A lipid nanoparticle composition and a drug delivery system prepared therefrom

Abstract

The present invention provides novel ionizable cationic lipid molecules, lipid nanoparticles composed of these lipid molecules, neutral lipid molecules, cholesterol-like lipid molecules, and PEGylated lipid molecules, and compositions containing these lipid nanoparticles. These lipid nanoparticles, as delivery vehicles for active ingredients, offer advantages such as small and uniform particle size, high encapsulation efficiency, and high cell transfection efficiency, making them particularly suitable for delivering nucleic acid molecules (e.g., mRNA).

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical preparations, and in particular relates to a lipid nanoparticle containing a novel lipid compound, and a pharmaceutical composition or drug delivery system carrying an active ingredient prepared therefrom, such as an mRNA vaccine. Background Art

[0002] Different types of nucleic acid agents are being developed to treat a variety of major diseases, including infectious diseases, cancer, and rare diseases. These include DNA, antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamers, and ribozymes. Among them, mRNA vaccines offer disruptive advantages in terms of safety, rapid preparation, and immunogenicity. Advances in mRNA modification and delivery tools allow for rapid design and production of clinical-scale mRNA vaccines within weeks once the viral antigen sequence is obtained, enabling standardized production and making them highly attractive for pandemic response. Furthermore, mRNA vaccines do not present the potential risk of reversion associated with attenuated vaccines, nor do they present the reversion mutations associated with inactivated vaccines. Regarding immunogenicity, mRNA vaccines can induce both B and T cell immune responses, induce immune memory, deliver more effective antigens, and express multiple antigens simultaneously. Furthermore, mRNA only needs to cross the cell membrane to efficiently express antigenic proteins in the cytoplasm, and there is no risk of gene integration into the genome. Again, mRNA is easily degraded after being translated into protein. Its transient expression characteristics not only ensure the safety of mRNA drugs, but also make their dosage controllable, avoiding antigen immune tolerance (a state of no response to specific antigens) caused by long-term exposure to vaccine drugs.

[0003] However, nucleic acid preparations are negatively charged and often have large molecular weights, making them difficult to directly enter cells. Furthermore, RNA is unstable and easily degraded by nucleases during introduction into the body, thus losing its biological function. Therefore, developing efficient, safe, and universal nucleic acid delivery systems is a pressing challenge in the transformation of nucleic acid drugs.

[0004] Currently, nucleic acid delivery methods include chemical modification, bioconjugation technology, nanocarrier technology, lipid-based formulations, exosomes, spherical nucleic acids, DNA nanostructures, and stimulus-responsive polymer nanomaterials. Among them, lipid nanoparticles (LNPs) are relatively mature nucleic acid delivery vehicles. In 2018, the LNP-encapsulated siRNA drug Onpattro (patisiran) was approved for marketing. In 2021, the LNP-encapsulated mRNA vaccine was officially approved by the FDA for use in the prevention and control of the COVID-19 pandemic. Clinical results have shown high efficacy, with no serious adverse reactions to date.

[0005] The main components of this lipid formulation include cationic / ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol-lipid conjugates. Of these four lipid components, the charged head of the cationic lipids can bind to negatively charged nucleic acids and also to phospholipid molecules on cell membranes, playing a key role in nucleic acid encapsulation and membrane fusion. Given the potential toxicity of permanent cationic lipids, lipid nanoparticles containing ionizable cationic lipids offer greater application potential.

[0006] Ionizable cationic lipids consist of three key structural components: a hydrophilic polar head containing an amine group; a hydrophobic lipid chain; and a linker chain connecting the polar head and the nonpolar tail. Currently, commercially available ionizable cationic lipids primarily include the MC3 series and ALC-0315 and SM-102, which are used in COVID-19 mRNA vaccines. MC3 has strong liver targeting, which limits its application for potentially hepatotoxic nucleic acid formulations. Furthermore, MC3 was developed for the delivery of smaller siRNAs, potentially limiting its loading capacity for larger nucleic acid formulations. The delivery efficiency of ALC-0315 and SM-102 needs to be further improved. Therefore, to further advance the development of lipid formulations in my country and their application in nucleic acid drug delivery, we need to develop new ionizable cationic lipids and screen and optimize new nanodelivery systems for the safe and efficient delivery of nucleic acids, such as those used in mRNA vaccines. Summary of the Invention

[0007] To address the structural instability, susceptibility to nuclease degradation, and difficulty cellular entry of active ingredients, such as nucleic acids (e.g., mRNA molecules), during biological applications, new delivery technologies are needed. Furthermore, existing delivery technologies often suffer from poor lysosomal escape and low delivery efficiency. The present invention utilizes newly synthesized ionizable cationic lipid molecules to form a diverse lipid-based delivery system with diverse functions.

[0008] In a first aspect, the present invention provides a lipid nanoparticle composition, wherein the lipid nanoparticle composition contains lipid nanoparticles, and the lipid nanoparticles contain: ionizable cationic lipid molecules of formula I.

[0009] According to the present invention, the lipid nanoparticle composition further comprises other lipid molecules. The other lipid molecules can be lipid molecules known or conventionally used in the art for constructing lipid nanoparticles, including but not limited to neutral lipid molecules, cholesterol-like lipid molecules, and PEGylated lipid molecules.

[0010] According to the present invention, the lipid nanoparticle composition further comprises an active ingredient, which may be a small molecule compound, a nucleic acid, a protein, a polypeptide, etc. The active ingredient is located in the lipid nanoparticle. The nucleic acid includes but is not limited to DNA, antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, etc.

[0011] According to the present invention, in the lipid nanoparticle composition, the lipid nanoparticles contain 30-60 mol% of lipid molecules of formula I, preferably 32-55 mol%, and more preferably 34-46 mol%, of the total lipid molecules.

[0012] According to the present invention, in the lipid nanoparticle composition, the lipid nanoparticles may contain 5-30 mol% of neutral lipid molecules, preferably 8-20 mol%, and more preferably 9-16 mol%, of the total lipid molecules.

[0013] According to the present invention, in the lipid nanoparticle composition, the lipid nanoparticles may contain cholesterol lipid molecules accounting for 30-50 mol% of the total lipid molecules, preferably 35-50 mol%, and more preferably 37-49 mol%.

[0014] According to the present invention, in the lipid nanoparticle composition, the lipid nanoparticles may contain 0.4-10 mol% of PEGylated lipid molecules, preferably 0.5-5 mol%, and more preferably 1.3-2.7 mol%, of the total lipid molecules.

[0015] According to the present invention, when the active ingredient is nucleic acid, the ratio of the total mass of lipid molecules to the mass of nucleic acid in the lipid nanoparticle composition is 5-20:1.

[0016] According to the present invention, the structural formula of the ionizable cationic lipid molecule of formula I is in:

[0017] Q is a substituted or unsubstituted straight-chain C2-20 alkylene, wherein one or more C atoms of the alkylene 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, wherein the ring atoms of the 4-6 membered ring optionally contain one or more heteroatoms independently selected from O, S and N; the substituted substituent is selected from halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C1-20 alkoxy, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, -CH2CH(OH)R5,

[0018] R1, R2, R3, and R4 may be the same or different and are independently selected from hydrogen, substituted or unsubstituted linear or branched C1-30 alkyl, substituted or unsubstituted linear or branched C2-30 alkenyl, or substituted or unsubstituted linear or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl, or alkynyl group are optionally replaced by a heteroatom independently selected from O, S, and N, or -CH2CH(OH)R5; the substituted substituent is selected from halogen, -OH, linear or branched C1-10 alkyl, or linear or branched C1-10 alkoxy;

[0019] The condition is that at least one of R1, R2, R3, and R4 is

[0020] R5 is selected from hydrogen, substituted or unsubstituted straight or branched C1-30 alkyl, substituted or unsubstituted straight or branched C2-30 alkenyl, substituted or unsubstituted straight or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl group are optionally replaced by heteroatoms independently selected from O, S and N; the substituted substituents are selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy;

[0021] R6 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, -OH;

[0022] n is an integer selected from 1 to 8, m is an integer selected from 0 to 8, n and m are independent of each other and may be the same or different;

[0023] When at least two of R1, R2, R3, and R4 are When n and m in each of the groups are independent of each other, they may be the same or different.

[0024] In a preferred embodiment of the present invention, Q is a substituted or unsubstituted straight-chain C2-20 alkylene group, wherein one or more C atoms of the alkylene group are optionally replaced by heteroatoms independently selected from O, S and N;

[0025] Preferably, Q is wherein R8 and R9 are independently selected from substituted or unsubstituted straight-chain C1-10 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; R7 is hydrogen, halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, or -CH2CH(OH)R5, or The substituted substituent group is halogen, -OH, linear or branched C1-10 alkyl, linear or branched C1-10 alkoxy;

[0026] Preferably, Q is wherein: x and y may be the same or different and independently selected from integers of 1 to 8; R7 is defined as above; preferably, x or y are the same or different and are selected from integers of 1 to 3, for example, 1, 2 or 3; preferably, R7 is a linear or branched C1-4 alkyl group, for example, methyl, ethyl, n-propyl, n-butyl, etc.

[0027] In some embodiments of the present invention, the saturated or unsaturated 4-6 membered ring is piperazinyl or cyclohexyl.

[0028] In a preferred embodiment of the present invention, R6 is -OH.

[0029] In a preferred embodiment of the present invention, n is selected from an integer of 4 to 8, and m is selected from an integer of 4 to 8.

[0030] In a preferred embodiment of the present invention, the compound of formula I is the following formula A, B, C or D:

[0031] wherein each n1 is independent of each other and may be the same or different, each n1 is selected from an integer of 1 to 8, each m1 is independent of each other and may be the same or different, each m1 is selected from an integer of 0 to 8; preferably, each n1 is selected from an integer of 4 to 8, each m1 is selected from an integer of 4 to 8; preferably, each n1 is the same as each other, and each m1 is the same as each other.

[0032] Wherein each n2 is independent of each other and may be the same or different, each n2 is selected from an integer of 1 to 8, each m2 is independent of each other and may be the same or different, each m2 is selected from an integer of 0 to 8; preferably, each n2 is selected from an integer of 4 to 8, each m2 is selected from an integer of 4 to 8; preferably, each n2 is the same as each other, and each m2 is the same as each other.

[0033] Wherein each n3 is independent of each other and may be the same or different, each n3 is selected from an integer of 1 to 8, each m3 is independent of each other and may be the same or different, each m3 is selected from an integer of 0 to 8; preferably, each n3 is selected from an integer of 4 to 8, each m3 is selected from an integer of 4 to 8; preferably, each n3 is the same as each other, and each m3 is the same as each other.

[0034] wherein each n4 is independent of each other and may be the same or different, each n4 is selected from an integer of 1 to 8, each m4 is independent of each other and may be the same or different, each m4 is selected from an integer of 0 to 8; preferably, each n4 is selected from an integer of 4 to 8, each m4 is selected from an integer of 4 to 8; preferably, each n4 is the same as each other, and each m4 is the same as each other.

