Pharmaceutical composition containing cationic lipid and use thereof

Abstract

A pharmaceutical composition containing a cationic lipid and the use thereof. Specifically, provided are a pharmaceutical composition comprising a carrier, the carrier comprising a cationic lipid, and the molar percentage of the cationic lipid to the carrier being greater than or equal to 10% and less than 50%.

Description

A pharmaceutical composition containing cationic lipid and its use Technical Field

[0001] The present invention belongs to the field of medicine and relates to the preparation of a pharmaceutical composition containing cationic lipids and its use. Background Art

[0002] Nucleic acid-based drugs, such as messenger RNA (mRNA), antisense oligonucleotides, small interfering RNA (siRNA), plasmids, etc., have broad application prospects. How to safely and effectively deliver them to target organs and target cells in the body is a difficult problem that restricts the development of this technology.

[0003] Currently, nucleic acid drug delivery vectors can be divided into two categories: viral vectors and non-viral vectors. Lipid nanoparticle-mediated nucleic acid drug delivery is the main method of non-viral delivery vectors.

[0004] Lipid nanoparticles have been demonstrated as excellent carriers of nucleic acids for treating various diseases in gene therapy and vaccine applications. Lipid nanoparticles, formed from cationic lipids and other auxiliary lipids such as cholesterol, phospholipids, and PEGylated lipids, encapsulate nucleic acids, protecting them from degradation, promoting cellular uptake, and reducing immune responses. Furthermore, lipid nanoparticles offer other advantages for cellular delivery of bioactive components, including good targeting, minimal side effects, excellent stability, and high transfection efficiency.

[0005] Although lipid nanoparticles have demonstrated advantages in delivery, safety, efficacy, and specificity remain to be improved. There remains a need to develop improved cationic lipid compositions that facilitate delivery of therapeutic and / or prophylactic agents, such as nucleic acids, to cells.

[0006] Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition comprising a carrier, wherein the carrier comprises a cationic lipid, wherein the cationic lipid comprises a compound represented by Formula I or a pharmaceutically acceptable salt thereof, wherein the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%.

[0008] In some embodiments, the cationic lipid in the pharmaceutical composition accounts for 15-49% of the molar amount of the carrier, including but not limited to 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or any value therebetween.

[0009] In some embodiments, the cationic lipid in the pharmaceutical composition accounts for 42-49% of the molar amount of the carrier.

[0010] In some embodiments, the cationic lipid in the pharmaceutical composition comprises 45% by molar amount of the carrier.

[0011] In some embodiments, the cationic lipid in the pharmaceutical composition comprises 46% of the molar amount of the carrier.

[0012] In some embodiments, the cationic lipid in the pharmaceutical composition comprises 47% by molar amount of the carrier.

[0013] In some embodiments, the cationic lipid in the pharmaceutical composition comprises 48% by molar amount of the carrier.

[0014] In some embodiments, the cationic lipid in the pharmaceutical composition comprises 49% by molar amount of the carrier.

[0015] In other embodiments, the carrier in the pharmaceutical composition further contains a non-cationic lipid, and the non-cationic lipid is selected from at least one of phospholipids, cholesterol, or derivatives thereof.

[0016] In some embodiments, the non-cationic lipid in the pharmaceutical composition is selected from a mixture of phospholipids and cholesterol or a derivative thereof.

[0017] In some embodiments, the content of phospholipids in the pharmaceutical composition is 5-40% of the molar amount of the carrier, including but not limited to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or any value between two numbers.

[0018] In some embodiments, the phospholipid content in the pharmaceutical composition is 15% by molar amount of the carrier.

[0019] In some embodiments, the phospholipid content in the pharmaceutical composition is 20% by molar amount of the carrier.

[0020] In some embodiments, the content of phospholipids in the pharmaceutical composition is 10-20% of the molar amount of the carrier.

[0021] In some embodiments, the phospholipid content in the pharmaceutical composition is 10% by molar amount of the carrier.

[0022] In other embodiments, the pharmaceutical composition provided herein comprises a phospholipid selected from the group consisting 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-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-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 16.0PE), 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-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0023] In some embodiments, the phospholipid in the pharmaceutical composition is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0024] In some embodiments, the phospholipid in the pharmaceutical composition is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0025] On the other hand, in the pharmaceutical composition of the present invention, cholesterol or its derivatives account for 30-60% of the molar amount of the carrier, including but not limited to 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or any value between two numbers.

[0026] In some embodiments, the content of cholesterol or its derivatives in the pharmaceutical composition is 35-45% of the molar amount of the carrier.

[0027] In some embodiments, the content of cholesterol or its derivatives in the pharmaceutical composition accounts for 40.5% of the molar amount of the carrier.

[0028] In some embodiments, the content of cholesterol or its derivative in the pharmaceutical composition is 45% of the molar amount of the carrier.

[0029] In other embodiments, cholesterol derivatives include but are not limited to sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, and ursolic acid.

[0030] In some embodiments, the non-cationic lipid in the pharmaceutical composition accounts for 35-70% of the molar amount of the carrier, including but not limited to 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% or any value therebetween.

[0031] In some embodiments, the non-cationic lipid in the pharmaceutical composition accounts for 50-65% of the molar amount of the carrier.

[0032] In some embodiments, the non-cationic lipid in the pharmaceutical composition accounts for 40-55% of the molar amount of the carrier.

[0033] In some embodiments, the non-cationic lipid in the pharmaceutical composition accounts for 55-62% of the molar amount of the carrier.

[0034] On the other hand, the molar ratio of cholesterol or its derivatives to phospholipids in the pharmaceutical composition of the present disclosure is 1.0 to 5.0, including but not limited to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 or any value therebetween.

[0035] In some embodiments, the molar ratio of cholesterol or its derivative to phospholipid in the pharmaceutical composition is 2.0 to 5.0.

[0036] On the other hand, the carrier in the pharmaceutical composition described in the present disclosure further contains a conjugated lipid, and the conjugated lipid includes but is not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0037] In some embodiments, the conjugated lipid in the pharmaceutical composition is selected from distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0038] In some embodiments, the conjugated lipid in the pharmaceutical composition is selected from dimyristoylglycerol-3-methoxypolyethylene glycol 2000.

[0039] In some embodiments, the conjugated lipid in the pharmaceutical composition is selected from distearoylphosphatidylethanolamine polyethylene glycol 2000.

[0040] In some embodiments, the conjugated lipid acts to inhibit aggregation of the carrier (particles). In some embodiments, the content of the conjugated lipid in the pharmaceutical composition accounts for 0.5-4% of the molar amount of the carrier, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0% or any value between two numbers.

[0041] In some embodiments, the content of the conjugated lipid in the pharmaceutical composition is 0.5-1.0% of the molar amount of the carrier.

[0042] In some embodiments, the content of the conjugated lipid in the pharmaceutical composition is 1.5-2.5% of the molar amount of the carrier.

[0043] In some embodiments, the content of the conjugated lipid in the pharmaceutical composition is 1-2% of the molar amount of the carrier.

[0044] In some embodiments, the content of the conjugated lipid in the pharmaceutical composition is 1.5% by molar amount of the carrier.

[0045] In some embodiments, the conjugated lipid is present in the pharmaceutical composition in an amount of 2.0% by mole of the carrier.

[0046] The pharmaceutical composition of the present disclosure further comprises an active agent, wherein the active agent is selected from nucleic acids, including ribonucleic acids or deoxyribonucleic acids.

[0047] In some embodiments, the active agent is selected from any one of small interfering RNA (siRNA), micro RNA (miRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), circular RNA, self-replicating RNA, or any combination thereof.

[0048] In some embodiments, the active agent is selected from mRNA.

[0049] In some embodiments, the mRNA comprises an ORF, and the ORF expresses a protein or polypeptide.

[0050] In some embodiments, the mRNA encodes a protein or polypeptide.

[0051] In some embodiments, the mRNA encodes an antigen.

[0052] In some embodiments, the mRNA encodes a protein or polypeptide derived from an infectious agent, or is an infectious agent antigen.

