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WO2025002352A1 - 一种含有阳离子脂质的药物组合物及其用途 - Google Patents

一种含有阳离子脂质的药物组合物及其用途 Download PDF

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Publication number
WO2025002352A1
WO2025002352A1 PCT/CN2024/102386 CN2024102386W WO2025002352A1 WO 2025002352 A1 WO2025002352 A1 WO 2025002352A1 CN 2024102386 W CN2024102386 W CN 2024102386W WO 2025002352 A1 WO2025002352 A1 WO 2025002352A1
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Prior art keywords
glycero
pharmaceutical composition
phosphocholine
carrier
cationic lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/102386
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English (en)
French (fr)
Inventor
刘崇懿
唐晓娇
孙海伟
匡婧文
王勤
祝令建
姜军
黄建
宁威
廖成
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Shanghai Regenelead Therapies Co Ltd
Original Assignee
Shanghai Regenelead Therapies Co Ltd
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Filing date
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Publication of WO2025002352A1 publication Critical patent/WO2025002352A1/zh
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids

Definitions

  • the present invention belongs to the field of medicine and relates to the preparation of a pharmaceutical composition containing cationic lipids and its use.
  • 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.
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • plasmids etc.
  • 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.
  • lipid nanoparticles have been shown to be excellent carriers of nucleic acids for the treatment of various diseases.
  • Lipid nanoparticles formed by cationic lipids and other auxiliary lipids, such as cholesterol, phospholipids and PEGylated lipids encapsulate nucleic acids, protect nucleic acids from degradation, promote cellular uptake, and reduce immune responses.
  • lipid nanoparticles have other advantages for cellular delivery of bioactive ingredients, such as good targeting, few side effects, good stability and high transfection efficiency.
  • 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.
  • the present disclosure provides a pharmaceutical composition, which comprises 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, and the molar percentage of the cationic lipid to the carrier is greater than or equal to 10% and less than 50%.
  • 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 between two numbers.
  • the cationic lipid in the pharmaceutical composition accounts for 42-49% of the molar amount of the carrier.
  • the cationic lipid in the pharmaceutical composition comprises 45% of the molar amount of the carrier.
  • the cationic lipid in the pharmaceutical composition comprises 46% of the molar amount of the carrier.
  • the cationic lipid in the pharmaceutical composition accounts for 47% of the molar amount of the carrier.
  • the cationic lipid in the pharmaceutical composition accounts for 48% of the molar amount of the carrier.
  • the cationic lipid in the pharmaceutical composition accounts for 49% of the molar amount of the carrier.
  • 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 their derivatives.
  • the non-cationic lipid in the pharmaceutical composition is selected from a mixture of phospholipids and cholesterol or a derivative thereof.
  • 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.
  • the content of phospholipids in the pharmaceutical composition accounts for 15% of the molar amount of the carrier.
  • the content of phospholipids in the pharmaceutical composition accounts for 20% of the molar amount of the carrier.
  • the content of phospholipids in the pharmaceutical composition is 10-20% of the molar amount of the carrier.
  • the content of phospholipids in the pharmaceutical composition accounts for 10% of the molar amount of the carrier.
  • the phospholipids in the pharmaceutical composition are 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-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-ole ...distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DUPC), 1-sn-sn-sn-sn-sn-sn-sn-sn-sn-s
  • the phospholipid in the pharmaceutical composition is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the phospholipid in the pharmaceutical composition is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • 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.
  • the content of cholesterol or its derivatives in the pharmaceutical composition is 35-45% of the molar amount of the carrier.
  • the content of cholesterol or its derivatives in the pharmaceutical composition accounts for 40.5% of the molar amount of the carrier.
  • the content of cholesterol or its derivatives in the pharmaceutical composition accounts for 45% of the molar amount of the carrier.
  • cholesterol derivatives include but are not limited to sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid.
  • 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 between two numbers.
  • the non-cationic lipid in the pharmaceutical composition accounts for 50-65% of the molar amount of the carrier.
  • the non-cationic lipid in the pharmaceutical composition accounts for 40-55% of the molar amount of the carrier.
  • the non-cationic lipid in the pharmaceutical composition accounts for 55-62% of the molar amount of the carrier.
  • the molar ratio of cholesterol or its derivatives to phospholipids in the pharmaceutical composition of the present invention is 1.0-5.0, 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 between two numbers.
  • the molar ratio of cholesterol or its derivative to phospholipid in the pharmaceutical composition is 2.0-5.0.
  • the carrier in the pharmaceutical composition described in the present disclosure also contains conjugated lipids
  • the conjugated lipids include but are not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.
  • 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).
  • DSPE-PEG2000 distearoylphosphatidylethanolamine polyethylene glycol 2000
  • DMG-PEG2000 dimyristoylglycerol-3-methoxy polyethylene glycol 2000
  • AAC-0159 methoxy polyethylene glycol ditetradecanoyl acetamide
  • the conjugated lipid in the pharmaceutical composition is selected from distearoylphosphatidylethanolamine polyethylene glycol 2000.
  • the content of the conjugated lipid in the pharmaceutical composition is 0.5-1.0% of the molar amount of the carrier.
  • the content of the conjugated lipid in the pharmaceutical composition is 1.5-2.5% of the molar amount of the carrier.
  • the content of the conjugated lipid in the pharmaceutical composition is 1-2% of the molar amount of the carrier.
  • the content of the conjugated lipid in the pharmaceutical composition is 1.5% of the molar amount of the carrier.
  • the content of the conjugated lipid in the pharmaceutical composition is 2.0% of the molar amount of the carrier.
  • the pharmaceutical composition of the present disclosure further contains an active agent, which is selected from nucleic acids, including ribonucleic acid or deoxyribonucleic acid.
  • 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.
  • siRNA small interfering RNA
  • miRNA micro RNA
  • shRNA small hairpin RNA
  • mRNA messenger RNA
  • mRNA circular RNA
  • self-replicating RNA or any combination thereof.
  • the active agent is selected from mRNA.
  • the mRNA encodes a protein or polypeptide.
  • the mRNA encodes a protein or polypeptide derived from an infectious agent, or is an infectious agent antigen.
  • the protein or polypeptide is a viral antigen and/or a bacterial antigen.
  • the virus is selected from adenovirus, herpes virus (e.g., herpes simplex virus type I, herpes simplex virus type II, 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, coxsackievirus, rhinovirus, severe acute respiratory syndrome virus, 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-Con
  • the virus is selected from influenza virus (e.g., influenza A virus, influenza B virus), coronavirus, or a combination thereof.
  • influenza virus e.g., influenza A virus, influenza B virus
  • coronavirus is SARS-COV-2
  • the coronavirus is The original is a spike protein
  • the spike protein is selected from the spike protein of any virus strain of SARS-COV-2, SARS-COV-2Alpha, SARS-COV-2Beta, SARS-COV-2Gamma, SARS-COV-2Kappa, SARS-COV-2Delta or SARS-COV-2Omicron.
  • influenza virus is selected from influenza A virus or influenza B virus;
  • influenza virus antigen is hemagglutinin protein (HA) and/or neuraminidase (NA);
  • influenza virus antigen is selected from the hemagglutinin protein and/or neuraminidase of any virus strain of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria, and influenza B virus Yamagata.
  • the virus is RSV.
  • 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 pre-fusion glycoprotein F.
  • the RSV antigenic polypeptide or its immunogenic fragment and its sequence in WO2017070622A, WO2014160463A, WO2012158613A, WO2017109629A, and pre-fusion glycoprotein F and its sequence in WO2017172890A are introduced in full herein.
  • the virus is VZV.
  • the VZV antigen is a VZV glycoprotein.
  • 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.
  • the VZV glycoprotein is a VZV gE protein, such as a wild-type VZV gE protein or a truncate, fragment, or mutant thereof.
  • the wild-type VZV gE protein is from the Oka strain.
  • the wild-type VZV gE protein comprises (or is) the amino acid sequence shown in SEQ ID NO:1 and/or comprises (or is) the nucleotide sequence shown in SEQ ID NO:2.
  • the truncate 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.
  • 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.
  • 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 based on amino acids 1-574 of the wild-type gE protein, including a Y569A mutation.
  • 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.
  • 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.
  • the protein or polypeptide is selected from fructose bisphosphate aldolase A (ALDOA), ⁇ -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 ⁇ polypeptide (BCKDHA), colony stimulating factor 2 (granulocyte-macrophage) (GM-CSF), colony stimulating factor 3 (granulocyte) (GCSF), deficido
  • 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 that are specific to tumors or cancer antigens that are specific to each subject, the cancer antigen is referred to as a new epitope or subject-specific epitope or antigen).
  • the cancer antigen is a subject-specific cancer antigen.
  • the subject-specific cancer antigen may represent the exome of a tumor sample of a subject, or a transcriptome representing a tumor sample of a subject.
  • the subject-specific cancer antigen may represent an exosome of a subject.
  • the cancer antigen is a traditional cancer antigen.
  • a traditional cancer antigen is a non-mutated antigen.
  • a traditional cancer antigen is a mutated antigen.
  • the cancer antigen may be carcinoembryonic antigen, ⁇ 1-fetoprotein, isoferritin, and fetal sulphoglycoprotein, ⁇ 2-H-ferritin, and ⁇ -fetoprotein.
  • the protein or polypeptide is an antibody or an antigen-binding fragment thereof.
  • 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.
  • the antibody or its antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody peptibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv).
  • the mRNA comprises:
  • (b) untranslated region element (UTR) may include (b1) 5'UTR and/or (b2) 3'UTR;
  • the mRNA further comprises a 5' Cap structure.
  • the RNA molecule as described in any of the above contains any of i)-v):
  • the ORF and the 5'UTR and/or the 3'UTR are derived from the same and/or different genes.
  • the 5'UTR is selected from ⁇ -globin 5'UTR, 5'UTR containing a strong Kozak translation initiation signal, cytochrome b-245 alpha polypeptide (CYBA) 5'UTR, hydroxysteroid (17- ⁇ ) dehydrogenase (HSD17B4) 5'UTR, tobacco etch virus (TEV) 5'UTR, heat shock protein 70 (Hsp70) 5'UTR, eIF4G5'UTR, ACTG1 5'UTR, CTSB 5'UTR or ARHGAP15 gene 5'UTR.
  • CYBA cytochrome b-245 alpha polypeptide
  • HSD17B4 hydroxysteroid (17- ⁇ ) dehydrogenase
  • Hsp70 heat shock protein 70
  • the 5'UTR is derived from or is a 5'UTR sequence of ACTG1 or ARHGAP15 gene or a derivative sequence thereof.
  • the ARHGAP15 gene is derived from any species, such as human ARHGAP15, baboon ARHGAP15, mouse ARHGAP15, etc.
  • the 5'UTR is derived from or is the 5'UTR sequence of the human ARHGAP15 gene or a derivative thereof.
  • 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%, 100% identity thereto.
  • the 3'UTR is selected from the group consisting of ⁇ -globin 3'UTR, ⁇ -globin (hemoglobin subunit beta, HBB) 3'UTR, ⁇ -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.
  • the 3'UTR is derived from or is the 3'UTR sequence of the HBB, CTSB or ARHGAP15 gene or a derivative sequence thereof.
  • the 3'UTR comprises or is a nucleotide sequence as shown in SEQ ID NO: 10 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.
  • the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA or SV40late polyA.
  • 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.
  • the nucleotide sequence of the RNA molecule is as shown in any one of SEQ ID NO: 13-SEQ ID NO: 20.
  • 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 in the sugar, backbone or nucleobase portion of the nucleotide and/or nucleoside as recognized in the art.
  • the modified nucleobase in the mRNA comprises 1-methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C) and/or pseudouridine ( ⁇ ).
  • the modified nucleobase in the mRNA comprises 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine and/or 5-methoxycytidine.
  • the mRNA includes a combination of at least two (e.g., 2, 3, 4 or more) any of the above-mentioned modified nucleobases, including but not limited to chemical modifications.
  • the 5' Cap structure is located upstream of the 5' UTR, for example, at the 5' end of the 5' UTR.
  • the 5' cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first nucleobase, such as m7GpppN), Cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN, such as m7G(5')pp(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-
  • the pharmaceutical composition comprises: 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 40-49%,
  • the pharmaceutical composition contains:
  • 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%,
  • 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;
  • conjugated lipids said conjugates accounting for 0.5-4% of the molar amount of the carrier.
  • the pharmaceutical composition contains:
  • an active agent comprising mRNA
  • 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%,
  • 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;
  • conjugated lipids said conjugates accounting for 0.5-4% of the molar amount of the carrier.
  • the pharmaceutical composition contains:
  • an active agent comprising mRNA
  • 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%,
  • 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;
  • conjugating lipid DMG-PEG wherein the conjugate accounts for 0.5-2% of the molar amount of the carrier.
  • the pharmaceutical composition contains:
  • an active agent comprising mRNA
  • 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 40-49%,
  • 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;
  • the pharmaceutical composition contains:
  • an active agent comprising mRNA
  • 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 45 to 49%,
  • 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;
  • the pharmaceutical composition of the present disclosure is a nanoparticle preparation, and the average particle size of the nanoparticle preparation is 10nm to 220nm.
  • the average particle size of the nanoparticle preparation is 100-150nm.
  • the average particle size of the nanoparticle preparation is 150-200nm.
  • the average particle size of the nanoparticle preparation is 80-160nm.
  • the average particle size of the nanoparticle preparation is 80-150nm.
  • the average particle size of the nanoparticle preparation is 70-160nm.
  • the average particle size of the nanoparticles is measured using dynamic light scattering.
  • freeze drying includes a freezing step, a primary drying step, and a secondary drying step.
