Vaccine adjuvant lipid compounds based on RLRs receptor agonists, compositions containing the same, and uses thereof

Abstract

The present invention provides a vaccine adjuvant lipid compound based on RLRs receptor agonists and its applications, and specifically discloses the compounds shown in formula (I-1), formula (I-2) and formula (I-3), their stereoisomers, their N-oxides, their solvates, or their pharmaceutically acceptable salts. As a component of the mRNA or drug molecule delivery carrier, the compounds provided by the present invention enter the cytoplasm of immune cells in the body and can directly participate in the RIG-I-activated IRF3 and NF-κB-dependent innate immune responses.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to a vaccine adjuvant lipid compound based on RLRs receptor agonists, a composition containing the same, and uses thereof. Background Art

[0002] Adjuvant lipids are substances that can improve the immunogenicity of mRNA vaccines. They can enhance the ability of vaccines to stimulate the body to produce antibodies, thereby improving the protective effect of vaccines. Compared with traditional aluminum adjuvants, oil-in-water emulsion adjuvants, and natural polysaccharide adjuvants that require relatively large dosages, adjuvant lipids are a new type of adjuvant. They participate in the composition of the mRNA vaccine delivery vector LNP, form a stable complex with mRNA, and can exert a good adjuvant effect at low dosages.

[0003] Adjuvant lipids of the RLRs type combine the design concept of cationic lipid compounds with small molecule agonists that can activate the RLRs signaling pathway, such as benzothiazole compounds (A Small Molecule RIG-I Agonist Serves as an Adjuvant to Induce BroadMultifaceted Infuenza Virus Vaccine Immunity. J Immunol (2023) 210(9):1247-1256). As a structural component of LNP, adjuvant lipids of the RLRs type enhance the delivery of mRNA into the cytoplasm, and the further dissociated RLRs small molecule agonists can participate in the RIG-I activation of IRF3 and NF-kB-dependent innate immune responses. They can not only sense viral RNA but also activate the antiviral state of cells by inducing the production of IFNs and inflammatory factors, restricting virus replication and transmission.

[0004] mRNA vaccine technology has played a huge role in protecting human health during the COVID-19 pandemic. Currently, many mRNA vaccines still face problems such as a short protection period and a rapid decline in antibody levels. Therefore, there is an urgent need to develop adjuvant lipids that can enhance the immunogenicity of mRNA vaccines to improve the protective effect of vaccines. Summary of the Invention

[0005] The present invention provides an adjuvant lipid compound based on RLRs small molecule agonists, including its N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers. As a component of the mRNA or drug molecule delivery carrier, it enters the cytoplasm of immune cells in the body and can directly participate in the RIG-I activation of IRF3 and NF-kB-dependent innate immune responses. It exerts a highly efficient adjuvant effect at low dosages.

[0006] In a first aspect, the present invention provides a compound, its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, the structure of the compound being represented by formula (I-1), formula (I-2) or formula (I-3),

[0007] , , ;

[0008] G1 is selected from , , amino, halogen or H;

[0009] G2 is selected from or ;

[0010] G3 is ;

[0011] L 11 、L 12 、L 21 、L 22 、L 31 、L 32 are the same or different and each independently selected from C 1~10 alkylene (for example, it can be C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 ), or a single bond;

[0012] n1, n2, n3, m are the same or different and each independently selected from 0 or 1;

[0013] X1, X2, X3, Y1, Y2, Y3 are the same or different and each independently selected from NH, O or a single bond;

[0014] When n1 is 0, L 11 is directly covalently bonded to X1; when n2 is 0, L 21 is directly covalently bonded to X2; when n3 is 0, L 31 is directly covalently bonded to X3; when m is 0, L 32 is directly covalently bonded to N;

[0015] R 11 、R 21 、R 31 are the same or different and each independently selected from unsubstituted C 6~25 (for example, it can be C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C22 , C 25 , etc.) straight-chain or branched-chain hydrocarbon groups, where the C 6~25 straight-chain or branched-chain hydrocarbon group has 0 to 3 (e.g., it can be 0, 1, 2, or 3) C=C double bonds, where the double bond is in the E or Z configuration;

[0016] R 12 , R 22 , R 32 are the same or different and each independently selected from unsubstituted C 8~24 (e.g., it can be C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 , C 24 , etc.) straight-chain or branched-chain hydrocarbon groups, where the C 8~24 straight-chain or branched-chain hydrocarbon group has 0 to 3 (e.g., it can be 0, 1, 2, 3, etc.) C=C double bonds, where the double bond is in the E or Z configuration;

[0017] R 23 is selected from methyl, amino, or H;

[0018] R 33 , R 34 , R 35 , R 36 are the same or different and each independently selected from methoxy, ethoxy, amino, halogen, or H.

[0019] In some embodiments, G1 is .

[0020] In some embodiments, G2 is .

[0021] In some embodiments, R 33 , R 34 , R 35 , R 36 are the same or different and each independently selected from methoxy or H.

[0022] In some embodiments, m is 0.

[0023] In some embodiments, m is 1.

[0024] In some embodiments, n1 is 0.

[0025] In some embodiments, n1 is 1.

[0026] In some embodiments, n2 is 0.

[0027] In some embodiments, n2 is 1.

[0028] In some embodiments, n3 is 0.

[0029] In some embodiments, n3 is 1.

[0030] In some embodiments, L 11 、L 21 、L 31 are the same or different and each independently selected from C 2~7 alkylene or a single bond.

[0031] In some embodiments, L 12 、L 22 、L 32 are the same or different and each independently selected from C 2~7 alkylene or a single bond.

[0032] In some embodiments, R 11 、R 21 、R 31 are the same or different and each independently selected from unsubstituted C 10~18 linear hydrocarbon groups having 0 to 2 Z-form C=C double bonds or unsubstituted C 14~18 branched alkyl groups.

[0033] In some embodiments, R 11 、R 21 、R 31 are the same or different and each independently selected from 、 、 、 、 or 。

[0034] In some embodiments, R 12 、R 22 、R 32 are the same or different and each independently selected from unsubstituted C 10~19 linear hydrocarbon groups having 0 to 2 Z-form C=C double bonds or unsubstituted C 14~18 branched alkyl groups.

[0035] In some embodiments, R 12 、R 22 、R 32 are the same or different and each independently selected from 、 、 、 、 or 。

[0036] In some embodiments, the compound of formula (I-1) is selected from any one of the following structures:

[0037] , , , , wherein each group has the same defined scope as in claim 1.

[0038] In some embodiments, the compound has one of the following structures:

[0039] ,

[0040] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] ,

[0046] ,

[0047] ,

[0048] ,

[0049] , .

[0050] In some embodiments, the compound is the compound YK-1406 having the following structure:

[0051] .

[0052] In some embodiments, the compound is the compound YK-1407 having the following structure:

[0053] .

[0054] In some embodiments, the compound is the compound YK-1410 having the following structure:

[0055] .

[0056] Second aspect, the present invention provides a composition, the composition comprising a lipid composition, the lipid composition comprising an adjuvant lipid; the adjuvant lipid comprising the compound described in the first aspect, its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0057] In some embodiments, the molar ratio of the adjuvant lipid in the lipid composition is 0.1% - 20% (for example, it can be 1%, 5%, 10%, 15%, 20%, etc.).

[0058] In some embodiments, the lipid composition further comprises a cationic lipid and a neutral lipid.

[0059] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 - 15:1 (for example, it can be 1:1, 2:1, 4:1, 6:1, 10:1, 15:1, etc.), preferably 4:1 - 6:1.

[0060] In some embodiments, the cationic lipid is selected from any one or a combination of at least two of the following compounds (1) to (7): (1) the compound represented by formula (II), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein, G1 is C 1～6 alkylene; G2 is C 2～8 alkylene; G3 is C 1～3 alkylene; L1 is C 6～15 linear alkyl; L2 is C 12～25 branched alkyl;

[0061] (II)

[0062] (2) the compound represented by formula (III), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein, G1 is C 2~8 alkylene; G2 is C 2~8 alkylene; L1 is selected from -C(O)O- or -OC(O)-; L2 is selected from -C(O)O- or -OC(O)-; R1 is C 6~25 linear or branched alkyl; R2 is C 6~25 linear or branched alkyl; G3 is selected from HO(CH2)2- or HO(CH2)3-; G4 is selected from HO(CH2)2- or HO(CH2)3-; L is selected from -(CH2)2-, -(CH2)3- or -(CH2)4-;

[0063] (III)

[0064] (3) The compound shown in formula (IV), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein: G1 is C 1~6 alkylene; G2 is C 2~8 alkylene; R1 is C 6~20 straight-chain or branched-chain alkyl; R2 is C 12~25 branched-chain alkyl; G3 is selected from HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-;

[0065] (IV)

[0066] (4) The compound shown in formula (V), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein G1 is C 1~8 alkylene; G2 is C 2~8 alkylene; R1 is C 6~25 straight-chain or branched-chain alkyl; R2 is C 12~25 straight-chain or branched-chain alkyl; G3 is selected from HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3, -CH2CH3 or -CH2CH2OH ;

[0067] (V)

[0068] (5) The compound shown in formula (VI), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein, G 1 and G 2 are each independently C6-C 10 alkylene; G 3 is C1-C 12 alkylene; R 1 and R 2 are each independently selected from C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is selected from OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R4 is C1-C 12 hydrocarbyl; R 5 is selected from H or C1-C6 hydrocarbyl;

[0069] (VI)

[0070] The compound shown in formula (VII), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein R4 is selected from -(CH2) n Q or -(CH2) n CHQR; Q is selected from -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocycle; n is selected from 1, 2 or 3; R is C 1-8 alkyl; X is selected from H or C 1-8 alkyl;

[0071] (VII)

[0072] The compound shown in formula (VIII), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt,

[0073] (VIII).

[0074] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide or sterol and its derivatives.

[0075] In some embodiments, the lipid composition further comprises any one or a combination of at least two of cationic lipids, neutral lipids, structural lipids or polymer-conjugated lipids. In some embodiments, the cationic lipid is selected from any one or a combination of at least two of YK-009, YK-401, YK-305, ALC0315, SM102 or DLIN-MC3-DMA;

[0076] 、 、

[0077] 、 , , .

[0078] In some embodiments, the cationic lipid is YK-009.

[0079] In some embodiments, the molar percentage of the cationic lipid in the lipid composition is 30-47.5%, and can be, for example, 35%, 40%, 45%, etc.

[0080] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine (LPE).

[0081] In some embodiments, the neutral lipid is DOPE and / or DSPC, preferably DSPC.

[0082] In some embodiments, the molar percentage of the neutral lipid in the lipid composition is 5-25%, for example, it can be 5%, 10%, 15%, 20%, 25%, etc.

[0083] In some embodiments, the structural lipid is selected from any one or a combination of at least two of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, or corticosteroid.

[0084] In some embodiments, the structural lipid is cholesterol.

[0085] In some embodiments, the molar percentage of the structural lipid in the lipid composition is 15-65%, for example, it can be 15%, 20%, 38.5%, 50%, 65%, etc.

[0086] In some embodiments, the polymer-conjugated lipid is selected from any one or a combination of at least two of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), or methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0087] In some embodiments, the polymer-conjugated lipid is DMG-PEG2000.

[0088] In some embodiments, the molar percentage of the polymer-conjugated lipid in the lipid composition is 0.5-10%, for example, it can be 1%, 1.5%, 5%, 8%, 10%, etc.

[0089] In some embodiments, the lipid composition includes adjuvant-like lipid, cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid.

[0090] In some embodiments, the molar dosage ratio of the adjuvant-like lipid, cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid is (1~20):(25~75):(5~25):(15~65):(0.5~10).

[0091] The values in (1~20) above can be, for example, 1, 5, 10, 15, 20, etc.; the values in (25~75) can be, for example, 25, 45, 65, 75, etc.; the values in (5~25) can be, for example, 5, 10, 15, 20, 25, etc.; the values in (15~65) can be, for example, 15, 35, 55, 65, etc.; the values in (0.5~10) can be, for example, 0.5, 1, 4, 8, 10, etc.

