CN115957187B - A lipid nanoparticle composition and a drug delivery system prepared therefrom - Google Patents

Abstract

The present invention provides a novel ionizable cationic lipid molecule, and a lipid nanoparticle composed of the same together with a neutral lipid molecule, a cholesterol lipid molecule, a pegylated lipid molecule, and a composition containing the same. The lipid nanoparticle serving as a delivery carrier of an active ingredient has the advantages of small and uniform particle size, high encapsulation efficiency and high cell transfection efficiency, and is particularly suitable for a delivery carrier of nucleic acid molecules (such as mRNA).

Description

Lipid nanoparticle compositions and drug delivery systems prepared therefrom

Technical Field

The invention belongs to the technical field of biological medicine preparations, and particularly relates to a lipid nanoparticle containing a novel lipid compound, and a pharmaceutical composition or a drug delivery system, such as an mRNA vaccine, which is prepared from the lipid nanoparticle and carries an active ingredient.

Background

Different types of nucleic acid formulations are being developed for the treatment of various major diseases such as infectious diseases, cancers, rare diseases, etc. The nucleic acid preparation comprises DNA, antisense nucleic Acid (ASO), small interfering RNA (siRNA), micro RNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, ribozyme (ribozyme) and the like. Among them, mRNA vaccines have a subverted advantage in terms of safety, rapid preparation and immunogenicity. Based on the development of mRNA modification and delivery tools, once the viral antigen sequences are obtained, mRNA vaccines with clinical scale can be rapidly designed and manufactured within weeks, and standardized production can be achieved, making them very attractive in coping with pandemic outbreaks. The mRNA vaccine has no potential reversion danger of the attenuated vaccine and no recovery mutation problem of the inactivated vaccine. In immunogenicity, mRNA vaccines can induce B-cell and T-cell immune responses, can elicit an immune memory effect, deliver more potent antigens, and can express multiple antigens at once. In addition, mRNA can efficiently express antigen proteins in cytoplasm only by penetrating cell membrane, and mRNA has no risk of gene integration into genome. And thirdly, mRNA is easily degraded after being translated into protein, and the transient expression characteristic of the mRNA ensures the safety of the mRNA drug, enables the dosage to be controllable, and avoids antigen immune tolerance (a state of no response to specific antigens) caused by long-term exposure of vaccine drugs.

However, on the one hand, nucleic acid preparations are negatively charged and have a large molecular weight, so that the nucleic acid preparations are difficult to directly enter cells, and on the other hand, RNA is unstable and is extremely easily degraded by nucleases during the process of being introduced into the body, so that the biological functions are lost. Therefore, the development of efficient, safe and versatile nucleic acid delivery systems is a major challenge in nucleic acid drug transformation processes.

Currently, methods of delivery of nucleic acids include chemical modification, bioconjugate techniques, nanocarrier techniques, lipid-based formulations, exosomes, spherical nucleic acids, DNA nanostructures, stimulus-responsive polymeric nanomaterials, and the like. The mature nucleic acid delivery carrier is lipid nano-particles (LNP), LNP-coated siRNA drugs Onpattro (patisiran) are approved and marketed in 2018, and 2021 LNP-coated mRNA vaccine is formally approved and applied to prevention and control of new coronary epidemic situation, and clinical results show higher effectiveness and no serious adverse reaction at present.

The lipid preparation comprises cation/ionizable lipid, auxiliary lipid, cholesterol and polyethylene glycol-lipid conjugate. Of these four lipid components, the charged head of the cationic lipid is capable of binding to negatively charged nucleic acids and also to phospholipid molecules on the cell membrane, playing a critical role in both the nucleic acid encapsulation and membrane fusion processes. In view of the potential toxicity of permanent cationic lipids, lipid nanoparticles of ionizable cationic lipids have greater application value.

The ionizable cationic lipid comprises three important structural components, namely a hydrophilic polar head containing an amino group, a hydrophobic lipid chain and a connecting chain responsible for connecting the polar head and a nonpolar tail. Currently, the commercial ionizable cationic lipids are mainly the MC3 series and ALC-0315, SM-102 for new crown mRNA vaccines. Among them, MC3 has strong liver targeting, and its application will be limited for nucleic acid formulations that may have potential hepatotoxicity, and MC3 was developed for siRNA delivery of smaller molecular weight, and its load may be limited for nucleic acid formulations of larger molecular weight. The delivery efficiency of ALC-0315 and SM-102 needs to be further improved. Therefore, in order to further advance the development of lipid formulations in China and the application thereof in nucleic acid drug delivery and the like, we need to develop new ionizable cationic lipids, and screen and optimize new nano delivery systems to realize safe and efficient delivery of nucleic acids, for example, mRNA vaccine components.

Disclosure of Invention

To solve the problems of unstable structure, susceptibility to nuclease degradation and difficulty in cell entry of active ingredients such as nucleic acids (e.g., mRNA molecules) in biological applications, new delivery technologies are required. In addition, the problems of poor lysosomal escape and low delivery efficiency are common in the prior art of delivery, and the invention forms a plurality of delivery systems based on lipid with various functions based on newly synthesized ionizable cationic lipid molecules.

In a first aspect of the present invention there is provided a lipid nanoparticle composition comprising lipid nanoparticles comprising ionizable cationic lipid molecules of formula I.

According to the present invention, the lipid nanoparticle composition further comprises other lipid molecules. The additional lipid molecules may be lipid molecules known or conventionally used in the art for constructing lipid nanoparticles, including but not limited to neutral lipid molecules, cholesterol lipid molecules, pegylated lipid molecules.

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, or the like. The active ingredient is located in a lipid nanoparticle. Such nucleic acids include, but are not limited to, DNA, antisense nucleic Acids (ASO), small interfering RNAs (siRNA), micrornas (miRNA), small activating RNAs (saRNA), messenger RNAs (mRNA), aptamers, and the like.

According to the present invention, the lipid nanoparticle composition comprises 30-60mol% of lipid molecules of formula I, preferably 32-55mol%, and more preferably 34-46mol% of the total lipid molecules.

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

According to the present invention, the lipid nanoparticle composition may contain 30 to 50mol% of cholesterol lipid molecules, preferably 35 to 50mol%, and more preferably 37 to 49mol% of the total lipid molecules in the lipid nanoparticle.

According to the present invention, the lipid nanoparticle composition may contain 0.4 to 10mol% of the pegylated lipid molecules, preferably 0.5 to 5mol%, and more preferably 1.3 to 2.7mol% of the total lipid molecules.

According to the present invention, when the active ingredient is a 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.

According to the invention, the ionizable cationic lipid molecules of formula I are of the formulaWherein:

q is a substituted or unsubstituted straight chain C2-20 alkylene group, 1 or more C atoms of which 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, the ring atoms of which optionally contain 1 or more heteroatoms independently selected from O, S, N, the substituted substituent being selected from halogen, -OH, straight or branched C1-20 alkyl, straight or branched C1-20 alkoxy, straight or branched C2-20 alkenyl, straight or branched C2-20 alkynyl, -CH ₂CH(OH)R₅,

R ₁、R₂、R₃、R₄, which may be the same or different, are each independently selected from hydrogen, substituted or unsubstituted linear or branched C1-30 alkyl, substituted or unsubstituted linear or branched C2-30 alkenyl, substituted or unsubstituted linear or branched C2-30 alkynyl, 1 or more C atoms of the alkyl, alkenyl or alkynyl being optionally replaced by heteroatoms independently selected from O, S and N, or-CH ₂CH(OH)R₅;

provided that at least one of R ₁、R₂、R₃、R₄ is

R ₅ 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, 1 or more C atoms of said alkyl, alkenyl or alkynyl being optionally replaced by heteroatoms independently selected from O, S and N, said substituted substituent being selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy;

R ₆ is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, -OH;

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

when at least two of R ₁、R₂、R₃、R₄ are When n and m in each of the groups are independent of each other, they may be the same or different.

In a preferred embodiment of the invention, Q is a substituted or unsubstituted linear C2-20 alkylene group, 1 or more C atoms of which are optionally replaced by heteroatoms independently selected from O, S and N;

Preferably, Q is Wherein R ₈、R₉ is independently selected from a substituted or unsubstituted linear C1-10 alkylene group, 1 or more C atoms of which are optionally replaced by heteroatoms independently selected from O, S and N, R ₇ is hydrogen, halogen, -OH, linear or branched C1-20 alkyl, linear or branched C2-20 alkenyl, linear or branched C2-20 alkynyl, or-CH ₂CH(OH)R₅, orThe substituted substituent groups are halogen, -OH, linear or branched C1-10 alkyl, linear or branched C1-10 alkoxy;

Preferably, Q is Wherein x and y may be the same or different and are independently selected from integers of 1 to 8, R ₇ is the same as defined above, preferably x or y is the same or different and is selected from integers of 1 to 3, for example, 1, 2 or 3, preferably R ₇ is a straight chain or branched C1-4 alkyl group, for example, methyl, ethyl, n-propyl, n-butyl, etc.

