Lipid nanoparticles and lipid nanoparticle composition

Abstract

The present application provides lipid nanoparticles comprising an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid. The lipid nanoparticles of the present application enable encapsulation and delivery of a therapeutic / prophylactic agent, safely deliver the therapeutic / prophylactic agent to a targeted position, and enable high expression, thereby exerting the effect of the therapeutic / prophylactic agent.

Description

Lipid nanoparticles and lipid nanoparticle compositions Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a lipid nanoparticle and a lipid nanoparticle composition. Background Art

[0002] The effective targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids is an ongoing medical challenge. The key to the success of gene therapy lies in whether the therapeutic drugs can be safely and effectively delivered into the target cells through the vector in vivo. Due to the relative instability of nucleic acids and the low cell permeability of such substances, the delivery of nucleic acids to cells becomes difficult. Therefore, it is necessary to develop methods and compositions to promote the delivery of therapeutic and / or preventive drugs such as nucleic acids to cells. Gene therapy vectors are divided into viral vectors and non-viral vectors. Although viral vectors are efficient delivery systems to achieve target gene transfection and therapeutic purposes, viral vectors contain immunogenic viral proteins, have limited target gene loading capacity and are expensive. As a result, lipid nanoparticles (LNPs) as non-viral vectors have received widespread attention due to their good in vitro stability, degradability in vivo, safety and reliability, and are widely used in gene therapy research for congenital and acquired genetic defects.

[0003] Lipid-containing nanoparticles, or lipid nanoparticles, liposomes, and lipid complexes have been demonstrated to be effective delivery vehicles for bioactive substances such as small molecule drugs, proteins, and nucleic acids into and / or within cells. LNPs, small vesicles formed from one or more lipid components, can effectively encapsulate and deliver a variety of nucleic acid molecules, from DNA and RNA to chromosomes and even cells. Their defined construction scheme and ease of modification with targeting ligands facilitate large-scale production. Summary of the Invention

[0004] The purpose of this application is to provide a lipid nanoparticle and a lipid nanoparticle composition.

[0005] Specifically, this application involves the following aspects:

[0006] 1. A lipid nanoparticle comprising an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid, wherein the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (35-65): (35-65): (0.5-5), wherein the ionizable lipid is a compound of formula (I), or a salt thereof or an isomer thereof,

[0007] in,

[0008] R1 is Among them, R 1a 、R 1b R2 and R3 are independently selected from H and C1-C6 alkyl;

[0009] R4, R5, and R6 are independently selected from C1-C14 alkyl groups;

[0010] X and Y are independently selected from O or S;

[0011] X1 and X2 are independently selected from C=O or O, and Y1 and Y2 are independently selected from C=O or O, provided that X1 and Y1, X2 and Y2 are not C=O or O at the same time;

[0012] m and n are independently selected from 0, 1, and 2, provided that m and n are not 0 at the same time;

[0013] o and p are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0014] q is selected from 0, 1, 2, 3, 4, 5, 6.

[0015] 2. The lipid nanoparticle according to item 1, wherein the compound of formula (I) is formula (IA),

[0016] Wherein, q is selected from 0, 1, 2, 3, 4.

[0017] 3. The lipid nanoparticle according to item 1 or 2, wherein R4 is a C10-C11 alkyl group.

[0018] 4. The lipid nanoparticle of claim 3, wherein R4 is a C10-C11 straight chain alkyl group.

[0019] 5. The lipid nanoparticle according to any one of items 1 to 4, wherein R5 and R6 are C8 alkyl groups.

[0020] 6. The lipid nanoparticle according to claim 5, wherein R5 and R6 are C8 straight chain alkyl groups.

[0021] 7. The lipid nanoparticle of any one of items 1-6, wherein o is 5 or 6.

[0022] 8. The lipid nanoparticle of any one of items 1-7, wherein p is 7 or 8.

[0023] 9. The lipid nanoparticle according to any one of items 1 to 8, wherein

[0024] o is 5, R4 is a C11 straight chain alkyl, p is 7 and R5 and R6 are C8 straight chain alkyl or

[0025] o is 6, R4 is a C10 linear alkyl group, p is 7, and R5 and R6 are C8 linear alkyl groups.

[0026] 10. The lipid nanoparticle according to any one of items 1 to 9, wherein X1 and X2 are C═O, and Y1 and Y2 are O.

[0027] 11. The lipid nanoparticle according to any one of items 1 to 9, wherein X1 is O, Y1 is C=O, X2 is C=O, and Y2 is O.

[0028] 12. The lipid nanoparticle according to any one of items 1 to 9, wherein X1 is C=O, Y1 is O, X2 is O, and Y2 is C=O.

[0029] 13. The lipid nanoparticle according to any one of items 1 to 9, wherein X1 and X2 are O, and Y1 and Y2 are C=O.

[0030] 14. The lipid nanoparticle according to any one of items 1-9, wherein X and Y are both O.

[0031] 15. The lipid nanoparticle of any one of items 1-9, wherein n is 0 and m is 1, or n is 1 and m is 0, or n is 1 and m is 1.

[0032] 16. The lipid nanoparticle of any one of items 1-9, wherein the C1-C6 alkyl group is selected from -CH3, -CH2CH3 or -CH(CH3)2.

[0033] 17. The lipid nanoparticle according to claim 1, wherein the compound of formula (I) and its stereoisomers are selected from:

[0034] 18. The lipid nanoparticle according to any one of items 1 to 17, wherein the phospholipid is selected from one or more of the following compounds:

[0035] Dilauroyl phosphatidylcholine (DLPC),

[0036] Dimyristoylphosphatidylcholine (DMPC),

[0037] Dioleoylphosphatidylcholine (DOPC),

[0038] Dipalmitoylphosphatidylcholine (DPPC),

[0039] Distearoylphosphatidylcholine (DSPC),

[0040] Dioleoylphosphatidylcholine (DUPC),

[0041] Palmitoyloleoylphosphatidylcholine (POPC),

[0042] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0043] 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0044] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0045] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0046] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0047] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0048] 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME 16.0PE),

[0049] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0050] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0051] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0052] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0053] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0054] 1,2-Diol-sn-glycero-3-phosphate-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

[0055] 19. The lipid nanoparticle of claim 18, wherein the phospholipid is DSPC.

[0056] 20. The lipid nanoparticle according to items 1-19, wherein the structural lipid is selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol.

[0057] 21. A lipid nanoparticle according to claim 20, wherein the structural lipid is cholesterol.

[0058] 22. The lipid nanoparticle of any one of items 1-21, wherein the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

[0059] 23. The lipid nanoparticle of claim 22, wherein the PEG lipid is DMG-PEG2000.

[0060] 24. The lipid nanoparticle according to any one of items 1 to 23, wherein the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (40-50):(35-65):(1-3).

[0061] 25. The lipid nanoparticle according to any one of items 1 to 23, wherein the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (50-65):(35-65):(1-3).

[0062] 26. The lipid nanoparticles according to claim 24, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(30-50):(1-3).

[0063] 27. The lipid nanoparticles according to claim 26, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(10-15):(35-45):(1.5-2.5).

[0064] 28. The lipid nanoparticles according to claim 25, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(30-50):(1-3).

[0065] 29. The lipid nanoparticles according to claim 28, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(10-15):(35-45):(1.5-2.5).

[0066] 30. The lipid nanoparticle of items 1-29, wherein

[0067] The content of ionizable lipids is 35 mol%-65 mol%,

[0068] The total content of phospholipids and the structural lipids is 35 mol%-65 mol%,

[0069] The content of PEG lipid is 0.5mol%-5mol%,

[0070] The mol% is calculated based on the total moles of lipid in the lipid nanoparticle composition.

[0071] 31. The lipid nanoparticle according to any one of items 1 to 30,

[0072] The content of ionizable lipids is 40 mol% to 50 mol%, preferably 45 mol% to 49 mol%, and more preferably 48 mol% to 49 mol%.

[0073] 32. The lipid nanoparticle according to any one of items 1 to 30,

[0074] The content of ionizable lipids is 50 mol% to 65 mol%, preferably 51 mol% to 55 mol%, and more preferably 52 mol% to 53 mol%.

[0075] 33. The lipid nanoparticle according to any one of items 1 to 32,

[0076] The total content of phospholipids and the structural lipids is 35 mol% to 65 mol%, preferably 35 mol% to 55 mol%, and more preferably 45 mol% to 50 mol%.

[0077] 34. The lipid nanoparticle according to any one of items 1 to 33,

[0078] The content of phospholipids is 5 mol% to 15 mol%, preferably 10 mol% to 15 mol%.

[0079] 35. The lipid nanoparticle according to any one of items 1 to 34,

[0080] The content of structural lipids is 30 mol% to 50 mol%, preferably 35 mol% to 45 mol%.

[0081] 36. A lipid nanoparticle composition comprising the lipid nanoparticles of any one of items 1-35 and a therapeutic and / or prophylactic agent, wherein the therapeutic and / or prophylactic agent is selected from a vaccine or a compound capable of eliciting an immune response, or a nucleic acid.

[0082] 37. The lipid nanoparticle composition of claim 36, wherein the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, or mRNA.

[0083] 38. A lipid nanoparticle composition according to claim 37, wherein the nucleic acid is mRNA.

[0084] 39. The lipid nanoparticle composition of item 37, wherein the coding sequence of the mRNA is as shown in SEQ ID NO: 12.

[0085] 40. The lipid nanoparticle composition of any one of items 36-39, wherein the therapeutic and / or prophylactic agent is mRNA and the N / P ratio of the ionizable lipid to mRNA is 3.5-5.5.

[0086] 41. A lipid nanoparticle composition according to any one of items 36-40, wherein the encapsulation efficiency of the therapeutic agent and / or prophylactic agent is ≥50%; or ≥80%; or ≥90%.

[0087] 42. A lipid nanoparticle composition according to any one of items 36-41, wherein the average particle size of the nanoparticles is 50nm-150nm, preferably 50nm-80nm.

[0088] 43. A lipid nanoparticle composition according to any one of items 36-42, wherein the dispersibility index of the nanoparticles is 0.001-0.16.

[0089] 44. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of items 36-43 and a pharmaceutically acceptable excipient or auxiliary ingredient.

[0090] 45. A method for delivering a therapeutic and / or prophylactic agent to a mammalian cell, the method comprising administering to a subject the lipid nanoparticle composition of any one of items 36-43 or the pharmaceutical composition of item 44, wherein the administration comprises contacting the cell with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or prophylactic agent to the cell.

[0091] 46. ​​A method according to claim 45, wherein the mammalian cell is in a mammal.

[0092] 47. A method according to item 45 or 46, wherein the mammal is a human.

[0093] 48. A method according to any one of items 45-47, wherein the lipid nanoparticles are administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0094] 49. A method for producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the lipid nanoparticle composition of any one of items 36-43 or the pharmaceutical composition of item 44 to deliver a therapeutic and / or prophylactic agent to the cell, wherein the therapeutic and / or prophylactic agent is mRNA encoding the polypeptide of interest, whereby the mRNA can be translated in the cell to produce the polypeptide of interest.

[0095] 50. The method of claim 49, wherein the mammalian cell is in a mammal.

[0096] 51. The method of item 49 or 50, wherein the mammal is a human.

[0097] 52. The method of any one of items 49-51, wherein the lipid nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0098] 53. A method of treating a disease or condition in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the lipid nanoparticle composition of any one of items 36-43 or the pharmaceutical composition of item 44.

[0099] 54. A method according to claim 53, wherein the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0100] 55. The method of claim 53 or 54, wherein the disease or condition is selected from an infectious disease, cancer and proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease.

[0101] 56. A method according to any one of items 53-55, wherein the mammal is a human.

[0102] 57. A method according to any one of items 53-56, wherein the lipid nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0103] 58. A method for specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, the method comprising administering to the mammal the lipid nanoparticle composition of any one of items 36-43 or the pharmaceutical composition of item 44, wherein the administration comprises contacting the mammalian organ with the nanoparticles, thereby delivering the therapeutic and / or prophylactic agent to the organ.

[0104] 59. A method according to claim 58, wherein the mammal is a human.

[0105] 60. The method of claim 58 or 59, wherein the lipid nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0106] 61. The method of any one of items 58-60, wherein the mammal is pretreated 24 hours or less prior to the contacting or administering step.

[0107] 62. The method of any one of items 58-61, wherein the mammal is pretreated about one hour prior to the contacting or administering step.

[0108] 63. The lipid nanoparticle according to any one of items 1-35, or the lipid nanoparticle composition according to any one of items 36-43, or the pharmaceutical composition according to item 44, further comprising a divalent metal cation, preferably Ca 2+ Mg 2+ 、Zn 2+ or Mn 2+ .

[0109] 64. The lipid nanoparticle or lipid nanoparticle composition or pharmaceutical composition according to item 63, wherein the molar ratio of the divalent metal cation to the total lipid in the lipid nanoparticle composition is 0.5-400:100, preferably 10-50:100 or 30-60:100 or 100-200:100 or 300-400:100.

[0110] 65. The lipid nanoparticle composition of any one of items 63-64, wherein the preparation method comprises:

[0111] A certain concentration of divalent metal cation solution is prepared with an acidic buffer, which is then used to dilute mRNA to obtain an mRNA working solution. The final concentration of mRNA in the mRNA working solution is 100-300 μg / ml, and the final concentration of the divalent metal cation solution is 0.8-20 mM. The mRNA working solution is mixed with the mixed total lipid ethanol phase at a volume ratio of 1-5:1 to prepare LNP. After mixing, the sample is diluted, concentrated, and replaced, the ethanol is removed, and the volume is fixed and the pH value is adjusted to 7-8.

[0112] 66. The method according to claim 65, wherein the acidic buffer is an acetate buffer.

[0113] 67. The method according to items 65-66, wherein the pH of the acidic buffer is about 4.0-6.0, preferably about 5.0-5.5.

[0114] 68. According to the method described in items 65-67, the final concentration of mRNA in the mRNA working solution is 200 μg / ml.

[0115] 69. According to the method described in items 65-68, the volume ratio of the mRNA working solution to the mixed total lipid ethanol phase is 2.5:1-4:1.

[0116] 70. According to the method described in items 65-69, the mRNA working solution and the mixed total lipid ethanol phase are mixed using a T mixing device.