[0053] In some embodiments, the protein or polypeptide is a viral antigen and / or a bacterial antigen.

[0054] In some embodiments, the virus is selected from adenovirus, herpes virus (e.g., herpes simplex virus type 1, herpes simplex virus type 2, human herpes virus type 8), encephalitis virus, papillomavirus, varicella zoster virus (VZV), Epstein-Barr virus, human cytomegalovirus, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis virus (e.g., hepatitis A, B, C, D, or E), human bocavirus, parvovirus (e.g., B19), human astrovirus, norwalk virus, coxsackie virus, rhinovirus, severe acute respiratory syndrome , yellow fever virus, dengue virus, West Nile virus, rubella virus, human immunodeficiency virus (HIV), influenza A or B virus, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus (RSV), human metapneumovirus, Hendra virus, Nipah virus, rabies virus, rotavirus, orbivirus, Colorado tick fever virus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or Banna virus.

[0055] In some embodiments, the virus is selected from influenza virus (e.g., influenza A virus, influenza B virus), coronavirus, or a combination thereof. For example, the coronavirus is SARS-COV-2; the coronavirus antigen is a spike protein; the spike protein is selected from the spike protein of any one of SARS-COV-2, SARS-COV-2Alpha, SARS-COV-2Beta, SARS-COV-2Gamma, SARS-COV-2Kappa, SARS-COV-2Delta, or SARS-COV-2Omicron. For example, the influenza virus is selected from influenza A virus or influenza B virus; the influenza virus antigen is a hemagglutinin protein (HA) and / or a neuraminidase (NA); the influenza virus antigen is selected from the hemagglutinin protein and / or a neuraminidase of any one of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria, and influenza B virus Yamagata.

[0056] In some embodiments, the virus is RSV. For example, the RSV antigen is RSV attachment protein (G) or an immunogenic fragment thereof, RSV fusion (F) glycoprotein or an immunogenic fragment thereof, or a combination thereof; for example, the RSV antigen is prefusion glycoprotein F. The RSV antigenic polypeptides or immunogenic fragments thereof and their sequences in WO2017070622A, and prefusion glycoprotein F and their sequences in WO2014160463A, WO2012158613A, WO2017109629A, and WO2017172890A are incorporated herein in their entirety.

[0057] In some embodiments, the virus is VZV. For example, the VZV antigen is a VZV glycoprotein. For example, the VZV glycoprotein can be a VZV gE, gI, gB, gH, gK, gL, gC, gN, or gM polypeptide, or an immunogenic fragment or epitope thereof. In some embodiments, the VZV glycoprotein is a VZV gE protein, such as a wild-type VZV gE protein, or a truncate, fragment, or mutant thereof. In some embodiments, the wild-type VZV gE protein is from the Oka strain. In some embodiments, the wild-type VZV gE protein comprises (or is) the amino acid sequence of SEQ ID NO:1 and / or comprises (or is) the nucleotide sequence of SEQ ID NO:2.

[0058] In some embodiments, the truncation can be any truncated polypeptide, for example, a truncated VZV gE polypeptide comprising amino acids 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 540, 1 to 544, 1 to 545, 1 to 546, 1 to 550, 1 to 560, 1 to 561, 1 to 570, 1 to 573, 1 to 574, 1 to 575. In some embodiments, the VZV gE protein further comprises a Y569A point mutation. The VZV antigenic polypeptides and sequences thereof in WO2017070601A are incorporated herein in their entirety.

[0059] In some embodiments, the amino acid sequence of the truncate is amino acids 1-544, 1-546, 1-561, or 1-574 of the wild-type gE protein. In some embodiments, the amino acid sequence of the truncate is amino acids 1-574 of the wild-type gE protein, including the Y569A mutation.

[0060] In some embodiments, the amino acid sequence of the VZV gE protein is as shown in SEQ ID NO: 1 or any one of SEQ ID NO: 21-SEQ ID NO: 25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0061] In some embodiments, the nucleotide sequence of the open reading frame encoding the VZV gE protein is as shown in any one of SEQ ID NO:3-SEQ ID NO:8, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0062] In some embodiments, the protein or polypeptide is selected from fructose bisphosphate aldolase A (ALDOA), α-methylacyl-CoA racemase (AMACR), serum amyloid P component (APCS), angiopoietin 1 (ANGPT1), apolipoprotein AI (APOA1) Milano, apolipoprotein AI (APOA1) Paris, apolipoprotein AI (APOA1), argininosuccinate lyase (ASL), artemin (ARTN), arylsulfatase B (ARSB), bactericidal / permeability-increasing protein (rBPI-21), bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 7 (BMP7), branched-chain ketoacid dehydrogenase E1 α-polypeptide (BCKDHA), colony stimulating factor 2 (granulocyte-macrophage) (GM-CSF), colony stimulating factor 3 (granulocyte) (GCSF), deoxyribonuclease I (DNase 1), erythropoietin (EPO), factor IX, factor VII, factor XI, fibrinogen A (FGA), fibroblast growth factor 18 (FGF18), fibroblast growth factor 23 (FGF23), fibroblast growth factor 7 (FGF7 or KGF), follistatin (FST), fumarylacetoacetate hydrolase (fumarylacetoacetase) (FAH), galactokinase 1 (GALK1), galactosidase α (GLA), glucan (1,4-α-branching enzyme 1 (GBE1), glycoprotein hormone alpha polypeptide (CGA or FSH-α), hemoglobin beta (HBB), hepatocyte growth factor (HGF), human growth hormone (hGH), insulin aspart, insulin glargine, insulin glulisine, insulin lispro, interferon beta (IFNB), interferon alpha 2 (IFNA2), interleukin 10 (IL-10), interleukin 15 (IL-15), interleukin 7 (IL-7), klotho (KL), lecithin-cholesterol acyltransferase (LCAT), lysosomal acid lipase A cholesterol esterase (LIPA), lipoprotein lipase (LPL), low-density lipoprotein receptor (LDLR), mannosidase alpha 2B class member 1 (MAN2B1), microsomal triglyceride transfer protein (MTTP), N-acetylglutamate synthetase (NAGS), neuregulin 1 (NRG1), ornithine transcarbamylase (OTC), phosphorylase kinase alpha 2 (liver) (PHKA2), plasminogen activator (PLAT), cleavage protein 4 (ARTS or SEPT4), serpin peptidase inhibitor class C (antithrombin) member 1 (SERPINC1), serpin peptidase inhibitor class F (alpha-2 antiplasmin pigment epithelium-derived factor) member 2 (SERPINF2), sirtuin 1 (SIRT1), sirtuin 6 (SIRT6), solute carrier family 16 member 3 (monocarboxylate transporter 4) (SLC16A3), solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1 or GLUT1), sortilin 1 (SORT1), thrombopoietin (THPO), transforming growth factor β (TGFB1), amelogenin 1 (TUFT1), tumor protein p53 (TP53), tyrosinase (TYR), UDP-glucosyltransferase 1 family polypeptide A1 (UGT1A1), vascular endothelial growth factor (VEGF) and X-linked inhibitor of apoptosis (XIAP), or any combination thereof. The mRNA encoding the target polypeptide and its sequence in WO2013151736A are hereby incorporated in their entirety.

[0063] In some embodiments, the protein or polypeptide is a cancer antigen. The cancer antigen includes, but is not limited to, one or more traditional cancer antigens or subject-specific cancer antigens (including RNA encoding one or more known cancer antigens specific to tumors or cancer antigens specific to each subject, the cancer antigen is referred to as a new epitope or subject-specific epitope or antigen). In some specific embodiments, the cancer antigen is a subject-specific cancer antigen. In some specific embodiments, the subject-specific cancer antigen may represent the exon group of the subject's tumor sample, or the transcript group of the subject's tumor sample. In some embodiments, the subject-specific cancer antigen may represent the subject's exosome. The subject-specific cancer antigens and their identification methods described in WO2012159754A and WO2017070618A are incorporated herein in their entirety. In some specific embodiments, the cancer antigen is a traditional cancer antigen. In some embodiments, traditional cancer antigens are non-mutated antigens. In some specific embodiments, traditional cancer antigens are mutated antigens. For example, the cancer antigen may be carcinoembryonic antigen, α1-fetoprotein, isoferritin, and fetal sulphoglycoprotein, α2-H-ferritin, and γ-fetoprotein.