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

In a preferred embodiment of the invention, R ₆ is-OH.

In a preferred embodiment of the present invention, n is an integer of 4 to 8, and m is an integer of 4 to 8.

In a preferred embodiment of the invention, the compound of formula I is of formula A, B, C or D:

Wherein each n ₁ is independent of the other, and may be the same or different, each n ₁ is selected from an integer of 1 to 8, each m ₁ is independent of the other, and may be the same or different, each m ₁ is selected from an integer of 0 to 8, preferably each n ₁ is selected from an integer of 4 to 8, each m ₁ is selected from an integer of 4 to 8, preferably each n ₁ is the same as each other, and each m ₁ is the same as each other.

Wherein each n ₂ is independent of the other, and may be the same or different, each n ₂ is selected from an integer of 1 to 8, each m ₂ is independent of the other, and may be the same or different, each m ₂ is selected from an integer of 0 to 8, preferably each n ₂ is selected from an integer of 4 to 8, each m ₂ is selected from an integer of 4 to 8, preferably each n ₂ is the same as each other, and each m ₂ is the same as each other.

Wherein each n ₃ is independent of the other, and may be the same or different, each n ₃ is selected from an integer of 1 to 8, each m ₃ is independent of the other, and may be the same or different, each m ₃ is selected from an integer of 0 to 8, preferably each n ₃ is selected from an integer of 4 to 8, each m ₃ is selected from an integer of 4 to 8, preferably each n ₃ is the same as each other, and each m ₃ is the same as each other.

Wherein each n ₄ is independent of the other and can be the same or different, each n ₄ is selected from an integer of 1 to 8, each m ₄ is independent of the other and can be the same or different, each m ₄ is selected from an integer of 0 to 8, preferably each n ₄ is selected from an integer of 4 to 8, each m ₄ is selected from an integer of 4 to 8, preferably each n ₄ is the same as each other, and each m ₄ is the same as each other.

In some embodiments of the invention, the compound of formula I is selected from the following compounds shown in table 1:

TABLE 1

According to the invention, the mole percentage of lipid molecules of formula I in the lipid of the lipid nanoparticle is 30-60 mole%, e.g. 32-55 mole%, e.g. 30mol％,31mol％,32mol％,33mol％,34mol％,35mol％,36mol％, 37mol％,38mol％,39mol％,40mol％,41mol％,42mol％,43mol％,44mol％,45mol％,46mol％, 47mol％,48mol％,49mol％,50mol％,51mol％,52mol％,53mol％,54mol％,55mol％, etc.

According to the invention, the neutral lipid molecule is an uncharged lipid molecule or a zwitterionic lipid molecule, such as a phosphatidylcholine-like compound, or/and a phosphatidylethanolamine-like compound.

The structure of the phosphatidylcholine compound is shown as a formula E: The structure of the phosphatidylethanolamine compound is shown as a formula F: Wherein Ra, rb, rc, rd is independently selected from the group consisting of linear or branched C1-30 alkyl, linear or branched C2-30 alkenyl, preferably linear or branched C10-30 alkyl, linear or branched C10-30 alkenyl, e.g CH₃(CH₂)₁₇CH₂-、CH₃(CH₂)₁₅CH₂-、CH₃(CH₂)₁₃CH₂-、 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-.

Examples of neutral lipid molecules include, but are not limited to, 5-heptadecylphenyl-1, 3-diol (resorcinol), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), phosphorylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DAPC), phosphatidylethanolamine (PE), lecithin phosphatidylcholine (EPC), dilauryl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1, 2-Eicosyl Phosphatidylcholine (EPC), phosphatidylcholine (PE), stearoyl phosphatidylcholine (DPPC), stearoyl-2-palmitoyl phosphatidylcholine (DPPC), stearoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof.

In one embodiment, the neutral lipid molecule may be selected from the group consisting of distearyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), and distearoyl phosphatidylethanolamine (DSPE). In another embodiment, the neutral lipid molecule may be dimyristoyl phosphatidylethanolamine (DMPE). In another embodiment, the neutral lipid molecule may be dipalmitoyl phosphatidylcholine (DPPC).

According to the invention, the mole percentage of neutral lipid molecules in the lipid of the lipid nanoparticle is 5-30 mole%, such as 8-20 mole%, such as 8 mole%, 9 mole%, 10 mole%, 11 mole%, 12 mole%, 13 mole%, 14 mole%, 15 mole%, 16 mole%, 17 mole%, 18 mole%, 19 mole%, 20 mole%.

According to the present invention, cholesterol lipid molecules refer to sterols as well as lipids containing sterol moieties, including but not limited to cholesterol, 5-heptadecylresorcinol, stigmasterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycosyline, ursolic acid, alpha-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.

According to the invention, the mole percentage of cholesterol lipid molecules in the lipid of the lipid nanoparticle is 30-50 mole%, for example 30mol％,31mol％,32mol％,33mol％,34mol％,35mol％,36mol％,37mol％,38mol％,39mol％,40mol％,41mol％,42mol％,43mol％,44mol％,45mol％,46mol％,47mol％, 48mol％,49mol％,50mol％ etc.

According to the invention, the pegylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety. In some embodiments, the lipid moiety may be derived from diacylglycerols or diacylglycerol amides (DIACYLGLYCAMIDE), including those comprising a dialkylglycerol or dialkylglyceramide group having an alkyl chain length independently comprising from about C4 to about C30 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups, such as an amide or an ester. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglyceramide group may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical. As used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer, unless otherwise indicated. In one embodiment, the PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety may be substituted with, for example, one or more alkyl, alkoxy, acyl, hydroxy, 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)), or the PEG moiety does not comprise a PEG copolymer, e.g., it may be a PEG homopolymer. In one embodiment, the PEG has a molecular weight of about 130 to about 50,000, in a fruiting body embodiment, about 150 to about 30,000, in a fruiting body embodiment, about 150 to about 20,000, in a fruiting body embodiment, about 150 to about 15,000, in a fruiting body embodiment, about 150 to about 10,000, in a fruiting body embodiment, about 150 to about 6,000, in a fruiting body embodiment, about 150 to about 5,000, in a fruiting body embodiment, about 150 to about 4,000, in a fruiting body embodiment, about 150 to about 3,000, in a fruiting body embodiment, about 300 to about 3,000, in a fruiting body embodiment, about 1,000 to about 3,000, and in a fruiting body embodiment, about 1,500 to about 2,500. In certain embodiments, PEG is "PEG 2000" having an average molecular weight of about 2,000 daltons. In some embodiments of the invention, PEG is herein represented by the formulaMeaning that for PEG-2000 where n is 45, meaning that the number average degree of polymerization comprises about 45 subunits, other PEG embodiments known in the art can also be used, including, for example, those wherein the number average degree of polymerization comprises about 23 subunits (n=23) and/or 68 subunits (n=68). 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 unsubstituted C1-C30 alkyl, such as C1-C20 alkyl, C1-C10 alkyl, C1-C6 alkyl. In some embodiments, R may be H, methyl or ethyl.

In some embodiments, the pegylated lipid molecule may be represented as a "lipid fraction-PEG-number average molecular weight" or "PEG-lipid fraction" or "PEG-number average molecular weight-lipid fraction", which is a diacylglycerol or diacylglycerol amide selected from the group consisting of dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoyl glycerol, dilauroylglycerol amide, dimyristoylglycerol amide, dipalmitoylglycerol amide, distearoyl glyceramide, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine, and PEG has a number average molecular weight of about 130 to about 50,000, for example about 150 to about 30,000, about 150 to about 20,000, about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, about 150 to about 5,000, about 150 to about 4,000, about 3,000, about 3 to about 500, for example, about 3,000, about 500 to about 3,000.

In some embodiments, the pegylated lipid molecule may be selected from the group consisting of PEG-dilauroylglycerol, PEG-dimyristoylglycol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearylglycerol (PEG-DSPE), PEG-dilauryl glyceramide, PEG-dimyristoylglyceride, PEG-dipalmitoylglyceride and PEG-distearylglyceride, PEG-cholesterol (1- [8' - (cholest-5-ene-3 [ beta ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ omega ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetradecyloxybenzyl- [ omega ] -methyl-poly (ethylene glycol) ether), 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DMG-PEG 2000), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DSPE), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000) Poly (ethylene glycol) -2000-dimethacrylate (DMA-PEG 2000) and 1, 2-distearoyloxypropyl-3-amine-N- [ methoxy (polyethylene glycol) -2000] (DSA-PEG 2000). In one embodiment, the pegylated lipid molecule may be DMG-PEG2000. 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.