[0117] 71. According to the method described in items 65-70, the concentration of the divalent metal cation solution in the mRNA working solution can be selected from 0.8mM, 1.6mM, 3.2mM, 6.4mM, 8mM, 12.8mM, 16mM, and 20mM.

[0118] 72. A method for preparing the lipid nanoparticle composition according to items 65-71.

[0119] The lipid nanoparticle compositions of the present application can achieve the encapsulation and delivery of therapeutic / prophylactic agents, safely delivering the therapeutic / prophylactic agents to the targeted location, achieving high expression and exerting the therapeutic / prophylactic effect. The lipid nanoparticles of the present application have the advantages of small average particle size, good dispersibility, high encapsulation efficiency, and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG1 shows the analysis results of flow cytometry in Example 2.

[0121] Figure 2-3 shows the Elispot analysis results in Example 3. DETAILED DESCRIPTION

[0122] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0123] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0124] Terms and Definitions

[0125] As used herein, the term "alkyl" refers to a group containing one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms), which is optionally substituted. The term "C1-C14 alkyl" refers to an optionally substituted straight or branched saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, the alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0126] Unless otherwise indicated, the alkyl group may be optionally substituted. The optional substituents may be selected from, but are not limited to, a halogen atom (e.g., chloro, bromo, fluoro, or iodo), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl group, -OH), an ester (e.g., -C(O)OR or -OC(O)R), an aldehyde (e.g., -C(O)H), a carbonyl (e.g., -C(O)R, or represented by C=O), an acyl halide (e.g., -C(O)X, wherein X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy group (e.g., -OR), an acetal, a phosphate, a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfite (e.g., -S(O)R), a thiophene ...

[0015] In some embodiments, the substituents are alkyl, alkyl, alkylene ... 1-6 The alkyl group can be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0127] As used herein, the term "compound" is intended to include all isomers and isotopes of the described structure. "Isotopes" refer to atoms having the same atomic number but differing in mass due to the number of neutrons in their nuclei. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present application can be prepared by conventional methods by combining with solvents or water molecules to form sols and hydrates.

[0128] As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical connection. Methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology. For example, a nanoparticle composition can be contacted with a mammalian cell placed in a mammal by a variety of routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and can involve a variety of amounts of the nanoparticle composition. In addition, the nanoparticle composition can contact more than one mammalian cell.

[0129] As used herein, the term "specific delivery" or "specific transport" refers to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) of therapeutic and / or prophylactic drugs to a target tissue of interest (e.g., mammalian liver) by nanoparticles as compared to non-target tissues. The level of delivery of nanoparticles to a specific tissue can be measured by comparing the weight of protein produced in the tissue with the weight of the tissue, comparing the therapeutic and / or prophylactic amount in the tissue with the weight of the tissue, comparing the weight of protein produced in the tissue with the total protein weight in the tissue, or comparing the therapeutic and / or prophylactic amount in the tissue with the total therapeutic and / or prophylactic amount in the tissue.

[0130] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition, relative to the total amount of therapeutic and / or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of the therapeutic and / or prophylactic agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency can be 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial encapsulation, enclosure, surrounding, or encapsulation.

[0131] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0132] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism).

[0133] As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

[0134] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0135] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. Compounds may contain one or more chiral centers and / or double bonds and, therefore, may exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis-trans isomers). This application encompasses any and all isomers of the compounds described herein. Enantiomeric and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0136] As used herein, a "lipid component" is a component of a nanoparticle composition that comprises one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, PEGylated lipids, structured lipids, or other lipids, such as phospholipids.

[0137] As used herein, "ionizable lipid" or "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0138] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid comprising a polyethylene glycol component, for example, a PEG-conjugated lipid or a PEG-modified lipid or a PEG-modified lipid.

[0139] As used herein, " phospholipid " is a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipid can comprise one or more multiple (such as double or triple bonds) bonds (such as one or more unsaturated bonds). Specific phospholipid can promote fusion with membrane. For example, cationic phospholipid can interact with one or more negatively charged phospholipids of membrane (such as cell membrane or intracellular membrane). The fusion of phospholipid and membrane can allow one or more elements containing lipid composition to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to the cell.

[0140] As used herein, "lipid nanoparticles" are compositions comprising one or more lipids, including ionizable lipids, phospholipids, structural lipids, and PEG lipids. The particle size of lipid nanoparticles is generally on the order of micrometers, nanometers, or smaller.

[0141] As used herein, a "lipid nanoparticle composition" comprises a lipid nanoparticle (LNP) and a therapeutic and / or prophylactic agent, wherein the therapeutic and / or prophylactic agent is encapsulated by the lipid nanoparticle (LNP), wherein the therapeutic and / or prophylactic agent is selected from a vaccine or a compound, nucleic acid or protein capable of eliciting an immune response.

[0142] As used herein, "administration method" may include intravenous, intramuscular, intradermal, subcutaneous or other methods of delivering the composition to the subject. Any administration method can be selected to target delivery (e.g., specific delivery) to a specific area or system of the body.

[0143] As used herein, "patient" refers to a subject who may seek or need treatment, is in need of treatment, is receiving treatment, is about to receive treatment, or is under the care of a trained professional for a particular disease. The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0144] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) that is substantially non-toxic and non-inflammatory to the patient. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compressive aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (diacid), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

[0145] In this application, for convenience, the structural formulas of the compounds described herein represent certain isomers, but this application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. In addition, the compounds represented by the structural formulas of the compounds described herein may have crystal polymorphisms. Note that any crystal form, mixture of crystal forms, or anhydrides or hydrates thereof are included within the scope of this application.

[0146] The terms "crystal polymorph," "polymorph," or "crystalline form" refer to a crystal structure in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. The recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystalline form to predominate. Crystal polymorphs of a compound can be prepared by crystallization under different conditions.

[0147] The nanoparticle compositions of the present application may also include salts of one or more compounds. The salts may be pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glucoheptonate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmitate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate, and the like.

[0148] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharmaceutically acceptable salts of the present application include, for example, conventional non-toxic salts of the parent compound formed by non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing an alkaline or acidic part by conventional chemical methods. Typically, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent or in a mixture of the two. Typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0149] As used herein, the "polydispersity index" is a ratio that describes the uniformity of the particle size distribution of a system. Small values, such as less than 0.3, indicate a narrow particle size distribution.

[0150] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which can be produced naturally (eg, isolated or purified) or synthetically.

[0151] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, the RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. The RNA may have a nucleotide sequence that encodes a polypeptide of interest. For example, the RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, in vivo translation of the mRNA inside a mammalian cell, may produce the encoded polypeptide. The RNA may be selected from a non-limiting group including small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0152] As used herein, a "single unit dose" is a dose of any therapeutic agent that is administered in one dose / at one time / through a single route / at a single point of contact, ie, a single time.

[0153] As used herein, a "split dose" is a division of a single unit dose or total daily dose into two or more doses.

[0154] As used herein, the "total daily dose" is the amount given or prescribed over a 24-hour period. It may be administered in a single unit dose.

[0155] As used herein, "particle size" or "average particle size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0156] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present application can be administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0157] As used herein, "target cell" refers to any one or more cells of interest. The cells can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0158] As used herein, "target tissue" refers to any one or more tissue types of interest to which therapeutic and / or prophylactic delivery will result in a desired biological and / or pharmacological effect. Examples of target tissues include specific tissues, organs, and systems or groups thereof. In specific applications, the target tissue may be the kidney, lung, spleen, vascular endothelium in the kidney (e.g., intracoronary or intrafemoral) or in a blood vessel (e.g., by intratumoral injection). "Non-target tissue" refers to any one or more tissue types to which expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In specific applications, non-target tissues may include the liver and spleen.

[0159] The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents are also referred to as "active agents" or "active ingredients." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0160] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a nucleic acid, a drug, a composition, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) to be delivered that is sufficient when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0161] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can be performed, for example, in vitro, ex vivo, or in vivo.

[0162] As used herein, the term "treat" refers to partially or completely alleviating, relieving, ameliorating, resolving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment may be performed on subjects who do not exhibit the disease, disorder, and / or condition and / or on subjects who only exhibit early signs of the disease, disorder, and / or condition in order to reduce the risk of developing a pathological condition associated with the disease, disorder, and / or condition.

[0163] As used herein, "mol%" is calculated based on the total molar number of lipids in the lipid nanoparticles, that is, the molar percentage of the lipid components in the lipid nanoparticles to the total lipids present in the lipid nanoparticles.

[0164] As used herein, total lipids refer to all lipids in lipid nanoparticles. Specifically, total lipids may be composed of ionizable lipids, phospholipids, structural lipids, and PEG lipids.

[0165] As used herein, "coding sequence" may refer to a ribonucleotide sequence in a mature mRNA that can be translated into a protein, or may refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence that serves as a template for transcribing the ribonucleotide (RNA) sequence. Furthermore, the "coding sequence" of the present application may further include polynucleotide sequences encoding functional nucleic acids, such as miRNA, shRNA, dsRNA, and the like.

[0166] As used herein, "N:P ratio" or "N / P ratio" is the molar ratio of ionizable nitrogen atoms in the lipid to phosphate groups in the RNA.

[0167] The present invention provides a lipid nanoparticle comprising ionizable lipids, phospholipids, structural lipids and PEG lipids.

[0168] The ionizable lipid herein may be a compound of formula (I), or a salt or an isomer thereof,

[0169] in,

[0170] R1 is Among them, R 1a 、R 1b R2 and R3 are independently selected from H and C1-C6 alkyl;

[0171] R4, R5, and R6 are independently selected from C1-C14 alkyl groups;

[0172] X and Y are independently selected from O or S;

[0173] X1 and X2 are independently selected from C=O or O, and Y1 and Y2 are independently selected from C=O or O, provided that X1 and Y1, X2 and Y2 are not C=O or O at the same time;

[0174] m and n are independently selected from 0, 1, and 2, provided that m and n are not 0 at the same time;

[0175] o and p are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0176] q is selected from 0, 1, 2, 3, 4, 5, 6.

[0177] In a specific embodiment, the compound of formula (I) is formula (IA),

[0178] Wherein, q is selected from 0, 1, 2, 3, 4.

[0179] In a specific embodiment, in the above formula (I) or (IA), R4 is a C10-C11 alkyl group, for example, R4 is a C10 alkyl group or a C11 alkyl group. In a specific embodiment, in the above formula (I) or (IA), R4 is a C10-C11 straight-chain alkyl group, for example, R4 is a C10 straight-chain alkyl group or a C11 straight-chain alkyl group.

[0180] In a specific embodiment, in the above formula (I) or (IA), R5 and R6 are C8 alkyl. In a specific embodiment, in the above formula (I) or (IA), R5 and R6 are C8 straight-chain alkyl.

[0181] In a specific embodiment, in the above formula (I) or (IA), o is 5 or 6.

[0182] In a specific embodiment, in the above formula (I) or (IA), p is 7 or 8.

[0183] In a specific embodiment, in the above formula (I) or (IA), o is 5, R4 is a C11 linear alkyl group, p is 7, and R5 and R6 are C8 linear alkyl groups.

[0184] In a specific embodiment, in the above formula (I) or (IA), o is 6, R4 is a C10 straight-chain alkyl group, p is 7, and R5 and R6 are C8 straight-chain alkyl groups.

[0185] In a specific embodiment, in the above formula (I) or (IA), X1 and X2 are C═O, and Y1 and Y2 are O.

[0186] In a specific embodiment, in the above formula (I) or (IA), X1 is O, Y1 is C=O, X2 is C=O, and Y2 is O.

[0187] In a specific embodiment, in the above formula (I) or (IA), X1 is C=O, Y1 is O, X2 is O, and Y2 is C=O.

[0188] In a specific embodiment, in the above formula (I) or (IA), X1 and X2 are O, and Y1 and Y2 are C=O.

[0189] In a specific embodiment, in the above formula (I) or (IA), X and Y are both O.

[0190] In one specific embodiment, in the above formula (I) or (IA), n is 0 and m is 1. In one specific embodiment, in the above formula (I) or (IA), n is 1 and m is 0. In one specific embodiment, in the above formula (I) or (IA), n is 1 and m is 1.

[0191] In a specific embodiment, R 1a 、R 1b Selected from -CH3, -CH2CH3 or -CH(CH3)2.

[0192] In a specific embodiment, the compound of formula (I) is formula (IA),

[0193] Wherein, q is selected from 2, 3, R 1a 、R 1b Selected from -CH3,

[0194] R2 is H, R3 is H,

[0195] n is 1, m is 0,

[0196] X and Y are both 0,

[0197] o is 5 or 6,

[0198] p is 7,

[0199] X1 and X2 are C=O, Y1 and Y2 are O, or X1 is O, Y1 is C=O, X2 is C=O, Y2 is O, and R4 is a C10 straight chain alkyl or a C11 straight chain alkyl,

[0200] R5 and R6 are C8 straight chain alkyl groups.

[0201] In a specific embodiment, the C1-C6 alkyl group is selected from -CH3, -CH2CH3 or -CH(CH3)2.

[0202] In the lipid nanoparticles and lipid nanoparticle compositions of the present application, the ionizable lipids may further include, for example, one or more of the following: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2 , 2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[ 1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morpholinopropane (DLin-MA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morpholinopropane (DLin-MA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-DAC), 1,2-dilinoleylcarbamoyloxy-3-dimethylamino ... 2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazin-o)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylamino propane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 , 2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), dioctadecylaminoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane -3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',1-2'-octadienyloxy) propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), MC3, SM-102, ALC-0315.

[0203] In the present application, the phospholipids may be selected from one or more of the following compounds:

[0204] Dilauroyl phosphatidylcholine (DLPC),

[0205] Dimyristoylphosphatidylcholine (DMPC),

[0206] Dioleoylphosphatidylcholine (DOPC),

[0207] Dipalmitoylphosphatidylcholine (DPPC),

[0208] Distearoylphosphatidylcholine (DSPC),

[0209] Dioleoylphosphatidylcholine (DUPC),

[0210] Palmitoyloleoylphosphatidylcholine (POPC),

[0211] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0212] 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0213] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0214] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0215] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0216] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0217] 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME 16.0PE),

[0218] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0219] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0220] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0221] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0222] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0223] 1,2-Diol-sn-glycero-3-phosphate-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

[0224] In a specific embodiment, the phospholipid is DSPC.