[0035] In some embodiments of the present invention, the compound of formula I is selected from the following compounds shown in Table 1:

[0036] Table 1

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] According to the present invention, the molar percentage of the lipid molecules of Formula I in the lipids of the lipid nanoparticles is 30-60 mol%, for example, 32-55 mol%, for example, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, etc.

[0048] According to the present invention, the neutral lipid molecules are uncharged lipid molecules or zwitterionic lipid molecules, such as phosphatidylcholine compounds and / or phosphatidylethanolamine compounds.

[0049] The structure of phosphatidylcholine compounds is shown in Formula E: The structure of phosphatidylethanolamine compounds is shown in Formula F: Wherein Ra, Rb, Rc, and Rd are independently selected from a linear or branched C1-30 alkyl group, a linear or branched C2-30 alkenyl group, preferably a linear or branched C10-30 alkyl group, a linear or branched C10-30 alkenyl group, such as CH3(CH2) 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11 CH2-, CH3(CH2)9CH2-, CH3(CH2)7CH2-, CH3(CH2)7-CH=CH-(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)7-CH=CH-(CH2)9-.

[0050] Examples of neutral lipid molecules include, but are not limited to, 5-heptadecanethen-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (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-stearoylphosphatidylcholine (PSPC), 1,2-diacaproyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof.

[0051] In one embodiment, the neutral lipid molecule can be selected from the group consisting of: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE). In another embodiment, the neutral lipid molecule can be dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral lipid molecule can be dipalmitoylphosphatidylcholine (DPPC).

[0052] According to the present invention, the molar percentage of neutral lipid molecules in the lipids of the lipid nanoparticles is 5-30 mol%, for example 8-20 mol%, for example, it can be 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%.

[0053] According to the present invention, cholesterol lipid molecules refer to sterols and lipids containing sterol moieties, including but not limited to cholesterol, 5-heptadecanol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatidine, ursolic acid, α-tocopherol and mixtures thereof, cholesterol hemisuccinate. In one embodiment, the cholesterol lipid molecule is cholesterol (CHOL). In one embodiment, the cholesterol lipid molecule is cholesterol hemisuccinate.

[0054] According to the present invention, the molar percentage of cholesterol lipid molecules in the lipids of the 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%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, etc.

[0055] According to the present invention, the lipid molecules of PEGization include a lipid portion and a PEG-based polymer portion. In some embodiments, the lipid portion can be derived from diacylglycerol or diacylglycerol amide (diacylglycamide), including those comprising dialkylglycerol or dialkylglycerolamide groups with an alkyl chain length independently comprising about C4 to about C30 saturated or unsaturated carbon atoms, wherein the chain can include one or more functional groups, such as amides or esters. In some embodiments, the alkyl chain length includes about C10 to C20. The dialkylglycerol or dialkylglycerolamide groups can also include one or more substituted alkyl groups. The chain length can be symmetrical or asymmetrical. Unless otherwise indicated, as used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG portion is an optionally substituted straight or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG portion can be substituted by, for example, one or more alkyl, alkoxy, acyl, hydroxyl or aryl groups. In one embodiment, the PEG moiety comprises a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety does not comprise a PEG copolymer, e.g., it can be a PEG monomer. In one embodiment, the molecular weight of PEG is from about 130 to about 50,000, in a sub-embodiment, from about 150 to about 30,000, in a sub-embodiment, from about 150 to about 20,000, in a sub-embodiment, from about 150 to about 15,000, in a sub-embodiment, from about 150 to about 10,000, in a sub-embodiment, from about 150 to about 6,000, in a sub-embodiment, from about 150 to about 5,000, in a sub-embodiment, from about 150 to about 4,000, in a sub-embodiment, from about 150 to about 3,000, in a sub-embodiment, from about 300 to about 3,000, in a sub-embodiment, from about 1,000 to about 3,000, and in a sub-embodiment, from about 1,500 to about 2,500. In certain embodiments, the PEG is "PEG 2000," which has an average molecular weight of about 2,000 Daltons. In some embodiments of the invention, PEG is represented herein by the formula In some embodiments, n may be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl and unsubstituted alkyl. In some embodiments, R may be an unsubstituted C1-C30 alkyl, such as a C1-C20 alkyl, a C1-C10 alkyl, or a C1-C6 alkyl. In some embodiments, R may be H, methyl or ethyl.

[0056] In some embodiments, the PEGylated lipid molecule can be represented as "lipid moiety-PEG-number average molecular weight" or "PEG-lipid moiety" or "PEG-number average molecular weight-lipid moiety", wherein the lipid moiety is a diacylglycerol or a diacylglycerol amide selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylglycerol, dilaurylglyceramide, dimyristoylglyceramide, dipalmitoylglyceramide, distearoylglyceramide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG is From about 130 to about 50,000, for example, from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, from about 150 to about 5,000, from about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, from about 1,500 to about 2,500, for example, about 2000.

[0057] In some embodiments, the PEGylated lipid molecule can 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'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-di-tetradecylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-distearoyl-sn-glycero-methoxypolyethylene glycol (DSG-PEG2000), poly(ethylene glycol)-2000-dimethacrylate (DMA-PEG2000) and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (DSA-PEG2000). In one embodiment, the PEGylated lipid molecule may be DMG-PE In some embodiments, the PEGylated lipid molecule may be DSG-PEG2000. In one embodiment, the PEGylated lipid molecule may be DSPE-PEG2000. In one embodiment, the PEGylated lipid molecule may be DMA-PEG2000. In one embodiment, the PEGylated lipid molecule may be C-DMA-PEG2000. In one embodiment, the PEGylated lipid molecule may be DSA-PEG2000. In one embodiment, the PEGylated lipid molecule may be PEG2000-C11. In some embodiments, the PEGylated lipid molecule may be PEG2000-C14. In some embodiments, the PEGylated lipid molecule may be PEG2000-C16. In some embodiments, the PEGylated lipid molecule may be PEG2000-C18.

[0058] According to the present invention, the molar percentage of PEGylated lipid molecules in the lipid of lipid nanoparticles is 0.4-10 mol%, such as 0.5-5 mol%, for example, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%, 3.1 mol%, 3.2 mol%, 3.3 mol%, 3.4 mol%, 3.5 mol%, 3.6 mol%, 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%, etc.

[0059] In some embodiments of the present invention, the lipid nanoparticles contain lipid molecules represented by formula C, neutral lipid molecules, cholesterol lipid molecules, and PEGylated lipid molecules, wherein:

[0060] Formula C wherein each n3 is independent of each other and may be the same or different, each n3 is selected from an integer of 1 to 8, each m3 is independent of each other and may be the same or different, each m3 is selected from an integer of 0 to 8; preferably, each n3 is selected from an integer of 4 to 8, each m3 is selected from an integer of 4 to 8; preferably, each n3 is the same as each other, each m3 is the same as each other. The molar percentage of the ionizable cationic lipid molecules represented by Formula C in the lipid nanoparticles is 32-55 mol%, preferably 34-46 mol%.

[0061] The neutral lipid molecule is selected from the phosphatidylcholine compound shown in formula E Phosphatidylethanolamine compound represented by formula F Wherein Ra, Rb, Rc, and Rd are independently selected from a linear or branched C10-30 alkyl group, a linear or branched C10-30 alkenyl group, preferably CH3(CH2) 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11CH2-, CH3(CH2)9CH2-, CH3(CH2)7CH2-, CH3(CH2)7-CH=CH-(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)7-CH=CH-(CH2)9-. The molar percentage of neutral lipid molecules in the lipid nanoparticles is 8-20 mol%, preferably 9-16 mol%;

[0062] The cholesterol lipid molecules are selected from cholesterol and cholesterol hemisuccinate. The cholesterol lipid molecules account for 30-50 mol% of the lipids in the lipid nanoparticles, preferably 35-50 mol%, and more preferably 37-49 mol%.

[0063] The PEGylated lipid molecules are represented by "lipid moiety-PEG-number average molecular weight", wherein the lipid moiety is a diacylglycerol or a diacylglycerol amide selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylglycerol, dilaurylglyceramide, dimyristoylglyceramide, dipalmitoylglyceramide, distearoylglyceramide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG is 130 to 50,000, for example, 150 to 30,000, 150 to 20,000, 150 to 15,000, 150 to 10,000, 150 to 6,000, 150 to 5,000, 150-4,000, 150-3,000, 300-3,000, 1,000-3,000, 1,500-2,500, about 2000. The molar percentage of the PEGylated lipid molecules in the lipid nanoparticles is 0.5-5 mol%, preferably 1.3-2.7 mol%.

[0064] In one embodiment of the invention, the nucleic acid is mRNA.

[0065] According to the present invention, 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.

[0066] According to the present invention, the cap structure can be a Cap1 structure, a Cap2 structure or a Cap3 structure. In one embodiment of the present invention, the cap structure is a Cap1 structure.

[0067] According to the present invention, the 5'UTR may comprise the 5'UTR of β-globin or α-globin, or a homolog or fragment thereof. In some embodiments of the present invention, the 5'UTR comprises a nucleotide sequence that is 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 to the 5'UTR nucleotide sequence of β-globin as set forth in SEQ ID NO:6. In a specific embodiment of the present invention, the 5'UTR comprises the 5'UTR nucleotide sequence of β-globin as set forth in SEQ ID NO:6.

[0068] In some embodiments of the present invention, the 5'UTR further comprises a Kozak sequence. In one embodiment of the present invention, the Kozak sequence is GCCACC.

[0069] According to the present invention, the 3'UTR may comprise the 3'UTR of β-globin or α-globin, or a homolog, fragment, or combination of fragments thereof. In some embodiments of the present invention, the 3'UTR comprises a nucleotide sequence that is 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 approximately 100% homologous to the fragment of the α2-globin 3'UTR set forth in SEQ ID NO:7. In other embodiments of the present invention, the 3'UTR comprises two end-to-end nucleotide sequences that 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 approximately 100% homologous to the fragment of the α2-globin 3'UTR set forth in SEQ ID NO:7. In a specific embodiment of the present invention, the 3'UTR comprises two end-to-end nucleotide sequences set forth in SEQ ID NO:7.

[0070] According to the present invention, the length of the poly-A tail may 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.

[0071] In one embodiment of the present invention, the open reading frame (ORF) is an open reading frame (ORF) encoding a 2019-nCov S protein mutant, and its nucleic acid sequence is a nucleotide sequence that is 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 to the nucleotide sequence shown in SEQ ID NO: 8. 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 linked from the N-terminus to the C-terminus. In a specific embodiment of the present invention, the nucleotide sequence of the open reading frame (ORF) of the S protein mutant is as shown in SEQ ID NO: 8.