According to the invention, the mole percentage of pegylated lipid molecules in the lipid of the lipid nanoparticle is 0.4-10 mole%, such as 0.5-5 mole%, e.g. 0.4mol％,0.5mol％,0.6mol％,0.7mol％,0.8mol％,0.9mol％, 1.0mol％,1.1mol％,1.2mol％,1.3mol％,1.4mol％,1.5mol％,1.6mol％,1.7mol％,1.8mol％, 1.9mol％,2.0mol％,2.1mol％,2.2mol％,2.3mol％,2.4mol％,2.5mol％,2.6mol％,2.7mol％, 2.8mol％,2.9mol％,3.0mol％,3.1mol％,3.2mol％,3.3mol％,3.4mol％,3.5mol％,3.6mol％, 3.7mol％,3.8mol％,3.9mol％,4.0mol％,4.1mol％,4.2mol％,4.3mol％,4.4mol％,4.5mol％, 4.6mol％,4.7mol％,4.8mol％,4.9mol％,5.0mol％ etc.

In some embodiments of the invention, the lipid nanoparticle comprises a lipid molecule represented by formula C, a neutral lipid molecule, a cholesterol lipid molecule, a pegylated lipid molecule, wherein:

C (C) Wherein each n ₃ is independent of the other and can be the same or different, each n ₃ is selected from an integer of 1 to 8, each m ₃ is independent of the other and can be the same or different, each m ₃ is selected from an integer of 0 to 8, preferably each n ₃ is selected from an integer of 4 to 8, each m ₃ is selected from an integer of 4 to 8, preferably each n ₃ is the same as each other, and each m ₃ is the same as each other. The mole percentage of the ionizable cationic lipid molecules of formula C to the lipids in the lipid nanoparticle is 32-55 mole%, preferably 34-46 mole%.

The neutral lipid molecule is selected from phosphatidylcholine compounds shown in formula EPhosphatidylethanolamine compound shown in formula FWherein Ra, rb, rc, rd is independently selected from linear or branched C10-30 alkyl, linear or branched C10-30 alkenyl, preferably CH₃(CH₂)₁₇CH₂-、CH₃(CH₂)₁₅CH₂-、 CH₃(CH₂)₁₃CH₂-、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-. neutral lipid molecules comprising from 8 to 20 mole percent, preferably from 9 to 16 mole percent of the lipids in the lipid nanoparticle;

the cholesterol lipid molecule is selected from cholesterol and cholesterol hemisuccinate. The cholesterol lipid molecules comprise 30-50 mole%, preferably 35-50 mole%, more preferably 37-49 mole% of the lipids in the lipid nanoparticle.

The pegylated lipid molecule is expressed as "lipid fraction-PEG-number average molecular weight", said lipid fraction is diacylglycerol or diacylglycerol amide selected from dilauryl glycerol, dimyristoyl glycerol, dipalmitoyl glycerol, distearoyl glycerol, dilauryl glycerol amide, dimyristoyl glycerol amide, dipalmitoyl glycerol amide, distearoyl glycerol amide, 1, 2-ditearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, and the PEG has a number average molecular weight of 130 to 50,000, for example 150～30,000,150～20,000,150～15,000,150～10,000,150～6,000,150～5,000, 150～4,000,150～3,000,300～3,000,1,000～3,000,1,500～2,500, about 2000. The PEGylated lipid molecules comprise 0.5 to 5mol%, preferably 1.3 to 2.7mol% of the lipid in the lipid nanoparticle.

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

According to the invention, the mRNA may comprise, from the 5' end to the 3' end, a 5' cap structure, a 5' UTR, an Open Reading Frame (ORF), a 3' UTR and a poly-A tail.

According to the present invention, the Cap structure may be a Cap1 structure, a Cap2 structure or a Cap3 structure. In one embodiment of the invention, the Cap structure is a Cap1 structure.

According to the invention, the 5'UTR may comprise a 5' UTR of β -globin or α -globin or a homologue, fragment thereof. In some embodiments of the 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 the β -globin shown in SEQ ID NO. 6. In a specific embodiment of the invention, the 5'UTR comprises the 5' UTR nucleotide sequence of the β -globin as shown in SEQ ID NO. 6.

In some embodiments of the invention, the 5' utr further comprises a Kozak sequence. In one embodiment of the invention, the Kozak sequence is GCCACC.

According to the invention, the 3'UTR may comprise a 3' UTR of β -globin or α -globin or a homologue, fragment or combination of fragments thereof. In some embodiments of the invention, the 3'UTR comprises a nucleotide sequence which 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 a fragment of the α2-globin 3' UTR shown in SEQ ID NO. 7. In other embodiments of the invention, the 3'UTR comprises 2 nucleotide sequences joined end to end 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 about 100% homologous to a fragment of the α2-globin 3' UTR shown in SEQ ID NO. 7. In a specific embodiment of the invention, the 3' UTR comprises 2 nucleotide sequences as shown in SEQ ID NO. 7, joined end to end.

According to the invention, the poly-A tail may be 50-200 nucleotides, preferably 100-150 nucleotides, for example 110-120 nucleotides, such as about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides in length.

In one embodiment of the invention, the Open Reading Frame (ORF) is an Open Reading Frame (ORF) encoding a mutant of the S protein of 2019-nCov, the nucleic acid sequence of which 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 set forth in SEQ ID NO. 8. The amino acid sequence of the S protein mutant after ORF translation consists of an amino acid sequence shown in SEQ ID NO. 2 and an amino acid sequence shown in SEQ ID NO. 3 which are directly connected from the N end to the C end. In one embodiment of the present invention, the nucleotide sequence of the Open Reading Frame (ORF) of the S protein mutant is shown in SEQ ID NO. 8.

In one embodiment of the 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 set forth in SEQ ID NO. 9. In one embodiment of the invention, the mRNA comprises the nucleotide sequence set forth in SEQ ID NO. 9.

The mRNA of the present invention can be prepared by methods known in the art. In some embodiments of the invention, a nucleic acid sequence encoding an mRNA may be synthesized artificially, cloned into a vector, and constructed into a plasmid for in vitro transcription. And (3) transforming the constructed plasmid into host bacteria for culture and amplification, and extracting the plasmid. The extracted plasmid was digested into linear molecules using restriction enzymes immediately following the polyA tail. mRNA was prepared using in vitro transcription using the prepared linearized plasmid molecule as a template. The cap analogue may be added during in vitro transcription to directly obtain mRNA having a cap structure, or the cap analogue may be added to mRNA after in vitro transcription is completed by using a capping enzyme and a dimethyltransferase. The resulting mRNA may be purified by methods conventional in the art, such as chemical precipitation, magnetic bead, affinity chromatography, and the like.

According to the invention, one or more nucleotides in the mRNA may be modified. For example, one or more nucleotides (e.g., all nucleotides) in the mRNA can each independently be replaced with a naturally occurring nucleotide analog or an artificially synthesized nucleotide analog, e.g., selected from pseudouridine (pseudouridine), 2-thiouridine (2-thiouridine), 5-methyluridine (5-methyluridine), 5-methylcytidine (5-METHYLCYTIDINE), N6-methyladenosine (N6-methyladenosine), N1-methylpseudouridine (N1-methylpseudouridine), and the like.