[0092] In some embodiments, the lipid composition comprises the adjuvant-like lipid, the YK-009, DSPC, cholesterol, and DMG-PEG2000; the molar percentages of the adjuvant-like lipid, the YK-009, DSPC, cholesterol, and DMG-PEG2000 are (2.5-20):(30-47.5):10:38.5:1.5; preferably 5:45:10:38.5:1.5.

[0093] In some embodiments, the composition is a nanoparticle formulation, and the average particle size of the nanoparticle formulation is 10 nm to 300 nm (e.g., it can be 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, etc.); the polydispersity index (PDI) of the nanoparticle formulation is ≤50% (e.g., it can be 1%, 5%, 15%, 20%, 40%, 50%, etc.).

[0094] In some embodiments, the average particle size of the nanoparticle formulation is 50 nm to 200 nm; the polydispersity index (PDI) of the nanoparticle formulation is ≤20%.

[0095] In some embodiments, the cationic lipid further comprises one or more other ionizable lipid compounds.

[0096] In some embodiments, the lipid composition further comprises one or more cell-penetrating peptides.

[0097] In some embodiments, the composition further comprises a therapeutic agent and / or a prophylactic agent.

[0098] In some embodiments, the mass ratio of the lipid composition to the therapeutic agent and / or the prophylactic agent is 10:1 to 30:1 (e.g., it can be 10:1, 15:1, 20:1, 25:1, 30:1, etc.), preferably 12.5:1 to 25:1, more preferably 14:1 to 20:1.

[0099] In some embodiments, the dosages of the therapeutic agent and / or the prophylactic agent and the lipid composition are such that the charge ratio of positive charges to negative charges in the composition is 1:(2-5) (e.g., it can be 1:2, 1:3, 1:4, 1:5, etc.).

[0100] In some embodiments, the therapeutic agent and / or the prophylactic agent comprises any one or a combination of at least two of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0101] In some embodiments, the composition is used to deliver the therapeutic agent and / or the prophylactic agent to antigen-presenting cells in a target organ or tissue.

[0102] In some embodiments, the target organ or tissue is selected from any one or a combination of at least two of spleen, liver, lymph, muscle, or lung.

[0103] In some embodiments, the antigen-presenting cell is selected from any one or a combination of at least two of B cells, NK cells, cDC cells, pDC cells, or macrophages.

[0104] In some embodiments, the therapeutic agent and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

[0105] In some embodiments, the antigen is a disease-related antigen, or the nucleic acid molecule or antigen is capable of eliciting an immune response against a disease-related antigen or cells expressing a disease-related antigen.

[0106] In some embodiments, the nucleic acid molecule is an RNA encoding one or more antigens.

[0107] In some embodiments, the therapeutic agent and / or prophylactic agent is ribonucleic acid (RNA).

[0108] In some embodiments, the therapeutic agent and / or prophylactic agent is deoxyribonucleic acid (DNA).

[0109] In some embodiments, the RNA is selected from any one or a combination of at least two of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), or small hairpin RNA (shRNA), messenger RNA (mRNA).

[0110] In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the composition further comprises at least one auxiliary component. The auxiliary component can be a pharmaceutical carrier, diluent, or excipient.

[0111] In some embodiments, the composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface modifiers, functionalized lipids, or cytokines.

[0112] In a third aspect, the present invention provides the use of a compound according to the first aspect, its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, or a composition according to the second aspect in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs.

[0113] Fourth aspect, the present invention provides the use of a compound according to the first aspect, its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or a composition as described in the second aspect in the preparation of a medicament for treating a disease or disorder. The disease or disorder is preferably characterized by a malfunctioning or abnormal protein or polypeptide activity.

[0114] In some embodiments, the disease or disorder is selected from any one or a combination of at least two of infectious diseases, tumors, proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, or metabolic diseases.

[0115] In some embodiments, the infectious disease is selected from any one or a combination of at least two of diseases caused by coronavirus, influenza virus or HIV virus, Rift Valley fever, yellow fever, rabies, or various herpes.

[0116] In some embodiments, the tumor is selected from any one or a combination of at least two of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer, or prostate cancer.

[0117] In some embodiments, in the said use, the medicament is a medicament for treating a disease or disorder of a mammal in need thereof. The mammal is selected from any one or a combination of at least two of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

[0118] In some embodiments, the administration route of the medicament is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

[0119] In some embodiments, the administration route of the medicament is intravenous or intramuscular.

[0120] In some embodiments, the administration dose of the medicament is 0.001 - 10 mg / kg (for example, it can be 0.001 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, etc.).

[0121] The adjuvant lipid compound and lipid composition provided by the present invention can be used for encapsulating nucleic acids (such as mRNA, etc.) to form corresponding nucleic acid drugs.

[0122] Term Definitions

[0123] All publications and patents mentioned in this invention are hereby incorporated by reference in their entirety into this invention. If there are any conflicts between the uses or terms used in any of the publications and patents incorporated by reference and the uses or terms used in this invention, then the uses and terms of this invention shall prevail.

[0124] The chapter headings used in this invention are for the purpose of organizing the article only and should not be construed as limiting the subject matter described.

[0125] Unless otherwise specified, all technical and scientific terms used in this invention have the ordinary meanings in the field to which the claimed subject matter pertains. If there are multiple definitions for a term, the definition in this invention shall prevail.

[0126] Except as otherwise indicated in the examples or otherwise noted, all numbers expressing quantitative properties such as dosages stated in the specification and claims should be understood to be modified in all instances by the term "about". It should also be understood that any numerical range recited in this invention is intended to include all sub-ranges within that range and any combination of the endpoints of that range or sub-ranges.

[0127] In this invention, " " in a structural fragment means that this structural fragment is connected to the rest of the molecule through this bond. For example, means that it is connected to the rest of the molecule through " ".

[0128] In this invention, the term "alkyl" refers to a straight-chain or branched-chain, saturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1, C2, C3, C4, C5, C6, C9, C 10 、C 11 、C 12 、C 16 、C 17 、C 18 、C 19 、C 20 ). Alkyl includes but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, 、 .

[0129] In this invention, the term "alkylene" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight-chain or branched-chain hydrocarbon group. Alkylene includes but is not limited to: methylene (-CH2-), ethylene {including -CH2CH2- or -CH(CH3)-}, isopropylidene {including -CH(CH3)CH2- or -C(CH3)2-}, 、 .

[0130] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having at least one double bond, consisting only of carbon and hydrogen atoms, having, for example, 10 to 20 (such as 16, 17, 18, 19) carbon atoms, and being connected to the rest of the molecule by a single bond. Alkenyl includes, but is not limited to, vinyl, , , , , etc.

[0131] In the present invention, the term "aryl" refers to a cyclic, unsaturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C6-C 10 ), which is monocyclic or polycyclic (e.g., 2), and when polycyclic, two atoms and one bond are shared between the monocycles, and each ring has aromaticity. The aryl is connected to the rest of the molecule through an aromatic ring. Aryl includes, but is not limited to: phenyl, naphthyl.

[0132] In addition, when referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximation within the typical tolerances in the art, within experimental variability or within statistical experimental error, and thus the number or numerical range can vary, for example, between 1% and 15% of the said number or numerical range. For example, "about" can be understood as about 2 standard deviations from the average value, and when "about" is present before a series of numbers or ranges, it should be understood that "about" can modify each number in the said series or range.

[0133] In addition, in the present invention, when a numerical range is used in a compound general formula and / or structural formula, it means that the number of corresponding groups in the numerical range can be any natural number within the numerical range. For example, "C A-B " means that the number of carbon atoms is any integer from the starting point to the end point of the range, where both A and B are integers; for another example, C 1-5 represents that the number of carbon atoms is 1, 2, 3, 4 or 5; that is, when combined with other groups in the compound general formula and / or structural formula to form various possible compounds, C A-B can be used in combination with any group containing carbon atoms to define the number of carbon atoms. For example, C 1-5 alkyl / alkylene represents various possibilities of alkyl / alkylene having 1 C, 2 C, 3 C, 4 C and / or 5 C.

[0134] As used in the present invention, words such as "comprising", "containing" or "including" and the like are intended to mean that the elements appearing before the word cover the elements listed after the word and their equivalents, without excluding elements not recited. The terms "containing" or "including (comprising)" used in the present invention can be open-ended, semi-closed and closed. In other words, the said terms also include "consisting essentially of...", or "consisting of...".

[0135] The term "pharmaceutically acceptable" in the present invention means that a compound or composition is chemically and / or toxicologically compatible with other components of the formulation and / or with a human or mammal for which it is used to prevent or treat a disease or disorder.

[0136] The term "subject" or "patient" in the present invention may include mammalian subjects. For example, the mammalian subject may be selected from any one or at least a combination of two of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

[0137] The term "treatment" as used in the present invention means administering one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease, for the purpose of curing, alleviating, reducing, improving or affecting the disease or the symptoms of the disease. In the context of the present invention, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0138] In the present invention, the term "antigen" includes any molecule containing at least one epitope capable of eliciting an immune response and / or an epitope against which an immune response is directed, preferably a peptide or protein. Preferably, the antigen in the context of the present invention is such a molecule that optionally induces an immune response preferably specific for the antigen or the cells expressing the antigen after processing. In particular, "antigen" refers to such a molecule that is optionally presented by MHC molecules after processing and specifically reacts with T lymphocytes (T cells).

[0139] Therefore, an antigen or a fragment thereof should be capable of being recognized by a T cell receptor. Preferably, if recognized by a T cell receptor, the antigen or fragment is capable of inducing clonal expansion of T cells carrying a T cell receptor specifically recognizing the antigen or fragment in the presence of a suitable co-stimulatory signal. In the context of the embodiments of the present invention, the antigen or fragment is preferably presented by a cell in the context of MHC molecules, preferably by an antigen-presenting cell and / or a diseased cell, which results in an immune response against the antigen or the cells expressing the antigen.

[0140] According to the present invention, any suitable antigen is contemplated as a candidate for an immune response, wherein the immune response is preferably a cellular immune response.

[0141] The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. The naturally occurring antigen may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a part thereof.

[0142] The term "pathogen" refers to pathogenic microorganisms and includes viruses, bacteria, fungi, single-celled organisms, and parasites. Examples of pathogenic viruses include, but are not limited to, human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), hepatitis A virus (HAV), HBV, HCV, papillomavirus, and human T-lymphotrophic virus (HTLV). Single-celled organisms include, but are not limited to, Plasmodium, Trypanosoma, Amoeba, etc.

[0143] The term "disease-related antigen" refers to all antigens with pathogenic significance and includes "tumor antigens". According to the present invention, it is desired to induce an immune response against disease-related antigens or cells that express disease-related antigens and preferably present disease-related antigens in the context of MHC molecules. Preferably, the disease-related antigen is a naturally occurring antigen. In one embodiment, the disease-related antigen is expressed in diseased cells and is preferably presented by the MHC molecules of the cells.

[0144] The antigen encoded by the RNA (i.e., the therapeutic and / or prophylactic agent) contained in the nanoparticles of the present invention (lipid composition) should induce an immune response against the disease-related antigen to be targeted or cells that express the disease-related antigen to be targeted. Therefore, the antigen encoded by the RNA contained in the nanoparticles of the present invention may correspond to or may contain a disease-related antigen or one or more immunogenic fragments thereof, such as one or more MHC-binding peptides of the disease-related antigen. Therefore, the antigen encoded by the RNA contained in the nanoparticles of the present invention may be a recombinant antigen.

[0145] Therapeutic and / or prophylactic agent

[0146] The lipid composition of the present invention can be used to deliver pharmaceutically active ingredients, such as therapeutic and / or prophylactic agents. Based on this, the present invention further provides a (pharmaceutical) composition comprising the lipid composition provided by the present invention for delivering pharmaceutically active ingredients. The composition of the present invention may include one or more therapeutic and / or prophylactic agents (as pharmaceutically active ingredients). The pharmaceutically active ingredient can be encapsulated within the lipid composition or combined with the lipid composition.

[0147] The therapeutic and / or prophylactic agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, and proteins. Preferably, it is a nucleic acid molecule.

[0148] For example, the therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response. Thus, in some preferred embodiments, the therapeutic and / or prophylactic agent can be a nucleic acid molecule capable of encoding one or more antigens.