[0072] In one embodiment of the present invention, the mRNA comprises a nucleotide sequence that is 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 to the nucleotide sequence shown in SEQ ID NO: 9. In a specific embodiment of the present invention, the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 9.

[0073] The mRNA of the present invention can be prepared by methods known in the art. In some embodiments of the present invention, a nucleic acid sequence encoding mRNA can be artificially synthesized, and the sequence can be cloned into a vector to construct a plasmid for in vitro transcription. The constructed plasmid is transformed into a host bacterium, cultured and amplified, and the plasmid is extracted. The extracted plasmid is digested into a linear molecule using a restriction endonuclease immediately following the polyA tail. Using the prepared linearized plasmid molecule as a template, mRNA is prepared using an in vitro transcription method. A cap structure analog can be added during the in vitro transcription process to directly obtain an mRNA with a cap structure; or a capping enzyme and a dimethyltransferase can be used to add a cap structure to the mRNA after the in vitro transcription is completed. The obtained mRNA can be purified using conventional methods in the art, such as chemical precipitation, magnetic bead method, affinity chromatography, and the like.

[0074] According to the present invention, one or more nucleotides in the mRNA may be modified. For example, one or more nucleotides (e.g., all nucleotides) in the mRNA may be independently replaced with naturally occurring nucleotide analogs or synthetic nucleotide analogs, such as pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, N6-methyladenosine, N1-methylpseudouridine, etc.

[0075] The present invention also provides an mRNA vaccine, comprising lipid nanoparticles, the lipid nanoparticles comprising a lipid molecule represented by Formula C, a neutral lipid molecule, a cholesterol lipid molecule, a PEGylated lipid molecule, and an mRNA encoding a 2019-nCoV S protein mutant, wherein the mRNA comprises a nucleotide sequence that is 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 to the nucleotide sequence represented by SEQ ID NO: 9; the mass ratio of the total mass of the lipid molecules to the mass of the mRNA is 5-20:1; wherein:

[0076] Formula C wherein each n3 is independent of each other and may be the same or different, each n3 is selected from an integer of 1 to 8, each m3 is independent of each other and may be the same or different, each m3 is selected from an integer of 0 to 8; preferably, each n3 is selected from an integer of 4 to 8, each m3 is selected from an integer of 4 to 8; preferably, each n3 is the same as each other, each m3 is the same as each other. The lipid molecules represented by Formula C account for 34-46 mol% of the lipid in the lipid nanoparticles;

[0077] The neutral lipid molecule is selected from the phosphatidylcholine compound shown in formula E Phosphatidylethanolamine compound represented by formula F Wherein Ra, Rb, Rc, and Rd are independently selected from a linear or branched C10-30 alkyl group, a linear or branched C10-30 alkenyl group, preferably CH3(CH2) 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11The molar percentage of neutral lipid molecules in the lipid nanoparticles is 9-16 mol%;

[0078] The cholesterol lipid molecules are selected from cholesterol and cholesterol hemisuccinate. The cholesterol lipid molecules account for 37-49 mol% of the lipids in the lipid nanoparticles;

[0079] The PEGylated lipid molecules are represented as "lipid moiety-PEG-number average molecular weight", wherein the lipid moiety is a diacylglycerol or a diacylglycerol amide selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylglycerol, dilaurylglyceramide, dimyristoylglyceramide, dipalmitoylglyceramide, distearoylglyceramide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG is from about 130 to about 50,000, for example, from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, from about 150 to about 5,000, from about 150 to about 4,000, from about 150 to about 3,000, The number of PEGylated lipid molecules is about 300 to about 3,000, about 1,000 to about 3,000, about 1,500 to about 2,500, for example about 2000. The molar percentage of the PEGylated lipid molecules in the lipid nanoparticles is 1.3-2.7 mol%.

[0080] In some embodiments of the present invention, the molar ratio of the lipid molecule represented by Formula C, the neutral lipid molecule, cholesterol, and the PEGylated lipid molecule is 35:15:48.5:1.5.

[0081] In some embodiments of the present invention, the molar ratio of the lipid molecule represented by Formula C, the neutral lipid molecule, cholesterol, and the PEGylated lipid molecule is 40:10:48.5:1.5.

[0082] In some embodiments of the present invention, the molar ratio of the lipid molecule represented by Formula C, the neutral lipid molecule, cholesterol, and the PEGylated lipid molecule is 45:15:38.5:1.5.

[0083] In one embodiment of the present invention, the lipid molecule of formula C is compound II-37.

[0084] In one embodiment of the present invention, the neutral lipid molecule is DOPE and / or DSPC.

[0085] In one embodiment of the present invention, the PEGylated lipid molecule is DMG-PEG2000 and / or DSPE-PEG2000.

[0086] The present invention also provides a method for preparing the ionizable cationic lipid molecule of formula I.

[0087] The ionizable lipid compounds of the present invention can be synthesized using methods known in the art, for example, by reacting one or more equivalents of an amine with one or more equivalents of an epoxy-terminated compound under suitable conditions. The synthesis of the ionizable lipid compounds can be carried out with or without a solvent, and the synthesis can be carried out at elevated temperatures ranging from 25°C to 100°C. The resulting ionizable lipid compound can optionally be purified. For example, a mixture of ionizable lipid compounds can be purified to obtain a specific ionizable lipid compound. Alternatively, the mixture can be purified to obtain a specific stereoisomer or regioisomer. The epoxides can be purchased commercially or prepared synthetically.

[0088] In some embodiments of the present invention, the ionizable lipid compound of the present invention can be prepared using the following general preparation method.

[0089]

[0090] Formula A, B, C or D

[0091] Step 1: Restore

[0092] In the presence of a reducing agent, the carboxyl group of Compound A1 is reduced to a hydroxyl group to obtain Compound A2. Examples of reducing agents include, but are not limited to, lithium aluminum hydride and diisobutylaluminum hydride. Examples of solvents used in the reaction include, but are not limited to, ethers (e.g., diethyl ether, tetrahydrofuran, and dioxane), halogenated hydrocarbons (e.g., chloroform, dichloromethane, and dichloroethane), hydrocarbons (e.g., n-pentane, n-hexane, benzene, and toluene), and mixed solvents of two or more of these solvents.

[0093] Step 2: Oxidation

[0094] In the presence of an oxidizing agent, the hydroxyl group of compound A2 is oxidized to an aldehyde group to obtain compound A3. Examples of oxidizing agents include, but are not limited to, 2-iodoxybenzoic acid (IBX), pyridinium chlorochromate (PCC), pyridinium dichlorochromate (PDC), Dess-Martin periodinane, manganese dioxide, and the like. Examples of solvents used in the reaction include, but are not limited to, halogenated hydrocarbons (such as chloroform, dichloromethane, and dichloroethane), hydrocarbons (such as n-pentane, n-hexane, benzene, and toluene), nitriles (such as acetonitrile), and mixed solvents of two or more of these solvents.

[0095] Step 3: Halogenation-reduction

[0096] First, under acidic conditions, the aldehyde α-hydrogen of compound A3 is halogenated with a halogenating agent to obtain an α-halogenated aldehyde intermediate, and then, in the presence of a reducing agent, the aldehyde group of the α-halogenated aldehyde is reduced to a hydroxyl group to obtain compound A4. Examples of providing acidic conditions include, but are not limited to, DL-proline. Examples of halogenating agents include, but are not limited to, N-chlorosuccinimide (NCS) and N-bromosuccinimide (NBS). Examples of reducing agents include, but are not limited to, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

[0097] Step 4: Epoxidation

[0098] Compound A4 is subjected to an intramolecular nucleophilic substitution reaction in the presence of a base to obtain epoxy compound A5. Examples of bases include, but are not limited to, alkali metal hydroxides or hydrides, such as sodium hydroxide, potassium hydroxide, and sodium hydride. Examples of solvents used in the reaction include, but are not limited to, a mixture of dioxane and water.

[0099] Step 5: Ring-opening reaction

[0100] Compound A5 is subjected to a ring-opening reaction with an amine (e.g., N,N-bis(2-aminoethyl)methylamine) to obtain the final compound. Examples of reaction solvents include, but are not limited to, ethanol, methanol, isopropanol, tetrahydrofuran, chloroform, hexane, toluene, ether, and the like.

[0101] The raw material A1 in the preparation method can be purchased commercially or synthesized using conventional methods.

[0102] The present invention also provides a method for preparing the lipid nanoparticle composition.

[0103] According to the present invention, the preparation method comprises: dissolving each lipid molecule in a molar ratio in an organic solvent to form a mixed lipid solution, using the mixed lipid solution as the organic phase, using an aqueous solution of a delivery substance (e.g., a nucleic acid) as the aqueous phase, and mixing the organic phase and the aqueous phase 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 skilled in the art.

[0104] In some embodiments, the organic solvent is an alcohol, such as ethanol.

[0105] In some embodiments, the volume ratio of the organic phase to the aqueous phase is (2-4):1.

[0106] In some embodiments, nanoparticles are prepared using a microfluidic platform.

[0107] According to the present invention, the preparation method further comprises the steps of separating and purifying to obtain the lipid nanoparticles.

[0108] According to the present invention, the preparation method further comprises the step of freeze-drying the lipid nanoparticles.

[0109] The ionizable lipid of Formula I of the present invention contains two adjacent cis double bonds in its molecular structure, which enables it to have a high encapsulation efficiency and good cell transfection efficiency when subsequently used in a delivery system to encapsulate active substances (e.g., nucleic acids such as mRNA). In addition, when preparing lipid nanoparticles, the resulting lipid nanoparticles have a more uniform particle size. The ionizable lipid compound of the present invention is particularly suitable for preparing solid nanoparticles.

[0110] In addition, for the mRNA vaccine of the present invention, since the mRNA of the present invention has high translation efficiency and stability, the S protein mutant encoded by it has high stability. In addition, the lipid nanoparticles of the present invention have a high encapsulation rate, a more uniform particle size, and a better cell transfection rate. The mRNA vaccine of the present invention has a high mRNA encapsulation rate, drug loading capacity, cell transfection efficiency, efficient and stable in vivo translation, antigen stability, and good immune effect.

[0111] The particle size of the lipid nanoparticles in the present invention ranges from 1 nm to 1000 nm, for example, 10 to 500 nm, 10 to 200 nm, etc.

[0112] The lipid nanoparticles of the present invention can also be modified with targeting molecules to make them targeting agents that can target specific cells, tissues or organs. The targeting molecules can be located on the surface of the particles. The targeting molecules can be proteins, peptides, glycoproteins, lipids, small molecules, nucleic acids, etc., and examples thereof include (but are not limited to) antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, asialoglycoprotein (asialycoprotein), receptor ligands, sialic acid, aptamers, etc. The targeting molecules can be attached to the cholesterol lipid molecules or PEGylated lipid molecules of the lipid nanoparticles.