[0225] In a specific embodiment, the phospholipid is DOPE.

[0226] In a specific embodiment, the phospholipid is a combination of DSPC and DOPE.

[0227] In the present application, the structured lipid can be selected from the group consisting of, but not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0228] In the present application, the PEG lipid is alternatively referred to as a PEGylated lipid. The PEG lipid is a lipid modified with polyethylene glycol. The PEG lipid can be selected from the non-limiting group of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

[0229] In a specific embodiment, the PEG lipid is DMG-PEG2000.

[0230] In some embodiments of the present application, in the lipid nanoparticles, the content of the ionizable lipid is 35 mol%-65 mol%, for example, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5m ol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol%, 48. 9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54.5m ol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 6 0 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.5 mol%, 65 mol%.

[0231] In some embodiments of the present application, the content of the sum of the phospholipids and the structural lipids in the lipid nanoparticles may be 35 mol%-65 mol%, for example, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol 2mol%, 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol% , 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mo l%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54. 5mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 60mol%, 60.5mol%, 61mol%, 61.5mol%, 62mol%, 62.5mol%, 63mol%, 63.5mol%, 64mol%, 64.5mol%, 65mol%.

[0232] In some embodiments of the present application, in the lipid nanoparticles, the phospholipid content can be 5 mol%-15 mol%, for example, it can be 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, 15 mol%; for example, it can be 10 mol%-15 mol%.

[0233] In some embodiments of the present application, in the lipid nanoparticles, the structural lipid content may be 30 mol%-50 mol%, for example, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 42.5 mol%, 43 mol%, 43.5 mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7m ol%, 48.8mol%, 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%; for example, it can be 35mol%-45mol%.

[0234] In some embodiments of the present application, the content of the PEG lipid in the lipid nanoparticles is 0.5 mol%-5 mol%, for example, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%. l%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8mol%, 2.9mol%, 3mol%, 3.1mol%, 3.2mol%, 3.3mol%, 3.4mol%, 3.5mol%, 3.6mol%, 3.7mo 1%, 3.8mol%, 3.9mol%, 4mol%, 4.1mol%, 4.2mol%, 4.3mol%, 4.4mol%, 4.5mol%, 4.6mol%, 4.7mol%, 4.8mol%, 4.9mol%, 5mol%.

[0235] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids, the content of ionizable lipids is 35 mol%-65 mol%, the content of the sum of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%.

[0236] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids, and the component ratios are: the content of ionizable lipids is 50 mol%, the content of the sum of phospholipids and cholesterol is 48.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0237] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%-50 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

[0238] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 48 mol%-49 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

[0239] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 42.5 mol%, and the content of PEG lipids is 2.5 mol%.

[0240] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0241] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 48.5%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 1.5 mol%.

[0242] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 44.3 mol%, the content of phospholipids is 9.3 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 1.5 mol%.

[0243] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 50 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 43 mol%, and the content of PEG lipids is 2.1 mol%.

[0244] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 40 mol%, the content of phospholipids is 12.5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2.5 mol%.

[0245] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 46 mol%, the content of phospholipids is 11 mol%, the content of cholesterol is 41 mol%, and the content of PEG lipids is 2 mol%.

[0246] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 47.5 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2.5 mol%.

[0247] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 13 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2 mol%.

[0248] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 12.5 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 2.5 mol%.

[0249] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 50 mol%, the content of phospholipids is 8 mol%, the content of cholesterol is 39.5 mol%, and the content of PEG lipids is 2.5 mol%.

[0250] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 42.3 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 40.3 mol%, and the content of PEG lipids is 2.5 mol%.

[0251] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 46 mol%, the content of phospholipids is 11 mol%, the content of cholesterol is 41 mol%, and the content of PEG lipids is 2 mol%.

[0252] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 47.5 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 2.5 mol%.

[0253] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 48.5 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 1.5 mol%.

[0254] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 50 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0255] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0256] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipids is 48 mol%, the content of phospholipids is 10.3 mol%, the content of cholesterol is 39.6 mol%, and the content of PEG lipids is 2.1 mol%.

[0257] In a specific embodiment, the lipid nanoparticles contain ionizable lipids, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the component ratio is: the ionizable lipid content is 50 mol%, the phospholipid content is 10 mol%, the cholesterol content is 38.5 mol%, and the PEG lipid content is 1.5 mol%.

[0258] In some embodiments of the present application, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is 35-65:35-65:0.5-5.

[0259] In the lipid nanoparticles of the present application, the molar ratio of the sum of the ionizable lipid, the phospholipid and the structural lipid, and the PEG lipid is (35-65): (35-65): (0.5-5), and 35-65 can take any value between 35-65, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39 .5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 57 6.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 0.5-5 can take any value between 0.5-5, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.

[0260] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (40-50): (35-65): (1-3).

[0261] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (50-65):(35-65):(1-3).

[0262] In a specific embodiment, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is not 50:48.5:1.5.

[0263] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-65):(5-15):(30-50):(1-3).

[0264] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-65):(10-15):(35-45):(1.5-2.5).

[0265] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(30-50):(1-3). Wherein, for the molar ratio (40-50): (5-15): (30-50): (1-3), 40-50 can take any value between 40-50, for example, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 5-15 can take any value between 5-15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can take any value between 30-50, for example, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46 , 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1-3, for example, it can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0266] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(35-45):(1.5-2.5). Wherein, for the molar ratio (40-50): (5-15): (35-45): (1.5-2.5), 40-50 can take any value between 40-50, for example, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 5-15 can take any value between 5-15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can take any value between 35-45, for example, it can be 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can take any value between 1.5-2.5, for example, it can be 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0267] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(30-50):(1-3). In the molar ratio (50-65):(5-15):(30-50):(1-3), 50-65 can take any value between 50-65, for example, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60. 5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can take any value between 5-15, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can take any value between 5-15, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; Take any value between 30-50, for example, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45 , 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1-3, for example, it can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0268] In a specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is not 50:10:38.5:1.5.

[0269] In a specific embodiment, in the lipid nanoparticles, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(35-45):(1.5-2.5). Wherein, for the molar ratio (50-65): (5-15): (35-45): (1.5-2.5), 50-65 can take any value between 50-65, for example, it can be 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can take any value between 5-15, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can take any value between 35-45, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can take any value between 1.5-2.5, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0270] In a specific embodiment, the lipid nanoparticles comprise Compound 2, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 2, the sum of DSPC and cholesterol, and DMG-PEG2000 is (35-65): (35-65): (0.5-5).

[0271] In a specific embodiment, the lipid nanoparticles comprise Compound 2, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 2, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0272] In a specific embodiment, the lipid nanoparticles comprise Compound 2, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 2, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (10-15): (35-45): (1.5-2.5).

[0273] In a specific embodiment, the lipid nanoparticles comprise Compound 2, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 2, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0274] In a specific embodiment, the lipid nanoparticles comprise Compound 2, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 2, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0275] In a specific embodiment, the lipid nanoparticles contain compound 2, DSPC, cholesterol, and DMG-PEG2000, the content of compound 2 is 35 mol%-65 mol%, the content of the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0276] In a specific embodiment, the lipid nanoparticles contain compound 2, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 2 in the lipid nanoparticles is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0277] In a specific embodiment, the lipid nanoparticles comprise compound 2, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 2 in the lipid nanoparticles is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0278] In a specific embodiment, the lipid nanoparticles comprise compound 4, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 4, the sum of DSPC and cholesterol, and DMG-PEG2000 is (35-65): (35-65): (0.5-5).

[0279] In a specific embodiment, the lipid nanoparticles comprise compound 4, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 4, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (5-15): (30-50): (1-3).

[0280] In a specific embodiment, the lipid nanoparticles comprise compound 4, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 4, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (10-15): (35-45): (1.5-2.5).

[0281] In a specific embodiment, the lipid nanoparticles comprise compound 4, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 4, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0282] In a specific embodiment, the lipid nanoparticles comprise compound 4, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 4, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0283] In a specific embodiment, the lipid nanoparticles contain compound 4, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticle composition, the content of compound 4 is 35 mol%-65 mol%, the content of the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0284] In a specific embodiment, the lipid nanoparticles contain compound 4, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticle composition, the content of compound 4 is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0285] In a specific embodiment, the lipid nanoparticles comprise compound 4, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticle composition, the content of compound 4 is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0286] In a specific embodiment, the lipid nanoparticles comprise Compound 5, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 5, the sum of DSPC and cholesterol, and DMG-PEG2000 is (35-65): (35-65): (0.5-5).

[0287] In a specific embodiment, the lipid nanoparticles comprise Compound 5, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 5, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0288] In a specific embodiment, the lipid nanoparticles comprise Compound 5, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 5, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (10-15): (35-45): (1.5-2.5).

[0289] In a specific embodiment, the lipid nanoparticles comprise Compound 5, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 5, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0290] In a specific embodiment, the lipid nanoparticles comprise Compound 5, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of Compound 5, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0291] In a specific embodiment, the lipid nanoparticles contain compound 5, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticles, the content of compound 5 is 35 mol%-65 mol%, the content of the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0292] In a specific embodiment, the lipid nanoparticles contain compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 in the lipid nanoparticles is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0293] In a specific embodiment, the lipid nanoparticles contain compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 in the lipid nanoparticles is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0294] In a specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of the sum of compound 5A, DSPC and cholesterol, and DMG-PEG2000 is (35-65): (35-65): (0.5-5).

[0295] In a specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (5-15): (30-50): (1-3).

[0296] In a specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (10-15): (35-45): (1.5-2.5).

[0297] In a specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0298] In a specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (51-60): (10-15): (35-45): (1.5-2.5).

[0299] In a specific embodiment, the lipid nanoparticles contain compound 5A, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticles, the content of compound 5A is 35 mol%-65 mol%, the content of the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0300] In a specific embodiment, the lipid nanoparticles contain compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A in the lipid nanoparticles is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0301] In a specific embodiment, the lipid nanoparticles contain compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A in the lipid nanoparticles is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0302] In a specific embodiment, the lipid nanoparticles comprise compound 8, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 8, the sum of DSPC and cholesterol, and DMG-PEG2000 is (35-65): (35-65): (0.5-5).

[0303] In a specific embodiment, the lipid nanoparticles comprise compound 8, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 8, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0304] In a specific embodiment, the lipid nanoparticles comprise compound 8, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 8, DSPC, cholesterol, and DMG-PEG2000 is (40-49): (10-15): (35-45): (1.5-2.5).

[0305] In a specific embodiment, the lipid nanoparticles comprise compound 8, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 8, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0306] In a specific embodiment, the lipid nanoparticles comprise compound 8, DSPC, cholesterol, and DMG-PEG2000, and the molar ratio of compound 8, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0307] In a specific embodiment, in the lipid nanoparticles, the lipid nanoparticle composition comprises compound 8, DSPC, cholesterol, and DMG-PEG2000, the content of compound 8 is 35 mol%-65 mol%, the content of the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0308] In a specific embodiment, the lipid nanoparticles contain compound 8, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticles, the content of compound 8 is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0309] In a specific embodiment, the lipid nanoparticles contain compound 8, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 8 in the lipid nanoparticles is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0310] In a specific embodiment, in the lipid nanoparticles, the lipid nanoparticle composition comprises compound 13, DSPC, cholesterol, and DMG-PEG2000, the content of compound 13 is 35 mol%-65 mol%, the content of the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0311] In a specific embodiment, the lipid nanoparticles contain compound 13, DSPC, cholesterol, and DMG-PEG2000. In the lipid nanoparticles, the content of compound 13 is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0312] In a specific embodiment, the lipid nanoparticles contain compound 13, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 13 in the lipid nanoparticles is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0313] In addition to those described in the preceding sections, the lipid nanoparticles or lipid nanoparticle compositions of the present application may further comprise one or more components. For example, the lipid nanoparticles or lipid nanoparticle compositions may comprise one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0314] The lipid nanoparticles or lipid nanoparticle compositions may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers or other components. The permeability enhancer molecules may be molecules described in U.S. Patent Application 2005 / 0222064. The carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0315] The lipid nanoparticles or lipid nanoparticle compositions may further comprise one or more polymers, which may be biodegradable and / or biocompatible. The polymers may be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrs, and polyarylates. For example, the polymer may include polycaprolactone (PCL), ethylene vinyl acetate polymer (EVA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), poly(lactic acid-glycolic acid) (PLGA), poly(L-lactic acid-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-caprolactone-glycolide), poly(D,L-lactide-PEO-co-D,L-lactide), poly(D,L-lactide) lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, polyhydroxy acids, polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene; polyalkylene glycols such as poly(ethylene glycol) (PEG); polyalkylene oxides (PEO); polyalkylene terephthalates such as polyethylene terephthalate; polyvinyl alcohol (PVA); polyvinyl ethers, polyvinyl esters such as poly (vinyl acetate), polyvinyl halides such as polyvinyl chloride (PVC), polyvinyl pyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic polymers such as poly(methyl (meth))acrylate) (PMMA), polyethyl (meth)acrylate, polybutyl (meth)acrylate, polyisobutyl (meth)acrylate, polyhexyl (meth)acrylate, polyisodecyl (meth)acrylate, polylauryl (meth)acrylate Esters, poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, polyethylenediamine, poly(orthoesters), poly(butyric acid), poly(valeric acid), poly(lactide-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ) and polyglycerol.

[0316] The lipid nanoparticles or lipid nanoparticle compositions may further comprise one or more surface-altering agents, which may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, scleroderma, bromcaprolactone, carboxytine, eprozinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). The surface-altering agent may be disposed within the nanoparticle and / or on the surface of the nanoparticle composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0317] In addition to the above components, the lipid nanoparticles or lipid nanoparticle compositions can include any substance useful in pharmaceutical compositions. For example, the nanoparticle composition can include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending agents, granulation aids, disintegrants, fillers, glidants, liquid carriers, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other substances. Excipients such as waxes, butters, colorants, coatings, flavorings, and fragrances may also be included. Pharmaceutically acceptable excipients are well known in the art.

[0318] Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, dibasic calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, sulfamate buffer, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and / or combinations thereof. The lubricant may be selected from the group consisting of magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium laurate, sulfates, sodium lauryl sulfate, and combinations thereof.