[0064] In some embodiments, the protein or polypeptide is an antibody or an antigen-binding fragment thereof. For example, an anti-PD-1 antibody or an antigen-binding fragment thereof. In some specific embodiments, the antibody or its antigen-binding fragment is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof. In some specific embodiments, the antibody or its antigen-binding fragment is, for example, a recombinant antibody or a fragment thereof. In some specific embodiments, the antibody or its antigen-binding fragment is, for example, a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody, and tetrabody, tandem two-scFv, tandem three-scFv).

[0065] In some embodiments, the mRNA comprises:

[0066] (a) open reading frame (ORF),

[0067] (b) untranslated region elements (UTR),

[0068] (c) polyadenylic acid (poly-A) tail;

[0069] Wherein, (b) untranslated region element (UTR) may comprise (b1) 5'UTR and / or (b2) 3'UTR;

[0070] Wherein, (b) untranslated region element (UTR) and (c) polyadenylic acid (poly-A) tail may be present at the same time, absent at the same time, or only one of them may be present.

[0071] In some embodiments, the mRNA further comprises a 5' Cap structure.

[0072] In some embodiments, from the 5' to 3' direction, the RNA molecule of any of the above items contains any one of i)-v):

[0073] i) 5'UTR, and open reading frame (ORF);

[0074] ii) open reading frame (ORF), and 3'UTR;

[0075] iii) 5'UTR, open reading frame (ORF), and 3'UTR;

[0076] iv) 5'UTR, open reading frame (ORF), 3'UTR, and poly-A tail;

[0077] v) 5' cap structure (5'Cap), 5'UTR, open reading frame (ORF), 3'UTR, and poly-A tail;

[0078] The ORF and the 5'UTR and / or the 3'UTR are derived from the same and / or different genes.

[0079] In some embodiments, the 5'UTR is selected from β-globin 5'UTR, 5'UTR containing a strong Kozak translation initiation signal, cytochrome b-245 alpha polypeptide (CYBA) 5'UTR, hydroxysteroid (17-β) dehydrogenase (HSD17B4) 5'UTR, tobacco etch virus (TEV) 5'UTR, heat shock protein 70 (Hsp70) 5'UTR, eIF4G 5'UTR, ACTG1 5'UTR, CTSB 5'UTR or ARHGAP15 gene 5'UTR. In some embodiments, the 5'UTR is derived from or is a 5'UTR sequence of the ACTG1 or ARHGAP15 gene or a derivative thereof. In some embodiments, the ARHGAP15 gene is derived from any species, such as human ARHGAP15, baboon ARHGAP15, mouse ARHGAP15, etc.

[0080] In some embodiments, the 5'UTR is derived from or is the 5'UTR sequence of the human ARHGAP15 gene or a derivative thereof. In some embodiments, the 5'UTR of the ARHGAP15 gene comprises or is the nucleotide sequence shown in SEQ ID NO: 9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0081] In some embodiments, the 3'UTR is selected from α-globin 3'UTR, β-globin (hemoglobin subunit beta, HBB) 3'UTR, β-actin 3'UTR, CYBA 3'UTR, albumin 3'UTR, growth hormone (GH) 3'UTR, VEEV 3'UTR, hepatitis B virus (HBV) 3'UTR, ACTG1 3'UTR, CTSB 3'UTR or 3'UTR of ARHGAP15 gene.

[0082] In some embodiments, the 3'UTR is derived from or is the 3'UTR sequence of the HBB, CTSB or ARHGAP15 gene or a derivative sequence thereof.

[0083] In some embodiments, the 3'UTR comprises or is the nucleotide sequence of SEQ ID NO: 10, or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. In some embodiments, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0084] In some specific embodiments, the poly-A tail is selected from 120A or A30L70, the sequence of A30L70 is shown in SEQ ID NO:11, and the sequence of 120A is shown in SEQ ID NO:12.

[0085] In some embodiments, the nucleotide sequence of the RNA molecule is as shown in any one of SEQ ID NO: 13-SEQ ID NO: 20. In some embodiments, the mRNA comprises modified nucleotides or nucleosides. The modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. The modifications may include those modifications of the sugar, backbone or core base moiety of nucleotides and / or nucleosides as generally recognized in the art. In some embodiments, the modified core base in the mRNA comprises 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C) and / or pseudouridine (ψ). In some embodiments, the modified core base in the mRNA comprises 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine and / or 5-methoxycytidine. In some embodiments, the mRNA includes a combination of at least two (e.g., 2, 3, 4, or more) of any of the above-mentioned modified nucleobases, including but not limited to chemical modifications.

[0086] In some specific embodiments, the 5' Cap structure is located upstream of the 5' UTR, for example, at the 5' end of the 5' UTR. In some specific embodiments, the 5' cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first nucleobase, for example, m7GpppN), Cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN, for example, m7G(5')ppp(5')(2'OMeA)pG), Cap2 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (for example, phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.

[0087] In some embodiments, the pharmaceutical composition comprises: a cationic lipid, wherein the cationic lipid comprises a compound represented by Formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is 40-49%,

[0088] In some embodiments, the pharmaceutical composition contains:

[0089] a) active agent;

[0090] b) a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%,

[0091] c) a non-cationic lipid selected from the group consisting of phospholipids and cholesterol or its derivatives, wherein the phospholipids account for 5 to 40% of the molar weight of the carrier, and the cholesterol or its derivatives account for 30 to 60% of the molar weight of the carrier;

[0092] and d) conjugated lipids, said conjugates comprising 0.5 to 4% of the molar amount of the carrier.

[0093] In some embodiments, the pharmaceutical composition contains:

[0094] a) an active agent comprising mRNA;

[0095] b) a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%,

[0096] c) a non-cationic lipid, wherein the non-cationic lipid is selected from the group consisting of phospholipid DSPC and cholesterol, wherein the phospholipid DSPC accounts for 5 to 40% of the molar amount of the carrier, and the cholesterol accounts for 30 to 60% of the molar amount of the carrier;

[0097] and d) conjugated lipids, said conjugates comprising 0.5 to 4% of the molar amount of the carrier.

[0098] In some embodiments, the pharmaceutical composition contains:

[0099] a) an active agent comprising mRNA;

[0100] b) a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%,

[0101] c) a non-cationic lipid, wherein the non-cationic lipid is selected from the group consisting of phospholipid DSPC and cholesterol, wherein the phospholipid DSPC accounts for 5 to 40% of the molar amount of the carrier, and the cholesterol accounts for 30 to 60% of the molar amount of the carrier;

[0102] and d) conjugating the lipid DMG-PEG, wherein the conjugate accounts for 0.5-2% of the molar amount of the carrier.

[0103] In some embodiments, the pharmaceutical composition contains:

[0104] a) an active agent comprising mRNA;

[0105] b) a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is 40-49%,

[0106] c) a non-cationic lipid, wherein the non-cationic lipid is selected from the group consisting of phospholipid DSPC and cholesterol, wherein the phospholipid DSPC accounts for 10-15% of the molar amount of the carrier, and the cholesterol accounts for 35-42% of the molar amount of the carrier;

[0107] and d) conjugating the lipid DMG-PEG, wherein the conjugate accounts for 1-2% of the molar amount of the carrier.

[0108] In some embodiments, the pharmaceutical composition contains:

[0109] a) an active agent comprising mRNA;

[0110] b) a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is 45 to 49%,

[0111] c) a non-cationic lipid, wherein the non-cationic lipid is selected from the group consisting of phospholipid DSPC and cholesterol, wherein the phospholipid DSPC accounts for 10-15% of the molar amount of the carrier, and the cholesterol accounts for 35-42% of the molar amount of the carrier;

[0112] and d) conjugating the lipid DMG-PEG, wherein the conjugate accounts for 1-2% of the molar amount of the carrier.