  • freeze drying of the present disclosure is performed with reference to the method in CN107567497A, and the relevant contents are introduced herein for illustration.
  • the present disclosure also provides a reconstituted solution, which is obtained by reconstituted the aforementioned lyophilized preparation through a reconstitution step, and the solvent used for reconstitution is water for injection.
  • the present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, lyophilized preparation 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.
  • the organic solvent is selected from ethanol.
  • the method for preparing the pharmaceutical composition is prepared by referring to the method in the journal literature (Pharm. Res. 22 (2005) 362-372) or US9504651, and the relevant contents are introduced herein for illustration.
  • 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.
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition, lyophilized preparation or reconstituted solution in the preparation of a medicament for treating a disease or dysfunction in a mammal.
  • 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.
  • 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.
  • 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).
  • 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.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-dodecanoylphosphatidylethanolamine N-succinylphosphatidylethanolamine
  • N-glutarylphosphatidylethanolamine N-glutarylphosphatidylethanol
  • neutral lipid refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.
  • amphiphilic lipid refers in part to any suitable material, wherein the hydrophobic portion of the lipid material is oriented to the hydrophobic phase, and the hydrophilic portion is oriented to the aqueous phase.
  • the hydrophilic nature comes from the presence of polar or charged groups such as sugars, 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.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.
  • 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.
  • mammal refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, including DNA and RNA.
  • nucleic acid molecule is synonymous with “nucleic acid” and “polynucleotide”.
  • DNA can take the following forms: for example, antisense molecules, plasmid DNA, pre-concentrated 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.
  • the antigen is preferably presented by a cell, preferably an antigen presenting cell (including a diseased cell, particularly a cancer cell, in the context of an MHC molecule), which results in an immune response against the antigen.
  • the antigen is a product corresponding to or derived from a natural antigen. This natural antigen includes a tumor antigen.
  • cancer antigen is interchangeable with “tumor antigen” or “tumor-associated antigen”, which is capable of stimulating an immune response, preferably a cellular response against an antigen or a cell characterized by expressing the antigen and preferably presenting the antigen (e.g., a diseased cell, especially a cancer cell).
  • a "cancer antigen” is specifically expressed in a limited number of tissues and/or organs or in a specific developmental stage under normal conditions (e.g., a tumor antigen may be specifically expressed in gastric tissue (preferably in gastric mucosa), in reproductive organs (e.g., in testes), in trophoblastic tissue (e.g., in placenta) or in germline cells under normal conditions), and in one or more tumors.
  • the "limited number" refers to no more than 3, such as no more than 2.
  • Polypeptide “protein” or “protein” are used interchangeably to refer to a polymer of amino acid residues, and are applicable to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • VZV Via-zoster virus
  • the term "Varicella-zoster virus (VZV)” refers to an alpha herpes virus that exists in the form of a spherical multilayer structure.
  • the viral genome is surrounded by a protein capsid structure covered with an amorphous layer of membrane proteins.
  • the membrane composed of virus-encoded proteins and enzymes is located in the space between the nucleus and the viral envelope, which is obtained from the host cell membrane and contains virus-encoded glycoproteins.
  • VZV glycoproteins encoding glycoproteins gE, gI, gB, gH, gK, gL, gC, gN and gM, functions in different steps of the viral replication cycle.
  • glycoprotein E glycoprotein E
  • Glycoprotein I gI, ORG 67
  • TGN trans-Galgia network
  • Glycoprotein I (gI) is required in the TGN for VZV envelopment and for efficient membrane fusion during VZV replication.
  • VZV gE and gI are found complexed together on the surface of infected host cells.
  • Glycoprotein B (ORF 31), which is the second most prevalent glycoprotein and is thought to play a role in viral entry, binds neutralizing antibodies.
  • Glycoprotein H is thought to have a fusogenic function, facilitating the spread of the virus from cell to cell.
  • Antibodies to gE, gB, and gH are ubiquitous after natural infection and after vaccination, and have demonstrated in vitro neutralizing viral activity.
  • the amino acid sequence of an exemplary gE protein is shown in any one of SEQ ID NO: 1 or SEQ ID NO: 21-SEQ ID NO: 25 of the present disclosure.
  • mutants of a wild-type VZV gE protein refer to a polypeptide that exhibits an introduced mutation relative to the wild-type VZV gE protein and is immunogenic against the wild-type VZV gE protein.
  • 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.
  • a replacement 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)".
  • the annotation Y75A refers to the replacement 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).
  • the amino acid code before the position number such as "75A” may be omitted in the annotation.
  • Codon optimization refers to replacing codons that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species in a target sequence, while the codons before and after the replacement encode the same amino acid.
  • Codon preference differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • identity refers to the relationship between the sequences of two or more polypeptides or polynucleotides as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between two or more amino acid residues or nucleic acid residues as determined by the number of matches between them.
  • 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") in two or more sequences.
  • % 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 the second sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of identity in a candidate amino acid or nucleic acid sequence. Methods and computer programs for comparison are well known in the art. It should be understood that identity depends on the calculation of the percentage of identity, but its value may be different due to gaps and penalties introduced in the calculation.
  • 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.
  • tools for alignment include those of the BLAST kit (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).
  • a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has recently been developed that is claimed 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.
  • vaccine refers to a pharmaceutical preparation (pharmaceutical composition) or product that, after 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 treat cancer.
  • 5' untranslated region element refers to the region of an mRNA that is located immediately upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript translated by the ribosome) and that does not encode a polypeptide.
  • 3' untranslated region element refers to the region of an mRNA that is located immediately downstream (ie, 3') of a stop codon (ie, the codon of an mRNA transcript that signals the termination of translation) and that does not encode a polypeptide.
  • ORF open reading frame
  • poly (A) tail is located downstream of a 3'UTR containing multiple consecutive adenosine monophosphates, for example, an mRNA region directly downstream (i.e., 3') thereof.
  • the poly (A) tail may contain 10 to 300 adenosine monophosphates.
  • 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.
  • the poly (A) tail contains 50 to 250 adenosine monophosphates.
  • the poly(A) tail serves to protect the mRNA from enzymatic degradation, such as in the cytoplasm, and facilitates transcription termination and/or nuclear export of the mRNA and translation.
  • the “average particle size” (Z-average Size) described in the present disclosure is the average value of light intensity, which is calculated from the light intensity contributed by different types of particles.
  • the “average particle size” can be measured by conventional particle size measurement techniques well known to those skilled in the art. Such techniques include sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, etc.
  • the polydispersity index "PDI" (Polydispersity Index) disclosed in the present invention reflects the uniformity of particle size and is an important index for characterizing particle size.
  • 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, or B is added to A.
  • the numerical values in this disclosure are instrumental measurements, which have a certain degree of error. Generally speaking, plus or minus 10% is within the reasonable error range. Of course, the context in which the numerical value is used needs to be considered.
  • the particle size of the active ingredient the numerical value is the error change after measurement does not exceed plus or minus 10%, which can 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%.
  • the compound of formula I disclosed in the present invention is prepared by referring to the method in WO2023125738, and the relevant contents are illustrated in this application.
  • the mRNA used for testing in the present disclosure is luciferase mRNA.
  • Figure 1 Luciferase protein expression levels of liposome nanoparticles corresponding to lipid compositions 1-15 after transfection of HEK293 cells.
  • Figure 2 Particle size stability of lipid compositions at 4°C.
  • Figure 3 Particle size stability of lipid compositions at 25°C.
  • Figure 5 Serum anti-VZV gE IgG detection.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • 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 mixed to prepare an ethanol lipid solution.
  • the molar ratios of the four components were prepared as shown in Table 1.
  • the mRNA encoding the luciferase protein (whose 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 dialyzing in a 20mM Tris solution, and finally replaced in a 20mM Tris 8% sucrose solution to obtain a liposome nanoparticle composition encapsulating mRNA.
  • the nanoparticle size and polydispersity index (PDI) of liposome nanoparticles were measured by dynamic light scattering using a Malvern Zetasizer Pro in 173° backscattering detection mode.
  • Quant-iT RiboGreen RNA Assay Kit was used to detect RNA quantification. Encapsulation rate.
  • Test Example 1 Evaluation of the mRNA delivery efficiency of lipid particle composition carriers
  • the protein expression levels of the liposome nanoparticles corresponding to lipid compositions 1-15 after delivering mRNA in cells were detected.
  • HEK 293 cells were inoculated into 96-well plates and cultured overnight.
  • the lipid nanoparticle solution encapsulating luciferase mRNA was added to the culture medium of the cell plate wells, and the mRNA dose was 100 ng/well.
  • 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 value 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.
  • Test Example 2 Evaluation of the stability of lipid particle compositions
  • 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 detected, and the size change during the storage process was compared, which represents the stability of the lipid nanoparticles. A large change in particle size means poor stability. Each group was tested 3 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 respectively. 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 being placed in 4°C and 25°C environments for 3 days, the polydispersity coefficient of the particle size was detected by dynamic light scattering. The size of the particle size change is shown in Table 4 and Figures 2-3.
  • this embodiment performed codon optimization and designed different C-terminal variant molecules encoding Oka strain gE protein, 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.
  • 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 1 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-terminal truncated gE molecules.
  • T7 promoter sequence (TAATACGACTCACTATAAG), 5'-UTR sequence (its corresponding RNA sequence is such as SEQ ID NO:9), 3'-UTR sequence (its corresponding RNA sequence is such as SEQ ID NO:10) and poly (A) sequence (its corresponding RNA sequence is such as SEQ ID NO:11) were also designed.
  • T7 promoter sequence 5'-UTR sequence, DNA ORF, 3'-UTR sequence and poly (A) sequence were connected in order, and full gene synthesis was performed using pUC57 as a vector (Suzhou Jinweizhi Biotechnology Co., Ltd.) to obtain a plasmid DNA template.
  • 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.
  • Compound 1 (Formula I compound), 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 50mM pH 4 acetic acid buffer to prepare an mRNA aqueous solution.
  • the ethanol lipid solution and the mRNA aqueous solution were mixed by microfluidics at a volume ratio of 1:3, and the weight of compound 1 to mRNA in the lipid was 12:1 to prepare lipid nanoparticles.
  • the ethanol was removed by dialyzing in a 20mM Tris pH7.5 solution, and finally replaced in a 20mM Tris 8% sucrose solution and frozen to obtain lipid nanoparticles encapsulating mRNA, which were frozen at -80°C until use.
  • HEK 293T cells (a human kidney 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.
  • DMEM Dulbecco’s modified Eagle’s medium
  • gE-mRNA LNPs liquid preparations containing 30L70A were prepared using (1-methyl)-pseudouridine triphosphate instead of UTP as in Example 3, and 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).
  • HEK-293T cells were plated in a six-well plate, with 1.2 ⁇ 10 6 cells per well of HEK-293T cells, and incubated in a 37°C incubator for 18-24 hours; gE-mRNA LNPs preparation containing 1 ⁇ g mRNA was directly added to HEK-293T cells, and incubated for another 24 hours, and the culture supernatant or cell pellet was collected for Western Blot detection.
  • the mouse anti-VZV gE Ab used in Western Blot detection was purchased from Abcam, and the Goat anti-mouse IgG HRP Ab was purchased from TransGen.
  • mice Female C57 mice aged 6 to 8 weeks were used in this example. Animal research was conducted in strict accordance with the recommendations of the Shanghai Laboratory Animal Care and Use Guide.
  • the six gE-LNP mRNAs prepared as in Example 4.1 were used to immunize C57 mice to evaluate the level of gE-specific antibodies induced in mice.
  • PBS phosphate buffered saline
  • Each group was administered by intramuscular injection on day 0 (primary immunization) and day 14 (secondary immunization). On the 14th day (i.e., day 28) after the second immunization of mice, blood was collected from the eye sockets to collect mouse serum for antibody detection.
  • Serum anti VZV gE IgG detection is as follows: VZV-gE antigen (ACROBiosystems) is diluted with ELISA coating buffer to 0.25 ⁇ g/mL, and 100 ⁇ L 0.25 ⁇ g/mL VZV-gE antigen is added to each well using a dispenser, and incubated overnight at 4°C; the samples to be tested are equilibrated at room temperature for 30 minutes, and the plates are washed in PBST (0.05% Tween-20) and blocked with 5% skim milk in PBST.
  • PBST 0.05% Tween-20
  • the sample serum is diluted with 2.5% skim milk powder, with an initial dilution titer of 1:50, and then the serum samples to be tested are diluted at a 2-fold ratio, and a negative serum control is set in each plate.
  • the endpoint titer is determined as the reciprocal of the last serum dilution.
  • the sample OD450 nm was greater than 2.1 times the OD450 nm of the negative control group and was judged as a positive value.
  • the results are shown in Figure 5, which show that these six gE LNP-mRNA molecules induced a comparable level of humoral immunity.