[0113] The lipid nanoparticle compositions and vaccines of the present invention may further contain one or more pharmaceutical excipients. The term "pharmaceutical excipient" means any type of non-toxic, inert solid, semisolid, 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, ethylcellulose, 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; buffers such as phosphate buffer solution, acetate buffer, and citrate buffer; colorants, sweeteners, flavorings, and fragrances, preservatives, and antioxidants.

[0114] In one embodiment of the present invention, the lipid nanoparticle composition and vaccine are liquid preparations, further comprising sucrose, and the mass percentage concentration of sucrose is 5-20%, preferably 8-10%.

[0115] The lipid nanoparticle compositions and vaccines of the present invention can be administered to humans and / or animals orally, rectally, intravenously, intramuscularly, vaginally, intranasally, intraperitoneally, buccally, or in the form of an oral or nasal spray.

[0116] The S protein mutants in the present invention are produced by amino acid mutations in the parent S protein. In one embodiment of the present invention, 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 has the following mutations compared to the S protein of the 2019-nCoV wild strain: L18F, D80A, D215G, L242_244L del, R246I, K417N, E484K, N501Y, D614G, A701V (the sites are described by the positions of the amino acid sequence shown in SEQ ID NO: 1).

[0117] In the present invention, the amino acid positions of the S protein mutants 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 from NCBI GeneID: 43740568, which has a total of 1273 amino acids. Its sequence is shown below and is indicated as SEQ ID NO: 1 in the present invention.

[0118] The S protein mutant of the present invention comprises at least an extracellular domain. Relative to the extracellular domain of the parental S protein, the extracellular domain comprises the following amino acid mutations: F817P, A892P, A899P, A942P, and KV986-987PP, as well as a mutation of amino acids RRAR at positions 682-685 to GSAS. The S protein mutant also lacks the transmembrane domain and cytoplasmic tail. The T4 Fibritin Foldon Trimerization Motif, a domain that assists in trimer formation, is directly fused to the C-terminus of the extracellular domain. 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 linked from the N-terminus to the C-terminus.

[0119] List of the above sequences of the present invention:

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Terminology Notes:

[0129] The term "alkyl" refers to a saturated hydrocarbon radical derived from a hydrocarbon moiety containing 1 to 30 carbon atoms by removing a single hydrogen atom. Examples of 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.

[0130] The term "alkenyl" refers to a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by removing a single hydrogen atom. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0131] The term "alkynyl" refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by removing a single hydrogen atom. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0132] The term "alkoxy" refers to an alkyl group as defined above attached to the parent molecular group through an oxygen atom. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy.

[0133] The terms "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0134] The term "saturated or unsaturated 4-6 membered ring" refers to a ring having 4-6 ring atoms, which may be C, N, S, or O. Examples include, but are not limited to, 4-6 membered saturated cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; 4-6 membered aryl groups, such as phenyl; 4-6 membered heterocyclic groups, such as pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl; and 4-6 membered heteroaryl groups, such as triazolyl, oxazolyl, isoxazolyl, and thiazolyl. In some embodiments of the present invention, the saturated or unsaturated 4-6 membered ring is preferably piperazinyl or cyclohexyl.

[0135] The terms "substituted" (whether or not preceded by the term "optionally") and "substituent" refer to the ability to change one functional group to another, provided that the valence of all atoms is maintained. When more than one position in any particular structure can be substituted with more than one substituent selected from a specified group, the substituent may be the same or different at each position.

[0136] “And / or” will be taken as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0137] "Comprising" and "including" have the same meaning and are intended to be open ended and permit, but not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "consisting essentially of" are also included and disclosed.

[0138] In this specification and claims, nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' direction. Nucleobases are represented by the commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee in this article. It will be understood by those skilled in the art that the T bases in the codons disclosed herein are present in DNA, and the T bases will be replaced by U bases in the corresponding RNA. For example, the codon-nucleotide sequences of the DNA forms disclosed herein, such as vectors or in vitro translation (IVT) templates, are transcribed as U bases in their corresponding transcription mRNA. In this regard, codon-optimized DNA sequences (comprising T) and their corresponding mRNA sequences (comprising U) are considered to be codon-optimized nucleotide sequences of the present disclosure. It will also be understood by those skilled in the art that equivalent codon maps can be produced by replacing one or more bases with non-natural bases.

[0139] The terms "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" are used interchangeably and refer to a contiguous sequence of nucleic acids. The sequence can be single-stranded or double-stranded DNA or RNA, such as mRNA.

[0140] A "nucleotide sequence encoding" refers to a nucleic acid (e.g., an 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 a polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0141] In this specification and claims, the conventional one-letter or three-letter codes for amino acid residues are used.Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation.

[0142] "About": The term "about" used in conjunction with numerical values ​​throughout the specification and claims indicates an interval of accuracy that is familiar and acceptable to those skilled in the art. Typically, this interval of accuracy is ±10%.

[0143] For ease of reference, the S protein mutants of the present invention are described using the following naming convention: original amino acid: position: substituted amino acid. According to this naming convention, for example, the substitution of asparagine at position 30 with alanine is represented as: Asn30Ala or N30A; the deletion of asparagine at the same position is represented as: Asn30* or N30*; the insertion of another amino acid residue, such as lysine, is represented as: Asn30AsnLys or N30NK; the deletion of a continuous stretch of amino acid residues, such as the deletion of amino acid residues 242-244, is represented as: (242-244)* or Δ(242-244) or 242_244del; if the S protein mutant contains both a "deletion" and an insertion at the same position compared to the other S protein parent, it is represented as: *36Asp or *36D, indicating a deletion and an insertion of aspartic acid at position 36. When one or more alternative amino acid residues can be inserted at a given position, it is represented as: N30A,E, or N30A or N30E.

[0144] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestor. In the context of the present disclosure, the term homology includes both identity and similarity.

[0145] In some embodiments, polymer molecules are considered to be "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the monomers in the molecule are identical (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0146] Identity: As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percent identity of two polynucleotide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0147] 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.

[0148] The terms "coding region" and "coding region" refer to an open reading frame (ORF) in a polynucleotide that, when expressed, produces a polypeptide or protein.

[0149] "Operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0150] Domain: As used herein, when referring to a polypeptide, the term "domain" refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding ability, serving as a site for protein-protein interactions).

[0151] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from the DNA sequence (e.g., by transcription); (2) processing of the mRNA transcript (e.g., by splicing, editing, 5' capping and / or 3' end processing); (3) translation of the mRNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0152] The term "protein mutant" or "polypeptide mutant" refers to a molecule whose amino acid sequence differs from a native or reference sequence. Compared to a native or reference sequence, an amino acid sequence mutant may have substitutions, deletions, and / or insertions at certain positions within the amino acid 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, at least about 95% identity, or at least about 99% identity with the native or reference sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 : The integrity of B1.351 mRNA was analyzed using an RNA 6000 nano chip on a 2100 bioanalyzer.

[0154] Figure 2 : The expression level of S protein in the supernatant of CHO-K1 cells after nucleic acid transfection was detected by ELISA.

[0155] Figure 3 : 3D structure diagram of S protein mutant.

[0156] Figure 4: The expression level of S protein in the supernatant of cells transfected with the mRNA of the present invention encapsulated by nanoparticles made of II-37 was detected by ELISA.

[0157] Figure 5 : The expression level of S protein in the supernatant after cells were transfected with LNPs prepared from II-37 and MC3 and encapsulated with the mRNA of the present invention.

[0158] Figure 6 : Statistical graph of protein expression in cells using Firefly Luc as reporter protein and mRNA prepared by different in vitro transcription vectors.

[0159] Figure 7 : Statistical graph of the production of binding antibodies in BALB / c mice after immunization with the S protein mutants of the present invention. Figure a shows the results for the wild-type S protein trimer, b shows the results for the S protein trimer translated from B1.351 mRNA, and c shows the blank control.

[0160] Figure 8 Figure 2: Statistical graph of the production of binding and neutralizing antibodies in BALB / c mice following immunization with lipid nanoparticles containing mRNA encoding the S protein mutant. Panels d, e, and f show the binding antibody assay results after immunization with lipid nanoparticles (LNPs) containing 5 μg, 1 μg, and 0.2 μg of mRNA, respectively. The ordinate represents concentration (μg / ml). Panels g, h, and i show the neutralizing antibody assay results after immunization with lipid nanoparticles (LNPs) containing 5 μg, 1 μg, and 0.2 μg of mRNA, respectively. The abscissa represents the log-transformed serum dilution factor, and the ordinate represents the inhibition rate (%). DETAILED DESCRIPTION

[0161] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0162] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods are conventional molecular biology methods in the art and can be performed with reference to the instructions of molecular biology experimental manuals in the art or kit product instructions.

[0163] Example 1 Synthesis of lipid II-37

[0164]

[0165] Synthesis of Linolenic Alcohol (a2): LiAlH₄ (7.20 g) and linoleic acid (50 g, a1) were added to 950 mL of tetrahydrofuran at 0°C, and the mixture was stirred at 25°C for 2 h. After completion of the reaction as determined by thin-layer chromatography (TLC), the reaction mixture was quenched by the addition of water (7.2 mL), aqueous NaOH (7.2 mL, 15% by mass), and water (21.6 mL). An appropriate amount of Na₂SO₄ was added, and the mixture was stirred for 15 minutes. After filtration through a Buchner funnel, the filter cake was washed with ethyl acetate. The filtrate was collected and concentrated by evaporation to yield 47.4 g of the desired product, linolenic alcohol (a2).

[0166] 1 H NMR (400MHz, CDCl3): δ5.27-5.44(m,4H),3.63(t,J=6.63Hz,2H),2.77(t,J=6.44 Hz,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)

[0167] Synthesis of (9Z,12Z)-octadeca-9,12-dienal (a3): Linolenic alcohol (25.0 g, a2) and 2-iodoacylbenzoic acid (39.4 g) were added to 170 mL of acetonitrile at room temperature, and the mixture was stirred at 85°C for 4 hours. The reaction mixture was filtered through a Buchner funnel, and the filter cake was washed with dichloromethane. The filtrate was collected and concentrated by evaporation to obtain 24.0 g of the desired product, (9Z,12Z)-octadeca-9,12-dienal (a3).

[0168] 1 H NMR (400MHz, CDCl3): δ9.76 (t, J=1.76Hz, 1H), 5.25-5.43 (m, 4H), 2.76 (t, J=6.17 Hz,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)

[0169] Synthesis of (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (a4): To 246 mL of acetonitrile was added (9Z,12Z)-octadeca-9,12-dienal (43.0 g, a3), DL-proline (5.62 g), and N-chlorosuccinimide at 0°C, followed by stirring at 0°C for 2 h. After completion, the reaction mixture was diluted with anhydrous ethanol (246 mL), sodium borohydride (8.8 g) was added, and the mixture was stirred at 0°C for 4 h. The reaction mixture was quenched with water (120 mL) and extracted with methyl tert-butyl ether. The combined organic phases were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation to afford the desired product, (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (a4, 46 g), which was used directly in the next step.