[0113] On the other hand, the pharmaceutical composition of the present disclosure is a nanoparticle formulation having an average particle size of 10 nm to 220 nm. In some embodiments, the average particle size of the nanoparticle formulation is 100 to 150 nm. In some embodiments, the average particle size of the nanoparticle formulation is 150 to 200 nm. In some embodiments, the average particle size of the nanoparticle formulation is 80 to 160 nm. In some embodiments, the average particle size of the nanoparticle formulation is 80 to 150 nm. In some embodiments, the average particle size of the nanoparticle formulation is 70 to 160 nm.

[0114] In some embodiments, the average particle size of the nanoparticles is measured using dynamic light scattering.

[0115] The present disclosure also provides a lyophilized formulation obtained by freeze-drying the aforementioned pharmaceutical composition. In some embodiments, freeze-drying includes a freezing step, a primary drying step, and a secondary drying step. In some embodiments, freeze-drying in the present disclosure is performed with reference to the method described in CN107567497A, and the relevant content is incorporated herein for illustration.

[0116] The present disclosure also provides a reconstitution solution, which is obtained by reconstitution of the aforementioned lyophilized preparation, wherein the solvent used for reconstitution is water for injection.

[0117] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, lyophilized formulation or reconstituted solution, the method comprising the step of mixing a cationic lipid, a non-cationic lipid and a conjugated lipid in an organic solvent. In some embodiments, the organic solvent is selected from ethanol.

[0118] In some embodiments, the method for preparing the pharmaceutical composition is prepared with reference to the method in the journal literature (Pharm. Res. 22 (2005) 362-372) or US9504651, and the relevant contents are introduced herein for illustration.

[0119] The present disclosure also provides use of the aforementioned pharmaceutical composition, lyophilized preparation or reconstituted solution in the preparation of a medicament for inducing a protective immune response in a vertebrate.

[0120] The present disclosure also provides the use of the aforementioned pharmaceutical composition, lyophilized formulation or reconstituted solution in the preparation of a medicament for treating a disease or dysfunction in a mammal. In some embodiments, the disease is preferably selected from viral infection (such as a disease caused by coronavirus, influenza virus or HIV virus), liver disease, cancer or herpes.

[0121] the term

[0122] The term "lipid" refers to a class of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids" such as steroids.

[0123] The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (eg, pH about 7.0).

[0124] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0125] The term "neutral lipid" refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0126] The term "amphiphilic lipid" refers, in part, to any suitable material, wherein the hydrophobic portion of the lipid material is oriented to the hydrophobic phase, while the hydrophilic portion is oriented to the aqueous phase. The hydrophilic nature results from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. Hydrophobicity can be imparted by including non-polar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbons and such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include but are not limited to phospholipids, amino lipids, and sphingolipids.

[0127] The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0128] The term "conjugated lipid" refers to a conjugated lipid that inhibits aggregation of lipid particles. The conjugated lipid includes, but is not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0129] The term "mammal" refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0130] "Active agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Active agents are also referred to as "active substances" or "therapeutic agents." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0131] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, including DNA and RNA. As used herein, the term "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". DNA can take the following forms: for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can take the following forms: small interfering RNA (siRNA), microRNA (miRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), circular RNA, self-replicating RNA.

[0132] "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated to produce the encoded polypeptide in vitro, in vivo, or ex vivo. Those skilled in the art will understand that, unless otherwise indicated, the polynucleotide sequences described in this application will indicate "T" in representative DNA sequences, but where the sequence represents RNA (e.g., mRNA), the "T" will be replaced by a "U." Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number can also include a corresponding RNA (e.g., mRNA) sequence encoded by the DNA, wherein each "T" of the DNA sequence is replaced by a "U." The basic components of an mRNA molecule generally include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' terminal cap, and a polyadenylic acid tail (poly-A tail). The polynucleotides of the present invention can function as mRNA, but are distinguished from wild-type mRNA in their functional and / or structural design features that are used to overcome existing problems of efficient polypeptide expression using nucleic acid-based processing.

[0133] The term "antigen" encompasses any substance that will trigger an immune response. In particular, "antigen" relates to any substance, preferably a peptide or protein that specifically reacts with an antibody or T lymphocyte (T cell). In the present disclosure, the term "antigen" includes any molecule comprising at least one epitope. Preferably, an antigen herein is a molecule that (optionally after processing) induces an immune response that is specific to the antigen (including cells expressing the antigen). Herein, any suitable antigen that is a candidate for an immune response can be used, wherein the immune response is preferably a cellular immune response. In the case of some embodiments, the antigen is preferably presented by cells, preferably by antigen-presenting cells (including diseased cells, particularly cancer cells, in the context of MHC molecules), which results in an immune response against the antigen. Preferably, the antigen is a product corresponding to or derived from a natural antigen. This natural antigen includes tumor antigens.

[0134] The term "cancer antigen" is interchangeable with "tumor antigen" or "tumor-associated antigen", which is capable of stimulating an immune response, preferably a cellular response to an antigen or a cell characterized by expressing the antigen and preferably presenting the antigen (e.g., a diseased cell, especially a cancer cell). In some embodiments, a "cancer antigen" is specifically expressed under normal conditions in a limited number of tissues and / or organs or in a specific developmental stage (e.g., a tumor antigen may be specifically expressed under normal conditions in gastric tissue (preferably in gastric mucosa), in reproductive organs (e.g., in testicles), in trophoblastic tissue (e.g., in placenta), or in germline cells), and is expressed or abnormally expressed in one or more tumors or cancerous tissues. In some embodiments, a "limited number" refers to no more than 3, e.g., no more than 2.

[0135] "Polypeptide", "protein" or "protein" are used interchangeably to refer to a polymer of amino acid residues and apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0136] The term "Varicella-zoster virus (VZV)" refers to an alphaherpesvirus with a spherical, multilayered structure. The viral genome is surrounded by a protein capsid covered with an amorphous layer of envelope proteins. The envelope, composed of virally encoded proteins and enzymes, is located between the nucleus and the viral envelope, which is derived from the host cell membrane and contains virally encoded glycoproteins.

[0137] The term "VZV glycoprotein" refers to the glycoproteins gE, gI, gB, gH, gK, gL, gC, gN and gM that encode the different steps of the viral replication cycle. The most abundant glycoprotein found in infected cells and in mature virions is glycoprotein E (gE, ORF 68), which is the main component of the virion envelope and is necessary for viral replication. Glycoprotein I (gI, ORG 67) forms a complex with gE in infected cells, which facilitates the endocytosis of glycoproteins and guides them to the trans-Galvan network (TGN), where the final viral envelope is obtained. Glycoprotein I (gI) is required in the TGN to carry out the VZV envelope and to perform effective membrane fusion during VZV replication. It is found that VZV gE and gI are combined on the surface of infected host cells. Glycoprotein B (ORF 31), which is the second most common glycoprotein and is believed to play a role in the viral entrance, is combined with neutralizing antibodies. Glycoprotein H is believed to have a fusion function, which promotes the spread of the virus between cells. Antibodies to gE, gB, and gH are ubiquitous following natural infection and vaccination, and have demonstrated in vitro viral neutralization activity. The amino acid sequence of an exemplary gE protein is set forth in SEQ ID NO: 1 or any one of SEQ ID NO: 21 to SEQ ID NO: 25 of the present disclosure.

[0138] The terms "mutant" of wild-type VZV gE protein, "mutant" of VZV gE protein, "VZV gE protein mutant" refer to polypeptides that exhibit introduced mutations relative to wild-type VZV gE protein and are immunogenic against wild-type VZV gE protein.

[0139] The term "mutation" refers to the deletion, addition or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the substitution of an amino acid at a specific position in a protein sequence is referred to using the annotation "(amino acid residue in wild-type protein)(amino acid position)(amino acid residue in engineered protein)". For example, the annotation Y75A refers to the substitution of the tyrosine (Y) residue at position 75 of the amino acid sequence of the reference protein by an alanine (A) residue (in a mutant of the reference protein). In cases where there is a variation in the amino acid residue at the same position between different wild-type sequences, the amino acid code before the position number, such as "75A", may be omitted from the annotation.