The invention also provides an mRNA vaccine comprising lipid nanoparticles, wherein the lipid nanoparticles comprise lipid molecules shown in a formula C, neutral lipid molecules, cholesterol lipid molecules, PEGylated lipid molecules and mRNA encoding 2019-nCoV S protein mutants, the mRNA comprises nucleotide sequences which are at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homologous to the nucleotide sequences shown in SEQ ID NO 9, and the mass ratio of the total mass of the lipid molecules to the mRNA is 5-20:1, wherein:

C (C) Wherein each n ₃ is independent of the other and can be the same or different, each n ₃ is selected from an integer of 1 to 8, each m ₃ is independent of the other and can be the same or different, each m ₃ is selected from an integer of 0 to 8, preferably each n ₃ is selected from an integer of 4 to 8, each m ₃ is selected from an integer of 4 to 8, preferably each n ₃ is the same as each other, and each m ₃ is the same as each other. The lipid molecule shown in the formula C accounts for 34-46mol% of the lipid in the lipid nanoparticle;

the neutral lipid molecule is selected from phosphatidylcholine compounds shown in formula E Phosphatidylethanolamine compound shown in formula FWherein Ra, rb, rc, rd is independently selected from linear or branched C10-30 alkyl, linear or branched C10-30 alkenyl, preferably CH₃(CH₂)₁₇CH₂-、CH₃(CH₂)₁₅CH₂-、 CH₃(CH₂)₁₃CH₂-、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-. neutral lipid molecules comprising 9-16 mole% of the lipid in the lipid nanoparticle;

the cholesterol lipid molecule is selected from cholesterol and cholesterol hemisuccinate. The cholesterol lipid molecules account for 37-49mol% of the lipid in the lipid nano-particles;

The pegylated lipid molecule is denoted "lipid fraction-PEG-number average molecular weight", which is a diacylglycerol or diacylglycerol amide selected from dilauryl glycerol, dimyristoyl glycerol, dipalmitoyl glycerol, distearoyl glycerol, dilauryl glyceramide, dimyristoyl glyceramide, dipalmitoyl glyceramide, distearoyl glyceramide, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, and PEG has a number average molecular weight of about 130 to about 50,000, for example about 150 to about 30,000, about 150 to about 20,000, about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, about 150 to about 5,000, about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000, about 1, 2000 to about 3,000, for example about 500, 500. The PEGylated lipid molecules comprise 1.3 to 2.7 mole percent of the lipids in the lipid nanoparticle.

In some embodiments of the invention, the lipid molecules of formula C, neutral lipid molecules, cholesterol, and pegylated lipid molecules are in a molar ratio of 35:15:48.5:1.5.

In some embodiments of the invention, the lipid molecules of formula C, neutral lipid molecules, cholesterol, and pegylated lipid molecules are in a 40:10:48.5:1.5 molar ratio.

In some embodiments of the invention, the lipid molecules of formula C, neutral lipid molecules, cholesterol, and pegylated lipid molecules are in a 45:15:38.5:1.5 molar ratio.

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

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

In one embodiment of the invention, the pegylated lipid molecule is DMG-PEG2000 and/or DSPE-PEG2000.

The invention also provides a method for preparing the ionizable cationic lipid molecules of formula I.

The ionizable lipid compounds of the invention may be synthesized by 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 is performed with or without a solvent, and the synthesis may be performed at a higher temperature in the range of 25-100 ℃. The resulting ionizable lipid compound may optionally be purified. For example, a mixture of ionizable lipid compounds may be purified to yield a particular ionizable lipid compound. Or the mixture may be purified to give the particular stereoisomer or regioisomer. The epoxide may be commercially available or synthetically prepared.

In some embodiments of the invention, the ionizable lipid compounds of the invention may be prepared using the following general preparation methods.

A, B, C or D

Step 1. Reduction

The carboxyl group of the compound A1 is reduced to a hydroxyl group in the presence of a reducing agent to obtain a compound A2. Examples of reducing agents include, but are not limited to, lithium aluminum hydride, diisobutylaluminum hydride, and the like. Examples of the solvent used in the reaction include, but are not limited to, ethers (such as diethyl ether, tetrahydrofuran, dioxane, etc.), halogenated hydrocarbons (such as chloroform, methylene chloride, dichloroethane, etc.), hydrocarbons (such as n-pentane, n-hexane, benzene, toluene, etc.), and mixed solvents of two or more of these solvents.

Step 2 oxidation

The hydroxyl group of the compound A2 is oxidized to an aldehyde group in the presence of an oxidizing agent to obtain a compound A3. Examples of oxidizing agents include, but are not limited to, 2-iodoxybenzoic acid (IBX), pyridinium chlorochromate (PCC), pyridinium Dichlorochromate (PDC), dess-martin oxidizing agent, manganese dioxide, and the like. Examples of the solvent used in the reaction include, but are not limited to, halogenated hydrocarbons (such as chloroform, methylene chloride, dichloroethane, etc.), hydrocarbons (such as n-pentane, n-hexane, benzene, toluene, etc.), nitriles (such as acetonitrile, etc.), and mixed solvents of two or more of these solvents.

Step 3 halo-reduction

First, the aldehyde α -hydrogen of the compound A3 is subjected to halogenation with a halogenating agent under acidic conditions to obtain an α -halogenated aldehyde intermediate, and then the aldehyde group of the α -halogenated aldehyde is reduced to a hydroxyl group in the presence of a reducing agent to obtain the compound A4. Examples of conditions that provide acidity include, but are not limited to, DL-proline. Examples of halogenated 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.

Step 4 epoxidation

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

Step 5 ring opening reaction

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

The raw material A1 in the preparation method can be obtained commercially or synthesized by a conventional method.

The invention also provides methods of preparing lipid nanoparticle compositions.

According to the present invention, the preparation method comprises dissolving lipid molecules in an organic solvent at a molar ratio to obtain a lipid-mixed solution, mixing the lipid-mixed solution with an aqueous solution of a substance to be delivered (e.g., nucleic acid) as an organic phase, and mixing the organic phase with the aqueous phase to obtain lipid nanoparticles. Lipid nanoparticles may be prepared using other methods including, but not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nano-precipitation, microfluidic, simple and complex coacervation, and others known to those of ordinary skill in the art.

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

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

In some embodiments, the nanoparticle is prepared using a microfluidic platform.

According to the present invention, the preparation method further comprises the step of separating and purifying the lipid nanoparticle.

According to the present invention, the preparation method further comprises a step of lyophilizing the lipid nanoparticle.

The ionizable lipid of the formula I contains two adjacent cis double bonds in the molecular structure, so that the ionizable lipid has higher encapsulation efficiency and better cell transfection efficiency when being subsequently applied to a delivery system for wrapping active substances (such as nucleic acid (e.g. mRNA)), and the obtained lipid nanoparticles have more uniform particle size when being prepared. The ionizable lipid compounds of the invention are particularly suitable for preparing nanoparticles of solid structure.

In addition, for the mRNA vaccine of the invention, because the mRNA of the invention has high translation efficiency and stability, the encoded S protein mutant has high stability, and the lipid nanoparticle of the invention has high encapsulation efficiency, more uniform particle size and better cell transfection efficiency, and the mRNA vaccine of the invention has high mRNA encapsulation efficiency, drug loading capacity, cell transfection efficiency, high-efficiency and stable in vivo translation, antigen stability and good immune effect.

The particle size of the lipid nanoparticle in the invention is in the range of 1nm to 1000nm, for example 10 to 500nm,10 to 200nm, etc.

The lipid nanoparticle of the present invention may also be modified with targeting molecules to render it a targeting agent capable of targeting a specific cell, tissue or organ. The targeting molecule may be located on the surface of the particle. The targeting molecule may be a protein, peptide, glycoprotein, lipid, small molecule, nucleic acid, etc., examples of which include, but are not limited to, antibodies, antibody fragments, low Density Lipoproteins (LDL), transferrin (transferrin), asialoglycoprotein (asialycoprotein), receptor ligands, sialic acid, aptamers, etc. The targeting molecule may be attached to a cholesterol lipid molecule or a pegylated lipid molecule of the lipid nanoparticle.

The lipid nanoparticle composition and vaccine of the present invention may further contain one or more pharmaceutical excipients. The term "pharmaceutical excipient" means any type of nontoxic, inert solid, semi-solid or liquid filler, diluent, etc., including but not limited to sugars such as lactose, trehalose, glucose and sucrose, starches such as corn starch and potato starch, celluloses and derivatives thereof such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, gelatin, talc, oils such as peanut oil, cottonseed oil, safflower oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurate, surfactants such as Tween 80 (Tween 80), buffers such as phosphate buffer solutions, acetate buffer solutions and citrate buffer solutions, colorants, sweeteners, flavoring agents and fragrances, preservatives and antioxidants, and the like.

In one embodiment of the invention, the lipid nanoparticle composition and vaccine are liquid formulations, further comprising sucrose, in a concentration of 5-20% by mass, preferably 8-10%.

The lipid nanoparticle compositions and vaccines of the present invention can be administered orally, rectally, intravenously, intramuscularly, intravaginally, intranasally, intraperitoneally, bucally, or in the form of oral or nasal spray, etc., to humans and/or animals.

The S protein mutant is generated by amino acid mutation of a parent S protein. In one embodiment of the invention, the parent S protein is the S protein of the 2019-nCoV B1.351 mutant strain, which has mutations in the S protein of the 2019-nCoV B1.351 mutant strain compared to the S protein of the 2019-nCoV wild strain of L18F, D80A, D215G, L242_244L del, R246I, K417N, E484K, N501Y, D614G, A701V (the positions of the amino acid sequence shown in SEQ ID NO:1 are depicted).