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Abstract

一种含有阳离子脂质的药物组合物及其用途，具体而言，提供一种药物组合物，其包含载体，载体包含有阳离子脂质，阳离子脂质与载体摩尔百分比大于等于10％，且小于50％。

Description

一种含有阳离子脂质的药物组合物及其用途 技术领域
本公开属于医药领域，涉及制备一种含有阳离子脂质的药物组合物及其用途。
背景技术
基于核酸类的药物，例如信使RNA(mRNA)、反义寡核苷酸、小干扰RNA(siRNA)、质粒等具有广阔的应用前景，如何安全有效地递送到体内靶器官和靶细胞是制约该技术发展的难题。
目前核酸药物递送载体可分为病毒载体载体和非病毒载体两大类，脂质纳米颗粒介导的核酸药物递送是属于非病毒递送载体的主要方法。
在基因治疗和疫苗应用中，脂质纳米颗粒已经被证明是治疗各种疾病的核酸的优良载体。由阳离子脂质和其他辅助脂质，例如胆固醇、磷脂和PEG化的脂质形成的脂质纳米颗粒包封核酸，保护核酸免于降解并且促进细胞摄取，降低免疫反应。另外，脂质纳米颗粒进行生物活性成分的细胞传递具有其他优点，如靶向性好、副作用小、稳定性好和转染效率较高等。
尽管脂质纳米颗粒已经展示递送的优势，但安全性、功效和特异性仍有待改良。仍需要开发有助于将治疗剂和/或预防剂如核酸递送至细胞的改进的阳离子脂质组合物。
发明内容
本公开提供了一种药物组合物，其包含载体，所述载体包含有阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，阳离子脂质与载体摩尔百分比大于等于10％，且小于50％，
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的15～49％，包括但不限于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％或任意两数之间的值。
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的42～49％。
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的45％。
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的46％。
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的47％。
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的48％。
在一些实施方案中，药物组合物中阳离子脂质占载体摩尔量的49％。
在另一些实施方案中，药物组合物中所述载体还含有非阳离子脂质，所述非阳离子脂质选自磷脂、胆固醇或其衍生物的中至少一种。
在一些实施方案中，药物组合物中非阳离子脂质选自磷脂与胆固醇或其衍生物的混合物。
一些实施方案中提供的药物组合物中磷脂的含量占载体摩尔量的5～40％，包括但不限于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％或任意两数之间的值。
在一些实施方案中，药物组合物中磷脂的含量占载体摩尔量的15％。
在一些实施方案中，药物组合物中磷脂的含量占载体摩尔量的20％。
在一些实施方案中，药物组合物中磷脂的含量占载体摩尔量的10～20％。
在一些实施方案中，药物组合物中磷脂的含量占载体摩尔量的10％。
另一些实施方案中提供的药物组合物中磷脂选自1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16 Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)以及其混合物。
在一些实施方案中，药物组合物中磷脂为1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱 (DSPC)。
在一些实施方案中，药物组合物中磷脂为1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)。
另一方面，本公开药物组合物中胆固醇或其衍生物占载体摩尔量的30～60％，包括但不限于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％或任意两数之间的值。
在一些实施方案中，药物组合物中胆固醇或其衍生物含量占载体摩尔量的35～45％。
在一些实施方案中，药物组合物中胆固醇或其衍生物含量占载体摩尔量的40.5％。
在一些实施方案中，药物组合物中胆固醇或其衍生物含量占载体摩尔量的45％。
在另一些实施方案中，胆固醇衍生物包括但不限于谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸。
在一些实施方案中，药物组合物中非阳离子脂质占载体摩尔量的35～70％，包括但不限于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％或任意两数之间的值。
在一些实施方案中，药物组合物中非阳离子脂质占载体摩尔量的50～65％。
在一些实施方案中，药物组合物中非阳离子脂质占载体摩尔量的40～55％。
在一些实施方案中，药物组合物中非阳离子脂质占载体摩尔量的55～62％。
另一方面，本公开药物组合物中胆固醇或其衍生物与磷脂的摩尔比为1.0～5.0，包括但不限于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或任意两数之间的值。
在一些实施方案中，药物组合物中胆固醇或其衍生物与磷脂的摩尔比2.0～5.0。
另一方面，本公开所述药物组合物中载体还含有缀合脂质，所述缀合脂质包括但不限于PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油。
在一些实施方案中，所述药物组合物中缀合脂质选自二硬脂酰基磷脂酰乙醇胺聚乙二醇2000(DSPE-PEG2000)，二肉豆蔻酰甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)和甲氧基聚乙二醇双十四烷基乙酰胺(ALC-0159)。
在一些实施方案中，所述药物组合物中缀合脂质选自二肉豆蔻酰甘油-3-甲氧基聚乙二醇2000。
在一些实施方案中，所述药物组合物中缀合脂质选自二硬脂酰基磷脂酰乙醇胺聚乙二醇2000。
在一些实施方案中，缀合脂质起抑制载体(颗粒)聚集。在一些实施方案中，药物组合物中缀合脂质的含量占载体摩尔量的0.5～4％，包括但不限于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％或任意两数之间的值。
在一些实施方案中，药物组合物中缀合脂质的含量占载体摩尔量的0.5～1.0％。
在一些实施方案中，药物组合物中缀合脂质的含量占载体摩尔量的1.5～2.5％。
在一些实施方案中，药物组合物中缀合脂质的含量占载体摩尔量的1～2％。
在一些实施方案中，药物组合物中缀合脂质的含量占载体摩尔量的1.5％。
在一些实施方案中，药物组合物中缀合脂质的含量占载体摩尔量的2.0％。
本公开药物组合物中还含有活性剂，所述活性剂选自核酸，包括核糖核酸或脱氧核糖核酸。
在一些实施方案中，所述活性剂选自小干扰RNA(siRNA)、微RNA(miRNA)、小发夹RNA(shRNA)、信使RNA(mRNA)、环状RNA、自复制RNA中的任一或其任意组合。
在一些实施方案中，所述活性剂选自mRNA。
在一些实施方案中，所述mRNA包含ORF，所述ORF表达蛋白或多肽。
在一些实施方案中，所述mRNA编码蛋白或多肽。
在一些实施方案中，所述mRNA编码抗原。
在一些实施方案中，所述mRNA编码蛋白或多肽源自传染因子，或为传染因子抗原。
在一些实施方案中，所述蛋白或多肽为病毒抗原和/或细菌抗原。
在一些实施方案中，所述病毒选自腺病毒、疱疹病毒(例如，I型单纯疱疹病毒、II型单纯疱疹病毒8型人疱疹病毒)、脑炎病毒、乳头瘤病毒、水痘带状疱疹病毒(VZV)、爱泼斯坦-巴尔病毒、人巨细胞病毒、人乳头瘤病毒、BK病毒、JC病毒、天花、脊髓灰质炎病毒、肝炎病毒(例如，甲型、乙型、丙型、丁型或戊型肝炎病毒)、人博卡病毒、细小病毒(例如，B19)、人星状病毒、诺沃克病毒、柯萨奇病毒、鼻病毒、严重急性呼吸道综合征病毒、黄热病毒、登革热病毒、西尼罗病毒、风疹病毒、人免疫缺陷病毒(HIV)、甲型或乙型流感病毒、瓜纳里托病毒、胡宁病毒、拉沙病毒、马丘波病毒、萨比亚病毒、克里米亚-刚果出血热病毒、埃博拉病毒、马尔堡病毒、麻疹病毒、腮腺炎病毒、副流感病毒、呼吸道合胞病毒(RSV)、人偏肺病毒、亨德拉病毒、尼帕病毒、狂犬病病毒、轮状病毒、环状病毒、科罗拉多壁虱热病毒、汉坦病毒、中东呼吸道冠状病毒、基孔肯亚病毒或版纳病毒。
在一些实施方案中，所述病毒选自流感病毒(例如甲型流感病毒、乙型流感病毒)、冠状病毒或其组合。例如，所述冠状病毒为SARS-COV-2；所述冠状病毒抗 原为刺突蛋白；所述刺突蛋白选自SARS-COV-2、SARS-COV-2Alpha、SARS-COV-2Beta、SARS-COV-2Gamma、SARS-COV-2Kappa、SARS-COV-2Delta或SARS-COV-2Omicron任一病毒株的刺突蛋白。例如，所述流感病毒选自A型流感病毒或B型流感病毒；所述流感病毒抗原为血凝素蛋白(HA)和/或神经氨酸酶(NA)；所述流感病毒抗原选自A型流感病毒H1N1、A型流感病毒H3N2、B型流感病毒Victoria、B型流感病毒Yamagata任一病毒株的血凝素蛋白和/或神经氨酸酶。
在一些实施方案中，所述病毒为RSV。例如，所述RSV抗原为RSV附着蛋白(G)或其免疫原性片段、RSV融合(F)糖蛋白或其免疫原性片段，或其组合；例如，所述RSV抗原为融合前糖蛋白F。此处全文引入WO2017070622A中的RSV抗原性多肽或其免疫原性片段及其序列，WO2014160463A、WO2012158613A、WO2017109629A、WO2017172890A中的融合前糖蛋白F及其序列。
在一些实施方案中，所述病毒为VZV。例如，所述VZV抗原为VZV糖蛋白。例如，VZV糖蛋白可以是VZV gE、gI、gB、gH、gK、gL、gC、gN或gM多肽或其免疫原性片段或表位。在一些实施方案中，所述VZV糖蛋白是VZV gE蛋白，例如野生型VZV gE蛋白或其截短体、片段、突变体。在一些实施方案中，所述野生型VZV gE蛋白来自Oka毒株。在一些实施方案中，野生型VZV gE蛋白包含(或为)SEQ ID NO:1所示氨基酸序列和/或包含(或为)SEQ ID NO:2所示核苷酸序列。
在一些实施方案中，所述截短体可以是任何截短多肽，例如包括氨基酸1至200、1至250、1至300、1至350、1至400、1至450、1至500、1至540、1至544、1至545、1至546、1至550、1至560、1至561、1至570、1至573、1至574、1至575的截短VZV gE多肽。在一些实施方案中，所述VZV gE蛋白进一步包含Y569A点突变。此处全文引入WO2017070601A中的VZV抗原性多肽及其序列。
在一些实施方案中，所述截短体的氨基酸序列为野生型gE蛋白的第1-544、1-546、1-561或1-574位氨基酸。在一些实施方案中，所述截短体的氨基酸序列为在野生型gE蛋白的第1-574位氨基酸的基础上，包含Y569A突变。
在一些实施方案中，所述VZV gE蛋白的氨基酸序列如SEQ ID NO:1或SEQ ID NO:21-SEQ ID NO:25中任一项所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％序列同一性的氨基酸序列。