[0140] "Codon optimization" refers to the replacement of a generally rare codon in a highly expressed gene of a given species with a generally common codon in the highly expressed gene of such species in a target sequence, and the codons before and after the replacement encode the same amino acid. Various species show specific preferences for certain codons of specific amino acids. Codon preference (the difference in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), and it is then believed that the messenger RNA is particularly dependent on the characteristics of the codons translated and the availability of specific transfer RNA (tRNA) molecules. The advantage of the selected tRNA in the cell is generally a reflection of the most commonly used codons in peptide synthesis. Therefore, based on codon optimization, genes can be modified for the optimal gene expression in a given organism. Therefore, the selection of optimal codons depends on the codon usage preference of the host genome.

[0141] As known in the art, the term "identity" or "homology" refers to the relationship between the sequences of two or more polypeptides or polynucleotides as determined by comparing sequences. In the art, identity also means the degree of sequence relatedness as determined by the number of matches between two or more amino acid residues or nucleic acid residue strings. Identity measures the percentage of consistent matches between the smaller of the gap alignments (if any) proposed by a specific mathematical model or computer program (e.g., "algorithm"). "% identity" is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are consistent with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and, if necessary, introducing gaps to achieve maximum percent identity, when applied to polypeptide or polynucleotide sequences. Methods and computer programs for comparison are well known in the art. It should be understood that identity depends on the calculation of percent identity, but its value may vary due to gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF and Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48: 443-453).Recently a fast optimized global sequence alignment algorithm (Fast Optimal Global Sequence Alignment Algorithm, FOGSAA) has been developed that is said to produce global alignments of nucleotide and protein sequences faster than other optimized global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0142] The term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that, upon administration, induces an immune response, particularly a cellular immune response, that recognizes and attacks pathogens (including viruses or bacteria) or diseased cells, such as cancer cells. Vaccines can be used to prevent or treat disease, for example, to prevent viral infection or to treat cancer.

[0143] The term "5' untranslated region element" (5'UTR) refers to the region of an mRNA immediately upstream (ie, 5') of the start codon (ie, the first codon of an mRNA transcript translated by ribosomes) that does not encode a polypeptide.

[0144] The term "3' untranslated region element" (3'UTR) refers to the region of an mRNA that is located immediately downstream (ie, 3') of a stop codon (ie, the codon that signals the termination of translation of an mRNA transcript) and that does not encode a polypeptide.

[0145] The term "open reading frame" (ORF) is a continuous stretch of DNA that begins with a start codon (eg, methionine (ATG)) and ends with a stop codon (eg, TAA, TAG, or TGA) and encodes a protein or polypeptide.

[0146] The term "poly (A) tail" refers to a region of an mRNA located downstream of a 3' UTR containing multiple consecutive adenosine monophosphates, such as a region directly downstream (i.e., 3') thereof. The poly (A) tail may contain 10 to 300 adenosine monophosphates. For example, the poly (A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the poly (A) tail contains 50 to 250 adenosine monophosphates. In the context of a relevant organism (e.g., in a cell, in vivo), the poly(A) tail serves to protect the mRNA from enzymatic degradation, such as in the cytoplasm, and facilitates transcription termination and / or mRNA export from the nucleus and translation.

[0147] The "average particle size" (Z-average Size) referred to in this disclosure, for example, "average particle size less than 2000 nm," is the average light intensity, calculated from the light intensity contributed by different types of particles. The "average particle size" can be measured using conventional particle size measurement techniques familiar to those skilled in the art. Such techniques include sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, and the like.

[0148] The polydispersity index (PDI) of the present disclosure reflects the uniformity of particle size and is an important index for characterizing particle size.

[0149] The terms "mixed" and "mixed" mean that the order of adding the components is not limited. For example, mixing A into B can mean A is added to B, or B is added to A. Mixing A and B can mean A is added to B and the mixture can also mean B is added to A and the mixture can also mean B is added to A and the mixture can also mean.

[0150] The numerical values ​​in this disclosure are instrumental measurements and are subject to a certain degree of error. Generally speaking, within a reasonable error range of plus or minus 10%. The context in which the numerical value is used must be considered. For example, the particle size of an active ingredient, where the error after measurement does not exceed plus or minus 10%, may be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2%, or plus or minus 1%, preferably plus or minus 5%.

[0151] The compound of formula I disclosed in the present invention is prepared by referring to the method in WO2023125738, and the relevant content is illustrated in this application.

[0152] The mRNA used for testing in the present disclosure is luciferase mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1: Luciferase protein expression levels of liposome nanoparticles corresponding to lipid compositions 1-15 after transfection in HEK293 cells.

[0154] Figure 2: Particle size stability of lipid compositions at 4°C.

[0155] Figure 3: Particle size stability of lipid compositions at 25°C.

[0156] Figure 4: Detection of VZV mRNA activity expression in vitro.

[0157] Figure 5: Serum anti-VZV gE IgG detection. DETAILED DESCRIPTION

[0158] The present disclosure is further described below with reference to examples, but these examples are not intended to limit the scope of the present disclosure.

[0159] Experimental methods in the examples of this disclosure that do not specify specific conditions are generally based on conventional conditions or the conditions recommended by the raw material or product manufacturers. Reagents without specific sources are conventional reagents purchased from the market.

[0160] Example 1

[0161] Step 1)

[0162] 6-Aminohexanoic acid (21.5 g, 100 mmol) was dissolved in a mixture of ethanol and water (EtOH / H2O, v / v = 2:1, 200 mL). CsOH·H2O (4.0 g, 100 mmol) and benzyloxyethane bromide (13.1 g, 100 mmol) were added. The mixture was reacted at room temperature for 40 h. Most of the ethanol was removed by concentration. The residue was directly subjected to reverse phase column chromatography (acetonitrile / water / 0.1% trifluoroacetic acid), directly concentrated, and drained to obtain 5.6 g of a white solid (yield: 21%).

[0163] MS: 266.2[M+H] + .

[0164] 1 H NMR(400MHz,D2O)δ7.36(s,5H),4.53(s,2H),3.72–3.67(m,2H),3.20–3.14(m,2H),2.95–2.88(m,2H) ,2.10(t,J=7.3Hz,2H),1.57(dd,J=15.3,7.7Hz,2H),1.47(dd,J=15.0,7.5Hz,2H),1.30–1.22(m,2H).

[0165] Step 2)

[0166] 6-((2-(Benzyloxy)ethyl)amino)hexanoic acid (5.6 g, 21.1 mmol) was weighed into a reaction flask, 1,4-dioxane (80 mL) and aqueous sodium hydroxide solution (42.2 mL, 1.0 M) were added, and Boc2O (5.5 g, 25.2 mmol) was added dropwise. The mixture was allowed to react at room temperature for 1 h. LC-MS indicated completion of the reaction. The dioxane was concentrated, and the aqueous phase was adjusted to pH 5 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried, and concentrated to afford 7.2 g of a nearly colorless gum (93% yield). This was used directly in the next step.

[0167] MS: 366.2[M+H] + .

[0168] Step 3)

[0169] 6-((2-(Benzyloxy)ethyl)(tert-butoxycarbonyl)amino)hexanoic acid (6.1 g, 16.7 mmol) was weighed into a reaction flask, and dichloromethane (150 mL) was added. The mixture was cooled to an ice bath, and undecanol (2.73 g, 15.9 mmol), DMAP (408 mg, 3.34 mmol), and DIPEA (4.3 g, 33.4 mmol) were added in sequence. EDCI (3.84 g, 20.0 mmol) was added, and the mixture was stirred for 10 minutes. The ice bath was removed, and the reaction was allowed to proceed at room temperature overnight. TLC monitoring was performed. After completion of the reaction, water (100 mL) was added to the reaction solution, and the layers were separated. The organic phase was washed sequentially with dilute hydrochloric acid, water, and sodium bicarbonate, dried, concentrated, and purified by column chromatography (PE:EtOAc = 20:1 to 10:1) to give 6.8 g of a nearly colorless gum with a yield of 82%.