In the present invention, the amino acid positions of both the S protein mutant and the parent S protein are described based on the amino acid sequence of the wild-type S protein, which can be obtained at NCBI GeneID:43740568, having a total of 1273 amino acids, the sequences of which are shown below and are designated as SEQ ID NO:1 in the present invention.

The S protein mutant at least comprises an extracellular domain, wherein the extracellular domain comprises amino acid mutations at positions F817P, A892P, A899P, A942P and KV986_987PP relative to the extracellular domain of a parent S protein, the amino acid RRAR at positions 682-685 is mutated into GSAS, the extracellular domain does not comprise a transmembrane domain and a cytoplasmic tail of the S protein, and a domain T4 Fibritin Foldon Trimerization Motif for directly fusing and assisting in forming a trimer at the C end of the extracellular domain. The S protein mutant comprises an amino acid sequence of SEQ ID NO.2 and an amino acid sequence of SEQ ID NO.3 which are directly connected from the N end to the C end.

List of the above sequences of the invention:

Description of the terminology:

The term "alkyl" refers to a saturated hydrocarbon group obtained by removing a single hydrogen atom from a hydrocarbon moiety containing 1 to 30 carbon atoms. 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.

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

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

The term "alkoxy" refers to an alkyl group, as defined above, attached to the parent molecule through an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, t-butoxy, neopentyloxy, and n-hexyloxy.

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

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

The terms "substituted" (whether the term "optional" is present or not) and "substituent" refer to the ability to change one functional group to another, provided that the valences of all atoms are maintained. When more than one position in any particular structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position.

And/or is to be taken as a specific disclosure of each of two specified features or components with or without the other. Thus, the term "and/or" as 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" as used in phrases such as "A, B and/or C" is intended to encompass each of A, B and C, A, B or C, A or B, B or C, A and B, B and C, A (alone), B (alone), and C (alone).

"Comprising" and "including" have the same meaning and are intended to be open and allow for the inclusion of additional elements or steps but not required. When the terms "comprising" or "including" are used herein, the term "consisting of" and/or "consisting essentially of" is therefore also included and disclosed.

In the present description and claims, nucleotides are referred to by their commonly accepted single letter codes. Unless otherwise indicated, nucleotide sequences are written in the 5 'to 3' direction from left to right. Nucleobases are herein indicated by commonly known single letter symbols recommended by the IUPAC-IUB biochemical naming committee. The skilled artisan will appreciate that the T base in the codons disclosed herein is present in DNA, whereas the T base will be substituted with a U base in the corresponding RNA. For example, a codon-nucleotide sequence in the form of DNA disclosed herein, such as a vector or an In Vitro Translation (IVT) template, has its T base transcribed into a U base in its corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (comprising T) and their corresponding mRNA sequences (comprising U) are considered codon-optimized nucleotide sequences of the present disclosure. Those skilled in the art will also appreciate that equivalent codon patterns can be generated by substituting one or more bases with non-natural bases.

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

"Nucleotide sequence encoding" refers to a nucleic acid (e.g., mRNA or DNA molecule) encoding a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements including promoters and polyadenylation signals capable of directing expression in cells of the individual or mammal to which the nucleic acid is administered.

In the present description and claims, conventional single-letter or three-letter codes for amino acid residues are used. Unless otherwise specified, amino acid sequences are written in an amino-to-carboxyl orientation from left to right.

The term "about" as used throughout the specification and claims in connection with a numerical value means an interval of accuracy that is familiar and acceptable to those skilled in the art. Typically, this accuracy is in the interval of + -10%.

For ease of reference, the S protein mutants of the present invention are described using the nomenclature of original amino acid: position: substituted amino acid. According to this naming convention, substitution of asparagine at position 30 with alanine is indicated as Asn30Ala or N30A, deletion of asparagine at the same position is indicated as Asn30 or N30, insertion of another amino acid residue, e.g. lysine, is indicated as Asn30AsnLys or N30NK, deletion of consecutive stretch of amino acid residues, e.g. deletion of amino acid residues 242-244, indicated as (242-244) or delta (242-244) or 242_244del, if the S protein mutant contains a "deletion" and an insertion at this position compared to the other S protein parent, is indicated as 36Asp or 36D, indicating deletion of the same time as insertion of aspartic acid at position 36. When one or more alternative amino acid residues may be inserted at a given position, this is denoted as N30A, E, or N30A or N30E.

Homology As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In general, the term "homology" means the 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 identity and similarity.

In some embodiments, polymer molecules are considered "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).

Identity the term "identity" as used herein refers to the overall monomer conservation between polymer molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. For example, calculation of percent identity of two polynucleotide sequences can be performed 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 abandoned for comparison purposes when comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

Suitable software programs are available from a variety of sources and are used for alignment of both protein and nucleotide sequences. For example, bl2seq, needle, STRETCHER, water or Matcher, etc.

The terms "coding region" and "coding region" refer to the Open Reading Frame (ORF) in a polynucleotide that, when expressed, produces a polypeptide or protein.

"Operably linked" refers to a functional linkage between two or more molecules, constructs, transcripts, entities, moieties, and the like.

Domain as used herein, the term "domain" when referring to a polypeptide refers to a motif of the polypeptide that has one or more identifiable structural or functional features or properties (e.g., binding capacity, serving as a site for protein-protein interaction).

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

The term "protein mutant" or "polypeptide mutant" refers to a molecule whose amino acid sequence differs from a native or reference sequence. Amino acid sequence mutants may have substitutions, deletions and/or insertions, etc., at certain positions within the amino acid sequence, as compared to the native or reference sequence. Typically, the 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, at least about 99% identity to the native or reference sequence.

Drawings

FIG. 1 analysis of B1.351 mM RNA integrity results on a 2100 bioanalyzer using an RNA 6000nano chip.

FIG. 2 ELISA method for detecting S protein expression level in supernatant after transfection of nucleic acid into CHO-K1 cells.

FIG. 3:S 3D Structure of protein mutants.

FIG. 4 ELISA assay for S protein expression levels in supernatants after encapsulation of mRNA of the invention with nanoparticles made of II-37.

FIG. 5 expression levels of S protein in supernatants after transfection of mRNA of the invention with LNP prepared by II-37 and MC 3.

FIG. 6 is a statistical chart of the amount of intracellular protein expressed by mRNA prepared from different in vitro transcription vectors using Firefly Luc as a reporter protein.

FIG. 7 is a statistical plot of the generation of binding antibodies in vivo after immunization of BALB/c mice with the S protein mutant of the present invention. In the figure, a is the result of wild type S protein trimer, B is the result of B1.351 mRNA translated S protein trimer, and c is blank control.

FIG. 8 is a statistical plot of the production of binding and neutralizing antibodies in vivo after immunization of BALB/c mice with lipid nanoparticles encoding mRNA of the S protein mutant. The results of detection of bound antibodies after immunization of Lipid Nanoparticles (LNP) of mRNA at 5. Mu.g, 1. Mu.g and 0.2. Mu.g are shown in the figures, the ordinate shows the concentration (. Mu.g/ml), and the results of detection of neutralized antibodies after immunization of Lipid Nanoparticles (LNP) of mRNA at 5. Mu.g, 1. Mu.g and 0.2. Mu.g are shown in the figures, the abscissa shows the log conversion value of serum dilution and the ordinate shows the inhibition percentage%.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The experimental method is a conventional molecular biological method in the field, and can be operated by referring to the manual of molecular biological experiments or the instruction of the kit product in the field.

EXAMPLE 1 Synthesis of lipid II-37

Synthesis of linolenol (a 2) LiAlH ₄ (7.20 g), linolenic acid (50 g, a 1) was added to 950mL tetrahydrofuran at 0℃and the mixture was stirred for 2h at 25 ℃. After the completion of the reaction, which was shown by Thin Layer Chromatography (TLC), the reaction mixture was quenched by adding water (7.2 mL), naOH aqueous solution (7.2 mL, mass fraction 15%) and water (21.6 mL) in this order, and adding an appropriate amount of Na ₂SO₄, stirring for 15 minutes, filtering through a buchner funnel and washing the filter cake with ethyl acetate, collecting the filtrate and concentrating by evaporation to obtain 47.4g of the target product linolenol (a 2).

¹H 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)

Synthesis of (9Z, 12Z) -octadeca-9, 12-dienal (a 3) to 170mL of acetonitrile at room temperature were added linolenol (25.0 g, a 2) and 2-iodoxybenzoic acid (39.4 g), after which the mixture was stirred at 85℃for 4h. The reaction solution was filtered through a buchner funnel and the filter cake was washed with methylene chloride, and the filtrate was collected and concentrated by evaporation to give 24.0g of the objective (9Z, 12Z) -octadeca-9, 12-dienal (a 3).