在一些实施方案中，所述编码VZV gE蛋白的开放阅读框的核苷酸序列如SEQ ID NO:3-SEQ ID NO:8中任一项所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％序列同一性的核苷酸序列。
在一些实施方案中，所述蛋白或多肽选自果糖二磷酸醛缩酶A(ALDOA)、α-甲基酰基-辅酶A消旋酶(AMACR)、血清淀粉样蛋白P成分(APCS)、血管生成素1(ANGPT1)、载脂蛋白A-I(APOA1)Milano、载脂蛋白A-I(APOA1)Paris、载脂蛋白 A-I(APOA1)、精氨基琥珀酸裂解酶(ASL)、artemin(ARTN)、芳基硫酸酯酶B(ARSB)、杀菌/通透性增加蛋白(rBPI-21)、骨形态发生蛋白2(BMP2)、骨形态发生蛋白7(BMP7)、支链酮酸脱氢酶E1α多肽(BCKDHA)、集落刺激因子2(粒细胞-巨噬细胞)(GM-CSF)、集落刺激因子3(粒细胞)(GCSF)、脱氧核糖核酸酶I(DNA酶1)、促红细胞生成素(EPO)、IX因子、VII因子、XI因子、纤维蛋白原A(FGA)、成纤维细胞生长因子18(FGF18)、成纤维细胞生长因子23(FGF23)、成纤维细胞生长因子7(FGF7或KGF)、卵泡抑素(FST)、延胡索酰乙酰乙酸水解酶(延胡索酰乙酰乙酸酶)(FAH)、半乳糖激酶1(GALK1)、半乳糖苷酶α(GLA)、葡聚糖(1,4-α-)分支酶1(GBE1)、糖蛋白激素α多肽(CGA或FSH-α)、血红蛋白β(HBB)、肝细胞生长因子(HGF)、人生长激素(hGH)、门冬胰岛素、甘精胰岛素、谷赖胰岛素、赖脯胰岛素、干扰素β(IFNB)、干扰素α2(IFNA2)、白细胞介素10(IL-10)、白细胞介素15(IL-15)、白细胞介素7(IL-7)、klotho(KL)、卵磷脂-胆固醇酰基转移酶(LCAT)、溶酶体酸性脂肪酶A胆固醇酯酶(LIPA)、脂蛋白脂肪酶(LPL)、低密度脂蛋白受体(LDLR)、甘露糖苷酶α2B类成员1(MAN2B1)、微粒体甘油三酯转移蛋白(MTTP)、N-乙酰谷氨酸合成酶(NAGS)、神经调节蛋白1(NRG1)、鸟氨酸氨甲酰基转移酶(OTC)、磷酸化酶激酶α2(肝脏)(PHKA2)、纤溶酶原(PLAT)、胞裂蛋白4(ARTS或SEPT4)、serpin肽酶抑制剂分化体C(抗凝血酶)成员1(SERPINC1)、serpin肽酶抑制剂分化体F(α-2抗纤维蛋白溶酶色素上皮衍生因子)成员2(SERPINF2)、sirtuin 1(SIRT1)、sirtuin 6(SIRT6)、溶质载体家族16成员3(单羧酸转运蛋白4)(SLC16A3)、溶质载体家族2(易化葡萄糖转运蛋白)成员1(SLC2A1或GLUT1)、分拣蛋白1(SORT1)、血小板生成素(THPO)、转化生长因子β(TGFB1)、釉丛蛋白1(TUFT1)、肿瘤蛋白p53(TP53)、酪氨酸酶(TYR)、UDP葡萄糖苷酰转移酶1家族多肽A1(UGT1A1)、血管内皮生长因子(VEGF)和X连锁细胞凋亡抑制剂(XIAP)，或其任意组合。此处全文引入WO2013151736A中mRNA编码目标多肽及其序列。
在一些实施方案中，所述蛋白或多肽为癌抗原。所述癌抗原包括但不限于：一种或多种传统癌抗原或受试者特异性癌抗原(包括编码一种或多种已知的为肿瘤所特有的癌抗原或为各受试者所特有的癌抗原的RNA，所述癌抗原被称为新表位或受试者特异性表位或抗原)。在一些具体实施方案中，所述癌抗原是受试者特异性癌抗原。在一些具体实施方案中，受试者特异性癌抗原可代表受试者的肿瘤样品的外显子组，或代表受试者的肿瘤样品的转录物组。在一些实施方案中，受试者特异性癌抗原可代表受试者的外来体。此处全文引入WO2012159754A和WO2017070618A中所描述的受试者特异性癌抗原及其鉴定方法。在一些具体实施方案中，所述癌抗原是传统癌抗原。在一些实施方案中，传统癌抗原是非突变抗原。在一些具体实施方案中，传统癌抗原是突变抗原。例如，癌抗原可以是癌胚抗原、α1-胎蛋白、异铁蛋白和胎儿磺基糖蛋白(fetal sulphoglycoprotein)、α2-H-铁蛋白以及γ-胎蛋白。
在一些实施方案中，所述蛋白或多肽为抗体或其抗原结合片段。例如，抗PD-1抗体或其抗原结合片段。一些具体实施方案中，所述抗体或其抗原结合片段例如为骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。一些具体实施方案中，所述抗体或其抗原结合片段例如为重组抗体或其片段。一些具体实施方案中，所述抗体或其抗原结合片段为线性抗体、单链抗体、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。
在一些实施方案中，所述mRNA包含：
(a)开放阅读框(ORF)，
(b)非翻译区元件(UTR)，
(c)多聚腺苷酸(poly-A)尾；
其中，(b)非翻译区元件(UTR)可包含(b1)5’UTR和/或(b2)3’UTR；
其中，(b)非翻译区元件(UTR)、(c)多聚腺苷酸(poly-A)尾可同时存在，同时不存在，或则一存在。
在一些实施方案中，所述mRNA还包含5’Cap结构。
在一些实施方案中，从5'至3'方向上，如上任一项所述的RNA分子含有i)-v)中任一：
i)5'UTR，和开放阅读框(ORF)；
ii)开放阅读框(ORF)，和3'UTR；
iii)5'UTR，开放阅读框(ORF)，和3'UTR；
iv)5'UTR，开放阅读框(ORF)，3'UTR，和poly-A尾巴；
v)5'帽结构(5'Cap)，5'UTR，开放阅读框(ORF)，3'UTR，和poly-A尾巴；
其中所述ORF与所述5'UTR和/或所述3'UTR源自相同和/或不同的基因。
在一些实施方案中，所述5’UTR选自β-珠蛋白5'UTR、含有强Kozak翻译起始信号的5’UTR、细胞色素b-245α多肽(CYBA)5'UTR、羟基类固醇(17-β)脱氢酶(HSD17B4)5'UTR、烟草蚀纹病毒(TEV)5'UTR、热休克蛋白70(Hsp70)5'UTR、eIF4G5'UTR、ACTG1 5'UTR、CTSB 5’UTR或ARHGAP15基因的5'UTR。一些实施方案中，其中所述的5'UTR是源自或为ACTG1或ARHGAP15基因的5'UTR序列或其衍生序列。在一些实施方案中，所述ARHGAP15基因是来源任意物种的，如人类ARHGAP15、狒狒ARHGAP15、小鼠ARHGAP15等。
一些实施方案中，其中所述的5'UTR是源自或为人类ARHGAP15基因的5'UTR序列或其衍生序列。一些实施方案中，所述的ARHGAP15基因的5'UTR包含或为如SEQ ID NO：9所示的核苷酸序列或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的核苷酸序列。
在一些实施方案中，所述3'UTR选自α-珠蛋白3'UTR、β-珠蛋白(hemoglobin subunit beta，HBB)3'UTR、β-肌动蛋白3'UTR、CYBA 3'UTR、白蛋白3'UTR、生 长激素(GH)3'UTR、VEEV 3'UTR、乙型肝炎病毒(HBV)3'UTR、ACTG1 3'UTR、CTSB 3’UTR或ARHGAP15基因的3'UTR。
一些实施方案中，其中所述的3'UTR是源自或为HBB、CTSB或ARHGAP15基因的3'UTR序列或其衍生序列。
一些实施方案中，所述3'UTR包含或为如SEQ ID NO：10所示的核苷酸序列或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的核苷酸序列。在一些实施方案中，所述poly-A尾选自A120、A30L70、HGH polyA、SV40polyA、BGH polyA、rbGlob polyA或SV40late polyA。
一些具体实施方案中，所述poly-A尾巴选自120A或A30L70，所述A30L70的序列如SEQ ID NO:11所示，所述120A的序列如SEQ ID NO:12所示。
一些具体实施方案中，所述RNA分子的核苷酸序列如SEQ ID NO:13-SEQ ID NO:20中任一项所示。在一些实施方案中，所述mRNA包含经修饰的核苷酸或核苷。所述经修饰的核苷酸和核苷可为天然存在的经修饰的核苷酸和核苷或非天然存在的经修饰的核苷酸和核苷。所述修饰可包括如本领域中公认的在核苷酸和/或核苷的糖、主链或核碱基部分的那些修饰。在一些具体实施方案中，所述mRNA中经修饰的核碱基包含1-甲基-假尿苷(m1ψ)、1-乙基-假尿苷(e1ψ)、5-甲氧基-尿苷(mo5U)、5-甲基-胞苷(m5C)和/或假尿苷(ψ)。在一些具体实施方案中，所述mRNA中的经修饰的核碱基包含5-甲氧基甲基尿苷、5-甲硫基尿苷、1-甲氧基甲基假尿苷、5-甲基胞苷和/或5-甲氧基胞苷。在一些具体实施方案中，所述mRNA包括至少两种(例如，2、3、4种或更多种)任何上文所提及的经修饰的核碱基的组合，包括但不限于化学修饰。
在一些具体实施方案中，所述5'Cap结构位于5'UTR的上游，例如，位于5'UTR的5'末端。在一些具体实施方案中，5'帽结构是本领域技术人员已知的帽结构，如Cap0(第一个核碱基的甲基化，例如m7GpppN)、Cap1(m7GpppN的相邻核苷酸的核糖的额外甲基化，例如m7G(5')ppp(5')(2'OMeA)pG)、Cap2(m7GpppN下游第3个核苷酸的核糖的额外甲基化)、Cap3(m7GpppN下游第3个核苷酸的核糖的额外甲基化)、Cap4(m7GpppN下游第4个核苷酸的核糖的额外甲基化)、ARCA(抗反向帽类似物)、修饰的ARCA(例如，硫代磷酸酯修饰的ARCA)、肌苷、N1-甲基-鸟苷、2’-氟代-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷和2-叠氮基-鸟苷。
在一些实施方案中，所述药物组合物含有：阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体的摩尔百分比40-49％，
在一些实施方案中，所述药物组合物含有：
a)活性剂；
b)阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体摩尔百分比大于等于10％，且小于50％，
c)非阳离子脂质，所述非阳离子脂质选自磷脂和胆固醇或其衍生物，其中所述磷脂占载体摩尔量的5～40％，所述胆固醇或其衍生物占载体摩尔量的30～60％；
和d)缀合脂质，所述缀合物占载体摩尔量的0.5～4％。
在一些实施方案中，所述药物组合物含有：
a)活性剂，所述活性剂包含mRNA；
b)阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体摩尔百分比大于等于10％，且小于50％，
c)非阳离子脂质，所述非阳离子脂质选自磷脂DSPC和胆固醇，其中所述磷脂DSPC占载体摩尔量的5～40％，所述胆固醇占载体摩尔量的30～60％；
和d)缀合脂质，所述缀合物占载体摩尔量的0.5～4％。
在一些实施方案中，所述药物组合物含有：
a)活性剂，所述活性剂包含mRNA；
b)阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体的摩尔百分比大于等于10％，且小于50％，
c)非阳离子脂质，所述非阳离子脂质选自磷脂DSPC和胆固醇，其中所述磷脂DSPC占载体摩尔量的5～40％，所述胆固醇占载体摩尔量的30～60％；
和d)缀合脂质DMG-PEG，所述缀合物占载体摩尔量的0.5～2％。
在一些实施方案中，所述药物组合物含有：
a)活性剂，所述活性剂包含mRNA；
b)阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体的摩尔百分比40-49％，