[0170] 1 H NMR (400MHz, CDCl3): 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)

[0171] Synthesis of 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (a5): To 450 mL of 1,4-dioxane were added (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (45 g, a4) and a sodium hydroxide solution (120 g of sodium hydroxide dissolved in 585 mL of water) at room temperature. After the addition was complete, the mixture was stirred at 35°C for 2 h. After TLC indicated the reaction was complete, the reaction solution was separated using a separatory funnel, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation. The residue was then purified by flash column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 29.11 g of the desired product, 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (a5).

[0172] 1 H NMR (400MHz, CDCl3): δ5.27-5.46(m,4H),2.87-2.98(m,1H),2.70-2.85(m,3H),2.4 6(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)

[0173] Synthesis of II-37: 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (5 g) and N,N-bis(2-aminoethyl)methylamine (739 mg) were added to 10 mL of ethanol at room temperature, and the mixture was stirred at 90°C for 36 hours. The reaction solution was concentrated by evaporation, and the residue was purified by flash column chromatography with dichloromethane / methanol to obtain crude product II-37 (4 g). The target product was further purified by flash column chromatography with dichloromethane / methanol to obtain II-37 (2.2 g).

[0174] 1 H NMR (400MHz, CDCl3): 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)

[0175] ESI-MS: m / z 910.8[M+H] + ,911.8[M+2H] + ,912.8[M+3H] +

[0176] Example 2B1.351 Preparation of mRNA and its translation

[0177] 1. A nucleic acid sequence encoding the mRNA set forth in SEQ ID No. 8 was synthesized and cloned into the T7 promoter of the pUC57-kana vector, which had been modified to contain sequences encoding SEQ ID NO: 6, a Kozak sequence, two end-to-end copies of SEQ ID NO: 7, and a polyA tail. The nucleic acid sequence encoding the mRNA set forth in SEQ ID NO: 8 was cloned into the multiple cloning site between the Kozak sequence and the two end-to-end copies of SEQ ID NO: 7 to construct a plasmid for in vitro transcription.

[0178] 2. Transform the constructed plasmid into Escherichia coli Dh5a, culture and amplify it, and extract the plasmid.

[0179] 3. The extracted plasmid was digested into a linear molecule using the restriction endonuclease SpeI immediately following the polyA tail.

[0180] 4. Using the prepared linearized plasmid as a template, mRNA was prepared using in vitro transcription (Thermo Fisher Scientific in vitro transcription kit A45975). The sequence of the mRNA is shown in SEQ ID NO: 9, hereinafter referred to as B1.351 mRNA. Translation of this mRNA yielded the S protein mutant of the present invention, whose amino acid sequence, from N-terminus to C-terminus, consisted of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, directly linked. After in vitro transcription, the mRNA was capped with a CAP1 capping enzyme and a dimethyltransferase.

[0181] 5. Purification of mRNA: The obtained mRNA stock solution was purified using affinity chromatography.

[0182] 6. mRNA quality control: The prepared mRNA was analyzed for mRNA integrity using RNA 6000 nano chip on a 2100 bioanalyzer. The results were as follows: Figure 1 As shown, the transcribed mRNA band is single and there is no obvious degradation.

[0183] Separately, a spike fragment was excised from the commercially available plasmid pCMV3-spike using the restriction enzymes HindIII and EcoRI and inserted between the HindIII and EcoRI sites of the IVT1 vector described in Example 5, generating the IVT1-spike plasmid. This plasmid was then point-mutated to generate the IVT1-spike-D614G plasmid. Using this plasmid as a template, spike-D614G mRNA was generated by in vitro transcription, expressing the full-length S protein containing the D614G mutation.

[0184] 7.B1.351 mRNA cell-level expression assay: Using the CHO-K1 cell line as the expression system, mRNA was transfected using Lipofectamine Messenger MAX Reagent (Invitrogen, Cat#1168-027). After 48 hours of culture, the cell culture supernatant was collected and the S protein expression level was detected using an enzyme-linked immunosorbent assay kit to determine whether the mRNA could be translated into protein. The results are as follows: Figure 2 shown. Figure 2In the assay, "spike DNA" is the commercialized plasmid pCMV3-spike (purchased from Sino Biological), which expresses the full-length wild-type S protein; "spike-D614G mRNA" is the aforementioned mRNA expressing the full-length S protein containing the D614G mutation; and "spike B1.351 mRNA" is the aforementioned B1.351 mRNA, which expresses the S protein mutant described in the present invention. The results show that the mRNA of the present invention can highly express the S protein mutant in cells.

[0185] After the S protein mutant was purified, the structure was analyzed using cryo-electron microscopy. The 3D structure of the S protein is shown in Figure 3 As shown, the S protein mutant is a stable structure of prefusion spike structure. The sequence of the B1.351 mutant strain and the sequence of the wild strain differ in 9 mutation sites, 3 of which are in the RBD region. The RBD region state of the prefusion 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 2 OPEN and 1 CLOSE. This structural difference is the structural basis for the enhanced binding ability of the virus 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, thereby significantly different antibodies, especially neutralizing antibodies, induced based on different structures.

[0186] Example 3 Preparation of Lipid Nanoparticle Compositions Containing Nucleic Acids

[0187] Accurately weigh compound II-37, DOPE, CHOL, DSPE-PEG2000, DSPC, DMG-PEG2000, etc., place each lipid in an appropriate container, and fully dissolve it in anhydrous ethanol for later use.

[0188] The lipids were mixed evenly according to the molar ratios listed in the table below to form the organic phase, and the nucleic acid (mRNA or DNA) was prepared into an aqueous solution (using pure water as the solvent) to form the aqueous phase at pH = 4.

[0189] The organic phase and aqueous phase are mixed in a 3:1 volume ratio and a lipid nanoparticle suspension is prepared on a microfluidic platform (e.g., PNI Ignite). The resulting lipid nanoparticle suspension is purified and concentrated by centrifugation through a 100 kDa ultrafiltration centrifuge tube, and the concentrated liquid is aliquoted.

[0190] The particle size, PDI, and potential of the prepared lipid nanoparticles were measured using a laser nanoparticle size analyzer, and the encapsulation efficiency (%) was measured using an ultraviolet spectrophotometer combined with a RiboGreen RNA kit. The exemplary results are as follows.

[0191]

[0192]

[0193] After optimizing the preparation process, lipid nanoparticles with improved physical and chemical quality control data can be obtained. The results for the II-37:DSPC:CHOL:DMG-PEG2000 formulation are exemplified in the following table. The lipid molar ratio of tri-009-BJ-LNP-21040601 is 40:10:48.5:1.5; the lipid molar ratio of tri-009-BJ-LNP-21040602 is 35:15:48.5:1.5; and the lipid molar ratio of tri-009-BJ-LNP-21040603 is 45:15:38.5:1.5. A portion of these samples were transfected into CHO cells according to the method of Example 2, and protein expression was measured by ELISA to assess cell transfection efficiency.

[0194] Sample number PDI Diameter (nm) EE% Zeta potential (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

[0195] Cell transfection results are shown in Figure 4 , Figure 4 In the above examples, “tri-009-BJ-LNP-21040601”, “tri-009-BJ-LNP-21040602” and “tri-009-BJ-LNP-21040603” are the B1.351 mRNA of Example 2 encapsulated with the corresponding formulations, and “lipoMax-spike mRNA” is the mRNA of lipoMax TM The B1.351 mRNA of Example 2 was encapsulated, and the “Nagative control” was a blank lipid nanoparticle without mRNA. Figure 4 It can be seen that after 48 hours of transfection of cells with lipid nanoparticles loaded with nucleic acid obtained based on II-37, antigen protein expression can be detected by ELISA test. Its cell transfection efficiency is comparable to that of commercial lipoMax TM Equal or even better.

[0196] Example 4 Comparison of the effects of II-37 and commercial ionizable cationic lipid molecule MC3

[0197] MC3 is: 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester.

[0198] According to the method described in Example 3, lipid nanoparticles were prepared using II-37 and MC3, respectively, with a specific molar ratio of: II-37:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; MC3:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; and the B1.351 mRNA in Example 2 was encapsulated.

[0199] The physical and chemical quality control data of the prepared lipid nanoparticles are shown in the following table:

[0200] Sample Information Particle size (nm) PDI Zeta potential Encapsulation efficiency 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

[0201] As can be seen from the above table, under the same preparation process, 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.

[0202] The same transfection method as in Example 2 was used to transfect cells with the prepared lipid nanoparticles to understand the expression of the protein. The results are as follows: Figure 5 As shown, after the lipid nanoparticles prepared by II-37 (represented by C2 in the figure) carried mRNA to transfect cells, the protein expression level in the cells was much higher than that of MC3, indicating that the cell transfection efficiency of lipid nanoparticles made of II-37 was very high.

[0203] Example 5 Efficiency comparison experiment of the IVT vector of the present invention

[0204] In this example, Firefly Luc was used as a reporter protein to construct different IVT vectors for in vitro transcription and synthesis of mRNA capable of translating Firefly Luc, and the translation efficiency of synthesized mRNAs with different sequence characteristics was compared.

[0205] The Firefly Luc coding sequence was cloned into the multiple cloning site of the corresponding vector using conventional plasmid vector construction technology in the art to obtain vectors numbered IVT1, IVT2, IVT3, and IVT4. The corresponding Firefly Luc mRNA samples were then prepared by in vitro transcription using the AM1344 kit based on the aforementioned vectors.

[0206] Vectors IVT1 to IVT4 were all modified based on the commercial vector psp73. The following sequences were inserted at the restriction enzyme sites XhoI / NdeI of the vector psp73. No UTR sequence was added to IVT1, and the polyA tail length was 64 A. IVT2 used the 5'UTR of SEQ ID NO:6 and the 3'UTR sequence of GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACA TTTATTTTCATTGC (3'UTR sequence of β-globin), with a polyA length of 120 A. IVT3 used the 5'UTR of SEQ ID NO:6 and the 3'UTR of SEQ ID NO:7, with a polyA length of 120 A. IVT4 used the 5'UTR of SEQ ID NO:6 and two tandem repeats of SEQ ID NO: The 3'UTR sequence shown in NO:7 has a poly A length of 120 A. A multiple cloning site containing the common restriction enzyme sites HindIII and EcoRI was inserted between the 5'UTR and 3'UTR sequences above. The Firefly Luc coding sequence was then cloned into these HindIII and EcoRI multiple cloning sites. All vectors were constructed using gene synthesis methods from GenScript.