[0319] The present application also provides a lipid nanoparticle, which comprises any one of the above-mentioned lipid nanoparticle compositions and a therapeutic agent and / or a preventive agent, wherein the therapeutic agent and / or the preventive agent is selected from a vaccine or a compound, nucleic acid or protein capable of inducing an immune response.

[0320] The therapeutic and / or prophylactic agents include biologically active substances and may alternatively be referred to as "active agents." A therapeutic and / or prophylactic agent can be a substance that, once delivered to a cell or organ, produces a desired change in a cell, organ, or other body tissue or system. Such substances can be used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition. Examples of drugs that can be used in the nanoparticle compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine), vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs (e.g., methotrexate, purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and laxatives), and sedatives. betadalafil), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonversion agents (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, and naftifine), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and imaging agents.

[0321] In some embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticle compositions described herein include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0322] In some embodiments, therapeutic agent is polynucleotide or nucleic acid (for example, ribonucleic acid or deoxyribonucleic acid).Term " polynucleotide ", in its broadest sense, includes any compound and / or substance that is combined or can be combined into oligonucleotide chain.Exemplary polynucleotide used according to the present application includes but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), its hybrid, RNAi inducer, RN-Ai reagent, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, RNA that induces triple helix formation, aptamer, carrier etc. one or more.In some embodiments, therapeutic and / or preventive is RNA.The RNA that can be used for compositions and methods described herein can be selected from but is not limited to short polymer, antigametocyte, antisense, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and its mixture.In certain embodiments, RNA is mRNA.

[0323] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any target polypeptide, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can have any size and can have any secondary structure or activity. In some embodiments, when expressed in a cell, the polypeptide encoded by the mRNA can have a therapeutic effect.

[0324] In some embodiments, some or all of the uridine in the mRNA molecule is 1-methyl-pseudouridine. In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the 5' cap structure is type O, type I, and type II. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a polyA tail. In some embodiments, the polyA tail sequence comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 adenylate residues. In some embodiments, the polyA tail comprises at most 500, at most 400, at most 300, at most 200, at most 150, at most 140, at most 130, at most 120, at most 110, at most 100, at most 90, at most 80, at most 70, at most 60 adenylate nucleotides (A), in particular about 120 A's.

[0325] In a specific embodiment, the coding sequence of the mRNA of the present invention is as shown in SEQ ID NO: 12 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to SEQ ID NO: 12.

[0326] In a specific embodiment, the coding sequence of the mRNA of the present invention comprises the sequence shown in SEQ ID NO: 10 or comprises a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to SEQ ID NO: 10.

[0327] In a specific embodiment, the coding sequence of the mRNA of the present invention is the sequence shown in SEQ ID NO: 10 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to SEQ ID NO: 10.

[0328] In a specific embodiment, the coding sequence of the mRNA of the present invention comprises the sequence shown in SEQ ID NO: 11 or comprises a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to SEQ ID NO: 11.

[0329] In a specific embodiment, the coding sequence of the mRNA of the present invention is the sequence shown in SEQ ID NO: 11 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to SEQ ID NO: 11.

[0330] In a specific embodiment, the mRNA of the present invention encodes a protein comprising the sequence shown in SEQ ID NO:3 or a protein comprising a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to SEQ ID NO:3.

[0331] In a specific embodiment, the mRNA of the present invention encodes a protein having a sequence as shown in SEQ ID NO: 3 or a protein having a sequence at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 3.

[0332] In a specific embodiment, the mRNA of the present invention encodes a protein comprising the sequence shown in SEQ ID NO:4 or a protein comprising a sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO:4.

[0333] In a specific embodiment, the mRNA of the present invention encodes a protein having a sequence as shown in SEQ ID NO: 4 or a protein having a sequence at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similar to SEQ ID NO: 4.

[0334] The present invention further provides an mRNA vaccine for treating and / or preventing diseases related to HPV virus infection, which comprises mRNA encoding HPV antigens, and the mRNA is encapsulated in the lipid nanoparticles (LNPs) of the present invention.

[0335] In a specific embodiment, the encoded HPV antigen sequence comprises or is a sequence as shown in SEQ ID NO:3 or SEQ ID NO:4.

[0336] In a specific embodiment, all uridine in the mRNA molecule is 1-methyl-pseudouridine.

[0337] In a specific embodiment, the mRNA comprises a 5' cap structure, wherein the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG,

[0338] The mRNA molecule sequence is sequentially 5' cap, 5' UTR, ORF, 3' UTR, and poly A tail from the 5' end to the 3' end, wherein the 5' cap, 5' UTR, ORF, 3' UTR, and poly A tail are operably connected to each other.

[0339] In a specific embodiment, the mRNA ORF sequence encoding the HPV antigen is shown as SEQ ID NO:10 or SEQ ID NO:11.

[0340] In a specific embodiment, the encoded HPV antigen sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 4.

[0341] In a specific embodiment, the 5'UTR sequence is SEQ ID NO: 5,

[0342] In a specific embodiment, the 3'UTR sequence is SEQ ID NO: 6,

[0343] In a specific embodiment, the poly A tail sequence is SEQ ID NO: 7.

[0344] In a specific embodiment, the full-length sequence of the mRNA encoding the HPV antigen is SEQ ID NO:12.

[0345] In a specific embodiment, the lipid nanoparticles contain compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A in the lipid nanoparticles is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0346] In a specific embodiment, the lipid nanoparticles contain ionizable lipid 5A, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipid is 48.5%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipid is 1.5 mol%.

[0347] In a specific embodiment, the lipid nanoparticles contain ionizable lipid 5A, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0348] In a specific embodiment, the lipid nanoparticles contain ionizable lipid 5A, phospholipids, cholesterol, and PEG lipids. In the lipid nanoparticles, the content of ionizable lipid is 48 mol%, the content of phospholipids is 10.3 mol%, the content of cholesterol is 39.6 mol%, and the content of PEG lipid is 2.1 mol%.

[0349] In other embodiments, the therapeutic and / or preventive agent is siRNA. siRNA may be able to selectively knock down or lower the expression of a target gene. For example, siRNA can be selected to silence a gene associated with a specific disease, disorder or condition when administering a nanoparticle composition comprising siRNA to a subject in need thereof. siRNA can comprise a sequence complementary to the mRNA sequence encoding a gene of interest or protein. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0350] In some embodiments, the therapeutic and / or preventive agent is a vector or plasmid comprising shRNA or the shRNA encoding the shRNA. After a suitable construct is delivered to the nucleus, shRNA can be produced inside the target cell. Constructs and mechanisms relevant to shRNA are well known in the art.

[0351] The amount of therapeutic and / or prophylactic agent in a nanoparticle can depend on the size, composition, desired target and / or application, or other properties of the nanoparticle and the properties of the therapeutic and / or prophylactic agent. For example, the amount of RNA that can be incorporated into a nanoparticle can depend on the size, sequence, and other characteristics of the RNA.

[0352] The lipid nanoparticles or lipid nanoparticle compositions or pharmaceutical compositions of the present application may further include divalent metal cations. Specifically, the divalent metal cations may be Ca 2+ Mg 2+ 、Zn 2+ 、Mn 2+ .

[0353] In some embodiments of the present application, the molar ratio of divalent metal cations to total lipids in the lipid nanoparticle composition, lipid nanoparticle or pharmaceutical composition is 0.1-400:100, which can be 1-30:100 or 10-50:100 or 30-60:100 or 30-35:100 or 100-200:100 or 150-180:100 or 300-320:100 or 300-400:100.

[0354] In some embodiments of the present application, a divalent metal cation solution of a certain concentration is prepared with an acidic buffer, which is then used to dilute the mRNA to obtain an mRNA working solution. The final mRNA concentration in the mRNA working solution is 100-300 μg / ml, preferably 200 μg / ml, and the final concentration of the divalent metal cation solution is 0.8-20 mM. The mRNA working solution is mixed with the mixed total lipid ethanol phase at a volume ratio of 1-5:1, preferably 2.5:1-4:1, to prepare LNPs. The mixing method is preferably mixing in a T-type mixing device. After mixing, the sample is diluted, concentrated, and replaced, the ethanol is removed, and the volume is adjusted to 7-8.

[0355] Preferably, the concentration of the divalent metal cation solution in the mRNA working solution can be 0.8 mM, 1.6 mM, 3.2 mM, 6.4 mM, 8 mM, 12.8 mM, 16 mM, or 20 mM.

[0356] Preferably, the acidic buffer solution is an acetate buffer solution.

[0357] In some embodiments of the present application, the pH of the acidic buffer is 5.0-6.0, preferably 5.0-5.5;

[0358] In some embodiments of the present application, the pH of the mRNA working solution is 5.0-6.0, preferably 5.0-5.5.

[0359] In some embodiments of the present application, a divalent metal cation solution with a concentration of 0.8-20mM is prepared with an acidic buffer, and then used to dilute mRNA to obtain an mRNA aqueous phase. The final concentration of the mRNA aqueous phase is 100-300μg / ml, preferably 200μg / ml; the mRNA aqueous phase is mixed with the mixed total lipid ethanol phase in a volume ratio of 1-5:1, preferably 2.5:1-4:1, and the mixing method is preferably T mixing device mixing. After mixing, the sample is diluted, concentrated, replaced, and ethanol is removed, and the volume is fixed and the pH value is adjusted to 7-8.

[0360] The concentration of the divalent metal cation solution prepared in the acidic buffer solution can be 0.8 mM, 1.6 mM, 3.2 mM, 6.4 mM, 8 mM, 12.8 mM, 16 mM, or 20 mM.

[0361] Preferably, the acidic buffer solution is an acetate buffer solution.

[0362] In some embodiments of the present application, the pH of the acidic buffer is 5.0-6.0, preferably 5.0-5.5;

[0363] In some embodiments of the present application, the pH of the mRNA aqueous phase is 5.0-6.0, preferably 5.0-5.5.

[0364] In a specific embodiment, the therapeutic and / or prophylactic agent is mRNA, and the N / P ratio of the ionizable lipid to the mRNA in the lipid nanoparticles is 3.5-5.5, for example, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5.

[0365] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent that is encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The higher the encapsulation efficiency, the better (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after decomposition of the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0366] The lipid nanoparticles can be characterized by a variety of methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticles. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size.

[0367] In a specific embodiment, the average particle size of the lipid nanoparticles is 50 nm-150 nm.

[0368] In a specific embodiment, the average particle size of the lipid nanoparticles is 50 nm-100 nm.

[0369] In a specific embodiment, the average particle size of the lipid nanoparticles is 50 nm-80 nm.

[0370] In a specific embodiment, the average particle size of the lipid nanoparticles is 50 nm-60 nm.

[0371] The lipid nanoparticles can be relatively uniform. A polydispersity index can be used to indicate the uniformity of the nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.

[0372] In a specific embodiment, the polydispersity index of the lipid nanoparticles is 0.001-0.16, for example, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, or 0.16.

[0373] The present application also provides a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein, except that any conventional excipients or adjuvants may be incompatible with one or more components of the nanoparticles. If the excipient or adjuvant is incompatible with a component of the nanoparticle, its combination with that component may result in any undesirable biological or other deleterious effects.

[0374] Depending on the one or more lipid nanoparticles in the pharmaceutical composition of the present application, the relative amount of one or more pharmaceutically acceptable excipients and / or auxiliary ingredients will vary, depending on the identity, size and / or disease condition of the subject being treated, and further depending on the route of administration of the composition. For example, the pharmaceutical composition may contain 0.1% to 100% (wt / wt) of one or more lipid nanoparticles.

[0375] The lipid nanoparticles and / or pharmaceutical compositions can be administered to any patient or subject, including those patients or subjects who may benefit from the therapeutic effect provided by delivering a treatment and / or preventive agent to one or more specific cells, tissues, organs, or their systems or tissues (e.g., renal system). Although the description of the lipid nanoparticles and pharmaceutical compositions provided herein is primarily directed to being suitable for administration to humans, it will be appreciated by those skilled in the art that such lipid nanoparticles and pharmaceutical compositions are typically suitable for administration to any other mammal. In order to make lipid nanoparticles and pharmaceutical compositions suitable for administration to various animals, it is well known that lipid nanoparticles and pharmaceutical compositions suitable for human administration are modified, and conventionally skilled veterinary pharmacologists can design and / or carry out such modifications, including but not limited to humans, other primates, and other artificial animals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and / or rats, only through common, if any, experimental design.

[0376] The pharmaceutical compositions of the present application can be prepared by any method known in the art of pharmacology or developed in the future. Generally, such preparation methods include combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if necessary or desired, dividing, shaping and / or packaging the product into the desired single or multiple dose units.

[0377] The pharmaceutical compositions of the present application can be prepared, packaged, and / or sold in batches of single unit doses and / or multiple single unit doses. As used herein, a "unit dose" is a precise amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient portion of the dose, such as half or one-third of the dose.

[0378] Pharmaceutical compositions can be prepared in various forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and medicaments), injectable forms, solid dosage forms (e.g., capsules, tablets, tablets, etc.), liquids, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or medicaments.

[0379] In addition to the active ingredient, the liquid dosage form can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters and mixtures thereof. In addition to the inert diluent, oral compositions can also include other therapeutic and / or prophylactic agents, other agents, such as wetting agents, emulsifying agents and suspending agents, sweeteners, flavorings and / or spices. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent (e.g., alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer and / or a combination thereof).

[0380] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants, wetting agents and / or suspending agents according to known techniques. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in nontoxic parenteral acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water. Sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injections.

[0381] The injectable formulations can be sterilized by, for example, filtration through a bacteria-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions prior to dissolution or dispersion in sterile water or other sterile injectable medium.

[0382] In order to prolong the effect of the active ingredient, it is generally desired to slow down the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn depends on the crystal size and crystalline form. Alternatively, delayed absorption of the drug form for parenteral administration can be achieved by dissolving or suspending the drug in an oil carrier. The injectable drug depot form is made by forming a microcapsule matrix of the drug in a biodegradable polymer (such as polylactic acid-polyethylene glycol). Depending on the ratio of the drug to the polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Reserve injection preparations are prepared by embedding the drug in liposomes or microemulsions compatible with body tissues.

[0383] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the composition with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity to release the active ingredient.

[0384] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic), humectants (e.g., glycerol), disintegrants (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate), solubilizers (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glyceryl monostearate), absorbents (e.g., kaolin and bentonite, silicates) and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0385] Solid compositions of similar types can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifiers and may have a composition that releases the active ingredient only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of similar types can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols.