[0149] The lipid composition of the present invention can (as a carrier) deliver a therapeutic and / or prophylactic agent to target cells and / or target organs in a subject (such as a mammal). Accordingly, the present invention also provides methods for treating a disease or disorder in a subject in need thereof, the methods comprising administering to the subject a composition comprising a therapeutic and / or prophylactic agent and / or contacting the cells of the subject with the composition.

[0150] Therapeutic and / or prophylactic agents include bioactive substances and are alternatively referred to as "active agents", "active ingredients", etc. A therapeutic and / or prophylactic agent can be a substance that causes a desired change in a cell or organ after being delivered to the cell or organ or other body tissues or systems. Such agents 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 composition include, but are not limited to, antineoplastics (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), β-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), antispasmodics (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and imaging agents.

[0151] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any reagent that is harmful to cells. Examples include but are not limited to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis and may include nucleic acid molecules (e.g., mRNA) encoding antigens and / or epitopes derived from infectious diseases. Vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0152] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present invention 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, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0153] In some embodiments, the therapeutic and / or prophylactic agent can be a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance in the form of an oligonucleotide chain or that can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with the present invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrids; RNAi-inducing factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA that can be used in the compositions and methods described in the present invention can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0154] In certain embodiments, the therapeutic and / or prophylactic agent is mRNA. The mRNA can encode any polypeptide of interest, including any native or non-native or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0155] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. The siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA can be selected such that after administering a composition comprising the siRNA to a subject in need, a gene associated with a particular disease, disorder, or condition is silenced. The siRNA can comprise a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0156] In certain embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. The sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of a cell gene.

[0157] In some embodiments, the therapeutic and / or prophylactic agent is an shRNA or its encoding vector or plasmid. The shRNA can be generated inside the target cell after delivering the appropriate construct to the nucleus. The constructs and mechanisms associated with shRNA are well known in the relevant art.

[0158] disease or disorder

[0159] The compositions / carriers of the present invention can deliver therapeutic and / or prophylactic agents to a subject or patient, thereby achieving the treatment and / or prevention of a disease or disorder. The therapeutic and / or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Thus, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of the above-mentioned therapeutic and / or prophylactic agents, the compositions of the present invention can be used to treat or prevent a variety of diseases or disorders.

[0160] In one embodiment, the disease or disorder is characterized by a malfunctioning or abnormal protein or polypeptide activity.

[0161] The reagents, compositions and methods described in the present invention can be used to treat a subject suffering from a disease (e.g., a disease characterized by the presence of diseased cells that express an antigen and present antigenic peptides), or to prevent a subject from suffering from a disease. Examples of diseases that can be treated and / or prevented cover all diseases that express one of the antigens described in the present invention. Particularly preferred diseases are infectious diseases (e.g., viral diseases) and cancer diseases. The reagents, compositions and methods described in the present invention can also be used for immunization or vaccination to prevent the diseases described in the present invention.

[0162] According to the present invention, the term "disease" refers to any pathological condition, including infectious diseases and cancer diseases, especially those forms of the infectious diseases and diseases described in the present invention.

[0163] The disease to be treated according to the present invention is preferably a disease involving an antigen. According to the present invention, "disease involving an antigen" or a similar expression means that the antigen is expressed in the cells of a diseased tissue or organ. The expression in the cells of the diseased tissue or organ can be increased compared to the state of a healthy tissue or organ. In one embodiment, the expression occurs only in the diseased tissue, while the expression in the healthy tissue is suppressed. According to the present invention, diseases involving an antigen include infectious diseases and cancer diseases, wherein the disease-related antigens are preferably the antigens of the infectious agent and tumor antigens, respectively. Preferably, the disease involving an antigen is preferably a disease involving cells that express an antigen and present the antigen in the context of MHC molecules (especially class I MHC).

[0164] For example, the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0165] Examples of the infectious diseases include: ① viral infectious diseases, such as AIDS (HIV), hepatitis A, hepatitis B or hepatitis C, herpes zoster (varicella), German measles (rubella virus), yellow fever, dengue fever, etc., flavivirus, coronavirus, influenza virus, rabies virus, hemorrhagic infectious diseases (Marburg virus or Ebola virus); ② bacterial infectious diseases, such as Legionnaire's disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), infections caused by Escherichia coli, Staphylococci, Salmonella or Streptococci (tetanus); ③ infections caused by protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis, toxoplasmosis, that is, infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or ④ fungal infections, which are caused by, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0166] The cancer or carcinoma (the medical term is malignant tumor) is a class of diseases in which a group of cells exhibits uncontrolled growth (division beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasis (spreading to other parts of the body through lymph or blood). These three harmful properties of cancer distinguish it from benign tumors that are self-limiting and do not invade or metastasize. Most cancers form tumors, that is, swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some (such as leukemia) are not. According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, embryonal carcinoma, neuroblastoma, glioma, glioblastoma, renal cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, gastric cancer, lung cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, multiple myeloma, lymph node cancer, esophageal cancer, colon cancer, rectal cancer, bladder cancer, prostate cancer, endometrial cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and their metastases.

[0167] Malignant melanoma is a serious type of skin cancer. It is caused by the uncontrolled growth of pigment cells called melanocytes.

[0168] According to the present invention, "epithelial cancer" is a malignant tumor derived from epithelial cells. This group accounts for the most common cancers, including common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0169] Lymphoma and leukemia are malignant tumors derived from hematopoietic (blood-forming) cells.

[0170] Sarcoma is a cancer of transformed cells that originate from one of the tissues developed from the embryonic mesoderm. Therefore, sarcomas include bone tumors, cartilage tumors, fat tumors, muscle tumors, vascular tumors, and hematopoietic tissue tumors.

[0171] Blastic tumor or embryonal carcinoma is a tumor (usually malignant) similar to immature or embryonic tissue. Most of these tumors are common in children.

[0172] Glioma is a type of tumor that begins in the brain or spinal cord. It is called glioma because it originates from glial cells. The most common site of glioma is the brain.

[0173] Other components

[0174] The pharmaceutical composition of the present invention may include one or more components other than those described in the foregoing part. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (such as vitamin A or vitamin E) or sterols.

[0175] The composition may also include one or more permeation enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeation enhancing molecules may be, for example, the molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include simple sugars (such as glucose) and polysaccharides (such as glycogen and its derivatives and analogs).

[0176] The surface modifiers may include, but are not limited to, anionic proteins (such as bovine serum albumin), surfactants (such as cationic surfactants, such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (such as cyclodextrin), nucleic acids, polymers (such as heparin, polyethylene glycol and poloxamer), mucolytics (such as acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine and erdosteine) and DNAse (such as rhDNAse). The surface modifiers may be disposed within and / or on the surface of the nanoparticles of the composition (such as by coating, adsorption, covalent attachment or other methods).

[0177] The composition may also contain one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer may be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition or imaging. The surface of the composition may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging and membrane permeation are well known in the art.

[0178] In addition to these components, the composition can include any substance that can be used in a pharmaceutical composition. For example, the composition can include one or more pharmaceutically acceptable (e.g., medicinally acceptable) excipients or auxiliary components, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening agents or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.

[0179] The term "medicinally acceptable" means that the material is non-toxic and does not affect the action of the active components of the pharmaceutical composition. Non-medicinally acceptable components can be used to prepare medicinally acceptable components and are included in the present invention.

[0180] Suitable buffers for the compositions of the present invention include the salt forms of acetic acid, citric acid, boric acid, and phosphoric acid.

[0181] When used in the present invention, the term "excipient" is intended to mean all substances that can be present in the pharmaceutical compositions of the present invention and that are not active ingredients, such as carriers, binders, lubricants, thickening agents, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents. Excipients include, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington’s The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0182] Suitable preservatives for the compositions of the present invention include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0183] Examples of diluents can include but are not limited to calcium carbonate, sodium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0184] Dosage Forms and Administration

[0185] The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present invention can be prepared by methods well known in the pharmaceutical art. For example, a sterile injectable solution can be prepared by incorporating the required amount of the therapeutic or prophylactic agent with the various other ingredients required above into a suitable solvent such as sterile distilled water and then filtering and sterilizing. Surfactants can also be added to facilitate the formation of a uniform solution or suspension.

[0186] For example, the compositions of the present invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation. In one embodiment, the composition is administered intravenously or subcutaneously.

[0187] Therapeutically effective amount

[0188] A "therapeutically effective amount" is the amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or symptom. A "prophylactically effective amount" is the amount of a prophylactic agent that, when administered to a subject, prevents a disease or symptom. The amount of the therapeutic agent that constitutes a "therapeutically effective amount" or the amount of the prophylactic agent that constitutes a "prophylactically effective amount" varies with the therapeutic and / or prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient and / or subject to be treated and / or prevented. One of ordinary skill in the art can routinely determine the therapeutically effective amount and prophylactically effective amount based on their knowledge and the present invention.

[0189] The compositions of the present invention are administered in a therapeutically effective amount, which amount can vary not only with the particular reagent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, a dose of about 0.0001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent can be administered to a mammal (such as a human).

[0190] Antigen-presenting cell

[0191] An antigen-presenting cell (APC) is a cell that presents (i.e., displays) an antigen on its surface in the context of a major histocompatibility complex (MHC). This includes cases where only one fragment or more than one fragment of the antigen is presented. T cells can recognize this complex with their T cell receptors (TCRs). Antigen-presenting cells process antigens and present them to T cells.

[0192] Professional antigen-presenting cells are very efficient at internalizing antibodies (by phagocytosis or by receptor-mediated endocytosis) and then presenting antigen fragments bound to class II MHC molecules on their membranes. T cells recognize the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell and interact with it. Then the antigen-presenting cell generates additional co-stimulatory signals, leading to T cell activation. Expression of co-stimulatory molecules is a typical feature of professional antigen-presenting cells.

[0193] The main types of professional antigen-presenting cells are dendritic cells (which have the broadest range of antigen presentation and are probably the most important antigen-presenting cells), macrophages, B cells, and certain activated epithelial cells.

[0194] Dendritic cells are a group of white blood cells that include plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs), which present antigens captured in peripheral tissues to T cells via two antigen presentation pathways, class II and class I MHC. Dendritic cells are powerful inducers of immune responses, and activation of these cells is a key step in inducing anti-tumor immunity.

[0195] Antigen-presenting cells can be loaded with MHC-presented peptides by transducing them with nucleic acids encoding peptides or proteins containing the peptides to be presented (e.g., nucleic acids encoding antigens (e.g., RNA)). Transfecting dendritic cells with mRNA is a promising antigen-loading technique for stimulating strong anti-tumor immunity.

[0196] The term "immunogenicity" refers to the relative efficiency of an antigen in inducing an immune response.

[0197] The terms "T cell" and "T lymphocyte" are used interchangeably in the present invention and include helper T cells (CD4+ T cells) and cytotoxic T cells of the cytolytic T cell (CTL, CD8+ T cells).

[0198] T cells belong to the group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types (e.g., B cells and natural killer cells) by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for T cell maturation. Several different T cell subsets have been identified, each with different functions.

[0199] Helper T cells assist other white blood cells during the immune process, including functions such as maturing B cells into plasma cells and activating cytotoxic T cells and macrophages. Because they express the CD4 protein on their surface, these cells are also called CD4 +T cells. Helper T cells are activated when class II MHC molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. After activation, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0200] Cytotoxic T cells destroy diseased cells, such as infected cells (e.g., virus-infected cells) and cancer cells, and also participate in transplant rejection. Because they express the CD8 glycoprotein on their surface, these cells are also called CD8 + T cells. These cells recognize their targets by binding to antigens associated with class I MHC, which is present on the surface of almost every cell in the body.

[0201] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two independent peptide chains, which are produced by the independent T cell receptor alpha and beta (TCRα and TCRβ) genes and are called the α-TCR chain and the β-TCR chain. Gamma-delta T cells represent a small subtype of T cells that have a unique T cell receptor (TCR) on their surface. However, in gamma-delta T cells, the TCR is composed of one gamma chain and one delta chain. This group of T cells is less common than alpha-beta T cells (2% of total T cells).