[0170] MS: 520.4[M+H] + .

[0171] 1 H NMR (400MHz, CDCl3) δ7.30–7.20(m,5H),4.44(s,2H),3.98(t,J=6.7Hz,2H),3.51(d,J=18.6Hz,2H),3.32(d,J=21.5Hz,2H),3.16( s,2H),2.21(t,J=7.5Hz,2H),1.55(tt,J=13.3,6.8Hz,6H),1.36(d,J=8.4Hz,9H),1.21(d,J=15.5Hz,18H),0.81(t,J=6.8Hz,3H).

[0172] Step 4)

[0173] A solution of 6-((2-(benzyloxy)ethyl)(tert-butoxycarbonyl)amino)hexanoic acid undecyl ester (7.6 g, 14.6 mmol) was weighed into dichloromethane, TFA (20 mL) was added, the reaction was carried out at room temperature for 2 h, and the mixture was directly concentrated. The residue was dissolved in dichloromethane (100 mL), saturated sodium bicarbonate (100 mL) was added, the liquids were separated, the organic phase was dried, and concentrated to obtain a gum, which was then dissolved in dichloromethane, HCl / dioxane (4.0 M, 4 mL) was added, and the mixture was directly concentrated to obtain 5.0 g of a white solid with a yield of 81%.

[0174] MS: 420.3[M+H] + .

[0175] 1H NMR (400MHz, CDCl3) δ9.51(s,2H),7.39–7.28(m,5H),4.57(s,2H),4.03(t,J=6.8Hz,2H),3.88(t,J=4.8Hz,2H),3.18(s,2H),3.03(d,J=3 .4Hz,2H),2.28(t,J=7.4Hz,2H),2.02(s,1H),1.93–1.84(m,2H),1.61(dd,J=13.8,6.7Hz,4H),1.42–1.17(m,18H),0.88(t,J=6.8Hz,3H).

[0176] Step 5)

[0177] Weigh 2-((5-(benzyloxy)pentyl)oxy)acetic acid (preparation document Chemical and Pharmaceutical Bulletin, 1992, vol. 40, # 3, p. 617-623) (7.6 g, 30.12 mmol) was added to a reaction flask, dichloromethane (150 mL) was added, and an ice bath was added. Heptadecanol (prepared with reference to document WO 2020 / 219876) (6.18 g, 24.1 mmol) and DMAP (3.68 g, 30.12 mmol) were added in sequence, and EDCI (6.93 g, 36.14 mmol) was added. The mixture was stirred for 10 minutes, the ice bath was removed, and the reaction was allowed to proceed at room temperature overnight. TLC monitoring was performed. After the reaction was completed, water (100 mL) was added to the reaction solution, the liquid was separated, and the organic phase was washed with dilute hydrochloric acid, water, and sodium bicarbonate in sequence, dried, concentrated, and purified by column chromatography (PE: EtOAc = 50: 1 to 10: 1) to give 10.9 g of a nearly colorless gum with a yield of 92%.

[0178] 1 H NMR (400MHz, CDCl3) δ7.36–7.31(m,4H),7.30–7.26(m,1H),4.96(p,J =6.3Hz,1H),4.50(s,2H),4.04(s,2H),3.52(t,J=6.6Hz,2H),3.47(t,J=6.6Hz,2H ),1.69–1.61(m,4H),1.56(d,J=16.5Hz,6H),1.25(s,24H),0.88(t,J=6.8Hz,6H).

[0179] Step 6)

[0180] Heptadec-9-yl 2-((5-(benzyloxy)pentyl)oxy)acetate (4.0 g, 8.15 mmol) was dissolved in THF (100 mL). Pd(OH)2 / C (400 mg, 20%) was added and hydrogen was introduced. The reaction was allowed to react at room temperature for 2 h. TLC monitoring indicated the reaction was complete. Filtration and concentration were performed to obtain 3.2 g of a colorless gum (98% yield). This was used directly in the next reaction.

[0181] 1 H NMR (400MHz, CDCl3) δ4.97 (dd, J=12.4, 6.1Hz, 1H), 4.04 (s, 2H), 3.66 (t, J=6.5Hz, 2H), 3.54 (t,J=6.5Hz,2H),1.71–1.57(m,4H),1.56–1.42(m,6H),1.25(s,24H),0.88(t,J=6.8Hz,6H).

[0182] Step 7)

[0183] Weigh heptadecan-9-yl 2-((5-hydroxypentyl)oxy)acetate (2.4 g, 6.0 mmol) and dissolve it in dichloromethane (100 mL). Add DMP (3.82 g, 9.0 mmol) under ice bath and react at room temperature for 1.5 h. Add saturated aqueous sodium thiosulfate solution (100 mL) and stir for 30 minutes. Separate the liquids and wash the organic phase with saturated aqueous sodium bicarbonate solution (100 mL*2), dry and concentrate to obtain 2.5 g of a light yellow gum, which is overweight and used directly in the next reaction.

[0184] 1 H NMR (400MHz, CDCl3) δ9.78 (s, 1H), 4.96 (p, J = 6.3Hz, 1H), 4.04 (s, 2H), 3.55 (t, J = 6.1Hz, 2H), 2.49 (td, J = 7.2, 1.5Hz,2H),1.81–1.72(m,2H),1.68(d,J=7.6Hz,2H),1.60–1.48(m,4H),1.26(s,24H),0.88(t,J=6.8Hz,6H).

[0185] Step 8)

[0186] Heptadec-9-yl 2-((5-oxopentyl)oxy)acetate (2.4 g, 6 mmol) was weighed and dissolved in dichloromethane (50 mL). Undecyl 6-((2-(benzyloxy)ethyl)amino)hexanoate hydrochloride (2.46 g, 5.4 mmol) was added, followed by DIPEA (1.16 g, 9.0 mmol) and acetic acid (1.08 g, 18.0 mmol). The mixture was stirred until clear, and then cooled on ice. NaBH(OAc)3 (3.18 g, 15.0 mmol) was added. The mixture was reacted at room temperature overnight and monitored by TLC. After completion of the reaction, water (100 mL) was added, the layers were separated, and the organic phase was washed with saturated aqueous sodium bicarbonate solution (50 mL*1), dried, concentrated, and purified by column chromatography (PE:EtOAc=5:1 to 2:1) to give 4.1 g of a colorless liquid (yield: 85%).

[0187] 1 H NMR(400MHz, CDCl3)δ7.33(d,J=4.5Hz,4H),7.30–7.26(m,1H),4.99–4.91(m,1H),4.52(s,2H),4.08–4.02(m,4H),3.57–3.47(m,4H),2.68(t, J=5.8Hz,2H),2.45(s,4H),2.28(t,J=7.5Hz,2H),1.67–1.57(m,6H),1.48(dd,J=37.5,6.0Hz,6H),1.38–1.18(m,46H),0.88(t,J=6.8Hz,9H).

[0188] Step 9)

[0189] Undecyl 6-((2-(benzyloxy)ethyl)(5-(2-(heptadecan-9-yloxy)-2-oxoethoxy)pentyl)amino)hexanoate (1 g, 1.25 mmol) was weighed into a reaction flask and dissolved in ethanol. Pd(OH)2 / C (500 mg, 20%) was added and hydrogen was introduced. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. Filtering and concentration were performed to obtain a colorless gum. Column chromatography (CH2Cl2:MeOH = 10:1) afforded 450 mg of the product, a 51% yield.

[0190] 1H NMR (400MHz, CDCl3) δ5.00–4.89(m,1H),4.09–4.01(m,4H),3.61(s,2H),3.52(t,J=6.4Hz,2H),2.68(s,2H),2 .57(s,4H),2.30(t,J=7.4Hz,2H),1.70–1.59(m,6H),1.53(s,8H),1.43–1.18(m,44H),0.88(t,J=6.7Hz,9H).