[0386] The dosage form for the topical and / or transdermal administration of compositions can include ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant and / or patch.Usually, active component is aseptically mixed with pharmaceutically acceptable excipient and / or any preservative and / or buffer that may be needed.In addition, the application has considered the use of transdermal patch, which generally has the additional advantage of providing the controlled delivery of compound to health.Such dosage form can be prepared, for example, by dissolving the compound and / or being distributed in a suitable medium.Alternatively or additionally, speed can be provided rate control membrane and / or by being dispersed in a polymer matrix and / or gel by any one of the following control.

[0387] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid formulations such as liniments, lotions, oil-in-water and / or water-in-oil emulsions such as creams, ointments and / or pastes and / or solutions and / or suspensions, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the other ingredients described herein.

[0388] Pharmaceutical compositions can be prepared, packaged and / or sold in formulations suitable for pulmonary administration via the oral cavity. Such formulations can comprise dry particles of the active ingredient. Such compositions are conveniently in dry powder form for administration using a device comprising a dry powder reservoir and / or using a self-propelled solvent / powder dispensing container (e.g., a device comprising the active ingredient dissolved and / or suspended in a low-boiling-point propellant in a sealed container). Powder compositions can comprise a solid fine powder diluent, such as sugar, and are conveniently provided in unit dose form.

[0389] Low-boiling-point propellants typically include liquid propellants having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may comprise 50% to 99.9% (wt / wt) of the composition, while the active ingredient may comprise 0.1% to 20% (wt / wt) of the composition. The propellant may further comprise other ingredients, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (whose particle size may be of the same order of magnitude as the particles comprising the active ingredient).

[0390] Pharmaceutical compositions formulated for pulmonary delivery can provide the active ingredient in the form of droplets of solution and / or suspension. Such preparations can be prepared, packaged and / or sold as aqueous and / or diluted alcohol solutions and / or suspensions, optionally sterile, containing the active ingredient, and can be conveniently administered using any atomization and / or atomizing device. Such preparations may further comprise one or more other ingredients, including but not limited to flavorings such as saccharin sodium, volatile oils, buffers, surfactants and / or preservatives such as methyl hydroxybenzoate. The average diameter of the droplets provided by this route of administration can be in the range of 1 nm to 200 nm.

[0391] The formulations described herein for pulmonary delivery can be used to deliver pharmaceutical compositions intranasally. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of 0.2 μm to 500 μm. Such a formulation is administered in the manner of snuff, i.e., by rapid inhalation through the nasal passages from a powder container held near the nose.

[0392] Preparations suitable for nasal administration can, for example, contain 0.1% (wt / wt) to 100% (wt / wt) of active ingredients, and can contain one or more other active ingredients as described herein. Pharmaceutical compositions can be prepared, packaged and / or sold in preparations suitable for buccal administration. Such preparations can, for example, be in the form of tablets and / or lozenges prepared using conventional methods, and can, for example, be 0.1% to 20% (wt / wt) of active ingredients, the remainder comprising an orally soluble and / or degradable composition, and optionally, one or more other ingredients as described herein. Alternatively, preparations suitable for oral administration can include a powder and / or atomized and / or sprayed solution and / or suspension containing the active ingredient. Such powdered, aerosolized and / or aerosolized preparations can have an average particle and / or droplet size in the range of 0.1nm to 200nm when dispersed, and can further include one or more other ingredients as described herein.

[0393] Pharmaceutical compositions can be prepared, packaged and / or sold in the form of formulations suitable for ophthalmic administration. Such formulations can, for example, be in the form of drops, including, for example, 0.1 / 1.0% (wt / wt) solutions and / or suspensions of the active ingredient in aqueous or oily liquid excipients. Such drops may further comprise a buffer, salts and / or one or more of any other additive ingredients described herein. Other formulations useful for ophthalmic administration include those comprising the active ingredient in microcrystalline form and / or liposomal formulations. Ear drops and / or eye drops are considered within the scope of the present disclosure as methods for producing polypeptides in cells.

[0394] The present application further provides a method for producing a target polypeptide in a mammalian cell. The method for producing the polypeptide comprises contacting the cell with a lipid nanoparticle containing an mRNA encoding the target polypeptide. After contacting the cell with the lipid nanoparticle, the mRNA can be taken up and translated in the cell to produce the target polypeptide.

[0395] Typically, the step of contacting mammalian cells with nanoparticles comprising mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of the nanoparticles contacted with the cells and / or the amount of the mRNA therein can depend on the type of cells or tissues being contacted, the mode of administration, the physicochemical properties of the nanoparticle compositions, and the mRNA (e.g., size, charge, and chemical composition), as well as other factors. Typically, an effective amount of nanoparticles will allow for efficient production of polypeptides in cells. Efficiency metrics may include polypeptide translation (represented by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0396] The step of contacting the nanoparticles comprising the mRNA with the cells may involve or cause transfection. The phospholipids contained in the lipid component of the nanoparticles can, for example, promote transfection and / or increase transfection efficiency by interacting and / or fusing with the cells or intracellular membranes. Transfection can allow translation of the mRNA within the cell.

[0397] In certain embodiments, the mRNA included in the nanoparticle can encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in the cell in contact with the nanoparticle. Due to genetic mutations in the coding gene or its regulatory pathway, one or more substantially absent polypeptides may be lacking. Alternatively, the recombinant polypeptide produced by mRNA translation can antagonize the activity of endogenous proteins present in the cell, on the cell surface, or secreted from the cell. Antagonistic recombinant polypeptides may be ideal to antagonize the harmful effects caused by the activity of endogenous proteins, such as the positioning caused by activity changes or mutations. In another alternative, the recombinant polypeptide produced by mRNA translation can indirectly or directly antagonize the activity of biological parts present in the cell, on the cell surface, or secreted from the cell. The biological parts of antagonism can include but are not limited to lipids (such as cholesterol), lipoproteins (such as low-density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by mRNA translation can be engineered to be located in the cell, such as in a specific compartment such as the nucleus, or can be engineered to be secreted from the cell or transported to the plasma membrane of the cell.

[0398] In some embodiments, contacting cells with nanoparticles comprising mRNA can reduce the innate immune response of cells to exogenous nucleic acids. Cells can be contacted with a first nanoparticle composition comprising a first exogenous mRNA of a first amount, wherein the first exogenous mRNA comprises a translatable region, and the level of the innate immune response of the cells to the first exogenous mRNA can be determined. Subsequently, cells can be contacted with a second composition comprising a second exogenous mRNA of a second amount, wherein the second amount is a smaller amount of the first exogenous mRNA than the first amount. Alternatively, the second composition can comprise a second exogenous mRNA of a first amount that is different from the first exogenous mRNA. The step of contacting cells with the first and second compositions can be repeated one or more times. In addition, the efficiency of polypeptide production (e.g., translation) in the cell can be optionally determined, and cells can be repeatedly contacted with the first and / or second compositions until the target protein production efficiency is reached.

[0399] The present application further provides a method for treating a disease or condition in a mammal, the method comprising administering to a mammal a therapeutically effective amount of any one of the lipid nanoparticles or pharmaceutical compositions of the present application, in particular, the lipid nanoparticles or pharmaceutical compositions can be used to treat diseases, conditions or illnesses characterized by missing or abnormal protein or polypeptide activity. For example, lipid nanoparticles or pharmaceutical compositions comprising mRNA encoding a missing or abnormal polypeptide can be administered or delivered to a cell. Subsequent translation of the mRNA can produce a polypeptide, thereby reducing or eliminating problems caused by the lack of or abnormal activity caused by the polypeptide. Because translation can occur rapidly, these methods and compositions can be used to treat acute diseases, conditions or illnesses such as sepsis, stroke and myocardial infarction. The therapeutic and / or prophylactic agents included in the nanoparticle composition may also be able to change the transcription rate of a given species, thereby affecting gene expression.

[0400] Diseases, disorders and / or conditions characterized by malfunction or aberrant protein or polypeptide activity include, but are not limited to, rare diseases, infectious diseases (such as vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0401] It will be understood by those skilled in the art that the specific dosage for a particular subject may vary depending on the type, age and general condition of the subject; the purpose of management; the specific ingredients; the mode of management; and so on. The lipid nanoparticles or pharmaceutical compositions of the present application can be formulated in a unit dosage form for ease of administration and uniform dosage. However, it should be understood that the total daily dosage of the lipid nanoparticles or pharmaceutical compositions of the present application will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective or other appropriate dosage level (e.g., for imaging) for any particular patient will depend on a variety of factors, including the severity and identification (if any) of the disease being treated; the one or more therapeutic agents and / or prophylactic agents used; the specific ingredients used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific drug ingredient used; the duration of treatment; drugs used in combination with or concurrently with the specific drug ingredient used; and factors well known to the medical community.

[0402] The application further provides a method for therapeutic and / or preventative specific delivery to mammalian organs, said method comprising administering any one of the application's lipid nanoparticles or pharmaceutical composition to mammals, said administration comprising contacting mammalian organs with lipid nanoparticles or pharmaceutical composition, thereby delivering therapeutic and / or preventative to organs. Therapeutic and / or preventative, for example, protein, cytotoxic agent, radioactive ion, chemotherapeutic agent or nucleic acid (for example RNA, for example mRNA) can be delivered to cells or organs. In the case where therapeutic and / or preventative is mRNA, when cell contacts with lipid nanoparticles or pharmaceutical composition, translatable mRNA can be translated in cells to produce target polypeptide. However, substantially non-translatable mRNAs also can be delivered to cells. Substantially non-translatable mRNA can be used as vaccine and / or can isolate the translation composition of cells to reduce the expression of other species in cells.

[0403] In some embodiments, the lipid nanoparticles can target cells of a particular type or category (e.g., cells of a particular organ or system thereof). For example, lipid nanoparticles comprising target treatment and / or prevention can be specifically delivered to the liver, kidney, spleen, femur, or lung of a mammal. Specific delivery to a particular category of cells, organs, or their systems or groups means that a higher proportion of lipid nanoparticles comprising therapeutic and / or preventive agents, including treatment and / or prevention, is delivered to the target destination (e.g., tissue) relative to other destinations, e.g., when nanoparticles are administered to a mammal. In some embodiments, the target tissue is selected from the group consisting of liver, kidney, lung, spleen, femur, eye tissue (e.g., by intraocular, subretinal, or intravitreal injection), vascular endothelium in a blood vessel (e.g., intracoronary or intrafemoral) or kidney, and tumor tissue (e.g., by intratumoral injection).

[0404] The lipid nanoparticle of the present application or pharmaceutical composition can be used by any approach.In some embodiments, one or more administrations in multiple approaches include, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, percutaneous or intradermal, intradermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, local (such as by powder, ointment, cream, gel, lotion and / or drops), mucosa, nose, cheek, intestine, vitreous, tumor, sublingual, intranasal; By intratracheal instillation, bronchial instillation and / or suction; As oral spray and / or powder, nasal spray and / or aerosol, and / or by portal vein catheter.In some embodiments, lipid nanoparticle or pharmaceutical composition can be intravenous, intramuscular, intradermal, intraarterial, tumor, subcutaneous, intraocular, subretinal, intravitreal or by inhalation administration.However, considering the possible progress in drug delivery science, the application is contained in delivering or describing lipid nanoparticle or pharmaceutical composition described herein by any suitable approach. Generally, the most appropriate route of administration depends on a variety of factors, including the properties of the lipid nanoparticle composition comprising one or more therapeutic and / or prophylactic measures (e.g., its stability in various bodily environments (e.g., blood and gastrointestinal tract)), the condition of the patient (e.g., whether the patient can tolerate a particular route of administration), and the like.

[0405] In certain embodiments, a therapeutic and / or prophylactic agent is administered to a mammal at a dose of 0.0001 mg / kg to 10 mg / kg. For example, 0.0001 mg / kg to 10 mg / kg, 0.001 mg / kg to 10 mg / kg, 0.005 mg / kg to 10 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.05 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg 1mg / kg to 10mg / kg, 2mg / kg to 10mg / kg, 5mg / kg to 10mg / kg, 0.0001mg / kg to 5mg / kg, 0.001mg / kg to 5mg / kg, 0.005mg / kg to 5mg / kg, 0.01mg / kg to 5mg / kg, 0.05mg / kg to 5mg / kg, 0.1mg / kg to 5mg / kg, 1mg / kg to 5mg / kg, 2mg / kg to 5mg / kg, 0.0001mg / kg to 2.5mg / kg, 0.001mg / kg to 2.5mg / kg, 0.005mg / kg to 2.5mg / kg, 0.01mg / kg to 2.5mg / kg 0.05mg / kg to 2.5mg / kg, 0.1mg / kg to 2.5mg / kg, 1mg / kg to 2.5mg / kg, 2mg / kg to 2.5mg / kg, 0.0001mg / kg to 1mg / kg, 0.001mg / kg to 1mg / kg, 0.005mg / kg to 1mg / kg, 0.01mg / kg to 1mg / kg, 0.05mg / kg to 1mg / kg, 0.1mg / kg to 1mg / kg, 0.0001mg / kg to 0.25mg / kg, 0.001mg / kg to 0.25mg / kg, 0.005mg / kg to 0.25mg / kg, 0.01mg / kg to 0.25mg / kg, 0.05mg / kg to 0.25mg / kg or 0.1mg / kg to 0.25mg / kg.

[0406] In some embodiments, the dosage can be administered once or repeatedly with the same or different amounts every day to obtain the mRNA expression and / or treatment, diagnosis, prevention or imaging effect of the desired level. The desired dosage can be, for example, three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (for example, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations). In some embodiments, for example, before or after surgery, or in the case of acute disease, illness or illness, single dose can be used.

[0407] The lipid nanoparticles of the present application can be used in combination with one or more other treatments, preventions, diagnosis or imaging agents. "In combination with ..." does not mean that the reagents must be administered and / or formulated to be delivered together at the same time, although these delivery methods are within the scope of the present application. For example, one or more nanoparticles comprising one or more different treatments and / or preventive agents can be administered in combination. The nanoparticles can be administered before or after one or more other desired treatments or medical procedures. Typically, each medicament will be administered with a dosage and / or schedule determined for the medicament. In some embodiments, the present application includes delivering the lipid nanoparticles of the present application in combination with an agent that improves its bioavailability, reduces and / or changes its metabolism, inhibits its excretion and / or regulates contraction in the body.