[0202] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells are present in the thymus and expand by cell division to produce a large number of immature thymocytes. Early thymocytes express neither CD4 nor CD8 and are therefore classified as double-negative (CD4-CD8-) cells. As they develop through the process of development, they become double-positive thymocytes (CD4+CD8+) and eventually mature into single-positive (CD4 + CD8 - or CD4 - CD8 + ) thymocytes, which are then released from the thymus into the peripheral tissues.

[0203] The first signal for T cell activation is provided by the binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only T cells with a TCR specific for that peptide are activated. The partnering cell is usually a professional antigen-presenting cell (APC), usually a dendritic cell in the case of a primary response, but B cells and macrophages can also be important APCs. The peptides presented by class I MHC molecules to CD8 + T cells are 8 to 10 amino acids in length; the peptides presented by class II MHC molecules to CD4 +The peptides of T cells are longer because the ends of the binding clefts of class II MHC molecules are open.

[0204] On the basis of conforming to the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0205] The reagents and raw materials used in the present invention are all commercially available.

[0206] The positive and progressive effects of the present invention are as follows: Compared with the prior art, the mRNA composition prepared by using the compounds represented by formula (I-1), formula (I-2) or formula (I-3) provided by the present invention has one or more of the following advantages:

[0207] 1. Good particle size and uniform particle distribution;

[0208] 2. As a structural component of LNP, it enhances the delivery of mRNA;

[0209] 3. The mRNA involved in delivery has significant protein expression in the liver and spleen of mice;

[0210] 4. It can stimulate the production of cytokines such as INF-γ and TNF-α in mouse serum and activate immune cells such as DCs and T cells;

[0211] 5. It improves the immunogenicity of mRNA vaccines, enhances the ability of vaccines to stimulate the body to produce antibodies, and improves the protective effect of vaccines; Description of the Drawings

[0212] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure and do not limit the present invention.

[0213] Figure 1 Showing the results of cell transfection experiments of LNP formulations of eGFP-mRNA prepared based on the molar ratio of YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipid of 45:10:38.5:1.5:5, where: a is YK-1406, b is YK-1407, c is YK-1408, d is YK-1410, e is YK-009 (without adjuvant lipid), and f is compound 23.

[0214] Figure 2Show the fluorescence images of LNP formulations of eGFP-mRNA prepared with YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipids at a molar ratio of 45:10:38.5:1.5:5 in live mice and mouse organs (liver, spleen). (Top: live mice, Bottom: mouse organs), where the adjuvant lipids are: YK-1406, YK-1407, YK-1410; also include the YK-009-LNP control without adjuvant addition.

[0215] Figure 3 Show the stimulation of cytokines IFN-γ and TNF-α in the serum of mice by OVA mRNA-LNP formulations prepared with YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipids at a molar ratio of 45:10:38.5:1.5:5. The adjuvant lipids are: YK-1406, YK-1407, YK-1408, YK-1410; also include the YK-009-LNP control without adjuvant addition.

[0216] Figure 4 Show the situation of OVA mRNA-LNP formulations prepared with YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipids at a molar ratio of 45:10:38.5:1.5:5 stimulating the production of antigen-specific cytotoxic T cells in the spleens of mice. The adjuvant lipids are: YK-1406, YK-1407, YK-1410, Compound 23; also include the YK-009-LNP control without adjuvant addition.

[0217] Figure 5 Show the ELISPOT (enzyme-linked immunospot) count map of cytokines IFN-γ produced by the spleens of mice stimulated by OVA mRNA-LNP formulations prepared with YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipids at a molar ratio of 45:10:38.5:1.5:5. The adjuvant lipids are: YK-1406, YK-1407, YK-1410, Compound 23; also include the YK-009-LNP control without adjuvant addition. Detailed implementation mode

[0218] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples.

[0219] The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. In the specific embodiments of the present invention, the raw materials used can all be obtained commercially. Unless otherwise specified, percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0220] The following abbreviated letters represent the following reagents respectively:

[0221] YK-009: 2-octyldecyl ((decoxy-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate; ( , prepared according to Example 1 in Patent CN114044741B)

[0222] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine;

[0223] DMG-PEG2000: 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000;

[0224] DCM: Dichloromethane;

[0225] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0226] DMAP: 4-Dimethylaminopyridine;

[0227] ACN: Acetonitrile;

[0228] K2CO3: Potassium carbonate;

[0229] TEA: Triethylamine;

[0230] HCl in 1,4-Dioxane: 4M hydrogen chloride / 1,4-dioxane solution;

[0231] toluene: Toluene;

[0232] xylene: Xylene;

[0233] Di-tert-butyl dicarbonate: Di-tert-butyl carbonate;

[0234] Pd2(dba)3: Tris(dibenzylideneacetone)dipalladium;

[0235] X-Phos: 2-Dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl.

[0236] Example 1: Synthesis of RLRs adjuvant lipid compounds

[0237] 1. Synthesis of YK-1401

[0238] The synthesis route is as follows:

[0239]

[0240] Step 1: Synthesis of YK-1401-PM1

[0241] Using 7-chlorobenzo[d]thiazol-2-amine (500.0 mg, 2.71 mmol), 2-naphthoic acid (606.1 mg, 3.52 mmol), EDCI (778.6 mg, 4.06 mmol), DMAP (165.4 mg, 1.35 mmol) and DMF (10.0 mL) as raw materials, dissolve them in dichloromethane (10.0 mL), under the protection of nitrogen atmosphere, stir and react at room temperature for 24 hours. After the reaction is completed, add water (10.0 mL) to the reaction solution, then add dichloromethane (50.0 mL×2) for extraction. Combine the organic phases and wash them with saturated brine (10.0 mL×2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to remove the solvent. Purify the residue by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1401-PM1 (816.7 mg, 2.41 mmol, 89.02%). C 18 H 11 ClN2OS, MS(ES): m / z(M+H + ) 339.0

[0242] Step 2: Synthesis of YK-1401-PM2

[0243] YK-1401-PM1 (400.0 mg, 1.18 mmol), tetradecylamine (1007.8 mg, 4.72 mmol), Pd2(dba)3 (540.6 mg, 0.59 mmol), X-Phos (562.8 mg, 1.18 mmol), potassium tert-butoxide (265.0 mg, 2.36 mmol) and 1,4-dioxane (8.0 mL) were placed in a single-necked flask. After purging with N2, the mixture was refluxed at 110 °C for 24 h. After the reaction was completed, water (10.0 mL) was added to the reaction solution, and then dichloromethane (50.0 mL × 2) was added for extraction. The organic phases were combined and washed with saturated brine (10.0 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1401-PM2 (228.2 mg, 0.44 mmol, 37.48%). C 32 H 41 N3OS, MS(ES): m / z(M+H + ) 516.3.

[0244] Step 3: Synthesis of YK-1401

[0245] Using YK-1401-PM2 (228.2 mg, 0.44 mmol), 3-hexylnonyl 6-bromohexanoate (538.2 mg, 1.33 mmol), K2CO3 (244.6 mg, 1.77 mmol), Cs2CO3 (43.2 mg, 0.13 mmol), KI (14.7 mg, 0.09 mmol) as raw materials, they were dissolved in acetonitrile (4.0 mL) and stirred at 70 °C for 2 days. After the reaction was completed, the mixture was concentrated, ethyl acetate (5.0 mL) and water (5.0 mL) were added, stirred and separated, and then extracted with ethyl acetate (5.0 mL × 2). The organic phases were combined and washed with saturated brine (5.0 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1401 (60.3 mg, 0.07 mmol, 16.22%). C 53 H 81 N3O3S, MS(ES): m / z(M+H + ) 840.6.

[0246] 11H NMR (CDCl3, 400 MHz, 298 K) δ 8.46 (d J = 7.6 Hz, 1H), 8.21 – 8.12(m, 3H), 7.89 – 7.41 (m, 5H), 6.72 (d, J = 7.3 Hz, 1H), 4.19 (d, J = 7.0 Hz,2H), 3.84 (d, J = 5.3 Hz, 4H), 2.38 (t, J = 5.0 Hz, 2H), 1.59 – 1.57 (m, 8H),1.32 – 1.25 (m, 45H), 0.94 (d, J =3.2 Hz, 9H).

[0247] 2. Synthesis of YK-1402

[0248] The synthetic route is as follows:

[0249]

[0250] Step 1: Synthesis of YK-1402

[0251] Using YK-1401-PM2 (200.0 mg, 0.39 mmol), 2-octyldecyl 6-bromohexanoate (202 mg, 0.45 mmol), K2CO3 (465.4 mg, 3.37 mmol), Cs2CO3 (146.3 mg, 0.45 mmol), KI (55.9 mg, 0.34 mmol) and acetonitrile (4.0 mL) as raw materials, YK-1402 (86.0 mg, 0.10 mmol, 25.13%) was obtained according to the method for synthesizing YK-1401. C 56 H 87 N3O3S, MS(ES): m / z(M+H + ) 882.7.

[0252] 11H NMR (CDCl3, 400 MHz, 298 K) δ 8.46 (d J = 7.6 Hz, 1H), 8.21 – 8.12(m, 3H), 7.89 – 7.41 (m, 5H), 6.72 (d, J = 7.3 Hz, 1H), 3.86 – 3.78, (m, 6H),2.32 (t, J = 5.0 Hz, 2H), 1.89 – 1.86 (m, 1H),1.59 – 1.51 (m, 6H), 1.32 –1.25 (m, 52H), 0.94 (d, J =3.2 Hz, 9H).

[0253] 3. Synthesis of YK-1403

[0254] The synthetic route is as follows:

[0255]

[0256] Step 1: Synthesis of YK-1403-PM1

[0257] Put YK-1401-PM1 (400.0 mg, 1.18 mmol), decyl 4-aminobutyrate (880.0 mg, 3.62 mmol), Pd2(dba)3 (540.6 mg, 0.59 mmol), X-Phos (562.8 mg, 1.18 mmol), potassium tert-butoxide (265.0 mg, 2.36 mmol) and 1,4-dioxane (8.0 mL) into a single-necked flask. After purging with N2, reflux the reaction mixture at 110 °C for 24 h. After the reaction is completed, add water (10.0 mL) to the reaction solution, then add dichloromethane (50.0 mL × 2) for extraction. Combine the organic phases and wash them with saturated brine (10.0 mL × 2). Dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to remove the solvent. Purify the residue by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1403-PM1 (260.0 mg, 0.48 mmol, 40.35%). C 32 H 39 N3O3S, MS(ES): m / z(M+H + ) 546.3.

[0258] Step 2: Synthesis of YK-1403

[0259] Using YK-1403-PM1 (260.0 mg, 0.48 mmol), 6-bromohexanoic acid heptadec-9-yl ester (550.0 mg, 1.27 mmol), K2CO3 (244.6 mg, 1.77 mmol), Cs2CO3 (43.2 mg, 0.13 mmol), and KI (14.7 mg, 0.09 mmol) as raw materials, dissolve them in acetonitrile (4.0 mL), and obtain YK-1403 (96.0 mg, 0.11 mmol, 22.79%) according to the method for synthesizing YK-1401. C 54 H 81 N3O5S, MS(ES): m / z(M+H + ) 884.6。

[0260] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.46 (d J = 7.6 Hz, 1H), 8.21 – 8.12(m, 3H), 7.89 – 7.41 (m, 5H), 6.72 (d, J = 7.3 Hz, 1H), 4.47 (m, 1H), 4.13(m, 2H), 3.78 (t, J = 5.0 Hz, 4H), 2.47 – 2.32 (m, 4H),2.01 (m, 2H), 1.60 –1.25 (m, 48H), 0.89(m, 9H).

[0261] 4. Synthesis of YK-1404

[0262] The synthesis route is as follows:

[0263]

[0264] Step 1: Synthesis of YK-1404

[0265] Using YK-1403-PM1 (220.0 mg, 0.40 mmol), 6-bromohexanoic acid 2-octyldecyl ester (202 mg, 0.45 mmol), K2CO3 (244.6 mg, 1.77 mmol), Cs2CO3 (43.2 mg, 0.13 mmol), and KI (14.7 mg, 0.09 mmol) as raw materials, dissolve them in acetonitrile (4.0 mL), and obtain YK-1404 (82.0 mg, 0.09 mmol, 22.64%) according to the method for synthesizing YK-1401. C 55 H 83N3O5S, MS(ES): m / z(M+H + ) 898.6。

[0266] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.46 (d J = 7.6 Hz, 1H), 8.21 – 8.12(m, 3H), 7.89 – 7.41 (m, 5H), 6.72 (d, J = 7.3 Hz, 1H), 4.13 (m, 2H), 3.86 - 3.78 (m, 6H), 2.47 – 2.32 (m, 4H), 2.01 - 1.89 (m, 3H), 1.60 – 1.26 (m, 48H), 0.88(m, 9H).