[0191] Example 2:

[0192] Preparation of LNP lipid particles

[0193] Compound 1 (Formula I compound), DSPC, cholesterol, and DMG-PEG 2000 solution were mixed at a certain molar percentage (mol%), that is, the percentage of the molar amount of each component to the total molar amount of the carrier, and an ethanol lipid solution was prepared. The molar ratios of the four components were prepared as shown in Table 1. The mRNA encoding the luciferase protein (its nucleotide sequence is shown in SEQ ID NO: 26) was dissolved in 50mM pH 4 acetate buffer to prepare an mRNA aqueous solution. The ethanol lipid solution and the mRNA aqueous solution were mixed by microfluidics in a volume ratio of 1:3, wherein the weight ratio of compound 1 to mRNA was 12:1 to prepare liposomes. The ethanol was removed by dialysis in a 20mM Tris solution, and finally replaced in a 20mM Tris 8% sucrose solution to obtain a liposome nanoparticle composition encapsulating mRNA.

[0194] Lipid particle composition characterization

[0195] Characterization methods

[0196] The particle size and polydispersity index (PDI) of the liposome nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Pro in 173° backscattering detection mode.

[0197] The liposome encapsulation efficiency was determined using the Quant-iT RiboGreen RNA Assay Kit.

[0198] Table 1

[0199] Test Example 1: Evaluation of mRNA delivery efficiency of lipid particle composition carrier

[0200] The protein expression levels of the liposome nanoparticles corresponding to lipid compositions 1-15 after mRNA delivery in cells were detected.

[0201] HEK 293 cells were seeded into 96-well plates and cultured overnight. When the cell density reached more than 80%, the lipid nanoparticle solution encapsulating luciferase mRNA was added to the cell plate well culture medium at an mRNA dose of 100 ng / well. After 24 hours, the fluorescence intensity of the expressed luciferase protein was detected using a luciferase reporter gene assay kit (Promega) and a microplate reader. The fluorescence intensity value is the fluorescence value detected by the microplate reader, which represents the expression level of the luciferase protein. The higher the intensity of the strong light, the higher the protein expression level. The average fluorescence intensity was calculated for at least 3 groups of lipid nanoparticles corresponding to each compound. The data are shown in Table 2 and Figure 1.

[0202] Table 2

[0203] Test Example 2: Evaluation of the stability of lipid particle compositions

[0204] The lipid nanoparticles corresponding to lipid compositions 1-15 were placed in 4°C and 25°C environments respectively. After 3 days, the particle size of each group of lipid nanoparticles was measured and the size change during storage was compared, which represents the stability of the lipid nanoparticles. Large particle size changes indicate poor stability. Each group was tested three times, and the average particle size was calculated, as shown in Table 3. The difference between the particle size at 4°C and 25°C and the initial particle size of the lipid nanoparticles was calculated. The smaller the difference deviates from 0, the smaller the particle size change during the storage period, and the more stable the lipid nanoparticles. As shown in Table 3, after 3 days of storage at 4°C and 25°C, the polydispersity coefficient of the particle size was detected by dynamic light scattering. The size change is shown in Table 4 and Figures 2-3.

[0205] Table 3

[0206] Table 4

[0207] Example 3: Preparation of mRNA vaccine

[0208] In this disclosure, we have prepared a variety of mRNA products, exemplified by the VZV mRNA vaccine. The following is an example of its preparation and validation.

[0209] 3.1 Design and synthesis of VZV mRNA vaccine DNA template

[0210] In order to improve the expression of VZV gE protein and its structural stability in human cells, codon optimization was performed in this example, and different C-terminal variant molecules encoding the Oka strain gE protein were designed, wherein the amino acid sequence of the Oka strain gE protein is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. We designed six gE molecules, namely: gE1 (Oka gE, 1-546aa), gE2 (Oka gE, 1-574aa & Y569A), gE3 (Oka gE, 1-544aa), gE4 (Oka gE, 1-574aa), gE5 (Oka gE, 1-561aa) and one full-length gE6 (Oka gE, 1-623aa). The amino acid sequences of the gE1-gE6 molecules and their encoding nucleic acid sequences are shown in Table 5, wherein gE1-gE5 are C-terminally truncated gE molecules.

[0211] Table 5 VZV mRNA vaccine sequences

[0212] Also designed were a T7 promoter sequence (TAATACGACTCACTATAAG), a 5'-UTR sequence (the corresponding RNA sequence of which is shown in SEQ ID NO: 9), a 3'-UTR sequence (the corresponding RNA sequence of which is shown in SEQ ID NO: 10), and a poly(A) sequence (the corresponding RNA sequence of which is shown in SEQ ID NO: 11).

[0213] Then, the T7 promoter sequence, 5'-UTR sequence, DNA ORF, 3'-UTR sequence and poly(A) sequence were connected in order, and whole gene synthesis was performed using pUC57 as a vector (Suzhou Jinweizhi Biotechnology Co., Ltd.) to obtain a plasmid DNA template.

[0214] 3.2 Preparation of VZV mRNA vaccine mRNA stock solution

[0215] The plasmid DNA obtained in Example 3.1 was digested with XbaI, purified to obtain linearized plasmid DNA, and co-transcription capping (Cap1 type) reaction was performed by T7 RNA polymerase, and in vitro transcription was performed to produce the desired RNA. (1-Methyl)-pseudouridine-triphosphate was used instead of uridine triphosphate (UTP) in in vitro transcription. After transcription, the DNA template was digested with DNaseI to reduce the risk of residual DNA template. The mRNA was purified using AKTA, dissolved in sterile water, and frozen at -80 ° C until use.

[0216] 3.3 Preparation of VZV mRNA vaccine mRNA-LNP formulation

[0217] Compound 1 (Formula I), DSPC, cholesterol, and DMG-PEG 2000 were dissolved in ethanol at a molar ratio of 48:10:40.5:1.5 to prepare a lipid solution. mRNA was dissolved in 50 mM acetate buffer, pH 4, to prepare an mRNA aqueous solution. The ethanolic lipid solution and the mRNA aqueous solution were mixed using microfluidics at a volume ratio of 1:3, resulting in a lipid nanoparticle ratio of Compound 1 to mRNA of 12:1. The solution was dialyzed against 20 mM Tris, pH 7.5, to remove ethanol. Finally, the solution was exchanged into 20 mM Tris 8% sucrose solution and frozen to obtain mRNA-encapsulated lipid nanoparticles. These were then stored frozen at -80°C until use.

[0218] Example 4: Expression of VZV mRNA vaccine in cells and its immunogenicity in mice

[0219] 4.1 In vitro expression activity detection of VZV mRNA vaccine

[0220] HEK 293T cells (a human renal epithelial cell line) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. First, we verified the in vitro expression of six gE-LNP mRNAs designed with 30L70A as the poly A in HEK293 cells. In this example, six gE-mRNA LNP liquid formulations containing 30L70A were prepared using (1-methyl)-pseudouridine-triphosphate instead of UTP as in Example 3. The nucleotide sequences were gE1-LNP (SEQ ID NO: 13), gE2-LNP (SEQ ID NO: 14), gE3-LNP (SEQ ID NO: 15), gE4-LNP (SEQ ID NO: 16), gE5-LNP (SEQ ID NO: 17), and gE6-LNP (SEQ ID NO: 18).

[0221] HEK-293T cells were plated in six-well plates, with 1.2×10 HEK-293T cells per well. 6Cells were incubated at 37°C for 18-24 hours. gE-mRNA LNPs containing 1 μg of mRNA were directly added to HEK-293T cells and incubated for an additional 24 hours. The culture supernatant or cell pellet was collected for Western blot analysis. Mouse anti-VZV gE Ab was purchased from Abcam, and goat anti-mouse IgG HRP Ab was purchased from TransGen. The Western blot results are shown in Figure 4, demonstrating that: 1) all six gE-mRNA-LNPs expressed the corresponding gE protein; 2) gE2, gE4, gE5, and gE6, which retained the C-terminal transmembrane region, showed relatively high in vitro expression activity, with gE2, gE5, and gE6 showing relatively high expression activity.

[0222] 4.2 VZV mRNA vaccine immunization of C57 mice

[0223] Female, 6-8 week old C57 mice were used in this example. Animal studies were conducted in strict accordance with the recommendations of the Shanghai Laboratory Animal Care and Use Guide.