[0408] The specific combination of therapies (treatments or procedures) to be used in a combination regimen will take into account the compatibility of the desired treatments and / or procedures and the desired therapeutic effect to be achieved. It will also be recognized that the therapies employed may achieve the desired effect for the same condition (e.g., a composition for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., controlling any side effects, such as infusion-related reactions).

[0409] In some embodiments, treatment has the experimenter in need or the method for delivering therapeutic and / or preventive medicine to experimenter (for example mammal) can be included in using nano-particle with one or more medicament pre-treatment experimenter before.For example, can use the dexamethasone of (for example, 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg or any other effective dose), methotrexate, acetaminophen, H1 receptor blocker or H2 receptor blocker to experimenter pre-treatment.Pre-treatment can occur 24 hours or less hours (for example, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes or 10 minutes) before using nano-particle, and can occur once, twice or more times when for example increasing dosage.

[0410] On the other hand, the present application provides a vaccine preparation comprising the mRNA vaccine of the present invention and a pharmaceutically acceptable carrier.

[0411] In a specific embodiment, the pharmaceutically acceptable carrier includes a cryoprotectant, a pH buffer, a pH adjuster, and water for injection.

[0412] In a specific embodiment, the cryoprotectant is sucrose.

[0413] In a specific embodiment, the pH buffer is glacial acetic acid and / or tromethamine.

[0414] In a specific embodiment, the pH adjuster is sodium hydroxide and / or hydrochloric acid.

[0415] In a specific embodiment, the pH of the formulation is 7.2-8.

[0416] In another aspect of the present application, a method for treating or preventing diseases associated with HPV infection is provided, comprising administering the lipid nanoparticle composition or pharmaceutical composition or mRNA vaccine or vaccine formulation of the present invention to an individual.

[0417] In some embodiments, the HPV infection-related disease is cervical cancer.

[0418] In some embodiments, the HPV infection-related disease is cervical precancerous lesions.

[0419] In some embodiments, the HPV infection-related disease is high-grade cervical intraepithelial lesion. In some embodiments, the HPV infection-related disease is high-grade squamous intraepithelial lesion (HSIL).

[0420] In some embodiments, the HPV infection-related disease is cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2 / 3).

[0421] In some embodiments, the HPV infection-related disease is low-grade LSIL intraepithelial neoplasia (CIN 1).

[0422] In some embodiments, the HPV infection-related disease is cervical adenocarcinoma in situ (AIS).

[0423] In some embodiments, the HPV infection-related disease is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the HPV infection-related disease is cervical cancer (CC).

[0424] In some embodiments, the HPV infection-related disease is oropharyngeal squamous cell carcinoma.

[0425] In some embodiments, the HPV infection-related disease is one of anal cancer, vaginal cancer, vulvar cancer, and penile cancer.

[0426] In the above embodiment, the HPV infection-related disease is a disease associated with human papillomavirus (HPV) type 16 and / or type 18.

[0427] Preferably, the HPV infection-related disease is human papillomavirus (HPV) type 16 and / or type 18-related grade 2 or grade 3 cervical intraepithelial neoplasia (CIN2 / 3).

[0428] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related cervical cancer.

[0429] Preferably, the HPV infection-related disease is human papillomavirus (HPV) type 16 and / or type 18-related human papillomavirus (HPV) 16 and / or HPV18-related head and neck squamous cell carcinoma.

[0430] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related anal cancer.

[0431] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related vaginal cancer.

[0432] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related vulvar cancer.

[0433] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related penile cancer.

[0434] In some embodiments, the administration is intratumoral or perilumphal injection or intramuscular injection. In some embodiments, the method further comprises administering to the individual an immunostimulatory factor, chemotherapy, radiotherapy, and / or targeted therapy. In some embodiments, the targeted therapy is an antibody or its functional domain directed against a cervical cancer-specific tumor target.

[0435] In some embodiments, the method further comprises administering an immune checkpoint inhibitor to the individual. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0436] In some embodiments, the PD-1 or PD-L1 inhibitor is one or more of pembrolizumab, nivolumab, tislelizumab, toripalimab, sintilimab, camrelizumab, penampalimab, sepalimab, envolimab, sugemalimab, slulizumab, putalimab, and adebelimumab. In some preferred embodiments, the PD-1 inhibitor is pembrolizumab.

[0437] It should be understood that the aspects and embodiments of the present application described herein include aspects and embodiments that "comprise," "consist of," and "consist essentially of." The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple variations of the technical solution of the present application may be made, including combining the various technical features in any other suitable manner. These simple variations and combinations should also be considered as disclosed in the present application and fall within the scope of protection of the present application.

[0438] Example 1 Synthesis of Ionizable Fat Compounds

[0439] The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Unless otherwise indicated, all temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. Except for synthetic intermediates or otherwise noted, all reagents and raw materials used in this application were obtained from commercial sources.

[0440] Synthesis of compound 1

[0441] The synthetic route is shown below.

[0442] Specifically, to a solution of compound A1 (2.0 g, 8.96 mmol) in dichloromethane (30 ml) were added compound B1 (1.9 g, 7.47 mmol), DCC (1.8 g, 8.96 mmol), and DMAP (437 mg, 3.58 mmol). The mixture was stirred at room temperature for 7 hours and the reaction was monitored by TLC. After completion of the reaction, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction solution. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford compound C1 (2.9 g, 85.00%).

[0443] To a solution of compound E1 (5.0 g, 25.63 mmol) in dichloromethane (60 ml) were added compound D1 (3.7 g, 21.36 mmol), DCC (5.29 g, 25.63 mmol), and DMAP (1.2 g, 10.25 mmol). The mixture was stirred at room temperature for 7 hours and monitored by TLC. After completion, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction solution. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford compound F1 (6.5 g, 88.00%).

[0444] To a solution of compound F1 (500 mg, 1.44 mmol) in DMF (15 ml) were added compound G (562 mg, 2.88 mmol), NBu₄I (795 mg, 2.15 mmol), and K₂CO₃ (597 mg, 4.32 mmol). The mixture was stirred at room temperature for 12 hours and monitored by TLC. Upon completion, the reaction mixture was diluted with an equal volume of water, extracted with ethyl acetate (10 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to afford compound H1 (580 mg, 87% yield).

[0445] To a solution of compound H1 (500 mg, 1.08 mmol) in DMF (15 ml) were added compound C1 (745 mg, 1.62 mmol), NBu4I (598 mg, 1.62 mmol), and Cs2CO3 (1.4 g, 4.32 mmol). The mixture was stirred at room temperature for 15 hours and monitored by TLC. After completion, an equal volume of water was added to dilute the mixture, followed by extraction with ethyl acetate (10 ml x 3), concentration, and silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to afford compound I1 (811 mg, 89% yield).

[0446] To a solution of compound I1 (10 g, 11.85 mmol) in dichloromethane (100 ml) was added concentrated hydrochloric acid (60 ml). The mixture was stirred at room temperature for 3 hours and the reaction was monitored by TLC. After completion of the reaction, the layers were separated and the organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford compound J1 (6.5 g, 81.00%).

[0447] To a solution of compound J1 (1 g, 1.48 mmol) in toluene (25 ml) were added compound K1 (470 mg, 4.44 mmol) and pyridinium 4-methylbenzenesulfonate (744 mg, 2.96 mmol). A Dean-Stark apparatus was used and the mixture was heated under reflux for 20 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature and diluted with an equal volume of water. The mixture was extracted with ethyl acetate (10 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound L1 (648 mg, 57% yield).

[0448] To a solution of compound L1 (750 mg, 0.98 mmol) in ultra-dry dichloromethane (10 ml) at 0°C was added methylsulfonic anhydride (340 mg, 1.96 mmol) and anhydrous triethylamine (0.45 ml). The mixture was stirred for 12 hours and the reaction was monitored by TLC. After the reaction was complete, the mixture was allowed to cool to room temperature and diluted with an equal volume of water. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound M1 (800 mg, 97% yield).

[0449] To compound M1 (750 mg, 0.89 mmol) was added 30 ml of dimethylamine (2 M in THF), sealed, and stirred at room temperature for 6 days. The reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to afford compound 1 (212 mg, 30% yield) as an oil. 1H NMR (600MHz, CDCl3) δ4.87-4.82(m,1H),4.05-4.02(m,2H),3.90-3.85(m,2H),3.59-3.56(m,2H),2.31-2.23(m,4H),2. 13C NMR (151MHz, CDCl3) δ173.98,173.77,100.76,74.22,64.55,62.87,62.81,59.34,59.3 1,45.97,35.37,35.28,34.86,34.49,34.46,34.28,32.47,32.39,32.03,31.99,29.99 29.72,29.65,29.46,29.36,28.80,26.06,25.44,25.29,25.16,23.49,23.26,23.16,22.92,22.79,14.22; MS-ESI(m / z):794.6(M+H)+.

[0450] Synthesis of compound 2

[0451] The preparation method is the same as compound 1, except that 1,2,4-butanetriol is used instead of compound K1 as the raw material to obtain oily compound 2. 1H NMR (600 MHz, CDCl3) δ 4.82-4.78 (m, 1H), 4.04-3.96 (m, 4H), 3.42-3.39 (m, 1H), 2.39-2.34 (brs, 1H), 2.29-2.25 (brs, 1H), 2.23-2.18 (m, 10H), 1.77-1.71 (brs, 1H), 1.66-1.65 (brs, 1H), 1.58-1.39 (m, 13H), 1.33-1.16 (m, 53H), 0.82-0.80 (m, 9H); 13C NMR (151MHz, CDCl3) δ173.62,173.41,111.82,74.63,74.61,73.88,69.89,64.25,56.18,45. 28,37.73,37.48,37.40,37.20,34.59,34.19,34.18,34.09,31.83,31.78,31.60,29.70,29.6 8,29.52,29.50,29.45,29.34,29.32,29.25,29.20,29.15,29.09,29.07,28.59,25.86,25.23 ,25.07,24.89,23.88,23.58,23.56,23.24,22.60,22.58,14.01; MS-ESI(m / z):794.6(M+H)+.

[0452] Synthesis of compound 2A

[0453] The preparation method is the same as compound 1, except that (S)-1,2,4-butanetriol is used as the raw material instead of compound K1 to obtain oily compound 2A. 1H NMR (400 MHz, CDCl3) δ 4.97-4.83 (m, 1H), 4.17-4.05 (m, 4H), 3.52 (t, J = 8.0 Hz, 1H), 2.58-2.37 (m, 2H), 2.35-2.23 (m, 10H), 1.91-1.70 (m, 2H), 1.69-1.50 (m, 14H), 1.48-1.24 (m, 52H), 0.92 (t, J = 8.0 Hz, 9H); 13C NMR (100MHz, CDCl3) δ174.04,173.69,112.10,74.59,74.12,69.94,64.34,56.22,45 .21,37.79,37.70,37.44,37.38,34.74,34.42,34.16,31.91,31.88,31.43,31.41,2 9.80,29.57,29.55,29.52,29.49,29.32,29.28,29.25,29.19,28.63,28.61,25.95, 25.33,25.17,25.00,23.89,23.71,23.59,22.68,14.12; MS-ESI(m / z):795.0(M+H)+.

[0454] Synthesis of compound 3

[0455] The preparation method is the same as compound 1, using 3-dimethylamino-1,2-propanediol and p-toluenesulfonic acid monohydrate instead of compound K1 and 4-methylbenzenesulfonic acid pyridinium as raw materials to obtain oily compound 3,1H NMR (600MHz, CDCl3) δ4.85-4.81(m,1H),4.20-4.16(m,1H),4.06-4.01(m,3H) ,3.49-3.46(m,1H),2.48-2.44(m,1H),2.35-2.32(m,1H),2.27-2.22(m,10H) ,1.60-1.54(m,8H),1.48-1.47(m,4H),1.31-1.20(m,54H),0.86-0.83(m,9H) ;13CNMR(151MHz,CDCl3)δ173.90,173.69,112.58,74.40,74.36,74.13,69.0 6,64.48,62.62,62.59,46.22,37.80,37.55,37.51,37.30,34.79,34.40,34.38,34.24,31.99,31.94,29.88,29.83,29.68,29.66,29.61,29.58,29.52,29 .47,29.41,29.38,29.33,29.31,29.27,29.25,28.74,26.01,25.39,25.24,2 5.06,25.06,24.03,23.70,23.35,22.74,14.17; MS-ESI(m / z):780.6(M+H)+.

[0456] Synthesis of compound 4

[0457] The preparation method is the same as compound 1, except that undecanoic acid and 6-bromo-1-hexanol are used as raw materials instead of compound D1 and compound E1 to obtain oily compound 4. 1H NMR (600 MHz, CDCl3) δ 4.86-4.81 (m, 1H), 4.04-4.01 (m, 2H), 3.90-3.84 (m, 2H), 3.59-3.55 (m, 2H), 2.27-2.22 (m, 4H), 2.20-2.17 (m, 8H), 1.94-1.85 (brs, 1H), 1.67-1.55 (m, 10H), 1.48-1.45 (m, 4H), 1.36-1.22 (m, 52H), 0.86-0.83 (m, 9H); 13C NMR (151MHz, CDCl3) δ174.01,173.70,100.73,74.14,64.40,64.36,62.83,62.75,59.31,59 .28,45.98,35.50,35.25,34.79,34.47,34.24,32.45,32.43,32.22,31.98,31.95,29.94,2 9.70,29.64,29.61,29.59,29.55,29.42,29.39,29.35,29.32,29.29,29.25,28.73,26.05, 25.40,25.24,25.10,23.45,23.32,23.21,23.08,22.75,14.18; MS-ESI(m / z):794.6(M+H)+.