[0267] 5. Synthesis of YK-1405

[0268] The synthesis route is as follows:

[0269]

[0270] Step 1: Synthesis of YK-1405-PM1

[0271] Dissolve m-aminobenzoic acid (5.00 g, 36.46 mmol) and triethylamine (11.07 g, 109.38 mmol) in DMF (10.0 mL). Slowly add Boc2O (9.55 g, 43.75 mmol) under ice bath and stir at room temperature for 6 hours. After the reaction is completed, purify directly by reverse chromatography (water / acetonitrile) to obtain YK-1405-PM1 (6.58 g, 27.73 mmol, 76.07%). C 12 H 15 NO4, MS(ES): m / z(M+H + ) 238.1。

[0272] Step 2: Synthesis of YK-1405-PM2

[0273] 5-Amino-2-methylbenzothiazole (1.00 g, 6.09 mmol), NH4SCN (1.85 g, 24.36 mmol), acetic acid (40.0 mL) were stirred at room temperature for 1 h, then bromine (1.27 g, 7.92 mmol) was slowly added, and the reaction was carried out at 45 °C for 1 day. After the reaction was completed, it was poured into ammonia water (160.0 mL), ethyl acetate (150.0 mL) was added, stirred and filtered, then extracted with ethyl acetate (150.0 mL × 2). The organic phases were combined and washed with saturated brine (100.0 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was slurried with DCM (10.0 mL), filtered, and the filter cake was washed twice with DCM (10.0 mL) to obtain YK-1405-PM2 (326.0 mg, 1.47 mmol, 24.19%). C9H7N3S2, MS(ES): m / z(M+H + ) 222.1.

[0274] Step 3: Synthesis of YK-1405-PM3

[0275] Using YK-1405-PM2 (326.0 mg, 1.47 mmol), YK-1405-PM1 (419.4 mg, 1.77 mmol), EDCI (564.8 mg, 2.94 mmol), DMAP (90.0 mg, 0.74 mmol) and DCM (20.0 mL) as raw materials, according to the method for synthesizing YK-1401-PM1, YK-1405-PM3 (365.4 mg, 0.83 mmol, 56.31%) was obtained. C 21 H 20 N4O3S2, MS(ES): m / z(M+H + ) 441.1.

[0276] Step 4: Synthesis of YK-1405-PM4

[0277] Dissolve YK-1405-PM3 (365.4 mg, 0.83 mmol) in THF (10.0 mL). Slowly add 4 M HCl / dioxane (3.4 mL, 13.28 mmol), and then stir the reaction at room temperature for 24 hours. After the reaction is completed, add ethyl acetate (50.0 mL) and saturated sodium bicarbonate solution to adjust the pH to 8. Wash with ethyl acetate (30.0 mL×2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to remove the solvent to obtain YK-1405-PM4 (403.4 mg, 1.18 mmol, crude). C 16 H 12 N4OS2, MS(ES): m / z(M+H + )341.1。

[0278] Step 5: Synthesis of YK-1405-PM5

[0279] Using YK-1405-PM4 (201.7 mg, calculated as 0.59 mmol), 2-octyldecyl 6-bromohexanoate (318.2 mg, 0.71 mmol), K2CO3 (245.7 mg, 1.78 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1405-PM5 (196.4 mg, 0.28 mmol, 46.88%) is obtained. C 40 H 58 N4O3S2, MS(ES): m / z(M+H + )707.4。

[0280] Step 6: Synthesis of YK-1405

[0281] Using YK-1405-PM5 (196.4 mg, 0.28 mmol), tetradecyl bromide (155.3 mg, 0.56 mmol), K2CO3 (115.2 mg, 0.83 mmol), Cs2CO3 (36.2 mg, 0.11 mmol), KI (13.8 mg, 0.08 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1405 (138.0 mg, 0.15 mmol, 54.99%) is obtained. C 54 H 86 N4O3S2, MS(ES): m / z(M+H + )903.6。

[0282] 11H NMR (CDCl3, 400 MHz, 298 K) δ 7.76 (dd, J = 8.2 Hz, 1H), 7.46 (dd, J = 7.8 Hz, 1H), 7.39 – 7.29 (m, 4H), 3.86 – 3.78 (m, 6H), 2.79 (s, 3H), 2.32 (t, J = 7.6 Hz, 2H), 1.89 (m, 1H), 1.53 – 1.26 (m, 60H), 0.90 (s, 9H).

[0283] 6. Synthesis of YK-1406

[0284] The synthetic route is as follows:

[0285]

[0286] Step 1: Synthesis of YK-1406-PM1

[0287] Using YK-1405-PM4 (200.0 mg, 0.59 mmol), 3-hexylnonyl 6-bromohexanoate (320.0 mg, 0.79 mmol), K2CO3 (245.7 mg, 1.78 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1406-PM1 (216.0 mg, 0.32 mmol, 55.29%) was obtained. C 37 H 52 N4O3S2, MS(ES): m / z(M+H + ) 665.4.

[0288] Step 2: Synthesis of YK-1406

[0289] Using YK-1406-PM1 (216.0 mg, 0.32 mmol), decyl 4-bromobutyrate (180.0 mg, 0.59 mmol), K2CO3 (115.2 mg, 0.83 mmol), Cs2CO3 (36.2 mg, 0.11 mmol), KI (13.8 mg, 0.08 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1406 (166.0 mg, 0.19 mmol, 57.33%) was obtained. C 51 H 78 N4O5S2, MS(ES): m / z(M+H + ) 891.5.

[0290] 1 1H NMR (CDCl3, 400 MHz, 298 K) δ 7.76 (dd, J = 8.2 Hz, 1H), 7.46 (dd,J = 7.8 Hz, 1H), 7.39 – 7.29 (m, 4H), 4.13 (t, J = 7.6 Hz,4H), 3.96 –3.88 (m,4H), 2.79 (s, 3H), 2.47-2.32 (m, 4H), 2.02 – 1.99 (m, 2H), 1.70 – 1.39 (m,45H), 0.88 (s, 9H).

[0291] 7. Synthesis of YK-1407

[0292] The synthetic route is as follows:

[0293]

[0294] Step 1: Synthesis of YK-1407-PM1

[0295] Using YK-1405-PM5 (196.0 mg, 0.28 mmol), decyl 4-bromobutyrate (155.3 mg, 0.56 mmol), K2CO3 (115.2 mg, 0.83 mmol), Cs2CO3 (36.2 mg, 0.11 mmol), KI (13.8 mg, 0.08 mmol) and acetonitrile (4.0 mL) as raw materials, YK-1407 (168.4 mg, 0.18 mmol, 64.95%) was obtained according to the method for synthesizing YK-1401. C 54 H 84 N4O5S2, MS(ES): m / z(M+H + )933.6。

[0296] 11H NMR (CDCl3, 400 MHz, 298 K) δ 7.76 (dd, J = 8.2 Hz, 1H), 7.46 (dd, J = 7.8 Hz, 1H), 7.39 – 7.29 (m, 4H), 4.23 (t, J = 7.6 Hz, 2H), 3.96 – 3.88 (m, 6H), 2.89 (s, 3H), 2.57 (dd, J = 5.2 Hz, 2H), 2.42 (dd, J = 4.2 Hz, 2H), 2.12 – 1.99 (m, 3H), 1.70 – 1.39 (m, 50H), 0.98 (s, 9H).

[0297] 8. Synthesis of YK-1408

[0298] The synthetic route is as follows:

[0299]

[0300] Step 1: Synthesis of YK-1408-PM1

[0301] Using YK-1405-PM4 (201.7 mg, 0.59 mmol), heptadec-9-yl 6-bromohexanoate (256.8 mg, 0.59 mmol), K2CO3 (245.6 mg, 1.78 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1408-PM1 (235.6 mg, 0.34 mmol, 57.40%) was obtained. C 39 H 56 N4O3S2, MS(ES): m / z(M+H + ) 693.4.

[0302] Step 2: Synthesis of YK-1408

[0303] Using YK-1408-PM1 (235.6 mg, 0.34 mmol), decyl 4-bromobutyrate (418.1 mg, 1.36 mmol), K2CO3 (282.2 mg, 2.04 mmol), Cs2CO3 (44.3 mg, 0.14 mmol), KI (16.9 mg, 0.10 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1408 (183.4 mg, 0.20 mmol, 58.62%) was obtained. C 53 H 82N4O5S2, MS(ES): m / z(M+H + ) 919.6。

[0304] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.76 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 6.8 Hz, 1H), 7.39 – 7.29 (m, 4H), 4.57 (t, J = 6.2 Hz, 1H), 4.23 (t, J = 5.8 Hz, 2H), 3.88 (t, J = 5.3 Hz, 4H), 2.89 (s, 3H), 2.57 (t, J = 5.2 Hz, 2H), 2.42 (t, J = 4.8 Hz, 2H), 2.11 (t, J = 4.6 Hz, 2H), 1.70 – 1.68 (m, 6H), 1.59 – 1.36 (m, 44H), 0.98 (d, J = 4.2 Hz, 9H).

[0305] 9. Synthesis of YK-1409

[0306] The synthetic route is as follows:

[0307]

[0308] Step 1: Synthesis of YK-1409-PM1

[0309] 5-Methoxybenzothiazol-2-amine (2.00 g, 11.10 mmol), quinolin-8-ol (1.61 g, 11.10 mmol), 4-(difluoromethoxy)benzaldehyde (1.91 g, 11.10 mmol) and ethanol (20.0 mL) were reacted at 80 °C for 2 days. After the reaction was completed, it was concentrated, ethyl acetate (50.0 mL) and water (50.0 mL) were added, stirred and separated, then extracted with ethyl acetate (50.0 mL × 2), the organic phases were combined and washed with saturated brine (50.0 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1409-PM1 (1.15 g, 2.40 mmol, 21.61%). C 25 H 19 F2N3O3S, MS(ES): m / z(M+H + ) 480.1。

[0310] Step 2: Synthesis of YK-1409

[0311] Using YK-1409-PM1 (300.0 mg, 0.63 mmol), decyl 4-bromobutyrate (769.0 mg, 2.50 mmol), K2CO3 (691.8 mg, 5.01 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1409 (243.3 mg, 0.26 mmol, 41.27%) was obtained. C 53 H 71 F2N3O7S, MS(ES): m / z(M+H + ) 932.5

[0312] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.94 (d J = 7.6 Hz, 1H), 8.47 (d, J = 7.3 Hz, 1H), 8.19 – 8.14 (m, 2H), 7.69 – 7.56 (m, 2H), 7.47 – 7.41 (m, 4H), 7.20 (d, J = 7.0 Hz, 1H), 7.09 (d, J = 5.3 Hz, 2H), 5.39 (t, J = 5.0 Hz, 1H), 4.53 (t, J = 4.8 Hz, 2H), 4.33 (t, J = 4.6 Hz, 4H), 4.07 (s, 3H), 3.65 (t, J = 4.5 Hz, 2H), 2.87 (t, J = 4.2 Hz, 4H), 2.68 (t, J = 4.0 Hz, 4H), 1.80 (t, J = 3.8 Hz, 4H), 1.63 (t, J = 3.6 Hz, 4H), 1.50 – 1.46 (m, 24H), 1.08 (d, J = 3.2 Hz, 6H).

[0313] 10. Synthesis of YK-1410

[0314] The synthesis route is as follows:

[0315]

[0316] Step 1: Synthesis of YK-1410

[0317] Using YK-1409-PM1 (300.0 mg, 0.63 mmol), linoleic acid (315.8 mg, 1.13 mmol), EDCI (239.9 mg, 1.25 mmol), DMAP (38.2 mg, 0.31 mmol) and dichloromethane (10.0 mL) as raw materials, according to the method for synthesizing YK-1401-PM1, YK-1410 (256.4 mg, 0.26 mmol, 40.80%) was obtained. C 61 H 79 F2N3O5S, MS(ES): m / z(M+H + ) 1004.6.

