WO2024098491A1 - Mrna for encoding anti-avian influenza h7n9 virus antibody, preparation method therefor, and use thereof - Google Patents
Mrna for encoding anti-avian influenza h7n9 virus antibody, preparation method therefor, and use thereof Download PDFInfo
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- WO2024098491A1 WO2024098491A1 PCT/CN2022/137375 CN2022137375W WO2024098491A1 WO 2024098491 A1 WO2024098491 A1 WO 2024098491A1 CN 2022137375 W CN2022137375 W CN 2022137375W WO 2024098491 A1 WO2024098491 A1 WO 2024098491A1
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- WIPO (PCT)
- Prior art keywords
- mrna
- monoclonal antibody
- sequence
- seq
- light chain
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
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- 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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
Definitions
- the invention belongs to the field of immunology and molecular biology, and specifically relates to mRNA encoding anti-avian influenza H7N9 virus antibodies and a preparation method and application thereof.
- the H7N9 virus is an avian influenza virus that is resistant to traditional antiviral drugs amantadine and rimantadine, and there is currently no effective treatment.
- the H7N9 avian influenza outbreak in 2013 killed 40% of infected patients.
- the H7N9 virus needs to rely on specific molecules expressed by the virus itself to bind to receptors on human cells in order to infect cells and further proliferate.
- Human antibodies that neutralize the virus are certain specific antibodies produced by human B lymphocytes, which can bind to antigens on the surface of the virus, thereby preventing the virus from adhering to target cell receptors and preventing the virus from invading cells. They can effectively prevent and treat H7N9 influenza.
- Therapeutic monoclonal antibodies are usually produced by cell lines, such as Chinese hamster ovary cells, and then the antibodies are purified in large quantities from the cell culture supernatant and pharmaceutical formulations are developed. There are many challenges in the production of antibodies, including misfolding or incorrect post-translational modifications that can lead to adverse events. The purification of each monoclonal antibody is specific, requiring the development of a new method for each antibody, making production costs high.
- mRNA vaccines have a short R&D cycle, high safety and effectiveness, simple production process, and large production capacity. They are the most advanced biomedical technology today.
- mRNA vaccines encoding neutralizing antibodies are an important development trend in the development of drugs for the treatment of infectious diseases: mRNA encoding VRC01, a broad-spectrum neutralizing HIV antibody, was successfully injected into mice to produce VRC01 antibodies in vivo and protect humanized mice from HIV-1 virus infection; for human RSV, Tiwari developed the existing drug palivizumab into mRNA encoding membrane-anchored neutralizing antibodies, which were more efficient than palivizumab and significantly inhibited RSV 7 days after transfection; mRNA encoding neutralizing antibodies against chikungunya virus was effectively expressed in mice and protected mice from arthritis and musculoskeletal tissue infection 2 days after inoculation, with viremia reduced to undetectable levels.
- Anti-avian influenza virus monoclonal antibodies can prevent and treat avian influenza virus infection.
- antibodies face problems of purification and post-translational modification, and the development and production costs are expensive. Therefore, the use of mRNA technology encoding anti-avian influenza antibodies for more efficient, safe, and low-cost monoclonal antibody therapy is a new solution for the prevention and treatment of avian influenza.
- Antibody drugs are effective drugs for treating major infectious diseases, but their high R&D and production costs and complex technical requirements limit their application worldwide.
- mRNA drugs have a short R&D cycle, high safety and effectiveness, simple production process, and large production capacity, and are the most advanced biotechnology today.
- the inventors of the present invention used the monoclonal antibody sequence against H7N9 avian influenza virus (Announcement No.: CN 109810189 B) developed in previous studies.
- the present invention transcribes this antibody gene into mRNA and encapsulates it in LNP to form an LNP-RNA vaccine, which can treat the H7N9 virus infected by the subject after vaccination.
- the first aspect of the present invention provides an isolated mRNA composition, which includes mRNA encoding the heavy chain of a monoclonal antibody against H7N9 and mRNA encoding the light chain of a monoclonal antibody against H7N9.
- amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the H7N9 monoclonal antibody are shown below:
- Heavy chain CDR1 region GFTFSSYA SEQ ID NO.1;
- Heavy chain CDR2 region ISGSGGST SEQ ID NO.2;
- Heavy chain CDR3 region AKNRRGSMIVSFLAKSRAGMDV SEQ ID NO.3;
- Light chain CDR3 region QQSYSTPWT SEQ ID NO.5;
- the mRNA further comprises:
- the mRNA encoding the heavy chain of the monoclonal antibody against H7N9 or the mRNA encoding the light chain of the monoclonal antibody against H7N9 respectively includes the following elements in sequence from 5' to 3' direction: a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, an mRNA corresponding to the open reading frame of the heavy chain or light chain of the monoclonal antibody against H7N9, a stop codon and a restriction site sequence, a 3' UTR sequence and a polyadenylation sequence.
- amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.6;
- amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.7:
- the light chain amino acid sequence of the antibody is shown in SEQ ID NO.9:
- RNA sequence corresponding to the open reading frame of the heavy chain of the monoclonal antibody of H7N9 is the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.10:
- RNA sequence corresponding to the open reading frame of the monoclonal antibody light chain of H7N9 is the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.11:
- the 5' cap structure is selected from at least one of m7GpppG, m27,3'-OGpppG, m7Gppp(5')N1 or m7Gppp(m2'-O)N1.
- the 5'UTR sequence is selected from the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.12.
- the 3'UTR sequence is selected from the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.13.
- poly(A) sequence comprises a sequence of 25-400 adenylic acids.
- poly(A) sequence comprises a sequence of 50-400 adenylic acids.
- poly(A) sequence comprises a sequence of 50-300 adenylic acids.
- poly(A) sequence comprises a sequence of 50-250 adenylic acids.
- poly(A) sequence comprises a sequence of 80-120 adenylic acids.
- sequence encoding the signal peptide is selected from the RNA sequence corresponding to the sequence shown in SEQ ID NO.16.
- restriction site sequence is selected from the RNA sequence corresponding to CTCGAG.
- the second aspect of the present invention provides a pharmaceutical composition comprising the mRNA composition described in the first aspect, and an optional delivery vector.
- the delivery vehicle is a nanoparticle.
- the delivery vehicle is a lipid nanoparticle.
- the lipid nanoparticles include cationic lipids and at least one selected from non-cationic lipids, sterols, and PEG-modified lipids.
- the lipid nanoparticles are cationic lipids, non-cationic lipids, sterols and PEG-modified lipids.
- the cationic lipid is an ionizable cationic lipid selected from one or more of the following components: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, dilinoleyl-methyl-4-dimethylaminobutyrate and 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester, preferably dilinoleyl-methyl-4-dimethylaminobutyrate.
- the non-cationic lipid is a neutral lipid selected from at least one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS).
- DSPC distearoylphosphatidylcholine
- DOPE dioleoylphosphatidylethanolamine
- DOPC dioleoylphosphatidylcholine
- DOPS dioleoylphosphatidylserine
- the sterol is cholesterol
- the PEG-modified lipid is selected from at least one of PEG-DMG, PEG-DSG and PEG-DMPE.
- the PEG length of the PEG-modified lipid is 0.5-200 KDa.
- the pharmaceutical composition optionally contains an adjuvant.
- the third aspect of the present invention provides a kit comprising the mRNA composition described in the first aspect of the present invention and/or the pharmaceutical composition described in the second aspect of the present invention.
- the fourth aspect of the present invention provides the use of the mRNA composition described in the first aspect of the present invention, the pharmaceutical composition described in the second aspect of the present invention, and the kit described in the third aspect of the present invention in the preparation of drugs for preventing and/or treating H7N9 virus infection diseases.
- the fifth aspect of the present invention provides a method for preparing the mRNA composition according to the first aspect of the present invention, the preparation method comprising the following steps:
- step S6 capping the RNA obtained in step S5) to obtain mRNA
- the H7N9 mRNA vaccine of the present invention has a short development cycle and is particularly suitable for the development of vaccines for emerging infectious diseases including H7N9; it has high safety.
- the mRNA composition of the present invention overcomes the problems that may occur when using antigen mRNA vaccines to treat viral infectious diseases in the prior art, such as different levels of immune response in different subjects after vaccination, not all subjects can produce corresponding antibodies, and even if antibodies are generated, the time for antibody production is relatively long, and there may be a problem that the production is not enough to inhibit viral infection.
- the mRNA composition of the present invention can be directly expressed as monoclonal antibodies in the subject, has a fast onset, can be directly used for the treatment of H7N9 infection, and has better effects than direct injection of monoclonal antibodies.
- Figure 1 is a photo of plasmid linearization gel electrophoresis.
- Figure 2 is a photograph of gel electrophoresis of transcribed mRNA.
- FIG3 is an ELISA to verify the antibody expression of LNP-mRNA in cells.
- the mRNA is not chemically modified or is chemically modified, and the chemical modification is to replace at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil in the mRNA, and replace uracil with pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4- At least one of methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-1-methyl-
- the 5' cap structure is selected from at least one of m7GpppG, m27,3'-OGpppG, m7Gppp(5')N1 or m7Gppp(m2'-O)N1.
- different 5’ cap structures can be flexibly added to the 5’ end of the mRNA.
- m7G represents 7-methylguanosine cap nucleoside
- ppp represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript
- N1 is the 5' most nucleotide
- G represents guanosine nucleoside
- m7 represents the methyl group at the 7-position of guanine
- m2'-O represents the methyl group at the 2'-O position of the nucleotide.
- the nanoparticles provided in the present invention can efficiently deliver mRNA and have the following characteristics and advantages: for example, when encapsulating mRNA, the acidic pH condition causes the ionizable cationic lipids to have a positive charge, compressing the negatively charged mRNA molecules, thereby obtaining a higher encapsulation rate; under physiological pH conditions, the ionizable lipid nanoparticles are electrically neutral and do not interact with the negatively charged cell membrane, and have high biocompatibility; after the ionizable lipid nanoparticles form endosomes and enter the cells through cellular endocytosis, the acidic conditions in the endosomes cause the nanoparticles to have a positive charge again, and electrostatically interact with the negatively charged endosomal membrane, thereby facilitating the release of mRNA.