[0458] Synthesis of compound 5

[0459] The preparation method is the same as compound 1, using undecanoic acid, 6-bromo-1-hexanol and 1,2,4-butanetriol instead of compound D1, compound E1 and compound K1 as raw materials to obtain oily compound 5,1H NMR (600MHz, CDCl3) δ4.81(p,J=6.2Hz,1H),4.03(t,J=6.6Hz,1H),3.99(t,J=6.6Hz,3H),3.42(t,J=7.6Hz,1H),2.41-2.36(brs,1H),2.31-2. 26(brs,1H),2.23-2.20(m,10H),1.78-1.73(brs,1H),1.66-1.60(brs, 1H),1.58-1.43(m,12H),1.31-1.20(m,54H),0.82(t,J=7.0Hz,9H); 13C NMR (151MHz, CDCl3) δ173.92,173.60,112.00,74.75,74.73,74.05,70.00,64.31,56.29 ,45.41,37.83,37.73,37.50,37.42,34.73,34.40,34.20,31.93,31.90,31.73,31.71,2 9.82,29.80,29.58,29.56,29.53,29.50,29.33,29.30,29.26,29.20,28.67,28.65,25. 96,25.35,25.19,25.05,23.90,23.71,23.59,22.69,14.13; MS-ESI(m / z):794.6(M+H)+.

[0460] Synthesis of compound 5A

[0461] The preparation method is the same as compound 1, using undecanoic acid, 6-bromo-1-hexanol and (S)-1,2,4-butanetriol instead of compound D1, compound E1 and compound K1 as raw materials to obtain oily compounds 5A, 1H NMR (400MHz, CDCl3) δ4.97-4.83(m,1H),4.17-4.06(m,4H),3.51(t,J=4.0Hz,1H),2.51-2.45(m,1H),2.44-2.32(m,1H),2. 13C NMR (100Hz, CDCl3) δ173.96,173.62,111.98,74.71,74.08,69.96,64.30,56.24,4 5.36,37.60,37.53,37.50,37.43,34.71,34.39,34.13,31.87,31.84,31.67,31.61 ,29.78,29.70,29.51,29.48,29.30,29.28,29.24,29.18,28.64,28.61,25.94,25 .32,25.17,25.03,23.89,23.70,23.59,22.67,14.11; MS-ESI(m / z):795.0(M+H)+.

[0462] Synthesis of compound 5B

[0463] The preparation method is the same as compound 1, using undecanoic acid, 6-bromo-1-hexanol and (R)-1,2,4-butanetriol instead of compound D1, compound E1 and compound K1 as raw materials to obtain oily compounds 5B, 1H NMR (400MHz, CDCl3) δ4.97-4.83(m,1H),4.17-4.05(m,4H),3.51(t,J=4.0Hz,1H),2.51-2.45(m,1H),2.44-2.32(m,1H),2. 13C NMR (100MHz, CDCl3) δ173.97,173.62,111.98,74.71,74.08,69.96,64.30,56.24, 45.36,37.60,37.43,37.50,37.44,34.71,34.39,34.13,31.87,31.84,31.67,31.6 1,29.79,29.70,29.51,29.48,29.30,29.28,29.24,29.18,28.64,28.61,25.94,25 .32,25.18,25.03,23.89,23.70,23.59,22.67,14.11; MS-ESI(m / z):795.0(M+H)+.

[0464] Synthesis of compound 6

[0465] The preparation method is the same as compound 1, using undecanoic acid, 6-bromo-1-hexanol and 3-dimethylamino-1,2-propanediol instead of compound D1, compound E1 and compound K1 as raw materials to obtain oily compound 6,1H NMR (500MHz, CDCl3) δ4.84(p,J=6.2Hz,1H),4.19(p,J=6.5Hz,1H),4.06-4.01(m,3H),3.49(t,J=7.8Hz,1H),2.48(dd,J=12.6,6.8Hz,1H) 13C NMR (126MHz, CDCl3) δ174.05,173.72,112.66,74.44,74.18,69.08,64.40,62. 65,46.25,37.82,37.71,37.49,37.44,34.83,34.50,34.26,32.00,31.97,29.9 1,29.86,29.63,29.61,29.40,29.37,29.33,29.27,28.73,26.04,25.42,25.26 ,25.12,24.06,23.95,23.73,23.61,22.77,14.19; MS-ESI(m / z):780.6(M+H)+.

[0466] Synthesis of compound 7

[0467] The preparation method is the same as compound 1, except that 8-bromo-1-octanol, 2-octyl-decanoic acid and 3-dimethylamino-1,2-propylene glycol are used instead of compound A1. Compound B1 and compound K1 are used as raw materials to obtain oily compound 7. 1H NMR (500 MHz, CDCl3) δ 4.22-4.17 (m, 1H), 4.06-4.01 (m, 5H), 3.48 (t, J = 7.8 Hz, 1H), 2.50-2.46 (m, 1H), 2.39-2.36 (m, 1H), 2.28-2.24 (m, 9H), 1.60-1.53 ​​(m, 13H), 1.42-1.37 (m, 2H), 1.30-1.22 (m, 53H), 0.85 (t, J = 6.8 Hz, 9H); 13C NMR (126MHz, CDCl3) δ176.64,173.92,112.62,74.26,74.21,68.96,64.29,64.10 ,62.48,46.07,45.85,37.75,37.60,37.42,37.32,34.40,32.56,31.90,31.87,2 9.91,29.86,29.57,29.46,29.31,29.25,29.18,28.75,28.64,27.47,25.99,25. 94,25.03,23.98,23.85,23.64,23.51,22.67,14.10; MS-ESI(m / z):794.6(M+H)+.

[0468] Preparation of compound 8

[0469] The preparation method is the same as compound 1, except that 8-bromo-1-octanol, 2-octyl-decanoic acid and 1,2,4-butanetriol are used instead of compound A1. Compound B1 and compound K1 are used as raw materials to obtain oily compound 8. 1H NMR (500 MHz, CDCl3) δ 4.07-4.00 (m, 6H), 3.44 (t, J = 7.5 Hz, 1H), 2.42-2.36 (brs 1H), 2.32-2.21 (m, 10H), 1.81-1.74 (brs, 1H), 1.68-1.51 (m, 14H), 1.42-1.34 (brs, 2H), 1.26-1.22 (m, 53H), 0.84 (t, J = 6.9 Hz, 9H); 13C NMR (126MHz, CDCl3) δ176.69,173.97,112.05,74.81,70.06,64.36,64.16,56 .36,45.92,45.50,37.93,37.78,37.59,37.48,34.46,32.63,31.97,31.94,3 1.82,29.63,29.53,29.37,29.32,29.24,28.81,28.71,27.54,26.06,26.02, 25.09,24.07,23.94,23.77,23.64,22.73,14.16; MS-ESI(m / z):808.6(M+H)+.

[0470] Preparation of compound 9

[0471] The preparation method is the same as compound 1, except that 8-bromo-1-octanol, 2-octyl-decanoic acid, undecanoic acid and 6-bromo-1-hexanol are used as raw materials instead of compound A1, compound B1, compound D1 and compound E1 to obtain oily compound 9, 1H NMR (500 MHz, CDCl3) δ 4.04-4.01 (m, 4H), 3.89-3.85 (m, 2H), 3.59-3.56 (m, 2H), 2.31-2.18 (m, 11H), 1.93-1.86 (brs, 1H), 1.67-1.52 (m, 12H), 1.43-1.23 (m, 56H), 0.85 (t, J = 6.9 Hz, 9H); 13C NMR (126MHz, CDCl3) δ176.71,174.00,100.75,64.40,64.36,64.18,62.83,62.76,5 9.34,59.31F,45.98,45.94,35.46,35.25,34.48,32.64,32.54,32.46,32.34,31.9 8,31.96,30.06,29.71,29.65,29.55,29.39,29.34,29.26,28.83,28.74,27.56,26 .08,25.11,23.46,23.32,23.23,23.09,22.75,14.18; MS-ESI(m / z):808.5(M+H)+.

[0472] Preparation of compound 10

[0473] The preparation method is the same as compound 1, except that 1,2,4-butanetriol and N-ethylmethylamine are used instead of compound K1 and dimethylamine as raw materials to obtain oily compound 10. 1H NMR (600MHz, CDCl3) δ4.83-4.75 (m, 1H), 4.05-3.93 (m, 4H), 3.42-3.35 (m, 1H), 2.46-2.30 (m, 4H), 2.23-2.15 (m, 7H), 1.79-1.70 (m, 1H), 1.67-1.59 (m, 1H), 1.56-1 .51(m,6H),1.43(d,J=7.2Hz,4H),1.29-1.14(m,56H),0.99(t,J=7.1Hz,3H),0.8 0(t,J=7.0Hz,9H);13CNMR(151MHz,CDCl3)δ173.72,173.51,111.83,74.85,74.8 1,73.96,69.99,64.33,53.70,51.43,41.49,37.80,37.56,37.48,37.28,34.66,34.26,34.25,34.15,31.90,31.85,31.30,29.77,29.59,29.57,29.51,29.49,29 .41,29.38,29.32,29.29,29.24,29.22,29.16,29.13,28.65,25.92,25.30,25.1 3,24.97,23.96,23.66,23.31,22.65,14.08,12.09; MS-ESI(m / z):808.7(M+H)+.

[0474] Preparation of compound 11

[0475] The preparation method is the same as compound 1, except that 1,2,4-butanetriol and diethylamine are used as raw materials instead of compound K1 and dimethylamine to obtain oily compound 11. 1H NMR (600 MHz, CDCl3) δ 4.86-4.80 (m, 1H), 4.08-4.00 (m, 4H), 3.49-3.43 (m, 1H), 2.77-2.62 (m, 7H), 2.25 (dt, J = 10.5, 7.5 Hz, 4H), 1.85-1.75 (m, 2H), 1.63-1.56 (m, 6H), 1.50-1.44 (m, 4H), 1.37-1.19 (m, 56H), 1.12 (t, J = 7.2 Hz, 6H), 0.85 (td, J = 7.1, 2.4 Hz, 9H); 13C NMR (151MHz, CDCl3) δ173.95,173.73,112.22,74.59,74.55,74.17,69.94,64.52,49.36,46 .95,37.84,37.59,37.42,37.21,34.80,34.39,34.24,31.99,31.95,30.30,29.87,29.69,2 9.67,29.62,29.59,29.50,29.47,29.42,29.35,29.32,29.29,29.26,28.74,26.02,25.40, 25.24,25.07,24.09,23.82,23.76,23.47,22.75,14.20,10.74; MS-ESI(m / z):822.9(M+H)+.

[0476] Preparation of compound 12

[0477] The preparation of compound 12 refers to the preparation of compound 2.

[0478] Preparation of compound 13

[0479] The preparation method is the same as compound 1, except that 1,2,5-pentanetriol is used instead of compound K1 as the raw material to obtain oily compound 13. 1H NMR (600 MHz, CDCl3) δ 4.81-4.74 (m, 1H), 4.00-3.92 (m, 4H), 3.38-3.33 (m, 1H), 2.25-2.17 (m, 6H), 2.15 (s, 6H), 1.59-1.46 (m, 15H), 1.46-1.37 (m, 5H), 1.29-1.12 (m, 50H), 0.79 (td, J = 7.1, 2.3 Hz, 9H); 13C NMR (151MHz, CDCl3) δ173.69,173.47,111.86,76.08,76.05,73.92,69.93,69.91,F64.29,59.48,53. 38,45.24,37.80,37.56,37.47,37.25,34.64,34.24,34.22,34.13,31.88,31.83,31.20,29.76,29.5 7,29.55,29.49,29.47,29.39,29.37,29.30,29.27,29.22,29.20,29.14,29.11,28.63,25.90,25.28 ,25.12,24.95,23.95,23.89,23.64,23.62,23.29,22.64,22.62,14.05; MS-ESI(m / z):808.8(M+H)+.

[0480] Example 2 Synthesis of mRNA

[0481] 2.1 Synthesis of HPV vaccine sequences and construction of recombinant vectors

[0482] In this embodiment, the antigenic sequences of the HPV mRNA vaccine are the E6 and E7 proteins of HPV types 16 and 18, and their coding fragments are concatenated to obtain a polynucleotide sequence. From the 5' end, the coding nucleotide sequence containing, in sequence: a T7 promoter with XbaI at the 5' end, a 5'UTR, tPA-SP, Flt3L, HPV E2, E6 / E7 protein or its variant, a 3'UTR and / or a poly A tail is double-digested with XbaI and NotI, and ligated with the pUC57-GW-Kan (Jinweizhi) vector backbone fragment digested with XbaI and NotI to construct a recombinant plasmid.

[0483] 2.2 mRNA preparation

[0484] 2.2.1 Plasmid linearization

[0485] The recombinant plasmid constructed in step 1 has a Sap I restriction site after the last A in the polyA tail sequence. Linearize the plasmid containing the target gene with the restriction endonuclease Sap I using the reaction system shown in Table 1. Incubate the enzyme digestion at 37°C for 3 hours.

[0486] Table 1. Plasmid linearization enzyme digestion system

[0487] 2 μL of the digested product was subjected to 1% agarose gel electrophoresis to check the linearization of the plasmid. The linearized plasmid was purified using a PCR product recovery kit (Comvison).

[0488] (2) In vitro transcription and purification

[0489] The linearized recombinant plasmid obtained in step (1) was used as a template for in vitro transcription using a high-yield T7 RNA transcription kit. The high-yield T7 RNA transcription kit, product name is High Yield T7 RNA Synthesis Kit, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number is ON-040; 5× Reaction Buffer, 100mM ATP Solution, 100mM CTP Solution, 100mM GTP Solution, Enzyme mix, DNase I, Ammonium Acetate Stop Solution, Lithium Chloride (LiCl) Precipitation Solution are all components of the high-yield T7 RNA transcription kit. 100mM ΨUTP Solution (pseudouridine triphosphate), full name N1-Me-pUTP, 100mM, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number is R5-027. Add each component according to the following system (Table 2) (taking 20μL reaction system as an example), mix well, and react at 37℃ for 3h.

[0490] Table 2. In vitro transcription system

[0491] Among them, CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG, product number is ON-134, Shanghai Zhaowei.