¹H 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)

Synthesis of (9Z, 12Z) -2-chloro-octadeca-9, 12-dien-1-ol (a 4) to 246mL of acetonitrile at 0℃were added (9Z, 12Z) -octadeca-9, 12-dienal (43.0 g, a 3), DL-proline (5.62 g) and N-chlorosuccinimide, followed by stirring at 0℃for 2h. After completion of the reaction, the reaction mixture was diluted with absolute ethanol (246 mL), and sodium borohydride (8.8 g) was added thereto, followed by stirring at 0 ℃ for 4 hours. 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 give the desired product (9 z,12 z) -2-chloro-octadeca-9, 12-dien-1-ol (a 4,46 g) which was used directly in the next step.

¹H 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)

Synthesis of 2- [ (7Z, 10Z) -hexadecane-7, 10-diene ] oxirane (a 5) to 450mL of 1, 4-dioxane were added (9Z, 12Z) -2-chloro-octadeca-9, 12-dien-1-ol (45 g, a 4) and aqueous sodium hydroxide solution (120 g of sodium hydroxide in 585mL of water) at room temperature, and the mixture was stirred at 35℃for 2h after the addition. TLC showed that after the reaction was completed, the reaction solution was separated by a separating funnel and washed with saturated brine, dried over sodium sulfate, filtered and concentrated by evaporation, and then the residue was purified by flash column chromatography eluting with petroleum ether/ethyl acetate to give the target product 2- [ (7 z,10 z) -hexadecane-7, 10-diene ] oxirane (a 5) 29.11g.

¹H NMR(400MHz,CDCl₃):δ5.27-5.46(m,4H),2.87-2.98(m,1H),2.70-2.85(m,3H), 2.46(dd,J＝5.00,2.75Hz,1H),1.94-2.21(m,4H),1.24-1.58(m,17H),0.78-1.00(m,3H)

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 10mL of ethanol at room temperature, and the mixture was stirred at 90℃for 36h. The reaction solution was concentrated by evaporation, and the residue was purified by flash column chromatography eluting with methylene chloride/methanol to give crude product II-37 (4 g). The target product was purified again by flash column chromatography with dichloromethane/methanol to give II-37 (2.2 g).

¹H 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)

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

Example 2B1.351 preparation of mRNA and translation thereof

1. A nucleic acid sequence encoding the mRNA shown in SEQ ID No.8 was synthesized and cloned into pUC57-kana vector behind the T7 promoter, which had been previously engineered to contain sequences encoding SEQ ID No. 6, a Kozak sequence, 2 end-to-end SEQ ID No. 7, and a polyA tail. The nucleic acid sequence encoding the mRNA shown in SEQ ID No.8 was cloned into the multiple cloning site between the Kozak sequence and 2 end-to-end SEQ ID No. 7, and a plasmid for in vitro transcription was constructed.

2. And (3) transforming the constructed plasmid into escherichia coli Dh5a, culturing and amplifying the plasmid, and extracting the plasmid.

3. The extracted plasmid was digested into linear molecules using the restriction enzyme SpeI immediately following the polyA tail.

4. The prepared linearized plasmid molecule is used as a template, an in vitro transcription method (in vitro transcription kit A45975 of Thermo company) is used for preparing mRNA, the sequence of the mRNA is shown as SEQ ID NO:9, the mRNA is hereinafter abbreviated as B1.351 mRNA, and the S protein mutant is obtained after translation of the mRNA, wherein the amino acid sequence of the S protein mutant is the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:3 which are directly connected from the N end to the C end. After the end of in vitro transcription, CAP structures of CAP1 are added to mRNA using capping enzymes and dimethyltransferase.

Purification of mRNA the mRNA stock solution obtained was purified by affinity chromatography.

MRNA quality control the prepared mRNA was analyzed for mRNA integrity on a 2100 bioanalyzer using an RNA 6000nano chip, and the results are shown in FIG. 1, the transcribed mRNA bands were single and no significant degradation was observed.

In addition, spike fragments were excised from the commercial plasmid pCMV3-Spike by restriction enzymes HindIII and EcoRI, and inserted between the HindIII and EcoRI sites of the IVT1 vector of example 5 to give an IVT1-Spike plasmid. And then carrying out point mutation on the plasmid to obtain IVT1-spike-D614G plasmid, and carrying out in vitro transcription by taking the plasmid as a template to obtain spike-D614G mRNA, thereby expressing the full-length S protein containing the D614G mutation.

Cell level expression assay of mRNA from B1.351 Using CHO-K1 cell line as expression system, using Lipofectamine Messenger MAX Reagent (Invitrogen, cat # 1168-027) to transfect mRNA, culturing for 48h, collecting cell culture supernatant, and detecting S protein expression level with ELISA assay kit for detecting S protein to evaluate whether mRNA can be translated into protein. The results are shown in FIG. 2. In FIG. 2, "spike DNA" is a commercial plasmid pCMV3-spike (purchased from Equipped with China) expressing full-length wild-type S protein, "spike-D614G mRNA" is the mRNA expressing the full-length S protein containing the D614G mutation, and "spike B1.351 mRNA" is the B1.351 mRNA, expressing the S protein mutant of the present invention, and the result shows that the mRNA of the present invention can highly express the S protein mutant in cells.

After purifying the obtained S protein mutant, carrying out structural analysis by adopting a cryoelectron microscope, wherein the 3D structure of the S protein is shown as a figure 3, and the S protein mutant is a stable structure of pre-fusion (prefusion spike structure). The sequence of the B1.351 mutant strain and the sequence of the wild strain differ by 9 mutation sites, 3 of which are in the RBD region. The RBD domain status of the pre-fusion S protein of the wild strain has been reported to be mainly 1 OPEN, 2 CLOSE structures. The structure of the S protein mutant of the invention is mainly in flexible state of 2 OPEN and1 CLOSE. This structural difference is the structural basis for the enhanced binding capacity and the enhanced infectivity of the virus to the receptor ACE2 and also leads to a significant difference in the immunogenic epitopes of the S protein and thus to a significant difference in antibodies, especially neutralizing antibodies, induced on the basis of the different structures.

Example 3 preparation of lipid nanoparticle compositions comprising nucleic acids

Accurately weighing the compounds II-37, DOPE, CHOL, DSPE-PEG2000, DSPC, DMG-PEG2000 and the like, and fully dissolving each lipid in absolute ethanol in a proper container for standby.

The lipids were mixed uniformly in the molar ratio shown in the following table, and nucleic acid (mRNA or DNA) was prepared as an organic phase in an aqueous solution (purified water as solvent) as aqueous phase ph=4.

Mixing the organic phase and the water phase in a volume ratio of 3:1, and preparing the lipid nanoparticle suspension on a microfluidic platform (e.g. PNI IGNITE). And centrifugally filtering the obtained lipid nanoparticle suspension through a 100kDa ultrafiltration centrifuge tube, purifying and concentrating, and sub-packaging the concentrated liquid.

The prepared lipid nanoparticle was measured for particle size, PDI, potential using a laser nanoparticle analyzer, and encapsulation efficiency (%) using an ultraviolet spectrophotometer in combination with a RiboGreen RNA kit, and the exemplary results are as follows.

After optimizing the preparation process, lipid nanoparticles with better physical and chemical control data can be obtained, and the formula result of II-37:DSPC: CHOL: DMG-PEG2000 is exemplified in the following table, wherein 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 the samples were transfected into cells CHO as in example 2 and protein expression levels were measured by Elisa to assess cell transfection efficiency.

Sample numbering	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

The results of cell transfection are shown in FIG. 4, in which "tri-009-BJ-LNP-21040601", "tri-009-BJ-LNP-21040602", "tri-009-BJ-LNP-21040603" are B1.351 mRNA of example 2 entrapped in the corresponding formulation described above, and "lipoMax-SPIKE MRNA" is B1.351 mRNA of example 2 entrapped in lipoMax ^TM, and "Nagative control" is empty white lipid nanoparticle without mRNA. From FIG. 4, it can be seen that the antigen protein expression was detected by ELISA detection 48 hours after the transfection of the cells with the nucleic acid-entrapped lipid nanoparticles obtained on the basis of II-37, and the cell transfection efficiency was comparable to or even better than that of commercially available lipoMax ^TM.

Examples 4II-37 and comparison of effects of commercial ionizable cationic lipid molecules MC3

MC3 is 4- (N, N-dimethylamino) butanoic acid (6Z, 9Z,28Z, 31Z) -heptanes thirty-carbon-6,9,28,31-tetralin-19-yl ester.