c)非阳离子脂质，所述非阳离子脂质选自磷脂DSPC和胆固醇，其中所述磷脂DSPC占载体摩尔量的10～15％，所述胆固醇占载体摩尔量的35～42％；
和d)缀合脂质DMG-PEG，所述缀合物占载体摩尔量的1～2％。
在一些实施方案中，所述药物组合物含有：
a)活性剂，所述活性剂包含mRNA；
b)阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体的摩尔百分比45～49％，
c)非阳离子脂质，所述非阳离子脂质选自磷脂DSPC和胆固醇，其中所述磷脂DSPC占载体摩尔量的10～15％，所述胆固醇占载体摩尔量的35～42％；
和d)缀合脂质DMG-PEG，所述缀合物占载体摩尔量的1～2％。
另一方面，本公开药物组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均粒径为10nm～220nm。在一些实施方案，所述纳米颗粒制剂的平均粒径为100～150nm。在一些实施方案，所述纳米颗粒制剂的平均粒径为150～200nm。在一些实施方案，所述纳米颗粒制剂的平均粒径为80～160nm。在一些实施方案，所述纳米颗粒制剂的平均粒径为80～150nm。在一些实施方案，所述纳米颗粒制剂的平均粒径为70～160nm。
在一些实施方案中，所述纳米颗粒平均粒径采用动态光散射法测定。
本公开还提供一种冻干制剂，其由前述药物组合物经冷冻干燥获得。在一些实施方案中，冷冻干燥包括冷冻步骤、初级干燥和二次干燥步骤。在一些实施方案中，本公开冷冻干燥参照CN107567497A中方法进行，并将相关内容引入本文以示说明。
本公开还提供一种复溶溶液，其由前述冻干制剂经复溶的步骤获得，其复溶所用溶剂为注射用水。
本公开还提供前述药物组合物、冻干制剂或复溶溶液的制备方法，所述方法包括将阳离子脂质、非阳离子脂质和缀合脂质在有机溶剂中混合的步骤。在一些实施 方案中，所述有机溶剂选自乙醇。
在一些实施方案中，制备药物组合物的方法参照期刊文献(Pharm.Res.22(2005)362–372)或US9504651中方法制备，并将相关内容引入本文中以示说明。
本公开还提供了前述药物组合物、冻干制剂或复溶溶液在制备用于在脊柱动物体内引起保护性免疫应答的药物中用途。
本公开还提供了前述药物组合物、冻干制剂或复溶溶液在制备用于在哺乳动物中治疗疾病或功能障碍的药物中用途。在一些实施方案中，所述疾病优选自病毒感染(如冠状病毒、流感病毒或HIV病毒引起的疾病)、肝疾病、癌症或疱疹。
术语
术语“脂质”是指一类有机化合物，其包括但不仅限于脂肪酸的酯，并且特征是在水中不溶，但是在许多有机溶剂中是可溶的。通常将它们分成至少三类：(1)“简单脂质”，其包括脂肪和油以及蜡；(2)“化合物脂质”，其包括磷脂和糖脂；和(3)“衍生的脂质”诸如类固醇。
术语“阳离子脂质”指在选定的pH值，诸如生理pH值(例如，pH为约7.0)下携带净正电荷的许多脂质物种中的任何一种。
术语“阴离子脂质”指在生理pH值下带负电荷的任何脂质。这些脂质包括，但不限于，磷脂酰甘油、心磷脂、二酰磷脂酰丝氨酸、二酰磷脂酸、N-十二烷酰磷脂酰乙醇胺、N-琥珀酰磷脂酰乙醇胺、N-戊二酰磷脂酰乙醇胺、赖氨酰磷脂酰甘油、棕榈酰油酰磷脂酰甘油(POPG)，和其它与中性脂质连接的阴离子修饰基团。
术语“中性脂质”指在选定的pH值下以不带电荷或中性两性离子形式存在的许多脂质物种中的任何一种。在生理pH值下，这样的脂质包括，例如，二酰磷脂酰胆碱、二酰磷脂酰乙醇胺、神经酰胺、鞘磷脂、脑磷脂、胆固醇、脑苷脂和二酰甘油。
术语“两亲性脂质”部分地指任何适合的材料，其中脂质材料的疏水部分定向到疏水相中，而亲水部分定向到水相。亲水性质来自极性或带电基团诸如糖类、磷酸根、羧基、硫酸根、氨基、巯基、硝基、羟基和其它类似基团的存在。疏水性可以通过包含非极性基团而赋予，所述基团包括但不限于长链饱和和不饱和脂族烃基和由一个或多个芳族、脂环族或杂环基团取代的这样的基团。两亲性化合物的实例包括但不限于磷脂、氨基脂和鞘脂类。
术语“非阳离子脂质”指任何两亲性脂质以及任何其他中性脂质或阴离子脂质。
术语“缀合脂质”指抑制脂质颗粒聚集的结合脂质。所述缀合脂质包括，但不仅限于PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油。
术语“哺乳动物”指任何哺乳动物物种诸如人、小鼠、大鼠、狗、猫、仓鼠、豚鼠、兔、家畜等。
“活性剂”是指当对受试者施用时具有治疗、诊断和/或预防作用和/或引起所需的生物学和/或药理学作用的任何药剂。活性剂也被称为“活性物质”或“治疗剂”。这类药剂包括但不限于细胞毒素、放射性离子、化疗剂、小分子药物、蛋白质和核酸。
术语“核酸”用于本文中时，指含有处于单链或双链形式的至少两个脱氧核糖核苷酸或核糖核苷酸的聚合物，包括DNA和RNA。如本文所用的术语“核酸分子”与“核酸”和“多核苷酸”同义。DNA可以采用下列形式：例如，反义分子、质粒DNA、预浓缩DNA、PCR产物、载体(P1，PAC，BAC，YAC，人工染色体)、表达盒、嵌合序列、染色体DNA、或这些组的衍生物和组合。RNA可以采用下列形式：小干扰RNA(siRNA)、微RNA(miRNA)、小发夹RNA(shRNA)、信使RNA(mRNA)、环状RNA、自复制RNA。
“信使RNA”(mRNA)指的是编码(至少一种)多肽(天然产生、非天然产生或改性的氨基酸聚合物)并且可通过转译以体外、体内或离体产生经过编码的多肽的任何多核苷酸。本领域技术人员将了解，除另外说明以外，本申请中所述的多核苷酸序列将在代表性DNA序列中表示“T”，但其中所述序列表示RNA(例如，mRNA)，“T”将被“U”取代。因此，由特定序列识别号码标识的DNA编码的任何RNA多核苷酸也可包含由所述DNA编码的相应RNA(例如，mRNA)序列，其中所述DNA序列的每个“T”被“U”取代。mRNA分子的基本组分通常包括至少一个编码区、5′非翻译区(UTR)、3′UTR、5′端帽和聚腺苷酸尾(聚-A尾)。本发明的多核苷酸可充当mRNA，但在其功能性和/或结构设计特征方面区别于野生型mRNA，所述特征用以使用基于核酸的处理来克服有效多肽表达的现有问题。
术语“抗原”涵盖将引发免疫应答的任何物质。特别是，“抗原”涉及任何物质，优选为与抗体或T淋巴细胞(T细胞)特异性反应的肽或蛋白质。本公开中，术语“抗原”包括包含至少一个表位的任何分子。优选地，本文中抗原是这样的分子，其(任选地在加工后)诱导优选地对抗原(包括表达该抗原的细胞)特异的免疫反应。本文中，可使用对于免疫反应而言为候选物的任何合适的抗原，其中所述免疫反应优选地为细胞免疫反应。在一些实施方案的情况下，抗原优选地被细胞、优选被抗原呈递细胞(包括病变细胞、特别是癌症细胞，在MHC分子的环境下)呈递，这导致针对该抗原的免疫反应。优选地，抗原是对应于或来源于天然抗原的产物。这种天然抗原包括肿瘤抗原。
术语“癌抗原”与“肿瘤抗原”或“肿瘤相关抗原”通用，其能够刺激免疫应答，优选针对抗原或特征为表达该抗原以及优选地呈递该抗原之细胞(例如病变细胞，尤其是癌症细胞)的细胞应答。一些实施方案中，“癌抗原”在正常条件下在有限数目的组织和/或器官中或者在特定的发育阶段中特异性表达(例如，肿瘤抗原可在正常条件下在胃组织中(优选在胃粘膜中)、在生殖器官中(例如，在睾丸中)、在滋养层组织(例如，在胎盘)或在生殖系细胞中特异性表达)，并且在一种或更多种肿 瘤或癌组织中表达或异常表达。一些实施方案中，“有限数目”是指不超过3，例如不超过2。
“多肽”、“蛋白”或“蛋白质”可互换使用，指氨基酸残基的聚合物，适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。除非另外说明，否则特定的多肽序列还隐含地涵盖其保守修饰的变体。
术语“水痘-带状疱疹病毒(VZV)”：是球形多层结构形式存在的α疱疹病毒，病毒基因体被覆盖有非晶形层被膜蛋白的蛋白衣壳结构包围。由病毒编码蛋白和酶组成的被膜位于核鞘与病毒包膜之间的空间，病毒包膜由宿主细胞膜获得并且含有病毒编码的糖蛋白。
术语“VZV糖蛋白”，编码糖蛋白gE、gI、gB、gH、gK、gL、gC、gN和gM在病毒复制周期的不同步骤中起作用。在感染细胞中以及在成熟病毒粒子中发现最多的糖蛋白是糖蛋白E(gE、ORF 68)，它是病毒粒子包膜的主要组分并且为病毒复制所必需。糖蛋白I(gI，ORG 67)与gE在感染细胞中形成复合物，这有利于糖蛋白的胞吞作用并且将其引导至反式高基氏体网络(TGN)，在此获得最终的病毒包膜。在TGN中需要糖蛋白I(gI)进行VZV包膜并且在VZV复制期间进行有效膜融合。发现VZV gE和gI在感染宿主细胞表面上复合在一起。作为第二普遍的糖蛋白并且认为在病毒入口中发挥作用的糖蛋白B(ORF 31)与中和抗体结合。认为糖蛋白H具有融合功能，促使病毒在细胞和细胞之间传播。gE、gB和gH的抗体在天然感染之后而且在疫苗接种后普遍存在，并且已证实在体外中和病毒活性。示例性的gE蛋白的氨基酸序列为本公开的SEQ ID NO:1或SEQ ID NO:21-SEQ ID NO:25中任一项所示。
术语野生型VZV gE蛋白的“突变体”、VZV gE蛋白的“突变体”、“VZV gE蛋白突变体”是指相对于野生型VZV gE蛋白展现引入的突变且具有针对野生型VZV gE蛋白的免疫原性的多肽。
术语“突变”是指与参照蛋白或多肽的氨基酸序列相比，蛋白或多肽的氨基酸序列中缺失、添加或取代氨基酸残基。在说明书和权利要求通篇中，在蛋白序列中的一个特定位置取代氨基酸使用注释“(野生型蛋白中的氨基酸残基)(氨基酸位置)(工程改造的蛋白中的氨基酸残基)”来提及。例如，注释Y75A是指参照蛋白的氨基酸序列的第75位的酪氨酸(Y)残基被丙氨酸(A)残基取代(在参照蛋白的突变体中)。在不同野生型序列间相同位置的氨基酸残基中存在变异的情况下，该注释中可省略位置编号之前的氨基酸代码，诸如“75A”。
“密码子优化”是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。各种物种对特定氨基酸的某些密码子表现出特定的偏好。密码子偏好(生物体之间密码子使用的差异)通常与信使RNA(mRNA)的翻译效率相关，继而认为所述信使RNA尤其取决于翻译的密码子的特性和具体 的转运RNA(tRNA)分子的利用度。选择的tRNA在细胞中的优势通常是肽合成中最常使用的密码子的反映。因此，基于密码子优化，基因可以针对给定生物体中的最佳基因表达进行修改。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。
如本领域中已知，术语“同一性”或“同源性”是指如通过对比序列所测定的两种或两种以上多肽或多核苷酸的序列之间的关系。在本领域中，同一性还意谓如由两个或两个以上氨基酸残基或核酸残基串之间的匹配数所测定的其间的序列相关程度。同一性测量两种或两种以上序列中具有由特定数学模型或计算机程序(例如，“算法”)提出之间隙比对(如果存在)的较小者之间的一致匹配百分比。“同一性％”在适用于多肽或多核苷酸序列时定义为在比对序列和必要时引入间隙以达成最大同一性百分比后，候选氨基酸或核酸序列中与第二序列的氨基酸序列或核酸序列中的残基一致的残基(氨基酸残基或核酸残基)百分比。用于比对的方法和计算机程序在本领域中熟知。应了解，同一性取决于同一性百分比的计算，但由于计算中引入之间隙和罚分而其值可不同。通常，如通过本文所述且本领域技术人员已知的序列比对程序和参数所测定，特定多核苷酸或多肽的变体与彼特定参考多核苷酸或多肽具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、但小于100％的序列同一性。用于比对的此类工具包括BLAST试剂盒的那些工具(Stephen F.Altschul，等人(1997)，“GappedBLAST and PSI-BLAST：a new generation of protein database search programs”，Nucleic Acids Res.25：3389-3402)。另一种受欢迎的局部比对技术是基于Smith-Waterman算法(Smith，T.F.和Waterman，M.S.(1981)“Identification of commonmolecular subsequences.”J.Mol.Biol.147：195-197)。基于动态程序设计的通用全局比对技术为Needleman-Wunsch算法(Needleman，S.B.和Wunsch，C.D.(1970)“A generalmethod applicable to the search for similarities in the amino acid sequencesof two proteins.”J.Mol.Biol.48：443-453)。最近已研发出一种快速优化全局序列比对算法(Fast Optimal Global Sequence Alignment Algorithm，FOGSAA)，据称其比其他优化全局比对方法(包括Needleman-Wunsch算法)更快地产生对核苷酸和蛋白序列的全局比对。本文中描述其他工具，尤其在下文“同一性”的定义中。