[0318] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.78 (d J = 7.6 Hz, 1H), 8.31 (d, J = 7.3 Hz, 1H), 7.97 – 7.94 (m, 2H), 7.54 – 7.21 (m, 6H), 7.00 (d, J = 7.0 Hz, 1H), 6.89 (d, J = 5.3 Hz, 2H), 6.16 (t, J = 5.0 Hz, 1H), 5.43 (t, J = 4.8 Hz, 4H), 5.29 (t, J = 4.6 Hz, 4H), 3.87 (s, 3H), 2.80 (t, J = 4.5 Hz, 4H), 2.52 (t, J = 4.2 Hz, 2H), 2.16 (t, J = 4.0 Hz, 8H), 2.05 (t, J = 3.8 Hz, 2H), 1.66 (t, J = 3.6 Hz, 2H), 1.53 (t, J = 3.6 Hz, 2H), 1.33 – 1.26 (m, 28H), 0.96 (d, J = 3.2 Hz, 6H).

[0319] 11. Synthesis of YK-1411

[0320] The synthesis route is as follows:

[0321]

[0322] Step 1: Synthesis of YK-1411

[0323] Using YK-1409-PM1 (300.0 mg, 0.63 mmol), 6-bromohexanoic acid heptadec-9-yl ester (813.7 mg, 1.88 mmol), K2CO3 (518.8 mg, 3.75 mmol) and DMF (3.0 mL) as raw materials, according to the method for synthesizing YK-1401, YK-1411 (103.8 mg, 0.09 mmol, 14.00%) was obtained. C 71 H 107 F2N3O7S, MS(ES): m / z(M+H + ) 1184.8.

[0324] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.79 (d J = 7.6 Hz, 1H), 8.32 (d, J = 7.3 Hz, 1H), 8.04 – 7.99 (m, 2H), 7.54 – 6.94 (m, 9H), 5.19 (d, J = 7.0 Hz, 1H), 4.52 (d, J = 5.3 Hz, 2H), 4.11 (t, J = 5.0 Hz, 2H), 3.92 (s, 3H), 3.04 (t, J = 4.6 Hz, 2H), 2.37 (t, J = 4.5 Hz, 4H), 1.82 – 1.49 (m, 16H), 1.38 – 1.26 (m, 52H), 0.93 (d, J = 3.2 Hz, 12H).

[0325] 12. Synthesis of YK-1412

[0326] The synthesis route is as follows:

[0327]

[0328] Step 1: Synthesis of YK-1412-PM1

[0329] Using benzothiazol-2-amine (500.0 mg, 3.33 mmol), quinolin-8-ol (483.2 mg, 3.33 mmol), 4-(difluoromethoxy)benzaldehyde (573.0 mg, 3.33 mmol) and ethanol (10.0 mL) as raw materials, according to the method for synthesizing YK-1409-PM1, YK-1412-PM1 (505.9 mg, 1.13 mmol, 33.81%) was obtained. C 24 H 17F2N3O2S, MS(ES): m / z(M+H + ) 450.1.

[0330] Step 2: Synthesis of YK-1412

[0331] Using YK-1412-PM1 (505.9 mg, 1.13 mmol), YK-1404-PM2 (1951.7 mg, 4.50 mmol), K2CO3 (933.4 mg, 6.75 mmol) and DMF (5.0 mL) as raw materials, following the method for synthesizing YK-1401-PM3, YK-1412 (276.7 mg, 0.24 mmol, 21.29%) was obtained. C 70 H 105 F2N3O6S, MS(ES): m / z(M+H + ) 1154.8.

[0332] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.76 (d J = 7.6 Hz, 1H), 8.29 – 8.20 (m, 2H), 8.04 (d, J = 7.3 Hz, 1H), 7.55 – 7.23 (m, 8H), 6.91 (d, J = 7.0 Hz, 2H), 5.21 (d, J = 5.3 Hz, 1H), 4.49 (t, J = 5.0 Hz, 2H), 4.08 (t, J = 4.8 Hz, 2H), 3.06 (t, J = 4.6 Hz, 2H), 2.34 (t, J = 4.6 Hz, 4H), 1.79 – 1.51 (m, 16H), 1.35 – 1.28 (m, 52H), 0.90 (d, J = 3.2 Hz, 12H).

[0333] 13. Synthesis of Compound 23

[0334]

[0335] The synthesis of Compound 23 refers to the synthesis route of Compound 23 in WO2021237055A1, and 30 mg of Compound 23 was obtained.

[0336] 14. Synthesis of C12-TLRa

[0337]

[0338] The synthesis of C12-TLRa refers to the synthesis route on the fourth page of the main text of Nature Nanotechnology Volume 18 September 2023 1105–1114, and 85 mg of C12-TLRa is obtained.

[0339] Example 2: Preparation of mRNA Lipid Composition

[0340] A) Preparation of Fluc DNA, eGFP DNA, and OVA DNA Templates

[0341] 1) The luciferase (Luciferase protein CDS), green fluorescent protein (GFP), and ovalbumin (OVA) circular plasmids were digested with EcoRV and ligated to the pVAX1 vector (purchased from Thermo Fisher Scientific).

[0342] 2) The plasmid constructed on the pVAX1 vector in step 1) was mixed with 50 μL of Escherichia coli competent cell Stbl2 (purchased from Thermo Fisher Scientific), and the mixture was incubated on ice for 30 minutes, heat-shocked at 42 °C for 90 seconds, immediately placed back on ice, and incubated on ice for 2 minutes.

[0343] 3) 400 μL of LB medium (purchased from Thermo Fisher Scientific) was added, and the mixture was incubated with slow shaking on a shaker at 30 °C for 45 - 60 minutes.

[0344] 4) 50 - 100 μL of the bacterial solution was spread on an LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yeasen Biotech Co., Ltd.), and incubated upside down at 37 °C overnight.

[0345] 5) The obtained monoclonal colony plate was sequenced to verify its correctness, and the monoclonal colony with correct sequencing was picked and incubated with slow shaking on a shaker at 30 °C overnight.

[0346] 6) The plasmid was extracted using an endotoxin-free large-scale plasmid extraction kit (purchased from Yeasen Biotech Co., Ltd.).

[0347] 7) The extracted plasmid was digested with a restriction enzyme to obtain a linearized plasmid for use as a transcription template. The specific enzyme digestion process steps are shown in steps ① - ③.

[0348] Step ①: Take 1 mg of the luciferase circular plasmid and digest it at 37 °C for 4 hours (BspQ Ⅰ enzyme, purchased from Yeasen Biotech Co., Ltd.) to obtain a linearized DNA transcription template (the enzyme digestion system is shown in Table 1).

[0349] Table 1

[0350]

[0351] Step ② After the reaction is completed, anhydrous ethanol and sodium acetate are added successively. According to the volume ratio of V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 = 1:3:1, anhydrous ethanol and 3M sodium acetate are added, and the mixture is placed at -20 °C for precipitation for 1 hour. Then, it is centrifuged at 12000 rmp to retain the precipitate;

[0352] Step ③ The precipitate in Step ② is washed twice with 70% ethanol. The centrifuged material is placed in an oven at 55 °C and dried for 10 minutes, and then 1.7 mL of injection water is added for dissolution;

[0353] The concentration of the linearized plasmid in the dissolution solution is 500 ng / µL, the linearization ratio is more than 90%, and the purification and recovery efficiency is 85%.

[0354] B) Preparation of Fluc mRNA, eGFP mRNA and OVA mRNA

[0355] 1) Co-transcriptional capping reaction:

[0356] Using the Fluc DNA, eGFP DNA and OVA DNA prepared in A) as templates, NTP solution (NTPs) and Cap1 cap analog (product number: 10678ES80, purchased from Yeasen Biotech Co., Ltd.) as starting materials, mRNA is transcribed and synthesized by T7 RNA polymerase. The specific reaction system is shown in Table 2. The prepared reaction system is placed in a constant temperature incubator at 37 °C and shaken for reaction for 3 h. The above Cap1 cap analog is Cap1-GAG, which has the structure of m7G (5') ppp (5') (2'-OMeA) pG, and its molecular formula is C 32 H 43 N 15 O 24 P4.

[0357] The co-transcriptional capping reaction system is shown in Table 2.

[0358] Table 2

[0359]

[0360] Note: All the above reagents are purchased from Yeasen Biotech Co., Ltd.

[0361] 2) Digest the template DNA:

[0362] DNase Ⅰ (purchased from Yeasen Biotech Co., Ltd.) is added to the co-transcriptional capping reaction system after the reaction in Step 1) is completed to make the final concentration 1 U / μg of the linearized plasmid. After mixing, it is centrifuged and digested at 37 °C for 1 hour to obtain the co-transcriptional capping product.

[0363] 3) Purification by lithium chloride precipitation method:

[0364] Purify the co-transcriptionally capped product obtained in step 2) above by the lithium chloride precipitation method as follows:

[0365] Step ① Add lithium chloride: Add a lithium chloride solution (purchased from Thermo Fisher Scientific) to the product of step 2) above to a final concentration of 2.8 M, and precipitate at low temperature for 2 hours;

[0366] Step ② Precipitation: Centrifuge at 12,000 rmp for 15 minutes and retain the precipitate;

[0367] Step ③ Washing: Wash twice with 75% ethanol, and dissolve with water for injection to obtain an mRNA solution. The purified mRNA solution is stored at -80 °C.

[0368] Example 3: Effects of different addition amounts of adjuvant lipids on the LNP-mRNA composition

[0369] This example investigated the effects of adjuvant lipids on the LNP composition.

[0370] Experimental procedure:

[0371] Weigh YK-009, YK-1407, DSPC, cholesterol, and DMG-PEG2000 according to the proportions in Table 3 and dissolve them in ethanol to prepare an ethanol lipid solution. Dilute eGFP-mRNA in citrate buffer (pH = 4 - 5) to obtain an mRNA aqueous solution. Use a microfluidic device to mix the ethanol lipid solution with the Fluc mRNA aqueous solution prepared from different capping structures above at a flow rate of 10 mL / min and a volume ratio of 1:3 to prepare LNP with a weight ratio of total lipid to mRNA of approximately 15:1. After diluting the obtained liposomes to 10 times the volume with PBS, ultrafiltrate to remove ethanol using a 300 KDa ultrafiltration tube. Then, make up the volume to a certain volume with PBS. Finally, filter the lipid nanoparticles through a 0.2 μm sterile filter to obtain the LNP drug composition with or without adjuvant lipids added. Use dynamic light scattering and a Malvern laser particle size analyzer to measure the particle size and polydispersity index (PDI). Take 10 μL of the liposome solution, dilute it to 1 mL with RNase-free deionized water, add it to the sample cell, and repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25 °C. According to the manufacturer's instructions, use the Quant it Ribogreen RNA Quantification Assay Kit (Thermo Fisher Scientific, UK) to determine the encapsulation efficiency of the lipid nanoparticles. The particle size, PDI, and encapsulation rate results of the compositions with different formulations are shown in Table 3.

[0372] Table 3

[0373]

[0374] The results showed that when YK-1407 was used to partially replace the component of cationic lipid YK-009 in the LNP composition at 1%, 2.5%, 5.0%, 10.0%, and 15.0%, the particle size, PDI, and encapsulation efficiency of the obtained LNP pharmaceutical composition were all within the qualified range (particle size < 200 nm, PDI < 0.3, encapsulation efficiency > 85%). When the adjuvant content was 5 mol%, the particle size, PDI, and encapsulation efficiency reached the best state, and then the adjuvant lipid was studied at an adjuvant lipid ratio of 5%.

[0375] Example 4: Effects of Different Adjuvant Lipids on LNP-mRNA Composition

[0376] The structures of RLRs adjuvant lipid compounds are shown in Tables 4-1 and 4-2.