Lipid nanoparticles were prepared using II37 and MC3, respectively, according to the method described in example 3, in specific molar ratios of II-37:dspc: chol: DMG-PEG 2000=45:15:38.5:1.5, mc3:dspc: chol: DMG-PEG 2000=45:15:38.5:1.5, and B1.351 mRNA in example 2 was entrapped.

The physical and chemical quality control data of the prepared lipid nanoparticle are shown in the following table:

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

As can be seen from the table, under the same preparation process, the encapsulation rate of the lipid nanoparticle prepared by II-37 is as high as 90.5%, which is far higher than that of the lipid nanoparticle of MC3, and the particle size is smaller and more uniform, and the potential is higher.

The same transfection method as in example 2 is adopted to transfect cells with the prepared lipid nanoparticle, the expression condition of the protein is known, and the result is shown in fig. 5, and after the lipid nanoparticle prepared by II-37 (shown as C2 in the figure) carries mRNA to transfect cells, the protein expression amount in the cells is far higher than MC3, which indicates that the cell transfection efficiency of the lipid nanoparticle prepared by II-37 is very high.

Example 5 efficiency comparison experiments with IVT vectors of the present invention

In the embodiment, firefly Luc is taken as a reporter protein, different IVT vectors are constructed for in vitro transcription synthesis of mRNA capable of translating the Firefly Luc, and the translation efficiency of the synthesized mRNA with different sequence characteristics is compared.

The coding sequence of Firefly Luc is cloned to the multiple cloning sites of the corresponding vector by adopting a plasmid vector construction technology conventional in the art to obtain vectors with the numbers of IVT1, IVT2, IVT3 and IVT4 respectively, and then corresponding Firefly Luc mRNA samples are prepared by using an AM1344 kit according to the in vitro transcription of the vectors.

The vectors IVT 1-IVT 4 are all modified on the basis of a commercial vector psp73, the following sequences are inserted at the cleavage site XhoI/NdeI of the vector psp73, wherein UTR sequences are not added in the IVT1, the polyA tail length is 64A, the 3' UTR sequences (3 ' UTR sequences of beta globin) of 5' UTR and GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACA TTTATTTTCATTGC shown in SEQ ID NO:6 are used in the IVT2, the polyA length is 120A, the 5' UTR sequences shown in SEQ ID NO:6 and the 3' UTR sequences shown in SEQ ID NO:7 are used in the IVT3, the polyA length is 120A, the 5' UTR shown in SEQ ID NO:6 and the 3' UTR sequences shown in 2 tandem repeats in SEQ ID NO:7 are used in the IVT4, and the polyA length is 120A. A multiple cloning site comprising the common cleavage sites HindIII and EcoRI is inserted between the sequences of the above 5'UTR and 3' UTR, and the coding sequence of Firefly Luc is cloned into the multiple cloning sites HindIII and EcoRI. All vectors were constructed by the company Jinsri using the method of gene synthesis.

Each Firefly Luc mRNA sample was transfected into CHO cells using Lipofectamine2000 (cat# 11668030, available from Semer femto, inc.) as a transfection reagent and luciferase was detected using the Dual-Lumi ^TM double luciferase reporter assay kit (at#RG088S, available from Shanghai Biyunshii Biotechnology Co., ltd.). The DNA of Firefly Luc was transferred into psicheck plasmid as a positive control (psicheck plasmid, cat#60908-6151, available from Beijing Tian Enzem Gene technologies Co., ltd.). Specifically, the method comprises the steps of seeding CHO cells into 96-well plates on the first day, culturing 1.5X10- ⁴ cells per well with F12K+10% FBS overnight, replacing the culture medium with serum-free F12K culture medium before the second day, transfecting mRNA or DNA into CHO cells with Lipofectamine2000 on the second day, culturing overnight with 100ng of nucleic acid per well and 0.3 μl of liposome per well total volume, replacing the serum-free culture medium with complete culture medium (F12K+10% FBS) on the third day, culturing for 24 hours, and detecting Firefly Luc fluorescence on the fourth day (48 hours after transfection).

The results are shown in FIG. 6. In the figure, "DNA" is a positive control (psicheck plasmid of DNA carrying Firefly Luc), "IVT1-Luc", "IVT2-Luc", "IVT3-Luc", "IVT4-Luc" represent the corresponding Firefly Luc mRNA transcribed in vitro from the vectors of IVT1, IVT2, IVT3 and IVT4, respectively, "Nagative control" is a negative control. As can be seen from FIG. 6, the protein expression level of IVT4-Luc is far higher than that of the other three mRNAs by 2-3 times under the same transfection level of the mRNAs, which indicates that the stability of IVT4-Luc is good and the translation efficiency is high.

EXAMPLE 6 determination of immunogenicity of protein mutants

BALB/c mice were used to evaluate the production of S protein mutants induced binding and neutralizing antibodies 6 week old female BALB/c mice were immunized for 2 weeks at intervals of primary and secondary immunization, and blood was collected 14 days after immunization. ELISA method detects the expression of the binding antibody against the S protein mutant, and chemiluminescence detects the neutralizing antibody titer against the S protein mutant.

ELISA method for detecting the conjugated antibody by coating commercial S protein on ELISA plate to capture conjugated antibody against S protein mutant in immune mouse plasma, and detecting absorbance with biotin labeled detecting antibody. Chemiluminescence detection of neutralizing antibody titres against S protein mutants plasma from mice after immunization was neutralized with SPIKE lentivirus carrying a luciferase reporter gene (well-Gekko; trade name: SRAS-CoV-2 pseudovirus (B.1.351) -LUC; trade name: DZPSC-L-0; batch: K05202102) to infect 293T cells highly expressing ACE-2 (well-Gekko; trade name: "YJ1B09" hACE2-293T cell lines; trade name: YJ293T-01; batch: A23202001) plasma neutralizing antibody titres were assessed by chemiluminescence (Bright-Lumi II firefly luciferase reporter gene detection kit; trade name: biyun; trade name: RG 052M).

The control group consisted of 9 mice, each injected subcutaneously with 2ug of protein. Wherein 3 injected proteins were trimeric purified of S protein translated from B1.351 mRNA of example 2, 3 injected proteins were trimeric of wild type S protein, 3 injected were blank lipid nanoparticles, and the lipid formulation of the lipid nanoparticles was tri-009-BJ-LNP-21040602 of example 3.

The experimental group consisted of 18 mice injected subcutaneously with lipid nanoparticles of mRNA, wherein No. 1-6 injected with lipid nanoparticles of 0.2. Mu.g of mRNA (LNP), no. 7-12 injected with lipid nanoparticles of 1. Mu.g of mRNA (LNP), no. 13-18 injected with lipid nanoparticles of 5. Mu.g of mRNA (LNP), wherein mRNA was B1.351 mRNA of example 2, and the formulation of lipid nanoparticles was tri-009-BJ-LNP-21040602 of example 3.

The levels of bound and neutralizing antibodies in mice after primary and secondary immunization are shown in FIGS. 7-8.

As can be seen from the results of a, B and c in FIG. 7, both the S protein trimer translated from the B1.351 mRNA in example 2 and the wild-type S protein trimer induced in mice, the antibodies against the S protein were raised to a higher concentration in the experimental mice after the second immunization, and the concentration of the antibodies remained high after 8 weeks of the second immunization, and the concentration of the antibodies remained about 2.2. Mu.g/ml after the second immunization and about 1.6. Mu.g/ml after 8 weeks of the second immunization, respectively.