术语“疫苗”涉及药物制备物(药物组合物)或产品，其在施用后诱导识别并攻击病原体(包括病毒或细菌)或病变细胞如癌症细胞的免疫应答，特别是细胞免疫应答。疫苗可用于预防或治疗疾病，例如用于预防病毒感染或治疗癌症。
术语“5'非翻译区元件”(5'UTR)指的是直接位于起始密码子(即，由核糖体转译的mRNA转录物的第一密码子)上游(即，5’)的不编码多肽的mRNA区域。
术语“3'非翻译区元件”(3'UTR)指的是直接位于终止密码子(即，表示转译终止的mRNA转录物的密码子)下游(即，3')的不编码多肽的mRNA区域。
术语“开放阅读框架”(ORF)是由起始密码子(例如，甲硫氨酸(ATG))开始并以终止密码子(例如，TAA、TAG或TGA)结束的DNA连续延伸段并且编码蛋白或多肽。
术语“聚腺苷酸(polyA)尾”是位于含有多个连续单磷酸腺苷的3’UTR下游，例如直接位于其下游(即3’)的mRNA区域。聚腺苷酸尾可含有10至300个单磷酸腺苷。例如，聚腺苷酸尾可含有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或300个单磷酸腺苷。在一些实施方案中，聚腺苷酸尾含有50至250个单磷酸腺苷。在一种相关生物的情境中(例如，在细胞中、体内)，聚(腺苷酸)尾用来保护mRNA以免酶促降解，例如在细胞质中，并且有助于转录终止和/或由核输出mRNA并转译。
本公开所述“平均粒径”(Z-average Size)例如“平均粒径小于2000nm”是光强平均值，由不同种类粒子所贡献的光强计算而得。所述“平均粒径”可通过本领域技术人员熟知的常规粒度测量技术测量颗粒的平均粒径。这样的技术包括沉降场流分级，光子相关光谱，光散射等。
本公开多分散系数“PDI”(Polydispersity Index)体现了粒子粒径均一程度，是粒径表征的一个重要指数。
术语“混入”、“混合”表示的意思是不限定组分的加入顺序，例如将A混入B，可以表达A加入B的意思，也可以表达B加入A的意思，将A和B混合，可以表达A加入B混合的意思，也可以表达B加入A混合的意思。
本公开中数值为仪器测量值，存在一定程度的误差，一般而言，正负10％均属于合理误差范围内。当然需要考虑该数值所用之处的上下文，例如，活性成分的粒径，该数值为测量后误差变化不超过正负10％，可以为正负9％、正负8％、正负7％、正负6％、正负5％、正负4％、正负3％、正负2％或正负1％，优选正负5％。
本公开式I化合物参照WO2023125738中方法制备，并将相关内容本申请以示说明。
本公开用于测试的mRNA为荧光素酶mRNA。
附图说明
图1：脂质组合物1-15对应的脂质体纳米颗粒的体在HEK293细胞转染后的荧光素酶蛋白表达量。
图2：脂质组合物4℃粒径稳定性。
图3：脂质组合物25℃粒径稳定性。
图4：VZV mRNA体外活性表达检测。
图5：血清anti-VZV gE IgG检测。
具体实施方式
以下结合实施例进一步描述本公开中，但这些实施例并非限制本公开中的范围。
本公开中实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1
步骤1)
称取6-氨基己酸(21.5g,100mmol)溶于乙醇和水的混合液(EtOH/H2O,v/v＝2:1,200mL)，加入CsOH·H2O(4.0g,100mmol)，加入苄氧溴乙烷(13.1g,100mmol)，室温反应40h，浓缩除去绝大部分乙醇，残留物直接反相柱层析(乙腈/水/0.1％三氟乙酸)，直接浓缩，抽干，得到白色固体5.6g，收率：21％。
MS：266.2[M+H]+。
1H 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).
步骤2)
称取6-((2-(苄氧)乙基)氨基)己酸(5.6g，21.1mmol)于反应瓶中，加入1,4-二氧六环(80mL)，氢氧化钠水溶液(42.2mL,1.0M)，滴加Boc2O(5.5g,25.2mmol)，室温反应1h，LC-MS显示反应完毕，浓缩掉二氧六环，水相用稀盐酸调PH至5左右，乙酸乙酯萃取(50mL*3)，合并有机相，干燥，浓缩，得到近无色胶状物7.2g，收率93％。直接用于下一步反应。
MS：366.2[M+H]+。
步骤3)
称取6-((2-(苄氧)乙基)(叔丁氧羰基)氨基)己酸(6.1g，16.7mmol)于反应瓶 中，加二氯甲烷(150mL)，冰浴，依次加入十一醇(2.73g，15.9mmol)，DMAP(408mg，3.34mmol)及DIPEA(4.3g,33.4mmol)，加入EDCI(3.84g,20.0mmol)，搅拌10分钟，撤冰浴，室温反应过夜，TLC监测，反应完毕，向反应液加入水(100mL)，分液，有机相依次用稀盐酸洗，水洗，碳酸氢钠洗，干燥，浓缩，柱层析(PE:EtOAc＝20:1～10:1)，得到近无色胶状物6.8g，收率82％。
MS：520.4[M+H]+。
1H 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).
步骤4)
称取6-((2-(苄氧)乙基)(叔丁氧羰基)氨基)己酸十一酯(7.6g，14.6mmol)溶液二氯甲烷中，加入TFA(20mL)，室温反应2h，直接浓缩，残留物用二氯甲烷(100mL)溶解，加入饱和碳酸氢钠(100mL)，分液，有机相干燥，浓缩，得到胶状物，再用二氯甲烷溶解，加入HCl/dioxane(4.0M,4mL),直接浓缩，得到白色固体5.0g，收率81％。
MS：420.3[M+H]+。
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).
步骤5)
称取2-((5-(苄氧基)戊基)氧基)乙酸(制备文献Chemical and Pharmaceutical Bulletin,1992,vol.40,#3,p.617-623)(7.6g，30.12mmol)于反应瓶中，加二氯甲烷(150mL)，冰浴，依次加入十七-9-醇(参照文献WO2020/219876制备)(6.18g，24.1mmol)，DMAP(3.68g，30.12mmol)，加入EDCI(6.93g,36.14mmol)，搅拌10分钟，撤冰浴，室温反应过夜，TLC监测，反应完毕，向反应液加入水(100mL)，分液，有机相依次用稀盐酸洗，水洗，碳酸氢钠洗，干燥，浓缩，柱层析(PE:EtOAc＝50:1～10:1)，得到近无色胶状物10.9g，收率92％。
1H 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).
步骤6)
称取十七烷-9-基2-((5-(苄氧基)戊基)氧基)乙酸酯(4.0g，8.15mmol)溶于THF(100mL)中，加入Pd(OH)2/C(400mg,20％)，通入氢气，室温反应2h，TLC监测，反应完毕。过滤，浓缩。得到无色胶状物3.2g，收率98％。直接用于下一步反应。
1H 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).
步骤7)
称取十七烷-9-基2-((5-羟基戊基)氧基)乙酸酯(2.4g，6.0mmol)溶于二氯甲烷(100mL)中，冰浴下，加入DMP(3.82g,9.0mmol)，室温反应1.5h，加入饱和硫代硫酸钠水溶液(100mL)，搅拌30分钟，分液，有机相用饱和碳酸氢钠水溶液洗(100mL*2)，干燥，浓缩，得到浅黄色胶状物2.5g，超重，直接用于下一步反应。
1H 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).
步骤8)
称取十七烷-9-基2-((5-氧代戊基)氧基)乙酸酯(2.4g，6mmol)溶于二氯甲烷(50mL)，加入6-((2-(苄氧)乙基)氨基)己酸十一酯盐酸盐(2.46g,5.4mmol)，加入DIPEA(1.16g,9.0mmol)，乙酸(1.08g,18.0mmol)，搅拌溶清，冰浴，加入NaBH(OAc)3(3.18g,15.0mmol)，室温反应过夜，TLC监测，反应完毕，加水(100 mL)，分液，有机相用饱和碳酸氢钠水溶液洗(50mL*1)，干燥，浓缩，柱层析(PE:EtOAc＝5:1～2:1)，得到无色液体4.1g，收率：85％。
1H 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).
步骤9)
称取十一烷基6-((2-(苄氧基)乙基)(5-(2-(十七烷-9-基氧基)-2-氧代乙氧基)戊基)氨基)己酸酯(1g,1.25mmol)于反应瓶中，加入乙醇溶解，加入Pd(OH)2/C(500mg,20％)，通入氢气，室温反应过夜，TLC监测，反应完毕。过滤，浓缩。得到无色胶状物，柱层析(CH2Cl2:MeOH＝10:1)，得到无色胶状物450mg，收率51％。
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)。
实施例2：
LNP脂质颗粒的制备
分别将化合物1(式I化合物)、DSPC、胆固醇、DMG-PEG 2000溶液，以一定摩尔百分比混合(mol％)，即各组分摩尔量占载体总摩尔量的百分比，混合配制乙醇脂质溶液。四组分的摩尔比按表1中所示进行配制。将编码荧光素酶蛋白的mRNA(其核苷酸序列如SEQ ID NO:26所示)溶于50mM pH 4乙酸盐缓冲液，配制mRNA水溶液。将乙醇脂质溶液和mRNA水溶液通过微流控按1:3体积比混合，其中化合物1与mRNA的重量比为12:1制备脂质体。在20mM Tris溶液中透析除去乙醇，最后置换到20mM Tris 8％蔗糖溶液中，得到包封mRNA的脂质体纳米颗粒组合物。
脂质颗粒组合物表征
表征方法
使用Malvern Zetasizer Pro，以173°反向散射检测模式，利用动态光散射检测脂质体纳米颗粒的纳米粒径和多分散系数PDI。
使用Quant-iT RiboGreen RNA Assay Kit RNA定量检测试剂盒，测定脂质体 包封率。
表1
测试例1：评估脂质颗粒组合物载体递送mRNA效率
检测脂质组合物1-15对应的脂质体纳米颗粒的在细胞中递送mRNA后的蛋白表达量。
将HEK 293细胞接种到96孔板培养过夜，待细胞密度达到80％以上，将包封荧光素酶mRNA的脂质纳米颗粒溶液加入到细胞板孔培养基中，mRNA剂量为100ng/孔。24小时后，使用荧光素酶报告基因检测试剂盒(Promega)和酶标仪检测表达荧光素酶蛋白的荧光强度。荧光强度值即酶标仪检测到的荧光数值，代表荧光素酶蛋白的表达量，强光强度越高即蛋白表达量越高。每个化合物对应的脂质纳米颗粒至少3组重复计算平均荧光值强度，数据见表2和图1。
表2