[0207] Each Firefly Luc mRNA sample was transfected into CHO cells using Lipofectamine 2000 (cat#11668030, purchased from Thermo Fisher Scientific) as a transfection reagent and then transfected using Dual-Lumi TM Luciferase was detected using a dual-luciferase reporter gene assay kit (at#RG088S, purchased from Shanghai Biotech Biotechnology Co., Ltd.). Firefly Luc DNA was transferred into the psicheck2 plasmid as a positive control (psicheck2 plasmid, cat#60908-6151, purchased from Beijing Tianenze Gene Technology Co., Ltd.). The specific steps were as follows: On the first day, CHO cells were seeded into 96-well plates at 1.5×10 cells per well. 4Cells were cultured overnight in F12K + 10% FBS. On the second day, before transfection, the culture medium was replaced with serum-free F12K medium, and mRNA or DNA was transfected into CHO cells using Lipofectamine 2000. The amount of nucleic acid used in each well was 100 ng, the amount of liposomes used was 0.3 μl, and the total volume per well was 100 μl. The cells were cultured overnight. On the third day, the serum-free medium was replaced with complete medium (F12K + 10% FBS) and cultured for another 24 hours. On the fourth day (48 hours after transfection), the Firefly Luc fluorescence value was detected.

[0208] The results are as follows Figure 6 As shown in Figure 6, "DNA" represents the positive control (psicheck2 plasmid carrying Firefly Luc DNA), "IVT1-Luc," "IVT2-Luc," "IVT3-Luc," and "IVT4-Luc" represent the corresponding Firefly Luc mRNAs transcribed in vitro from the IVT1, IVT2, IVT3, and IVT4 vectors, respectively. "Nagative control" is the negative control. As shown in Figure 6, at the same mRNA transfection dose, IVT4-Luc protein expression is significantly higher than the other three mRNAs, by 2-3 times, demonstrating the good stability and high translation efficiency of IVT4-Luc.

[0209] Example 6 Determination of immunogenicity of S protein mutants

[0210] BALB / c mice were used to evaluate the production of binding and neutralizing antibodies induced by the S protein mutants. Six-week-old female BALB / c mice were immunized 2 weeks apart between the primary and secondary immunizations. Blood was collected 14 days after immunization. Binding antibody expression against the S protein mutants was assessed by ELISA, and neutralizing antibody titers against the S protein mutants were measured by chemiluminescence.

[0211] ELISA method to detect binding antibodies: The commercial S protein is coated on the enzyme-labeled plate to capture the binding antibodies against the S protein mutants in the plasma of immunized mice, and then the absorbance is detected using a biotin-labeled detection antibody. Chemiluminescence detection of neutralizing antibody titers against S protein mutants: After immunization, the mouse plasma was neutralized with the SPIKE lentivirus carrying the luciferase reporter gene (Zhongji Dangkang; trade name: SRAS-CoV-2 pseudovirus (B.1.351)-LUC; product number: DZPSC-L-0; batch: K05202102) and then infected with 293T cells that highly express ACE-2 (Zhongji Dangkang; trade name: "YJ1B09" hACE2-293T cell lines; product number: YJ293T-01; batch: A23202001). The neutralizing antibody titer of the plasma was assessed using chemiluminescence (Bright-Lumi II Firefly Luciferase Reporter Gene Detection Kit, brand: Biyuntian; product number: RG052M).

[0212] A total of 9 mice were injected subcutaneously with 2 μg of protein per mouse in the control group. Three mice were injected with the purified trimer of S protein translated from the B1.351 mRNA described in Example 2, three mice were injected with the trimer of wild-type S protein, and three mice were injected with blank lipid nanoparticles. The lipid formulation of the lipid nanoparticles was tri-009-BJ-LNP-21040602 described in Example 3.

[0213] A total of 18 mice in the experimental group were subcutaneously injected with lipid nanoparticles containing mRNA; mice 1-6 were injected with 0.2 μg mRNA lipid nanoparticles (LNP), mice 7-12 were injected with 1 μg mRNA lipid nanoparticles (LNP), and mice 13-18 were injected with 5 μg mRNA lipid nanoparticles (LNP). The mRNA was B1.351 mRNA in Example 2, and the formula of the lipid nanoparticles was tri-009-BJ-LNP-21040602 in Example 3.

[0214] The expression levels of binding antibodies and neutralizing antibodies in mice after primary and secondary immunizations were as follows: Figure 7-8 shown.

[0215] Depend on Figure 7As can be seen from a, b, and c in Figure 2, the S protein trimer translated from the B1.351 mRNA of Example 2 and the wild-type S protein trimer can both induce anti-S protein binding antibodies in mice: a relatively high concentration of binding antibodies can be produced in the experimental mice during the second immunization, and the concentration of the binding antibodies remains at a relatively high level 8 weeks after the second immunization. It is calculated that the binding antibody concentration after the second immunization is around 2.2 μg / ml, and 8 weeks after the second immunization, it can still be maintained at around 1.6 μg / ml.

[0216] Depend on Figure 8 As shown in Figures d, e, and f, after secondary immunization with LNP-encapsulated mRNA preparations, even the low-dose (0.2 μg) injection group can induce anti-S protein binding antibodies in mice. The binding antibody level is calculated to be approximately 0.1-0.3 μg / ml. Figure 8 As shown in g, h, and i, good neutralizing antibodies were induced in mice after secondary immunization with LNP-encapsulated mRNA preparations, and the GMT values ​​of neutralizing antibodies were 78.69, 21.9, and 72.19, respectively.