[0492] After the transcription reaction is completed, add 1 μL of DNase I and react at 37°C for 15 minutes. Add 15 μL of Ammonium Acetate Stop Solution and mix well. Then add 1 / 3 volume of 7.5M Lithium Chloride (LiCl) Precipitation Solution (to a final concentration of 2.5M) and incubate at -20°C for 30 minutes. Centrifuge at 12,000g for 15 minutes to allow the RNA to precipitate at the bottom. Discard the supernatant. Add 1 mL of 70% ethanol to wash the RNA. Centrifuge at 12,000g for 5 minutes and discard the supernatant. After drying, add 50 μL of RNase-free water to dissolve the precipitate and quantify the mRNA using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0493] Example 3 Lipid Nanoparticle (LNP) Encapsulation and Determination

[0494] HPV mRNA (SEQ ID NO: 12) was prepared into a 200 μg / ml solution using acetic acid at a pH of 4.0-6.0. Four lipids were then mixed into a lipid-mixed solution using anhydrous ethanol according to the prescribed dosage. The oil and water phases were mixed using a plunger pump or syringe pump at a flow rate of 1:3 through a T-type mixing device. After LNP formation, the solution was diluted with 2-10 times sucrose or an aqueous solution. Ethanol was removed from the sample by ultrafiltration and replaced with acetic acid solution at a displacement factor of at least 3. After replacement, the pH of the product was adjusted to 7.0-8.0 with Tris, and the osmotic pressure was adjusted to near physiological values ​​with sucrose. The product was sterile filtered, sealed, and stored at -20°C. Finally, the total and free mRNA content in the LNPs was determined using a Ribogreen assay kit and 10% OTG as a demulsifier, and the LNP encapsulation efficiency was calculated. The final LNP product was diluted with diluent, 1 ml was added to the particle size pool, and placed on the Malvern ZetaSizer instrument to measure the particle size of the LNP. The results are shown in Table 3.

[0495] Table 3. Results of the HPV mRNA encapsulation efficiency test of different ratios of LNP formulations of compound 5A

[0496] As shown in Table 3, the results show that the LNPs formed by Example Compound 5A have excellent properties such as particle size uniformity and encapsulation efficiency, and are comparable to or better than the currently known SM-102 standard formulation.

[0497] Flow Cytometry

[0498] Flow cytometry is used to analyze the cellular immunity level in test animals to provide a scientific basis for evaluating the effectiveness of candidate vaccines. Cytotoxic T lymphocytes (CTL cells), commonly known as CD8+ T cells, play an important role in the immune system's defense against pathogens (such as viruses, bacteria, and tumors). IFN-γ, as a cytokine secreted by immune-active cells, plays an important immunomodulatory role in inducing cellular immunity, including activating cytotoxic T lymphocytes (CTL), natural killer cells (NK) and phagocytes. Currently, detecting IFN-γ levels has gradually replaced various traditional cellular immunity detection methods and has become an important method for detecting cellular immunity effects.

[0499] Experimental procedures

[0500] Immunization steps for C57BL / 6 mice: HPV mRNA sequences encapsulated in the formula in Table 3 were injected intramuscularly into the hind limbs of mice. Immunization dose: 12.5 μg, immunization volume: 50 μL. The test endpoint was 7 days after immunization.

[0501] At the end of the experiment, C57BL / 57 mice were sacrificed, and the spleens were removed under sterile conditions and placed in a 6-well cell culture plate containing 2 mL of RPMI 1640 complete medium. The spleens were ground to prepare a single-cell suspension, which was filtered through a cell sieve. The single-cell filtrate was transferred to a 50 mL sterile centrifuge tube and centrifuged at 300 g for 5 minutes at room temperature. The supernatant was discarded, 3 mL of red blood cell lysis buffer was added, the cells were fully resuspended, and the cells were placed in a 37°C 5% CO2 cell culture incubator and incubated for 3 minutes. After the incubation period, an equal volume of RPMI 1640 complete medium was added, mixed thoroughly, and the reaction was terminated. The single-cell suspension was then filtered again through a cell sieve. The single-cell filtrate was obtained and centrifuged at 300 g for 5 minutes. The supernatant was discarded, 2 mL of RPMI 1640 complete medium was added, the cells were fully resuspended, and the cells were counted. The cell concentration was adjusted to 1x10 7 Take a 96-well U-bottom cell culture plate and inoculate 1x10 cells / mL in each well. 6 Cell amount; HPV vaccine peptide library as stimulator, BFA + monensin as blocker: peptide library working concentration: 2 μg / ml / peptide; BFA dilution factor: 0.1 μL / test (1000-fold dilution); Monensin dilution factor: 0.1 μL / test (1000-fold dilution).

[0502] Place the 96-well U-bottom cell plate in a 37°C, 5% CO2 incubator and culture overnight. Remove the stimulated 96-well U-bottom cell plate and centrifuge at 300g for 5 minutes, discarding the supernatant.

[0503] Extracellular flow cytometry antibody staining

[0504] Preparation of extracellular staining antibodies (prepared in 1x PBS)

[0505] The concentrations of Anti-CD3-cy5.5 staining are: 1 μL / test

[0506] Anti-CD4-APC (Biolegend) staining concentrations are: 0.5 μL / test

[0507] The concentrations of Anti-CD8-FITC (MBL) staining are: 5 μL / test

[0508] Add 100 μL of extracellular antibody staining solution to each well and incubate at 4°C in the dark for 30 minutes. Add 100 μL of PBS and centrifuge at 300 g for 5 minutes, discarding the supernatant. Add 200 μL of fixative solution to each well and thoroughly resuspend the cells. Incubate at 4°C in the dark for 30 minutes. Centrifuge at 300 g for 5 minutes and discard the supernatant. Add 100 μL of 1x permeabilization buffer to each well and thoroughly resuspend the cells. Centrifuge at 300 g for 5 minutes and discard the supernatant.

[0509] Intracellular flow cytometry antibody staining

[0510] Intracellular staining antibody preparation (prepared in 1x permeabilization buffer)

[0511] The concentration of Anti-IFN-γ-PE (Biolegend) staining was 0.5 μL / test. 100 μL of intracellular antibody staining mixture was added to each well and incubated at room temperature in the dark for more than 1 h with shaking.

[0512] After incubation, centrifuge at 300g for 5 minutes and discard the supernatant; add 200μL PBS to each well, resuspend the cells thoroughly, centrifuge at 300g for 5 minutes, and discard the supernatant; 4.9.4 Add 200μL PBS to each well, resuspend the cells thoroughly, and analyze on the analyzer.

[0513] The expression levels of compound 5A in different formulations were compared with the CD8+IFN-γ+ double-positive cell count of the control group (SM-102 standard formulation sample) at 1. As shown in Figure 1, the different formulation ratios of compound 5A significantly outperformed the control group (SM-102 standard formulation sample) in terms of CD8+IFN-γ+ positive cells, with the CD8+IFN-γ+ positive cell ratio increasing by 1.2-1.7 times. This indicates that the compound 5A-based formulation, when delivered with mRNA sequences, can induce a stronger cellular immune response than the SM-102 standard formulation.

[0514] Example 4

[0515] Dissolve calcium chloride in acetate buffer to obtain a certain concentration of Ca 2+The solution was then used to dilute HPV mRNA to obtain mRNA working solution, with a final mRNA concentration of 200 μg / ml. 2+ The final concentration of the solution is shown in Table 4. The mRNA aqueous phase and the mixed lipid ethanol phase are mixed in a volume ratio of 3:1. After mixing, the sample is diluted, concentrated, and replaced, and the ethanol is removed. The volume is then fixed and the pH is adjusted to 7-8 (for specific steps, see Example 2). Sucrose is used to adjust the osmotic pressure to near physiological values. Sterile filter, seal, and store at -20°C.

[0516] Take 0.5 ml of HPV mRNA injection, add 0.5 ml of 10% Triton solution to break the emulsion, then dilute to 10 ml with 2% nitric acid aqueous solution and shake well. Detection was performed according to the Chinese Pharmacopoeia (2020 edition) 0411 inductively coupled plasma atomic emission spectrometry method.

[0517] Table 4: Recipe composition

[0518] Elispot analysis

[0519] Elispot is used to analyze the cellular immunity level in experimental animals to provide a scientific basis for the effectiveness evaluation of candidate vaccines. Cytotoxic T lymphocytes (CTL cells) are usually called CD8 + T cells play a vital role in the immune system's defense against pathogens, such as viruses, bacteria, and tumors. IFN-γ, a cytokine secreted by immune-active cells, plays a crucial immunomodulatory role in inducing cellular immunity, including activation of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and phagocytes. Currently, measuring IFN-γ levels has gradually replaced various traditional cellular immunity assays and become a key method for measuring cellular immunity effectiveness.

[0520] Mice were intramuscularly injected with HPV mRNA solution at 25 μg / mice, 5 mice / sample. Seven days after administration, the mice were sacrificed and spleen single cell suspensions were obtained at a concentration of 1.5 x 10 6 cells / ml, and CD8 + The number of IFN-γ positive secreting cells. The Elispot KIT kit uses a color reaction to make the positive cells that secrete the cytokine IFN-γ appear as clear spots. The spots are then counted using the ELISPOT analysis system. One spot represents one active cell. The frequency of cells secreting the cytokine is calculated based on this, usually 10 6 The number of active cells in cells is expressed.

[0521] The IFN-γ secretion expression effect of mouse spleen of each sample was detected using IFN-γ Elispot KIT kit. 6 The number of positive cells in cells was expressed as 2+ The IFN-γ positive cell test result of the sample with Ca was 1, and the other samples were compared with it. As shown in Figure 2 (Formula 21-1, 21-2, 21-3) and Figure 3 (Formula 13-1), the results were the same as those without Ca. 2+ The expression effects of the samples were compared, and within the scope of investigation, different concentrations of Ca 2+ The expression of samples was improved by 1 to 5 times.

[0522] The sequences used in the above examples of the present application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application and are not intended to limit the present application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When representing RNA sequences, "T" represents uridine.

[0523] Sequence Listing:

Claims

1. A lipid nanoparticle comprising an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(30-50):(1-3), wherein the ionizable lipid is compound 5, or a salt thereof or a stereoisomer thereof, 2. The lipid nanoparticle according to claim 1, wherein the structure of the stereoisomer of the ionizable lipid is selected from One or more of .

3. The lipid nanoparticle according to claim 1, wherein the ionizable lipid structure is selected from 4. The lipid nanoparticle according to any one of claims 1 to 3, wherein the phospholipid is selected from one or more of the following compounds: Dilauroyl phosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DUPC), Palmitoyloleoylphosphatidylcholine (POPC), 1,2-Di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Divinyl-sn-glycero-3-phosphocholine, 1,2-Diaryl acyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-Dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME 16.0PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-sn-glycero-3-phosphoethanolamine, 1,2-Diaryl-sn-glycero-3-phosphoethanolamine, 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine, 1,2-Diol-sn-glycero-3-phosphate-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

5. The lipid nanoparticle according to any one of claims 1 to 3, wherein the phospholipid is DSPC.

6. The lipid nanoparticle according to any one of claims 1 to 3, wherein the structural lipid is selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol.

7. The lipid nanoparticle of any one of claims 1-3, wherein the structural lipid is cholesterol.

8. The lipid nanoparticle according to any one of claims 1 to 3, wherein the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

9. The lipid nanoparticle of any one of claims 1-3, wherein the PEG lipid is DMG-PEG2000.

10. The lipid nanoparticle according to any one of claims 1-3, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(10-15):(35-45):(1.5-2.5) or (45-49):(10-15):(35-45):(1.5-2.5) or (48-49):(10-15):(35-45):(1.5-2.5).

11. The lipid nanoparticle according to any one of claims 1 to 3, wherein The content of ionizable lipids is 40mol%-50mol%, preferably 45mol%-49mol%, further preferably 48mol%-49mol%, The content of phospholipids is 5mol%-15mol%, preferably 10mol%-15mol%, The structural lipid content is 30mol%-50mol%, preferably 35mol%-45mol%, The content of PEG lipid is 1 mol%-3 mol%, preferably 1.5 mol%-2.5 mol%; The mol% is calculated based on the total moles of lipid in the lipid nanoparticle composition.

12. The lipid nanoparticle of any one of claims 1-3, wherein the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is not 50:10:38.5:1.

5.

13. A lipid nanoparticle composition comprising the lipid nanoparticles of any one of claims 1 to 12 and a therapeutic and / or prophylactic agent, wherein the therapeutic and / or prophylactic agent is selected from a vaccine or a compound, nucleic acid or protein capable of eliciting an immune response, and the therapeutic and / or prophylactic agent is encapsulated by the lipid nanoparticles.

14. The lipid nanoparticle composition according to claim 13, wherein the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

15. The lipid nanoparticle composition of claim 14, wherein the nucleic acid is mRNA.

16. The lipid nanoparticle composition of claim 15, wherein the mRNA encodes a protein comprising the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or wherein the mRNA encodes a protein comprising the sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

17. The lipid nanoparticle composition of claim 15, wherein the coding sequence of the mRNA comprises the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or wherein the coding sequence of the mRNA is shown in SEQ ID NO: 10 or SEQ ID NO:

11.

18. The lipid nanoparticle composition of claim 15, wherein the coding sequence of the mRNA comprises the sequence shown in SEQ ID NO: 12 or wherein the coding sequence of the mRNA is as shown in SEQ ID NO:

12.

19. The lipid nanoparticle composition according to any one of claims 15 to 18, wherein the coding sequence of the mRNA includes a 5'UTR coding sequence, preferably, the 5'UTR coding sequence is as shown in SEQ ID NO:

5.

20. The lipid nanoparticle composition according to any one of claims 15-18, wherein the coding sequence of the mRNA includes a 3'UTR coding sequence, preferably, the 3'UTR coding sequence is as shown in SEQ ID NO:

6.

21. The lipid nanoparticle composition according to any one of claims 15 to 18, wherein the coding sequence of the mRNA comprises a poly A tail coding sequence, preferably, the poly A tail coding sequence is as shown in SEQ ID NO:

7.

22. The lipid nanoparticle composition according to any one of claims 15-18, wherein the coding sequence of the mRNA includes a 5' cap, preferably, the 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG.

23. The lipid nanoparticle composition of any one of claims 15-18, wherein some or all of the uridine in the mRNA molecule is 1-methyl pseudouridine.

24. The lipid nanoparticle composition according to any one of claims 15 to 18, wherein the N / P ratio of the ionizable lipid to the mRNA is 3.5-5.5, preferably 5.

2.

25. The lipid nanoparticle composition according to any one of claims 15-18, further comprising a divalent metal cation, preferably Ca 2+ Mg 2+ 、Zn 2+ or Mn 2+ .

26. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1-12 or the lipid nanoparticle composition of any one of claims 13-25 and a pharmaceutically acceptable excipient or auxiliary component.

27. The pharmaceutical composition according to claim 26, further comprising a divalent metal cation, preferably Ca 2+ Mg 2+ 、Zn 2+ or Mn 2+ .

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