测试例2：评估脂质颗粒组合物的稳定性
分别将脂质组合物1-15对应的脂质纳米颗粒置于4℃和25℃环境中，放置3天后分别检测各组脂质纳米颗粒的粒径，比较保存过程中的粒径变化大小，代表脂质纳米颗粒的稳定性。粒径变化大，即稳定性差。每组重复检测3次，计算平均粒径，如表3所示。分别计算4℃和25℃粒径与脂质纳米颗粒初始粒径的差值。差值偏离0点越小即在保存期内粒径变化越小，脂质纳米颗粒更稳定。如表3所示，4℃和25℃环境中放置3天后，动态光散射检测粒径的大小的多分散系数。表4和图2-3所示粒径变化的大小。
表3
表4

实施例3：mRNA疫苗的制备
本公开中，我们制备了多种mRNA产品，示例性的出示了VZV mRNA疫苗。以下为其制备和验证例。
3.1 VZV mRNA疫苗DNA模板的设计和合成
为了在人细胞中提高VZV gE蛋白表达和其结构的稳定性，本实施例进行了密码子优化，并设计了编码Oka株gE蛋白的不同C末端变异体分子，其中，所述Oka株gE蛋白的氨基酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示。我们设计了6个gE分子，分别为：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)和1个全长gE6(Oka gE，1-623aa)，所述gE1-gE6分子的氨基酸序列及其编码核酸序列如表5所示，其中gE1-gE5为C末端截短gE分子。
表5 VZV mRNA疫苗序列
还设计T7启动子序列(TAATACGACTCACTATAAG)、5'-UTR序列(其对应RNA序列如SEQ ID NO:9)、3'-UTR序列(其对应RNA序列如SEQ ID NO:10)和poly(A)序列(其对应RNA序列如SEQ ID NO:11)。
然后按照T7启动子序列、5'-UTR序列、DNA ORF、3'-UTR序列和poly(A)序列的顺序连接，以pUC57为载体进行全基因合成(苏州金唯智生物科技有限公司)，获得质粒DNA模板。
3.2 VZV mRNA疫苗mRNA原液的制备
以实施例3.1获得的质粒DNA经XbaI酶切，纯化获取线性化质粒DNA，通过T7 RNA聚合酶进行共转录加帽(Cap1型)反应，进行体外转录以生产所需RNA。体外转录中使用(1-甲基)-假尿苷-三磷酸代替三磷酸尿苷(UTP)，转录结束后，使用DNaseI消化DNA模板，以降低残余DNA模板带来的风险。使用AKTA对mRNA进行纯化，将纯化的mRNA溶解于无菌水中，并在-80℃下冷冻保存直至使用。
3.3 VZV mRNA疫苗mRNA-LNP制剂的制备
将化合物1(式I化合物)、DSPC、胆固醇、DMG-PEG 2000以48：10：40.5：1.5的摩尔比溶于乙醇配制脂质溶液。将mRNA溶于50mM pH 4乙酸缓冲液，配制mRNA水溶液。将乙醇脂质溶液和mRNA水溶液通过微流控按体积比1:3混合，脂质中化合物1与mRNA的重量为12:1制备脂质纳米粒。在20mM Tris pH7.5溶液中透析除去乙醇，最后置换到20mM Tris 8％蔗糖溶液中冻存，得到包封mRNA的脂质纳米粒，并在-80℃下冷冻保存直至使用。
实施例4：VZV mRNA疫苗在细胞中的表达及其在小鼠中的免疫原性
4.1 VZV mRNA疫苗的体外表达活性检测
HEK 293T细胞(人肾上皮细胞系)在添加了10％ FBS和1％青霉素-链霉素的Dulbecco改良Eagle培养基(DMEM)中，在37℃和5％ CO2条件下培养。首先地，我们在HEK293细胞中验证了以30L70A为poly A设计的6个gE-LNP mRNAs的体外表达。本实施例使用如实施例3制备了利用(1-甲基)-假尿苷-三磷酸代替UTP的含30L70A的6个gE-mRNA LNPs液体制剂，核苷酸序列分别为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)和gE6-LNP(SEQ ID NO:18)。
将HEK-293T细胞铺在六孔板中，HEK-293T细胞每孔种1.2×106个细胞，37℃培养箱孵育18-24小时；将含1μg mRNA的gE-mRNA LNPs制剂直接加入到HEK-293T细胞中，继续孵育24小时，收集培养上清或细胞沉淀进行Western Blot检测，Western Blot检测使用的mouse anti-VZV gE Ab购于Abcam，Goat anti-mouse IgG HRP Ab购于TransGen。Western Blot结果如图4所示，结果显示：1)6个gE-mRNA-LNPs均能表达出对应的gE蛋白；2)保留C端跨膜区的gE2，gE4，gE5和gE6以gE2，gE5和gE6的体外表达活性相对较高。
4.2 VZV mRNA疫苗免疫C57小鼠
本实施例使用雌性，6～8周龄的C57小鼠。动物研究严格按照《上海实验动物饲养管理和使用指南》中的建议进行。
本实施例使用如实施例4.1制备的6条gE-LNP mRNAs制剂免疫C57小鼠，评估其在小鼠中诱导的gE特异性抗体水平。
将小鼠随机分为7组(n＝7)，其中6组分别施用如实施例3制备的gE-LNP mRNAs制剂免疫(单次剂量为含2μg mRNA的gE-mRNA LNPs制剂)；另一组施用磷酸盐缓冲盐溶液(PBS)作为阴性对照组。各组通过肌肉注射分别在第0天(初免)和第14天(二免)施用。在小鼠二免后的第14天(即第28天)眼眶取血收集小鼠血清，进行抗体检测。
血清anti VZV gE IgG检测(gPELISA法)如下：使用ELISA coating Buffer稀释VZV-gE antigen(ACROBiosystems)，配置成0.25μg/mL，使用排枪，每孔加100μL 0.25μg/mL VZV-gE antigen，4℃，孵育过夜；待检测样品室温平衡30分钟，将板在PBST(0.05％ Tween-20)中洗涤，并用PBST中的5％脱脂奶封闭。使用含2.5％脱脂奶粉稀释样品血清，起始稀释滴度为1：50，后以2倍比稀释待检血清样品，在每块板中设置阴性血清对照。加入稀释的血清样品，每孔100μl，37℃(恒温箱)孵育1小时；弃去血清样品，使用排枪，每孔加入250μl PBST洗液，洗涤3次；每孔加入100μl稀释50000倍的goat anti-mouse IgG HRP(abcam)，37℃(恒温箱)孵育0.5小时；弃去二抗，使用排枪，每孔加入300μl PBST洗液，洗涤5次；清洗后，每孔加入100μl 3,3',5,5'-四甲基联苯胺(TMB)(ebioscience)显色液避光显色5分钟；每孔加入100μl 2N H2SO4终止反应并检测；使用酶标仪(BioTek)在450nm波长处读取OD值。将终点滴度确定为最后一次稀释血清的倒数。样品OD450 nm大于2.1倍阴性对照组OD450 nm判定为阳性值。结果如图5所示，结果显示这6种gE LNP-mRNA分子诱导了相当的体液免疫水平。
本公开部分序列如下所示：
>Oka株gE蛋白的氨基酸序列
>Oka株gE蛋白的核苷酸序列