[0217] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention. SEQUENCE LISTING <110> Beijing Qichensheng Biotechnology Co., Ltd. <120> A lipid nanoparticle composition and a drug delivery system prepared therefrom <130> CPCN21411053 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 1273 <212> PRT <213> Unknown <220> <223> 2019-nCoV wild-type S protein <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 2 <211> 1205 <212> PRT <213> Artificial Sequence <220> <223> 2019 - nCoV S protein mutant <400> 2 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Ala 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr 245 250 255 Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe 260 265 270 Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys 275 280 285 Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr 290 295 300 Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr 305 310 315 320 Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly 325 330 335 Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg 340 345 350 Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser 355 360 365 Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu 370 375 380 Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg 385 390 395 400 Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala 405 410 415 Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala 420 425 430 Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr 435 440 445 Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp 450 455 460 Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val 465 470 475 480 Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro 485 490 495 Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe 500 505 510 Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr 515 520 525 Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr 530 535 540 Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln 545 550 555 560 Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro 565 570 575 Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val 580 585 590 Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu 595 600 605 Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp 610 615 620 Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe 625 630 635 640 Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser 645 650 655 Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln 660 665 670 Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser Ser Val Ala Ser Gln Ser 675 680 685 Ile Ile Ala Tyr Thr Met Ser Leu Gly Val Glu Asn Ser Val Ala Tyr 690 695 700 Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr 705 710 715 720 Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr 725 730 735 Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln 740 745 750 Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala 755 760 765 Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln 770 775 780 Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser 785 790 795 800 Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Pro Ile Glu 805 810 815 Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys 820 825 830 Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys 835 840 845 Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp 850 855 860 Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr 865 870 875 880 Ser Gly Trp Thr Phe Gly Ala Gly Pro Ala Leu Gln Ile Pro Phe Pro 885 890 895 Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val 900 905 910 Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile 915 920 925 Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Pro Ser Ala Leu Gly Lys 930 935 940 Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val 945 950 955 960 Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp 965 970 975 Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg 980 985 990 Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln 995 1000 1005 Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 1010 1015 1020 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp 1025 1030 1035 Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala 1040 1045 1050 Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln 1055 1060 1065 Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys 1070 1075 1080 Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His 1085 1090 1095 Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr 1100 1105 1110 Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly 1115 1120 1125 Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp 1130 1135 1140 Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser 1145 1150 1155 Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val 1160 1165 1170 Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys 1175 1180 1185 Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr 1190 1195 1200 Glu Gln 1205 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Even if you have a snowflake <400> 3 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 1 5 10 15 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 20 25 <210> 4 <211> 3615 <212> DNA <213> Artificial Sequence <220> <223> The snowstorm <400> 4 atgttcgtgt tcctggtgct gcttcccctg gtctctagcc agtgcgtgaa cttcacgacc 120. cggacccaac tgccccccgc gtacacaaac tccttcacca gaggcgtgta ctaccctgac aaggtgttcc gcagcagcgt gctgcacagc acccaggacc tgttcctccc attcttcagc 180 240. aacgtgacct ggttccacgc catccacgtg tccggcacca atggaacaaa gagatttgcg aaccccgtgc tactttcaa cgacggcgtg tacttcgcct ccccgaga 300 atccggggct ggatcttcgg caccaccctg gactctaaaa cccagagcct gctgatcgtg 360 aataatgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccctttc 420 ctgggcgtct actaccacaa gaacaacaag agttggatgg aaagcgagtt cagagtgtac 480 tcttctgcta acaactgcac cttcgagtac gtgtcccagc ctttcctgat ggacctggaa 540 ggcaagcagg ggaacttcaa gaacctgcgg gagttcgtgt tcaagaacat cgacgggtat 600 ttcaagatct actccaagca cacacctatc aatctggtga gaggcctgcc ccagggcttc 660 agcgccctgg aacctctggt cgacctgcca atcggcatca acatcacccg gttccaaaca 720 ctgcatatca gctacctgac acctggcgat agctcctccg gctggaccgc cggcgctgcc 780 gcttattacg tcggctacct gcagcctaga acgttcctgc tgaagtacaa cgagaacggc 840 accatcaccg acgccgtcga ctgcgccctg gaccccctct ccgagacaaa atgcaccctg 900 aagagcttca ctgttgaaaa gggcatctac cagaccagca actttagagt gcagcctaca 960 gagtctatcg tgagattccc taacattacc aacctgtgtc cttttggaga agtgttcaac 1020 gccacaagat tcgcttctgt gtatgcctgg aaccggaaga gaatctcgaa ctgcgtggct 1080 gattacagcg tgctgtacaa cagcgctagc tttagcacat ttaagtgcta cggcgtgagc 1140 cccaccaagc tgaatgattt gtgcttcaca aatgtgtacg ccgactcttt cgtgataaga 1200 ggggacgagg tgcggcagat agctccaggc cagaccggca acatcgccga ttacaattac 1260 aagctgcctg acgactttac cggatgtgtg atcgcctgga acagcaacaa cctggatagc 1320 aaggtgggcg gaaactacaa ctacctgtac agactgttcc ggaaatctaa ccttaagcct 1380 tttgagcggg atatcagcac cgagatctac caagctggct ctacaccctg caacggcgtg 1440 aaggggttta attgttactt ccccctgcag agctacggct tccaaccgac ctacggagtg 1500 ggctaccagc cctaccgggt cgtggtgctg agctttgagc tgctgcacgc ccctgctaca 1560 gtgtgcggcc ccaagaagtc tacgaacctg gtgaagaaca agtgtgtgaa ttttaatttc 1620 aacggactga ccggcacagg cgtcctgacc gaatctaaca agaaattcct ccctttccag 1680 cagttcggga gagatatcgc cgacaccacc gacgccgtgc gggaccctca aacactggaa 1740 atcctggata tcaccccttg ttctttcgga ggcgtgtccg tgatcacccc aggtacgaac 1800 acatctaacc aggtggctgt gctgtaccag ggcgtgaact gcaccgaggt gcctgtggcc 1860 attcacgccg accagctgac tcctacctgg cgggtgtaca gcacgggctc caacgtgttt 1920 cagaccagag ctggctgtct gatcggagcc gagcacgtga acaactctta tgagtgcgat 1980 atccccatcg gcgctggaat ctgtgcctcc taccagactc aaaccaacag ccctggcagc 2040 gctagcagcg tggccagcca gagcatcatc gcctacacca tgagcctggg agtcgaaaac 2100 agcgtggcct actcaaacaa ctccatcgct atccctacca acttcaccat cagcgtaacg 2160 accgaaatcc tgcccgtgag catgaccaag accagcgtgg actgcacaat gtacatctgc 2220 ggcgatagca cagaatgcag caatctgcta ctgcagtacg gtagcttttg cacccaactg 2280 aatagagccc tgaccggcat cgccgtggaa caggataaaa acacccaaga ggtcttcgct 2340 caggtgaagc agatctacaa gacacctccc atcaaggact tcggaggatt caactttagc 2400 cagatcctgc ctgatccaag caaacctagc aagcggagtc ctatcgagga cctgctgttt 2460 aacaaggtga cactggccga cgccggcttc atcaagcagt atggcgactg tctgggcgac 2520 atcgccgcca gggatctgat ctgtgcccaa aaattcaacg gcctgacagt gctgccacct 2580 ctgctgaccg acgagatgat cgctcaatac accagcgccc tcctcgccgg cacgatcacc 2640 agcggctgga cattcggcgc cggccctgcc ctccagatcc ctttccctat gcagatggcc 2700 tacagattca acggcatcgg cgtgacacaa aacgtgctgt acgaaaacca gaagctgatc 2760 gccaatcagt ttaatagcgc catcgggaag atccaggata gcctgtcatc taccccttct 2820 gccctgggaa agctgcagga cgtggtgaac cagaacgccc aggccctgaa caccctggtg 2880 aaacagctgt ctagcaactt cggcgctatc agcagcgtgc tgaatgatat cctgagcaga 2940 ctggatcctc ctgaggccga ggtgcagatc gacagattga tcaccggccg gctgcagagc 3000 ctgcaaacct acgttacaca gcagctgatc agagccgctg aaatcagagc ctctgccaac 3060 ctggccgcca ccaaaatgag cgagtgcgtg ctgggacaga gcaaaagggt ggacttctgc 3120 gggaagggct accacctcat gagttttccc cagagcgccc cccacggcgt ggtgttcctg 3180 cacgtgacat atgtcccggc ccaggagaaa aactttacaa cagcccctgc catttgccat 3240 gacggaaagg cccacttccc tcgggaaggt gtgttcgtga gcaacggcac acactggttc 3300 gtgacccaga gaaacttcta cgagcctcaa atcatcacca cagacaacac cttcgttagt 3360 ggaaattgcg acgtggttat cggcatcgtg aacaacaccg tctacgaccc actgcagcct 3420 gaactggata gcttcaagga ggaactggat aagtatttca agaaccacac ctcccccgac 3480 gtggatctgg gcgacattag cggcatcaac gccagcgtgg tgaacatcca gaaagagatc 3540 gatagactta atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaggagctc 3600 ggcaaatacg agcag 3615 <210> 5 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 5 ggctatatcc cagaggcccc tagagatggc caggcctacg ttagaaagga cggcgagtgg 60 gtcctgctga gcacattcct gggc 84 <210> 6 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 acauuugcuu cugacacaac uguguucacu agcaaccuca aacagacacc 50 <210> 7 <211> 88 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc ccuccucccc 60 uccuugcacc ggcccuuccu ggucuuug 88 <210> 8 <211> 3702 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 auguucgugu uccuggugcu gcuuccccug gucucuagcc agugcgugaa cuucacgacc 60 cggacccaac ugccccccgc guacacaaac uccuucacca gaggcgugua cuacccugac 120 aagguguucc gcagcagcgu gcugcacagc acccaggacc uguuccuccc auucuucagc 180 aacgugaccu gguuccacgc cauccacgug uccggcacca auggaacaaa gagauuugcg 240 aaccccgugc uaccuuucaa cgacggcgug uacuucgccu ccaccgagaa gagcaacauc 300 auccggggcu ggaucuucgg caccacccug gacucuaaaa cccagagccu gcugaucgug aauaaugcca ccaacguggu gaucaaggug ugcgaguucc aguucugca cgacccuuuc cuggcgucu acuaccacaa covered aaagcgaguu cagaguguac 540. 540. 540. 540. 540. 540. 540. 540. 540. 540. 540 ggcaagcagg ggacuucaa ggaccugcgg gaguucgugu ucaagaacau cgacggguau 660. uucaagaucu acuccaagca cacaccuauc aaucugguga gaggccugcc ccagggcuuc agcgcccugg aaccucugg cgaccugcca aucggcauca acaucacccg 720. s.s.s.s.s.s.s.s.s.s.s.s.s.s.s.s.s.s cugcauauca gcuaccugac accuggcgau agcuccuccg gcuggaccgc cggcgcugcc 780 840. gcuuauuacg ucggcuaccu gcagccuaga acguuccugc ugaaguacaa cgagaacggc accaucaccg acgccgucga cugcgcccug gacccccucu ccgagacaaa augcacccug 960. aagagcuuca cuguugaaaa gggcaucuac cagaccagca acuuuagagu gcagccuaca 1020. 1020. 1020. 1020. 1020. 1020. 1020. 1020. 1020. 1020 gccacaagau ucgcuucugu guaugccugg aaccgggaaga gauucucgaa cugcguggcu 1080 gauuacagcg ugcuguacaa cagcgcuagc uuuagcacau uuaagugcua cggcgugagc 1140 cccaccaagc ugaaugauuu gugcuucaca aauguguacg ccgacucuuu cgugauaaga 1200 ggggacgagg ugcggcagau agcuccaggc cagaccggca acaucgccga uuacaauuac 1260 aagcugccug acgacuuuac cggaugugug aucgccugga acagcaacaa ccuggauagc 1320 aaggugggcg gaacuaacaa cuaccuguac agacuguucc ggaaaucuaa ccuuaagccu 1380 1440 aagggguua auuguuacuu cccccugcag agcuacggcu uccaaccgac cuacggagug 1500 ggcuaccagc ccuaccgggu cguggugcug agcuuugagc ugcugcacgc cccugcuaca 1560 gugugcggcc ccaagaaguc uacgaaccug gugaagaaca agugugugaa uuuaauuuc 1620 aacggacuga ccggcacagg cguccugacc gaaucuaaca agaaauuccu cccuuuccag 1680 caguucggga gagauaucgc cgacaccacc gacgccgugc gggacccuca aacacuggaa 1740 auccuggaua ucacccccuug uucuuucgga ggcguguccg ugaucacccc agguacgaac 1800 acaucuaacc agguggcugu gcuguaccag ggcgugaacu gcaccgaggu gccuguggcc 1860 1920 1980 auccccaucg gcgcuggau cgugcccucc uaccagacuc aaccaacag cccuggcagc 2040 gcuagcagcg uggccagcca gagcaucauc gccuacacca ugagccugggg agucgaaaac 2100 2160 2220 2280 aauagagccc ugaccggcau cgccguggaa caggauaaaa acacccaaga ggucuucgcu 2340 caggugaagc aguacuacaa gacaccuccc aucaaggacu ucggaggauu caacuuuagc 2400 2460 aacaagguga cacuggccga cgccggcuuc aucaagcagu auggcgacug ucugggcgac 2520 aucgccgcca gggaucugau cugugcccaa aaauucaacg gccugacagu gcugccaccu 2580 cugcugaccg acgagaugau cgcucaauac accagcgccc uccucgccgg cacgaucacc 2640 agcggcugga cauucggcgc cggcccugcc cuccagaucc cuuucccuau gcagauggcc 2700 uacagauuca acggcaucgg cgugacacaa aacgugcugu acgaaaacca gaagcugauc 2760 gccaaucagu uuaauagcgc caucgggaag auccaggaua gccugucauc uaccccuucu 2820 gcccugggaa agcugcagga cguggugaac cagaacgccc aggcccugaa cacccuggug 2880 aaacagcugu cuagcaacuu cggcgcuauc agcagcgugc ugaaugauau ccugagcaga 2940 cuggauccuc cugaggccga ggugcagauc gacagauuga ucaccggccg gcugcagagc 3000 cugcaaaccu acguuacaca gcagcugauc agagccgcug aaaucagagc cucugccaac 3060 cuggccgcca ccaaaaugag cgagugcgug cugggacaga gcaaaagggu ggacuucugc 3120 gggaagggcu accaccucau gaguuuuccc cagagcgccc cccacggcgu gguguuccug 3180 cacgugacau augucccggc ccaggagaaa aacuuuacaa cagccccugc cauuugccau 3240 gacggaaagg cccacuuccc ucgggaaggu guguucguga gcaacggcac acacugguuc 3300 gugacccaga gaaacuucua cgagccucaa aucaucacca cagacaacac cuucguuagu 3360 ggaaauugcg acgugguuau cggcaucgug aacaacaccg ucuacgaccc acugcagccu 3420 gaacuggaua gcuucaagga ggaacuggau aaguauuuca agaaccacac cucccccgac 3480 guggaucugg gcgacauuag cggcaucaac gccagcgugg ugaacaucca gaaagagauc 3540 gauagacuua augagguggc caagaaccug aacgagagcc ugaucgaccu gcaggagcuc 3600 ggcaaauacg agcagggcua uaucccagag gccccuagag auggccaggc cuacguuaga 3660 aaggacggcg aguggguccu gcugagcaca uuccugggcu ga 3702 <210> 9 <211> 4093 <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 9 gggagaccgg ccucgagaca uuugcuucug acacaacugu guucacuagc aaccucaaac 60 agacaccaag cuugccacca uguucguguu ccuggugcug cuuccccugg ucucuagcca 120 gugcgugaac uucacgaccc ggacccaacu gccccccgcg uacacaaacu ccuucaccag 180 aggcguguac uacccugaca agguguuccg cagcagcgug cugcacagca cccaggaccu 240 guuccuccca uucuucagca acgugaccug guuccacgcc auccacgugu ccggcaccaa 300 uggaacaaag agauuugcga accccgugcu accuuucaac gacggcgugu acuucgccuc 360 caccgagaag agcaacauca uccggggcug gaucuucggc accacccugg acucuaaaac 420 ccagagccug cugaucguga auaaugccac caacguggug aucaaggugu gcgaguucca 480 guucugcaac gacccuuucc ugggcgucua cuaccacaag aacaacaaga guuggaugga 540 aagcgaguuc agaguguacu cuucugcuaa caacugcacc uucgaguacg ugucccagcc 600 uuuccugaug gaccuggaag gcaagcaggg gaacuucaag aaccugcggg aguucguguu 660 caagaacauc gacggguauu ucaagaucua cuccaagcac acaccuauca aucuggugag 720 aggccugccc cagggcuuca gcgcccugga accucugguc gaccugccaa ucggcaucaa 780 caucacccgg uuccaaacac ugcauaucag cuaccugaca ccuggcgaua gcuccucccgg 840 cuggaccgcc ggcgcugccg cuuauuacgu cggcuaccug cagccuagaa cguuccugcu 900 960 cgagacaaaa ugcacccuga agagcuucac uguugaaaag ggcaucuacc agaccagcaa 1020 1080 uuuggagaa guguucaacg ccacaagauu cgcuucugug uaugccugga accggagaag 1140 aaucucgaac ugcguggcug auuacagcgu gcuguacaac agcgcuagcu uaagcacauu 1200 uaagugcuac ggcgugagcc ccaccaagcu gaaugauuug ugcuucacaa auguguacgc 1260 cgacucuuuc gugauaagag gggacgaggu gcggcagaua gcuccaggcc agaccggcaa 1320 1380 cagcaacaac cuggauagca agguggcggg aaacuacaac uaccuguaca gacuguuccg 1440 gaaucuaac cuuaagccuu uugagcggga uaucagcacc gagaucuacc aagcuggcuc 1500 uacaccguc aacggcguga agggguuuaa uuguuacuuc ccccugcaga gcuacggcuu 1560 ccaaccgacc uacggagugg gcuaccagcc cuaccggguc guggugcuga gcuuugagcu 1620 gcugcacgcc ccugcuacag ugugcggccc caagaagucu acgaaccugg ugaagaacaa 1680 gugugugaau uuuauuuca acggacugac cggcacaggc guccugaccg aaucuaacaa 1740 gaaauuccuc ccuuuccagc aguucgggag agauaucgcc gacaccaccg acgccgugcg 1800 ggacccucaa acacuggaaaa uccuggauau caccccuugu ucuuucggag gcguguccgu 1860 gaucacccca gguacgaaca caucuaacca gguggcugug cuguaccagg gcgugaacug 1920 caccgaggug ccuguggcca uucacgccga ccagcugacu ccuaccuggc ggguguacag 1980 cacgggcucc aacguuuuc agaccagagc uggcucucug aucggagccg agcacgugaa 2040 caacucuuau gagugcgaua uccccaucgg cgcuggaauc ugugccuccu accagacuca 2100 aaccaacagc ccuggcagcg cuagcagcgu ggccagccag agcaucaucg ccuacaccau 2160 gagccuggga gucgaaaaca gcguggccua cucaaacaac uccaucgcua ucccuaccaa 2220 cuucaccauc agcguaacga ccgaaauccu gcccgugagc augaccaaga ccagcgugga 2280 2340 uagcuuuugc acccaacuga auagagcccu gaccggcauc gccguggaac aggauaaaaa 2400 cacccaagag gucuucgcuc aggugaagca gaucuacaag acaccuccca ucaaggacuu 2460 cggaggauuc aacuuuagcc agauccugcc ugauccaagc aaaccuagca agcggagucc 2520 uaucgaggac cugcuguuua acaaggugac acuggccgac gccggcuuca ucaagcagua 2580 uggcgacugu cugggcgaca ucgccgccag ggaucugauc ugugcccaaa aauucaacgg 2640 2700 ccucgccggc acgaucacca gcggcuggac auucggcgcc ggcccugccc uccagauccc 2760 uuucccuaug cagauggccu acagauucaa cggcaucgg gugacacaaa acgugcugua 2820 cgaaaaccag aagcugaucg ccaaucaguu uaauagcgcc aucgggaaga uccaggauag 2880 ccugucaucu acccccuucug cccugggaaa gcugcaggac guggugaacc agaacgccca 2940 ggcccugaac acccugguga aacagcugc uagcaacuuc ggcgcuauca gcagcgugcu 3000 gaaugauauc cugagcagac uggauccucc ugaggccgag gugcagaucg acagauugau 3060 3120 aaucagagcc ucugccaacc uggccgccac caaaaugagc gagugcgugc uggcagag 3180 caaaagggug gacuucugcg ggaagggcua ccaccucaug aguuucccc agagcgcccc 3240 ccacggggug guguucgc acgugacaua ugucccggcc caggagaaaa acuuuacaac 3300 agccccugcc auuugccaug acggaaaggc ccacuuccu cgggaaggug uguucgugag 3360 3420 agacaacacc uucguuagug gaauugcga cgugguauc ggcaucguga acaacaccgu 3480 cuacgaccca cugcagccug aacuggauag cuucaaggag gaacuggaua aguauuucaa 3540 gaaccacc ucccccgacg uggaucuggg cgacauuagc ggcaucaacg ccagcguggu 3600 gaacaucccag aaagagaucg auagacuuaa ugagguggcc aagaaccuga acgagagccu 3660 gaucgaccug caggagcucg gcaaauacga gcagggcuau aucccagagg ccccuagaga 3720 uggccaggcc uacguuagaa aggacggcga guggguccug cugagcacau uccugggcug 3780 agaauucgcu ggagccucgg uagccguucc uccugcccgc ugggccuccc aacgggcccu 3840 ccuccccucc uugcaccggc ccuuccuggu cuuuggcugg agccucggua gccguuccuc 3900 cugcccgcug ggccucccaa cgggcccucc uccccuccuu gcaccggccc uuccuggucu 3960 uuguuaauua aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080 aaaaaaaaac uag 4093

