基间苯二酚和胆固醇半琥珀酸酯；
    PEG化的脂质分子包含脂质部分和基于PEG的聚合物部分，表示为“脂质部分-PEG-数均分子量”，所述脂质部分是二酰基甘油或二酰基甘油酰胺，选自二月桂酰甘油、二肉豆蔻酰甘油、二棕榈酰甘油、二硬脂酰甘油、二月桂基甘油酰胺、二肉豆蔻基甘油酰胺、二棕榈酰甘油酰胺、二硬脂酰甘油酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺；PEG的数均分子量为130～50,000，优选为150～10,000，进一步优选为300～3,000，最优选为1,500～2,500；例如DMG-PEG2000和/或DSPE-PEG2000。
  7. 如权利要求5所述的脂质纳米颗粒组合物，所述脂质纳米颗粒组合物中脂质分子的总质量与核酸质量之比为5-20:1。
  8. 如权利要求5所述的脂质纳米颗粒组合物，所述mRNA从5’端至3’端包含5’UTR，开放阅读框，3’UTR和poly-A尾；
    优选，所述5’UTR包含与SEQ ID NO:6所示的β-珠蛋白的5’UTR核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列；
    优选，所述3’UTR包含与SEQ ID NO:7所示α2-珠蛋白3’UTR的片段至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列；
    优选，所述3’UTR包含2个首尾相连的与SEQ ID NO:7所示的α2-珠蛋白3’UTR的片段至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列
    优选，所述poly-A尾的长度为50-200个核苷酸，优选为100-150个核苷酸；
    优选，所述开放阅读框是编码2019-nCov的S蛋白突变体的开放阅读框，其核酸序列是与SEQ ID NO:8所示核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列；
    优选，所述mRNA包含与SEQ ID NO:9所示的核苷酸序列至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或约100％同源性的核苷酸序列；
    优选，所述mRNA还具有5’帽结构。
  9. 如权利要求1-8任一项所述的脂质纳米颗粒组合物，进一步含有医药赋形剂；优选所述脂质纳米颗粒组合物是液体制剂，含有质量百分比浓度为5-20％的蔗糖。
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US20120295832A1 (en) * 2011-05-17 2012-11-22 Arrowhead Research Corporation Novel Lipids and Compositions for Intracellular Delivery of Biologically Active Compounds

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