>gE1核苷酸序列

>gE2核苷酸序列

>gE3核苷酸序列

>gE4核苷酸序列

>gE5核苷酸序列

>gE6核苷酸序列

>5'UTR
>3'UTR
>30L70A polyA
>120A polyA
>gE1(30L70A)mRNA序列

>gE2(30L70A)mRNA序列

>gE3(30L70A)mRNA序列

>gE4(30L70A)mRNA序列

>gE5(30L70A)mRNA序列

>gE6(30L70A)mRNA序列


>gE2 120A mRNA序列

>gE6 120A mRNA序列

>gE1氨基酸序列
>gE2氨基酸序列
>gE3氨基酸序列

>gE4氨基酸序列
>gE5氨基酸序列
>荧光素酶mRNA序列

Claims (19)

  1. 一种药物组合物，其包含载体，所述载体包含有阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，阳离子脂质与载体摩尔百分比大于等于10％，且小于50％，
  2. 根据权利要求1所述的药物组合物，其中所述载体还含有非阳离子脂质，所述非阳离子脂质选自磷脂、胆固醇或其衍生物中的至少一种。
  3. 根据权利要求2所述的药物组合物，其中所述磷脂选自1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0 Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16 Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0 PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)以及其混合物，优选1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)。
  4. 根据权利要求2或3所述的药物组合物，其中所述磷脂的含量占载体摩尔量的5～40％，优选10～20％，例如10％。
  5. 根据权利要求2-4任一项所述的药物组合物，其中所述胆固醇或其衍生物占载体摩尔量的30～60％，优选35～45％，例如40.5％。
  6. 根据权利要求1-5任一项所述的药物组合物，其中所述载体还含有缀合脂质，所述缀合脂质优选PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油，更优选二硬脂酰基磷脂酰乙醇胺聚乙二醇2000(DSPE-PEG2000)，二肉豆蔻酰甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)和甲氧基聚乙二醇双十四烷基乙酰胺(ALC-0159)。
  7. 根据权利要求6所述的药物组合物，其中所述缀合脂质占载体摩尔量的0.5～4％，优选1.5～2.5％，例如1.5％。
  8. 根据权利要求1-7任一项所述的药物组合物，其中含有活性剂，所述活性剂选自核酸，优选核糖核酸或脱氧核糖核酸，更优选小干扰RNA、微RNA、小发卡RNA、信使RNA(mRNA)、环状RNA、自复制RNA以及其混合物。
  9. 根据权利要求8所述的药物组合物，所述mRNA包含开放阅读框(ORF)，所述ORF编码蛋白或多肽；
    优选地，所述mRNA还包含5’非翻译区元件(5’UTR)、3’非翻译区元件(3’UTR)、多聚腺苷酸(poly-A)尾的任一或其任意组合；
    优选地，所述mRNA还包含5’帽结构(5'Cap)。
  10. 根据权利要求9所述的药物组合物，其中，所述蛋白或多肽为抗原，
    优选地，所述抗原选自病毒抗原、细菌抗原、癌抗原；
    更优选地，所述病毒毒株选自流感病毒、冠状病毒、水痘带状疱疹病毒(VZV)、呼吸道合胞病毒(RSV)。
  11. 根据权利要求10所述的药物组合物，其中，所述抗原为水痘带状疱疹病毒(VZV)糖蛋白E(gE)蛋白，其氨基酸序列如SEQ ID NO:1或SEQ ID NO:21-SEQ ID NO:25中任一项所示；
    优选地，所述抗原的核苷酸序列如SEQ ID NO:3-SEQ ID NO:8中任一项所示；
    更优选地，所述mRNA的核苷酸序列如SEQ ID NO:13-SEQ ID NO:20中任一项所示。
  12. 根据权利要求9所述的药物组合物，其中，所述蛋白或多肽为肝细胞生长 因子(HGF)、抗体或其抗原结合片段。
  13. 根据权利要求1-12任一项所述的药物组合物，其中含有
    a)活性剂，优选mRNA；
    b)阳离子脂质，所述阳离子脂质包含式I所示化合物或其可药用盐，所述阳离子脂质与载体摩尔百分比大于等于10％，且小于50％，
    c)非阳离子脂质，所述非阳离子脂质选自磷脂和胆固醇或其衍生物，其中所述磷脂占载体摩尔量的5～40％，所述胆固醇或其衍生物占载体摩尔量的30～60％；
    和d)缀合脂质，所述缀合物占载体的摩尔百分含量为0.5～4％。
  14. 根据权利要求1-13任一项所述的药物组合物，其为纳米颗粒制剂，所述纳米颗粒制剂的平均粒径为10nm～220nm。
  15. 一种冻干制剂，其由权利要求1-14任一项所述的药物组合物经冷冻干燥获得。
  16. 一种复溶溶液，其由权利要求15所述的冻干制剂经复溶的步骤获得，其复溶所用溶剂优选注射用水。
  17. 制备权利要求1-14任一项所述的药物组合物的方法，包括：将阳离子脂质、非阳离子脂质和缀合脂质在有机溶剂中混合的步骤。
  18. 权利要求1-14任一项所述的药物组合物、权利要求15所述的冻干制剂或、权利要求16所述的复溶溶液在制备用于在哺乳动物体内引起保护性免疫应答的药物中用途，优选制备疫苗的用途。
  19. 权利要求1-14任一项所述的药物组合物、权利要求15所述的冻干制剂或、权利要求16所述的复溶溶液在制备用于在哺乳动物中治疗疾病或功能障碍的药物中用途，所述疾病优选病毒感染、癌症。
PCT/CN2024/102386 2023-06-28 2024-06-28 一种含有阳离子脂质的药物组合物及其用途 Pending WO2025002352A1 (zh)

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