[0377] Table 4-1

[0378]

[0379] Table 4-2

[0380]

[0381] Experimental procedure:

[0382] According to the preparation method of Example 3, the RLRs adjuvant lipid compounds in Tables 4-1 and 4-2 were used to partially replace the proportion of YK-009 in the LNP composition at a ratio of 5 mol%, that is, the Fluc-mRNA-LNP pharmaceutical composition was prepared by mixing YK-009, DSPC, cholesterol, DMG-PEG2000, and adjuvant lipid at a molar ratio of 45:10:38.5:1.5:5. Evaluation method: First, the particle size, polydispersity index (PDI), and encapsulation efficiency were measured according to the method of Example 3; then the pharmaceutical composition was added to the cell culture medium in a 96-well plate, and after continuing to culture for 24 hours, the corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected by the IVIS fluorescence detection system; finally, 10 μL of CCK-8 solution was added to each well of the above-mentioned well plate after 24 hours of culture, and after incubating the culture plate in the incubator for 1 hour, the absorbance at 450 nm was measured by a microplate reader to detect the cell viability. The particle size and PDI detection results of the LNP composition containing different adjuvants are shown in Table 5.

[0383] Table 5

[0384]

[0385] The results showed that:

[0386] (1)Physicochemical properties of mRNA-LNP containing adjuvant lipids: In this application, the adjuvant lipids YK-1401 to 1412, the adjuvant lipid C12-TLRa and compound 23 disclosed in the prior art can all be used to prepare good mRNA-LNP compositions by replacing YK-009 at a ratio of 5%. The particle sizes of all lipid nanoparticles are between 67 and 89 nm, the PDI values are between 0.02 and 0.15, and the encapsulation rates are all above 85%;

[0387] (2)In vitro transfection activity of mRNA-LNP containing adjuvant lipids: There are significant differences in the relative fluorescence intensity (translation efficiency of mRNA) of the LNP compositions prepared above. The mRNA-LNP compositions prepared from YK-1406, YK-1407 and YK-1410 have significantly higher relative fluorescence intensity than those of the groups of YK-1401, YK-1402, YK-1403, YK-1404 and YK-1411, and are also significantly higher than the LNP group without adjuvant lipids and the mRNA-LNP groups added with C12-TLRa and compound 23;

[0388] (3)Cell activity of mRNA-LNP containing adjuvant lipids: There are significant differences in the cell viability (cytotoxicity) of the LNP compositions prepared above. The mRNA-LNP compositions prepared from YK-1406, YK-1407 and YK-1410 have significantly lower cytotoxicity than those of the groups of YK-1401, YK-1404 and YK-1405, and are at the same level as the mRNA-LNP group without adjuvant lipids and the mRNA-LNP groups added with C12-TLRa and compound 23;

[0389] Example 5: Animal expression of LNP-mRNA composition added with RLRs adjuvant lipid

[0390] Experimental procedure:

[0391] The LNP preparations containing 10 μg Fluc-mRNA with different adjuvant lipids prepared according to Example 4 were injected into female BALB / C mice aged 4 to 6 weeks and weighing 17 to 19 g via the tail muscle. At a specific time point (6 hours) after administration, the mice were intraperitoneally injected with a fluorescence imaging substrate. The mice were allowed to move freely for 5 minutes, and then the average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by the LNP in the mice was detected by an IVIS Spectrum small animal in vivo imager.

[0392] After sampling, the mice were euthanized with carbon dioxide, and then dissected to precisely isolate their internal organs: liver, spleen, and lung. The total radiation intensity (corresponding to the fluorescence expression intensity) of the proteins expressed by the mRNA carried by the LNP in each organ of the mice was detected by the IVIS Spectrum small animal in vivo imager. The results of the in vivo imaging of the mice are shown in Table 6.

[0393] Table 6

[0394]

[0395] Experimental results:

[0396] It can be seen that for the LNP formulations prepared from YK-1406, YK-1407, and YK-1410, the expression levels of the delivered mRNA in the injection site, abdominal cavity, liver, and spleen of the mice were significantly higher than those of the LNP without adjuvant lipid and the LNP with the representative adjuvant lipid compound 23 in the prior art. For example, in the mRNA-LNP group with YK-1410 added, the expression level at the injection site of the mice could reach 1.5 times that of the LNP without adjuvant lipid, and the expression in the spleen of the mice could reach 1.9 times that of the LNP with compound 23 added.

[0397] There are a large number of APC cells in the spleen and muscle (injection site). By increasing the expression levels of the delivered mRNA in the spleen and muscle, it is possible to rapidly induce an immune response in vivo and generate antibodies for the mRNA vaccine. Without changing the vaccine components, the preventive and therapeutic effects can be significantly improved, which has important clinical significance.

[0398] In addition, for the LNP formulations containing Fluc-mRNA with adjuvant lipid added, the expression differences in different organs of the mice are very large. The LNP formulations prepared from YK-1406, YK-1407, YK-1410, compound 23, and without adjuvant lipid added are all expressed in the liver and spleen, but not expressed in the lung.

[0399] Compared with the structurally similar adjuvant lipid YK-1408, for the mRNA-LNP formulations prepared from YK-1407 and YK-1410, the expression intensities of the mRNA in the liver and spleen of the mice were significantly increased. For example, for the LNP formulation with YK-1410 added, the expression level of the mRNA at the injection site could reach 1.8 times that of the LNP formulation with YK-1408 added, and the expression level in the spleen reached 3.0 times.

[0400] Example 6: Stimulation of cytokines IFN-γ and TNF-α in serum by the mRNA-LNP composition containing RLRs adjuvant lipid

[0401] Interferon-γ (IFN-γ) is an important cytokine, mainly produced by activated T cells and natural killer (NK) cells. It plays a key role in the immune response and has multiple functions such as antiviral, antitumor, immunomodulatory, and promoting inflammatory responses, and can treat various diseases. Tumor necrosis factor-α (TNF-α) is a key cytokine that links inflammation and the immune system, mainly produced by macrophages, and has multiple biological functions such as cell proliferation, metabolic activation, inflammatory response, and apoptosis. Detecting the stimulation of cytokines IFN-γ and TNF-α by the mRNA-LNP composition containing adjuvant lipids can reflect the improvement effect of adjuvant lipids on the innate immunity of the mRNA-LNP composition.

[0402] Experimental procedure:

[0403] Female BALB / C mice aged 4 - 6 weeks and weighing 17 - 19 g were euthanized by exsanguination from the orbital sinus 6 hours after intramuscular injection of the OVA-mRNA-LNP (10 µg) composition prepared according to the method of Example 4 to obtain as much serum as possible. The levels of IFN-γ and TNF-α in the serum were measured by ELISA. At the same time, mice injected with an equal volume of blank lipid solution were set as the blank group.

[0404] ELISA assay: Mouse IFN-γ and TNF-α were detected in mouse serum using a standard ELISA according to the manufacturer's instructions.

[0405] The comparison of the stimulating effects of the mRNA-LNP composition containing adjuvant lipids on cytokines IFN-γ and TNF-α is shown in Table 7.

[0406] Table 7

[0407]

[0408] Experimental results:

[0409] As shown in Table 7 and Figure 3As shown, there are significant differences in the levels of IFN-γ and TNF-α cytokines in the serum of mice 6 hours after injection with different RLRs adjuvant lipid-containing mRNA-LNP compositions in the present invention. Among them, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, YK-1408-LNP, and YK-1410-LNP is 2.6 times, 3.2 times, 1.9 times, and 3.3 times that of the mRNA-LNP composition of YK-1401-LNP; the serum content of cytokine TNF-α stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, YK-1408-LNP, and YK-1410-LNP is 2.0 times, 1.9 times, 1.6 times, and 2.4 times that of the mRNA-LNP composition added with YK-1403-LNP.

[0410] Compared with the mRNA-LNP composition without adjuvant lipid, the IFN-γ and TNF-α cytokines in the serum of the mRNA-LNP composition containing RLRs adjuvant lipid increase significantly 6 hours after injection. Among them, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, YK-1408-LNP, and YK-1410-LNP is 2.0 times, 2.4 times, 1.5 times, and 2.5 times that of the mRNA-LNP composition without adjuvant lipid; the serum content of cytokine TNF-α stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, YK-1408-LNP, and YK-1410-LNP is 1.6 times, 1.5 times, 1.3 times, and 2.0 times that of the mRNA-LNP composition without adjuvant lipid.

[0411] Compared with the mRNA-LNP composition with compound 23 as the adjuvant lipid in the prior art, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, YK-1408-LNP, and YK-1410-LNP is 1.5 times, 1.8 times, 1.1 times, and 1.9 times that of the mRNA-LNP composition added with the adjuvant lipid of compound 23; the serum content of cytokine TNF-α stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, YK-1408-LNP, and YK-1410-LNP is 1.4 times, 1.3 times, 1.2 times, and 1.7 times that of the mRNA-LNP composition added with the adjuvant lipid of compound 23.

[0412] The experimental results show that by detecting the IFN-γ and TNF-α cytokines stimulated by the mRNA-LNP composition containing adjuvant lipids, it can be demonstrated that the adjuvant lipids of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-LNP composition.

[0413] Example 7: The situation of antigen-specific cytotoxic T cells produced by the spleen stimulated by the mRNA composition containing RLRs adjuvant lipids

[0414] The strength of the T cell effect generated by the tumor mRNA-liposome complex can be judged by detecting antigen-specific CD8 + cytotoxic T cells (CD8 + T cells) in the spleen, which is crucial for the anti-tumor effect of lipid complexes delivering tumor mRNA.

[0415] Experimental procedure:

[0416] 1. Inject the OVA-mRNA-LNP (10 μg) composition with or without adjuvant lipids prepared in Example 6 into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein, injecting once each on days 0, 3, and 8, for a total of 3 injections. At the same time, set the mice injected with an equal volume of blank lipid solution after dilution as the control group, with 3 mice in each parallel group. On the 13th day, the mice were euthanized by cervical dislocation and dissected, and the spleens of the mice were precisely isolated. After preparing single cells, detect the percentage of OVA antigen-specific CD8 + T cells in the total CD8 + T cells by flow cytometry.

[0417] 2. Preparation of single cells

[0418] 1) Grind the isolated spleen tissue to make the spleen tissue single-celled and pass through a cell sieve;

[0419] 2) Add 10 times the volume (about 4 mL) of red blood cell lysate to lyse and remove the red blood cells in the tissue;

[0420] 3) Count the cells and take 5×10 6 cells into a flow tube (ensure that the number of cells taken is the same among each sample);

[0421] 3. Detection of immune cells in spleen tissue

[0422] 1) Add 100 μL of surface antibody MIX (the components of the surface antibody MIX are shown in Table 8) to each single cell suspension, and incubate in the dark at room temperature for 15 minutes (one negative control);

[0423] Detect OVA antigen-specific CD8 +The T cell experimental reagents and their sources are shown in Table 8.

[0424] Table 8

[0425]

[0426] 2), Add 2 mL of 1× RBC Lysis Buffer, incubate in the dark for 10 minutes, centrifuge at 500 g for 5 min, and discard the supernatant.

[0427] 3), Add 2 mL of 1× RBC Lysis Buffer, centrifuge at 500 g for 5 min, and discard the supernatant.

[0428] 4), Add 200 μL of PBS, transfer to a clean labeled EP tube, (filter through a 200-mesh nylon net), and detect on the flow cytometer Cytoflex S. Adjust the compensation with single-color compensation microspheres before loading. The detection sequence is as follows:

[0429] CD3 + →CD8 + →APC anti-mouse H-2Kb bound to SIINFEKL

[0430] The proportion of OVA antigen-specific T cells stimulated by the mRNA composition containing RLRs adjuvant lipid and the RNA composition without agonist cationic lipid is shown in Table 9.

[0431] Table 9

[0432]

[0433] As shown in Table 9 and Figure 4 as shown, compared with the mRNA-LNP composition without adjuvant lipid, after three inoculations in mice, the content of CD8 + T cells in the spleen cells of mice was significantly increased by the mRNA-LNP composition containing RLRs adjuvant lipid. Among them, the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, and YK-1410-LNP stimulated the production of CD8 + T cell ratios were 1.9 times, 1.6 times, and 2.6 times that of the mRNA-LNP composition without adjuvant lipid. Compared with the mRNA-LNP composition with compound 23 as the adjuvant lipid in the prior art, the mRNA-LNP compositions added with YK-1406-LNP and YK-1410-LNP stimulated the production of CD8 + T cell ratios were 1.3 times and 1.7 times that of the mRNA-LNP composition added with compound 23 adjuvant lipid.