Claims

1. A lipid nanoparticle composition comprising lipid nanoparticles, wherein the lipid nanoparticles comprise lipid molecules of formula C, C, wherein each n3 is independent of each other, the same or different, each n3 is selected from an integer of 1 to 8, each m3 is independent of each other, the same or different, each m3 is selected from an integer of 0 to 8; The lipid nanoparticles contain: lipid molecules of formula C accounting for 34-46 mol% of the total lipid molecules, neutral lipid molecules accounting for 9-16 mol% of the total lipid molecules, cholesterol lipid molecules accounting for 37-49 mol% of the total lipid molecules, and PEGylated lipid molecules accounting for 1.3-2.7 mol% of the total lipid molecules; Furthermore, the composition of the lipid nanoparticles is not: the molar ratio of (II-37:DSPC:CHOL:DMG-PEG2000) is: 45:10:43.5:1.5, 35:15:48.5:1.5 or 45:15:38.5:1.5; the II-37 is 2. The lipid nanoparticle composition according to claim 1, wherein Each n3 is selected from an integer of 4 to 8, and each m3 is selected from an integer of 4 to 8.

3. The lipid nanoparticle composition according to claim 1, wherein Each n3 is identical to each other, and each m3 is identical to each other.

4. The lipid nanoparticle composition according to claim 1, wherein the compound of formula C is 5. The lipid nanoparticle composition according to any one of claims 1 to 4, further comprising an active ingredient, wherein the active ingredient is located in the lipid nanoparticles.

6. The lipid nanoparticle composition of claim 5, wherein the active ingredient is a nucleic acid.

7. The lipid nanoparticle composition of claim 5, wherein the active ingredient is mRNA.

8. The lipid nanoparticle composition according to any one of claims 1 to 4, wherein the neutral lipid molecule is selected from the phosphatidylcholine compound represented by formula E E, phosphatidylethanolamine compound represented by formula F F, wherein Ra, Rb, Rc, and Rd are independently selected from a linear or branched C1-30 alkyl group, or a linear or branched C2-30 alkenyl group.

9. The lipid nanoparticle composition of claim 8, wherein Ra, Rb, Rc, and Rd are independently selected from a linear or branched C10-30 alkyl group, or a linear or branched C10-30 alkenyl group.

10. The lipid nanoparticle composition of claim 8, wherein Ra, Rb, Rc, and Rd are independently selected from CH3(CH2) 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11 CH2-, CH3(CH2)9CH2-, CH3(CH2)7CH2-, CH3(CH2)7-CH=CH-(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)7-CH=CH-(CH2)9-.

11. The lipid nanoparticle composition of claim 8, wherein the neutral lipid molecule is DOPE and / or DSPC.

12. The lipid nanoparticle composition of any one of claims 1 to 4, wherein the cholesterol lipid molecule is selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatidine, ursolic acid, α-tocopherol and mixtures thereof, 5-heptadecanol and cholesterol hemisuccinate.

13. The lipid nanoparticle composition according to any one of claims 1 to 4, wherein the PEGylated lipid molecule comprises a lipid portion and a PEG-based polymer portion, expressed as "lipid portion-PEG-number average molecular weight", wherein the lipid portion is diacylglycerol or diacylglycerol amide selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylglycerol, dilaurylglyceramide, dimyristoylglyceramide, dipalmitoylglyceramide, distearoylglyceramide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG is 130 to 50,000. The lipid nanoparticle composition according to claim 13 , wherein the number average molecular weight of the PEG is 150 to 10,000. The lipid nanoparticle composition according to claim 14 , wherein the number average molecular weight of the PEG is 300 to 3,000. The lipid nanoparticle composition of claim 15 , wherein the number average molecular weight of the PEG is 1,500 to 2,500.

17. The lipid nanoparticle composition of claim 13, wherein the PEGylated lipid molecule is DMG-PEG2000 and / or DSPE-PEG2000.

18. The lipid nanoparticle composition of claim 6, wherein the ratio of the total mass of lipid molecules to the mass of nucleic acids in the lipid nanoparticle composition is 5-20:

1.

19. The lipid nanoparticle composition of claim 7, wherein the mRNA comprises a 5'UTR, an open reading frame, a 3'UTR and a poly-A tail from the 5' end to the 3' end.

20. The lipid nanoparticle composition of claim 19, wherein the nucleotide sequence of the 5'UTR is shown in SEQ ID NO:

6.

21. The lipid nanoparticle composition according to claim 19, wherein the nucleotide sequence of the 3'UTR is two nucleotide sequences shown in SEQ ID NO: 7 connected end to end.

22. The lipid nanoparticle composition of claim 19, wherein the poly-A tail has a length of 50-200 nucleotides.

23. The lipid nanoparticle composition of claim 22, wherein the poly-A tail has a length of 100-150 nucleotides.

24. The lipid nanoparticle composition of claim 19, wherein the open reading frame is an open reading frame encoding the S protein mutant of 2019-nCov, and its nucleic acid sequence is shown in SEQ ID NO:

8.

25. The lipid nanoparticle composition of claim 19, wherein the nucleotide sequence of the mRNA is shown in SEQ ID NO:

9.

26. The lipid nanoparticle composition of claim 19, wherein the mRNA has a 5' cap structure.

27. The lipid nanoparticle composition according to any one of claims 1 to 4, further comprising a pharmaceutical excipient.

28. The lipid nanoparticle composition according to any one of claims 1 to 4, which is a liquid preparation containing sucrose at a mass percentage concentration of 5-20%.

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