As can be seen from d, e, f in FIG. 8, mice after the secondary immunization of LNP-coated mRNA preparation, even in the low dose (0.2. Mu.g) injection group, induced in the mice binding antibodies against S protein, which were calculated to be at about 0.1-0.3. Mu.g/ml. As can be seen from g, h, i in FIG. 8, mice after secondary immunization with LNP-coated mRNA preparation induced better neutralizing antibodies with GMT values 78.69,21.9 and 72.19, respectively.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

SEQUENCE LISTING

<110> Beijing Qihen 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> Domain aiding in trimer formation

<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> Artificial sequence

<400> 4

atgttcgtgt tcctggtgct gcttcccctg gtctctagcc agtgcgtgaa cttcacgacc 60

cggacccaac tgccccccgc gtacacaaac tccttcacca gaggcgtgta ctaccctgac 120

aaggtgttcc gcagcagcgt gctgcacagc acccaggacc tgttcctccc attcttcagc 180

aacgtgacct ggttccacgc catccacgtg tccggcacca atggaacaaa gagatttgcg 240

aaccccgtgc tacctttcaa cgacggcgtg tacttcgcct ccaccgagaa gagcaacatc 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 360

aauaaugcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgacccuuuc 420

cugggcgucu acuaccacaa gaacaacaag aguuggaugg aaagcgaguu cagaguguac 480

ucuucugcua acaacugcac cuucgaguac gugucccagc cuuuccugau ggaccuggaa 540

ggcaagcagg ggaacuucaa gaaccugcgg gaguucgugu ucaagaacau cgacggguau 600

uucaagaucu acuccaagca cacaccuauc aaucugguga gaggccugcc ccagggcuuc 660

agcgcccugg aaccucuggu cgaccugcca aucggcauca acaucacccg guuccaaaca 720

cugcauauca gcuaccugac accuggcgau agcuccuccg gcuggaccgc cggcgcugcc 780

gcuuauuacg ucggcuaccu gcagccuaga acguuccugc ugaaguacaa cgagaacggc 840

accaucaccg acgccgucga cugcgcccug gacccccucu ccgagacaaa augcacccug 900

aagagcuuca cuguugaaaa gggcaucuac cagaccagca acuuuagagu gcagccuaca 960

gagucuaucg ugagauuccc uaacauuacc aaccuguguc cuuuuggaga aguguucaac 1020

gccacaagau ucgcuucugu guaugccugg aaccggaaga gaaucucgaa 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 gaaacuacaa cuaccuguac agacuguucc ggaaaucuaa ccuuaagccu 1380

uuugagcggg auaucagcac cgagaucuac caagcuggcu cuacacccug caacggcgug 1440

aagggguuua auuguuacuu cccccugcag agcuacggcu uccaaccgac cuacggagug 1500

ggcuaccagc ccuaccgggu cguggugcug agcuuugagc ugcugcacgc cccugcuaca 1560

gugugcggcc ccaagaaguc uacgaaccug gugaagaaca agugugugaa uuuuaauuuc 1620

aacggacuga ccggcacagg cguccugacc gaaucuaaca agaaauuccu cccuuuccag 1680

caguucggga gagauaucgc cgacaccacc gacgccgugc gggacccuca aacacuggaa 1740

auccuggaua ucaccccuug uucuuucgga ggcguguccg ugaucacccc agguacgaac 1800

acaucuaacc agguggcugu gcuguaccag ggcgugaacu gcaccgaggu gccuguggcc 1860

auucacgccg accagcugac uccuaccugg cggguguaca gcacgggcuc caacguguuu 1920

cagaccagag cuggcugucu gaucggagcc gagcacguga acaacucuua ugagugcgau 1980

auccccaucg gcgcuggaau cugugccucc uaccagacuc aaaccaacag cccuggcagc 2040

gcuagcagcg uggccagcca gagcaucauc gccuacacca ugagccuggg agucgaaaac 2100

agcguggccu acucaaacaa cuccaucgcu aucccuacca acuucaccau cagcguaacg 2160

accgaaaucc ugcccgugag caugaccaag accagcgugg acugcacaau guacaucugc 2220

ggcgauagca cagaaugcag caaucugcua cugcaguacg guagcuuuug cacccaacug 2280

aauagagccc ugaccggcau cgccguggaa caggauaaaa acacccaaga ggucuucgcu 2340

caggugaagc agaucuacaa gacaccuccc aucaaggacu ucggaggauu caacuuuagc 2400

cagauccugc cugauccaag caaaccuagc aagcggaguc cuaucgagga ccugcuguuu 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 gcuccuccgg 840

cuggaccgcc ggcgcugccg cuuauuacgu cggcuaccug cagccuagaa cguuccugcu 900

gaaguacaac gagaacggca ccaucaccga cgccgucgac ugcgcccugg acccccucuc 960

cgagacaaaa ugcacccuga agagcuucac uguugaaaag ggcaucuacc agaccagcaa 1020

cuuuagagug cagccuacag agucuaucgu gagauucccu aacauuacca accugugucc 1080

uuuuggagaa guguucaacg ccacaagauu cgcuucugug uaugccugga accggaagag 1140

aaucucgaac ugcguggcug auuacagcgu gcuguacaac agcgcuagcu uuagcacauu 1200

uaagugcuac ggcgugagcc ccaccaagcu gaaugauuug ugcuucacaa auguguacgc 1260

cgacucuuuc gugauaagag gggacgaggu gcggcagaua gcuccaggcc agaccggcaa 1320

caucgccgau uacaauuaca agcugccuga cgacuuuacc ggauguguga ucgccuggaa 1380

cagcaacaac cuggauagca aggugggcgg aaacuacaac uaccuguaca gacuguuccg 1440

gaaaucuaac cuuaagccuu uugagcggga uaucagcacc gagaucuacc aagcuggcuc 1500

uacacccugc aacggcguga agggguuuaa uuguuacuuc ccccugcaga gcuacggcuu 1560

ccaaccgacc uacggagugg gcuaccagcc cuaccggguc guggugcuga gcuuugagcu 1620

gcugcacgcc ccugcuacag ugugcggccc caagaagucu acgaaccugg ugaagaacaa 1680

gugugugaau uuuaauuuca acggacugac cggcacaggc guccugaccg aaucuaacaa 1740

gaaauuccuc ccuuuccagc aguucgggag agauaucgcc gacaccaccg acgccgugcg 1800

ggacccucaa acacuggaaa uccuggauau caccccuugu ucuuucggag gcguguccgu 1860

gaucacccca gguacgaaca caucuaacca gguggcugug cuguaccagg gcgugaacug 1920

caccgaggug ccuguggcca uucacgccga ccagcugacu ccuaccuggc ggguguacag 1980

cacgggcucc aacguguuuc agaccagagc uggcugucug 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

cugcacaaug uacaucugcg gcgauagcac agaaugcagc aaucugcuac ugcaguacgg 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

ccugacagug cugccaccuc ugcugaccga cgagaugauc gcucaauaca ccagcgcccu 2700

ccucgccggc acgaucacca gcggcuggac auucggcgcc ggcccugccc uccagauccc 2760

uuucccuaug cagauggccu acagauucaa cggcaucggc gugacacaaa acgugcugua 2820

cgaaaaccag aagcugaucg ccaaucaguu uaauagcgcc aucgggaaga uccaggauag 2880

ccugucaucu accccuucug cccugggaaa gcugcaggac guggugaacc agaacgccca 2940

ggcccugaac acccugguga aacagcuguc uagcaacuuc ggcgcuauca gcagcgugcu 3000

gaaugauauc cugagcagac uggauccucc ugaggccgag gugcagaucg acagauugau 3060

caccggccgg cugcagagcc ugcaaaccua cguuacacag cagcugauca gagccgcuga 3120

aaucagagcc ucugccaacc uggccgccac caaaaugagc gagugcgugc ugggacagag 3180

caaaagggug gacuucugcg ggaagggcua ccaccucaug aguuuucccc agagcgcccc 3240

ccacggcgug guguuccugc acgugacaua ugucccggcc caggagaaaa acuuuacaac 3300

agccccugcc auuugccaug acggaaaggc ccacuucccu cgggaaggug uguucgugag 3360

caacggcaca cacugguucg ugacccagag aaacuucuac gagccucaaa ucaucaccac 3420

agacaacacc uucguuagug gaaauugcga cgugguuauc ggcaucguga acaacaccgu 3480

cuacgaccca cugcagccug aacuggauag cuucaaggag gaacuggaua aguauuucaa 3540

gaaccacacc ucccccgacg uggaucuggg cgacauuagc ggcaucaacg ccagcguggu 3600

gaacauccag 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 (28)

1. A lipid nanoparticle composition comprising lipid nanoparticles, wherein the lipid nanoparticles comprise lipid molecules of formula C, C, wherein each n ₃ is independent of each other, the same or different, each n ₃ is selected from an integer of 1 to 8, each m ₃ is independent of each other, the same or different, each m ₃ 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 of claim 1, wherein each n ₃ is selected from an integer of 4 to 8, and each m ₃ is selected from an integer of 4 to 8. 3. The lipid nanoparticle composition of claim 1 , wherein each n ₃ is the same as each other, and each m ₃ is the same as 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 ) _17CH2- , _CH3 ( _CH2 ) _15CH2- , _CH3 ( _CH2 ) _13CH2- , _CH3 ( _CH2 ) _11CH2- , _CH3 (CH2) _{9CH2- , CH3 ( CH2} ) _7CH2- , CH3( _CH2 ) _{7 -} CH＝CH- _{( CH2} ) _7- , _CH3 ( _CH2 ) _4CH ＝ _CHCH2CH ＝CH( _CH2 ) _7- , _{and 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 of 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 as shown in SEQ ID NO: 6. 21. The lipid nanoparticle composition of 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%.