[0224] In this example, the six gE-LNP mRNAs prepared as in Example 4.1 were used to immunize C57 mice to evaluate the gE-specific antibody levels induced in the mice.

[0225] Mice were randomly divided into seven groups (n=7). Six groups were immunized with the gE-LNP mRNA formulation prepared as in Example 3 (a single dose of 2 μg of mRNA in a gE-mRNA LNP formulation); another group was administered phosphate-buffered saline (PBS) as a negative control. Each group was administered intramuscularly on day 0 (primary immunization) and day 14 (secondary immunization). On day 14 (i.e., day 28) after the second immunization, mouse serum was collected from the eye sockets for antibody detection.

[0226] Serum anti-VZV gE IgG detection (gPELISA) was performed as follows: VZV-gE antigen (ACROBiosystems) was diluted in ELISA coating buffer to a concentration of 0.25 μg / mL. Using a dispenser, 100 μL of 0.25 μg / mL VZV-gE antigen was added to each well and incubated overnight at 4°C. The test samples were equilibrated at room temperature for 30 minutes. The plates were then washed in PBST (0.05% Tween-20) and blocked with 5% skim milk in PBST. The sample serum was diluted with 2.5% skim milk powder at an initial titer of 1:50. The test serum samples were then diluted in two-fold dilutions. A negative serum control was included on each plate. Diluted serum samples were added to each well (100 μl) and incubated at 37°C (incubator) for 1 hour. The serum samples were discarded, and the wells were washed three times with 250 μl of PBST buffer added to each well using a dispenser. 100 μl of a 50,000-fold diluted goat anti-mouse IgG HRP (Abcam) was added to each well and incubated at 37°C (incubator) for 0.5 hour. The secondary antibody was discarded, and the wells were washed five times with 300 μl of PBST buffer added to each well using a dispenser. After washing, 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) (eBioscience) colorimetric solution was added to each well for 5 minutes in the dark. The reaction was terminated by adding 100 μl of 2N H2SO4 to each well and the OD values ​​were read at 450 nm using a microplate reader (BioTek). The endpoint titer was determined as the reciprocal of the last serum dilution. The sample OD450 nm was determined to be positive if it was greater than 2.1 times the OD450 nm of the negative control group. The results are shown in Figure 5, which showed that the six gE LNP-mRNA molecules induced comparable humoral immunity levels.

[0227] The following is a partial sequence of the present disclosure:

[0228] >Amino acid sequence of Oka strain gE protein

[0229] >Nucleotide sequence of Oka strain gE protein

[0230] >gE1 nucleotide sequence

[0231] >gE2 nucleotide sequence

[0232] >gE3 nucleotide sequence

[0233] >gE4 nucleotide sequence

[0234] >gE5 nucleotide sequence

[0235] >gE6 nucleotide sequence

[0236] >5'UTR

[0237] >3'UTR

[0238] >30L70A polyA

[0239] >120A polyA

[0240] >gE1(30L70A) mRNA sequence

[0241] >gE2(30L70A) mRNA sequence

[0242] >gE3(30L70A) mRNA sequence

[0243] >gE4(30L70A) mRNA sequence

[0244] >gE5(30L70A) mRNA sequence

[0245] >gE6(30L70A) mRNA sequence

[0246] >gE2 120A mRNA sequence

[0247] >gE6 120A mRNA sequence

[0248] >gE1 amino acid sequence

[0249] >gE2 amino acid sequence

[0250] >gE3 amino acid sequence

[0251] >gE4 amino acid sequence

[0252] >gE5 amino acid sequence

[0253] >Luciferase mRNA sequence

Claims

1. A pharmaceutical composition comprising a carrier, wherein the carrier comprises a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, wherein the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%, 2. The pharmaceutical composition according to claim 1, wherein the carrier further contains a non-cationic lipid, and the non-cationic lipid is selected from at least one of phospholipids, cholesterol or derivatives thereof.

3. The pharmaceutical composition according to claim 2, wherein the phospholipid is selected from 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 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-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-dialinolenoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE ), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

4. The pharmaceutical composition according to claim 2 or 3, wherein the content of the phospholipid is 5-40% of the molar amount of the carrier, preferably 10-20%, for example 10%.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein the cholesterol or its derivatives account for 30 to 60% of the molar amount of the carrier, preferably 35 to 45%, for example 40.5%.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the carrier further contains a conjugated lipid, and the conjugated lipid is preferably PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, more preferably distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and methoxypolyethylene glycol ditetradecanoyl acetamide (ALC-0159).

7. The pharmaceutical composition according to claim 6, wherein the conjugated lipid accounts for 0.5-4%, preferably 1.5-2.5%, such as 1.5%, of the molar amount of the carrier.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein it contains an active agent, which is selected from nucleic acids, preferably ribonucleic acids or deoxyribonucleic acids, more preferably small interfering RNA, microRNA, small hairpin RNA, messenger RNA (mRNA), circular RNA, self-replicating RNA and mixtures thereof.

9. The pharmaceutical composition according to claim 8, wherein the mRNA comprises an open reading frame (ORF), and the ORF encodes a protein or a polypeptide; Preferably, the mRNA further comprises any one of a 5' untranslated region element (5'UTR), a 3' untranslated region element (3'UTR), and a polyadenylic acid (poly-A) tail or any combination thereof; Preferably, the mRNA further comprises a 5' cap structure (5'Cap).

10. The pharmaceutical composition according to claim 9, wherein The protein or polypeptide is an antigen, Preferably, the antigen is selected from viral antigens, bacterial antigens, cancer antigens; More preferably, the virus strain is selected from influenza virus, coronavirus, varicella zoster virus (VZV), respiratory syncytial virus (RSV).

11. The pharmaceutical composition according to claim 10, wherein The antigen is varicella zoster virus (VZV) glycoprotein E (gE) protein, and its amino acid sequence is shown in any one of SEQ ID NO: 1 or SEQ ID NO: 21-SEQ ID NO: 25; Preferably, the nucleotide sequence of the antigen is as shown in any one of SEQ ID NO:3-SEQ ID NO:8; More preferably, the nucleotide sequence of the mRNA is as shown in any one of SEQ ID NO: 13-SEQ ID NO:

20.

12. The pharmaceutical composition according to claim 9, wherein The protein or polypeptide is hepatocyte growth factor (HGF), antibody or antigen binding fragment thereof.

13. The pharmaceutical composition according to any one of claims 1 to 12, comprising a) an active agent, preferably mRNA; b) a cationic lipid, wherein the cationic lipid comprises a compound of formula I or a pharmaceutically acceptable salt thereof, and the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%, c) a non-cationic lipid, wherein the non-cationic lipid is selected from phospholipids and cholesterol or its derivatives, wherein the phospholipids account for 5-40% of the molar weight of the carrier, and the cholesterol or its derivatives account for 30-60% of the molar weight of the carrier; and d) conjugating the lipid, wherein the molar percentage of the conjugate to the carrier is 0.5-4%.

14. The pharmaceutical composition according to any one of claims 1 to 13, which is a nanoparticle preparation, and the average particle size of the nanoparticle preparation is 10 nm to 220 nm.

15. A lyophilized preparation obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 14.

16. A reconstituted solution, which is obtained by reconstituted the lyophilized preparation according to claim 15, wherein the solvent used for reconstitution is preferably water for injection.

17. A method for preparing the pharmaceutical composition according to any one of claims 1 to 14, comprising: The step of mixing the cationic lipid, the non-cationic lipid and the conjugated lipid in an organic solvent.

18. Use of the pharmaceutical composition according to any one of claims 1 to 14, the lyophilized preparation according to claim 15 or the reconstituted solution according to claim 16 in the preparation of a medicament for inducing a protective immune response in a mammal, preferably in the preparation of a vaccine.

19. Use of the pharmaceutical composition according to any one of claims 1 to 14, the lyophilized preparation according to claim 15, or the reconstituted solution according to claim 16 in the preparation of a medicament for treating a disease or dysfunction in a mammal, wherein the disease is preferably a viral infection or cancer.

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