[0434] The experimental results show that by detecting the generation of antigen-specific cytotoxic T cells in the spleen of mice stimulated by the mRNA composition containing RLRs adjuvant lipid, it can be demonstrated that the mRNA vaccine containing RLRs adjuvant lipid can produce a very strong T cell effect in the spleen of mice.

[0435] Example 8: Stimulation of spleen cytokine IFN-γ by the mRNA-LNP composition containing RLRs adjuvant lipid

[0436] Experimental procedure:

[0437] The OVA-mRNA-LNP (10 µg) composition with or without adjuvant lipid prepared in Example 6 was injected into female C57BL / 6 mice aged 4 - 6 weeks and weighing 17 - 19 g via the tail vein, once every 0, 3, and 8 days, for a total of 3 injections. At the same time, mice injected with the same volume of diluted blank lipid solution were set as the control group, with 3 mice in each parallel group. On the 13th day, the mice were sacrificed by cervical dislocation and dissected, and the spleens of the mice were precisely isolated. After preparing single cells, the secretion of interferon IFN-γ by activated immune cells (such as T cells) in the spleen was detected by ELISPOT.

[0438] ELISPOT counting assay: IFN-γ in mouse spleen cells was detected using a standard ELISPOT assay according to the manufacturer's instructions.

[0439] The comparison of the stimulation of cytokine IFN-γ by the mRNA-LNP composition containing adjuvant lipid is shown in Table 10.

[0440] Table 10

[0441]

[0442] Experimental results:

[0443] As shown in Table 10 and Figure 5As shown, compared with the mRNA-LNP composition without adjuvant lipid, the mRNA-LNP composition containing RLRs adjuvant lipid significantly increased the level of IFN-γ cytokine in mouse splenocytes after three inoculations in mice. Among them, the number of fluorescent spots of cytokines IFN-γ stimulated by the mRNA-LNP compositions added with YK-1406-LNP, YK-1407-LNP, and YK-1410-LNP was 1.5 times, 1.4 times, and 1.7 times that of the mRNA-LNP composition without adjuvant lipid. Compared with the mRNA-LNP composition with compound 23 as the adjuvant lipid in the prior art, the number of fluorescent spots of cytokines IFN-γ stimulated by the mRNA-LNP compositions added with YK-1406-LNP and YK-1410-LNP was 1.1 times and 1.5 times that of the mRNA-LNP composition added with compound 23 adjuvant lipid.

[0444] The experimental results show that by detecting the mRNA-LNP composition containing adjuvant lipid, it can well activate immune cells (such as T cells) in the spleen to secrete and produce interferon IFN-γ, which can prove that the adjuvant lipid of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-LNP composition.

[0445] The present invention designs a series of novel vaccine adjuvant lipids based on RLRs receptor agonists, such as YK-1406, YK-1407, and YK-1410. Compared with the adjuvant lipids disclosed in the prior art, they have the following advantages:

[0446] 1. The chemical structures of the adjuvant lipids designed by the present invention are all different from those of the adjuvant lipids disclosed in the prior art and are brand-new compounds.

[0447] 2. When preparing the mRNA-LNP composition by adding the adjuvant lipid (1% - 20 mol%) of the present invention, the particle size is good (<200 nm), and the particle distribution is uniform PDI (<0.3). Compared with the mRNA-LNP composition without adjuvant lipid, when preparing the mRNA-LNP composition by adding the adjuvant lipid of the present invention, the translation efficiency of mRNA is significantly increased, the cytotoxicity is significantly reduced, and the protein expression levels in the injection site, abdominal cavity, liver, and spleen of mice are significantly improved.

[0448] 3. The mRNA-TLP composition prepared from the adjuvant lipid of the present invention can significantly increase the content of cytokine IFN-γ in mouse serum and spleen, can initiate an immune stimulation program, and significantly improve the innate immunity of the mRNA-LNP composition.

[0449] 4. The mRNA vaccine containing RLRs adjuvant lipid can produce a very strong T cell effect in mice.

[0450] The above has described the present invention in detail, aiming to enable those skilled in this field of technology to understand the content of the present invention and implement it. However, this should not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A compound, a pharmaceutically acceptable salt or a stereoisomer thereof, characterized in that: The structure of the compound is shown in formula (I-1), formula (I-2) or formula (I-3), 、 、 ； G1 is selected from or ; G2 is selected from or , wherein a naphthalene ring or a benzene ring is connected to N; G3 , where O and L 32 connect; L 11 , L 12 , L 21 , L 22 , L 31 , L 32 The same or different, each independently selected from C 1~10 Alkylene or single bond; n1, n2, n3 are the same or different and are independently selected from 0 or 1; m is 1; X1, X2, X3, Y1, Y2, Y3 are the same or different and are each independently selected from NH, O or a single bond; When n1 is 0, L 11 Directly connected to X1 through a covalent bond; when n2 is 0, L 21 Directly connected to X2 through a covalent bond; when n3 is 0, L 31 Directly connected to X3 via a covalent bond; R 11 , R 21 , R 31 the same or different, each independently selected from unsubstituted C having 0 to 2 Z-type C=C double bonds 10~18 Straight chain hydrocarbon or unsubstituted C 14~18 Branched chain alkyl; R 12 , R 22 , R 32 the same or different, each independently selected from unsubstituted C having 0 to 2 Z-type C=C double bonds 10~19 Straight chain hydrocarbon or unsubstituted C 14~18 Branched chain alkyl; R 23 is selected from methyl, amino or H; R 33 , R 34 , R 35 , R 36 are the same or different and are each independently selected from methoxy, ethoxy, amino, halogen or H.

2. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: G1 is .

3. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: G2 is , in which the benzene ring is connected to N.

4. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: R 33 , R 34 , R 35 , R 36 are the same or different and are each independently selected from methoxy or H.

5. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: L 11 , L 21 , L 31 The same or different, each independently selected from C 2~7 Alkylene or a single bond.

6. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: L 12 , L 22 , L 32 The same or different, each independently selected from C 2~7 Alkylene or a single bond.

7. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: R 11 , R 21 , R 31 The same or different, each independently selected from , or .

8. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: R 11 , R 21 , R 31 The same or different, each independently selected from , or .

9. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: R 12 , R 22 , R 32 The same or different, each independently selected from , or .

10. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: R 12 , R 22 , R 32 The same or different, each independently selected from , or .

11. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: The compound of formula (I-1) is selected from any one of the following structures: , , , , wherein each group has the same definition as in claim 1.

12. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: The compound has one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 。 13. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: The compound is compound YK-1406 having the following structure: 。 14. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: The compound is compound YK-1407 having the following structure: 。 15. The compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that: The compound is compound YK-1410 having the following structure: 。 16. A composition, characterized in that The composition comprises a lipid composition, and the lipid composition comprises an adjuvant lipid; the adjuvant lipid comprises the compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt or stereoisomer thereof.

17. The composition according to claim 16, characterized in that The molar ratio of the adjuvant lipid to the lipid composition is 0.1% to 20%.

18. The composition according to claim 16, characterized in that The lipid composition also includes cationic lipids and neutral lipids.

19. The composition according to claim 18, characterized in that The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:

1.

20. The composition according to claim 19, characterized in that The molar ratio of the cationic lipid to the neutral lipid is 4:1 to 6:

1.

21. The composition according to claim 18, characterized in that The cationic lipid is selected from any one or a combination of at least two of the following compounds (1) to (7): (1) A compound represented by formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight chain alkyl; L2 is C 12～25 Branched chain alkyl; (II) (2) A compound represented by formula (III), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is selected from -C(O)O- or -OC(O)-; L2 is selected from -C(O)O- or -OC(O)-; R1 is C 6~25 Straight or branched alkyl; R2 is C 6~25 Straight or branched alkyl; G3 is selected from HO(CH2)2- or HO(CH2)3-; G4 is selected from HO(CH2)2- or HO(CH2)3-; L is selected from -(CH2)2-, -(CH2)3- or -(CH2)4-; (III) (3) A compound represented by formula (IV), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein: G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight or branched alkyl; R2 is C 12~25 Branched alkyl; G3 is selected from HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-; (IV) (4) A compound represented by formula (V), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight or branched alkyl; R2 is C 12~25 Straight or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is selected from -CH3, -CH2CH3 or -CH2CH2OH; (V) (5) A compound represented by formula (VI), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G 1 and G 2 Each independently is C6-C 10 Alkylene; G 3 C1-C 12 Alkylene; R 1 and R 2 Each independently selected from C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 Selected from OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 Hydrocarbon; R 5 Selected from H or C1-C6 hydrocarbon group; (WE) (6) A compound represented by formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from -(CH2) n Q or -(CH2) n CHQR; Q is selected from -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocycle; n is selected from 1, 2 or 3; R is C 1-8 Alkyl; X is selected from H or C 1-8 alkyl; (VII) (7) A compound represented by formula (VIII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, (VIII)。 22. The composition according to claim 18, characterized in that The neutral lipid is selected from any one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide or sterol and derivatives thereof.

23. The composition according to claim 16, characterized in that The lipid composition further comprises any one or a combination of at least two of cationic lipids, neutral lipids, structural lipids or polymer-conjugated lipids; The cationic lipid is selected from any one of YK-009, YK-401, YK-305, ALC0315, SM102 or DLIN-MC3-DMA, or a combination of at least two thereof; 、 、 、 、 、 ； The neutral lipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O -Octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl -sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl Any one of acylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine, or a combination of at least two thereof; The structured lipid is selected from any one or a combination of at least two of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol or corticosteroids; The polymer conjugated lipid is selected from any one of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000 or methoxy polyethylene glycol ditetradecyl acetamide or a combination of at least two thereof.

24. The composition according to claim 23, characterized in that The lipid composition includes adjuvant lipids, cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids.

25. The composition according to claim 24, characterized in that The molar ratio of the adjuvant lipid, cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is (1-20):(25-75):(5-25):(15-65):(0.5-10).

26. The composition according to claim 25, characterized in that The molar ratio of the adjuvant lipid, cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is (1-20):(25-75):(5-20):(15-65):(0.2-10).

27. The composition according to claim 16, characterized in that The composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm-300nm; the polydispersity coefficient of the nanoparticle preparation is ≤50%.

28. The composition according to claim 27, characterized in that The average particle size of the nanoparticle preparation is 50nm-200nm; the polydispersity coefficient of the nanoparticle preparation is ≤20%.

29. The composition according to claim 21, characterized in that The cationic lipid may also include one or more other ionizable lipid compounds.

30. The composition according to claim 16, characterized in that The composition also includes a therapeutic and / or prophylactic agent.

31. The composition according to claim 30, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 10:1 to 30:

1.

32. The composition according to claim 31, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 12.5:1 to 25:

1.

33. The composition according to claim 32, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 14:1 to 20:

1.

34. The composition according to claim 30, characterized in that The therapeutic agent and / or preventive agent includes any one of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

35. The composition according to claim 30, characterized in that The therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

36. The composition according to claim 34, characterized in that The therapeutic and / or prophylactic agent is a nucleic acid.

37. The composition according to claim 36, characterized in that The therapeutic and / or prophylactic agent is RNA.

38. The composition according to claim 36, characterized in that The therapeutic and / or prophylactic agent is DNA.

39. The composition according to claim 37, characterized in that The RNA is selected from any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA or messenger RNA.

40. The composition according to claim 39, characterized in that The RNA is messenger RNA.

41. The composition according to claim 16, characterized in that The composition may further comprise one or more pharmaceutically acceptable excipients.

42. Use of a compound as described in any one of claims 1 to 15, a pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described in any one of claims 16 to 41 in the preparation of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide or a protein drug.

43. Use of a compound as described in any one of claims 1-15, a pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described in any one of claims 16-41 in the preparation of a medicament for treating a disease or condition based on an RLRs receptor agonist in a mammal in need thereof.

44. The use according to any one of claims 42-43, characterized in that The subject of administration of the drug is human.

45. The use according to any one of claims 42-43, characterized in that The drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

46. ​​The use according to claim 45, characterized in that The drug is administered subcutaneously.

47. The use according to any one of claims 42-43, characterized in that The dosage of the drug is 0.001-10 mg / kg.

Images