- the H7N9 antibody heavy chain gene is shown in SEQ ID NO.10
- a signal peptide sequence SEQ ID NO.16 is also included before the gene sequence encoding the H7N9 antibody heavy chain; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC SEQ ID NO.16. After the signal peptide is expressed, it can guide the newly synthesized protein to transfer to the secretory pathway.
- the signal peptide used in the present invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
- H7N9 antibody heavy chain SEQ ID NO.8 Other gene sequences that can encode the H7N9 antibody heavy chain SEQ ID NO.8 can also be used.
- the H7N9 antibody heavy chain sequence is shown in SEQ ID NO.8:
- the H7N9 antibody light chain gene is shown in SEQ ID NO.11:
- a signal peptide sequence SEQ ID NO.16 is also included before the gene sequence encoding the light chain of the H7N9 antibody; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC SEQ ID NO.16. After the signal peptide is expressed, it can guide the newly synthesized protein to transfer to the secretory pathway.
- the signal peptide used in the present invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
- H7N9 antibody light chain SEQ ID NO.9 Other gene sequences that can encode the H7N9 antibody light chain SEQ ID NO.9 can also be used.
- the H7N9 antibody light chain sequence is shown in SEQ ID NO.9:
- Plasmid linearization Use Bsa I enzyme to digest the plasmid, add plasmid (2887 ⁇ L), enzyme (333 ⁇ L), buffer (1665 ⁇ L) and DEPC water (11765 ⁇ L) in a centrifuge tube in sequence, and incubate in a 37°C water bath overnight. Use a 100K ultrafiltration tube to replace the liquid and recover, and finally obtain a linearized plasmid concentration of 387.5 ng/ ⁇ L;
- RNA and 3424 ⁇ L water to a centrifuge tube, denature at 65°C for 5 minutes; add 600 ⁇ L buffer, 600 ⁇ L GTP, 150 ⁇ L SAM, 150 ⁇ L RNase inhibitor, 240 ⁇ L capping enzyme; 240 ⁇ L methyltransferase, react at 37°C for 1 hour; add 9 mL LiCl precipitation solution and 9 mL DEPC water, mix well and place in a -20°C refrigerator for 30 minutes; centrifuge at 12000 rpm for 15 minutes, wash twice with 70% ethanol to remove as much ethanol as possible from the centrifuge tube; open the tube and place in a clean bench to dry, add 3000 ⁇ L water to dissolve the RNA.
- the Nanodrop detection concentration was 715 ng/mL, the yield was 2.145 mg, and the yield was 71.5%.
- mRNAs containing the open reading frames of the heavy chain and light chain genes of the H7N9 monoclonal antibody were mixed at a mass ratio of 1:1 to obtain an mRNA composition.
- the experiment of LNP encapsulation of mRNA was completed by Shanghai Jinan Technology Co., Ltd.
- the appearance of LNP-mRNA is milky white liquid, the encapsulation rate is >98%, the dispersion coefficient is 0.103, and the particle size of the nanoparticles is 90.35 nm.
- LNP-mRNA was transfected into 293T cells using LIPO3000 (Invitrogen). After 48 hours, the supernatant and cell lysate were collected and diluted 20 times with PBS, and 100 ⁇ L/well was coated on the ELISA plate. The plates were incubated at 37°C for 2 hours, washed twice with PBS, blocked with 5% skim milk powder for 1 hour, washed twice with PBS, and 100 ⁇ L anti-his tag antibody was added to each well. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and 100 ⁇ L of color development solution was added to each well for 10 minutes. Finally, 100 ⁇ L of stop solution was added, and the OD450 data was recorded on a spectrophotometer.
- mice Use physiological saline to prepare a sodium pentobarbital solution with a final concentration of 1%, filter it with a 0.22 ⁇ m filter, and store it at 4°C.
- mice anesthetize them by intraperitoneal administration of 150 ⁇ l/20g, and infect them with 20 ⁇ L of H7N9 virus containing 10 ⁇ LD50 by intranasal drip.
- the mice were randomly divided into PBS group, LNP-mRNA experimental group and antibody experimental group, with 6 mice in each group, and PBS, LNP-mRNA (1.4 mg/kg) and antibody (30 mg/kg) were inoculated through the tail vein.
- the date of intranasal drip was day 0 of the mouse experiment. From the date of infection, the number of surviving mice was recorded until 2 weeks after infection.
- the LNP-mRNA encoding the antibody at a lower dose can more significantly protect mice from the lethal attack of the H7N9 virus.
- the LNP-mRNA encoding anti-avian influenza antibodies can directly express antibodies in vivo and significantly protect mice from the lethal attack of the H7N9 virus.
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Abstract
The present invention relates to an mRNA for encoding an anti-avian influenza H7N9 virus antibody, a preparation method therefor, and use thereof. Specifically, provided is an isolated mRNA composition, which comprises an mRNA comprising an open reading frame encoding a monoclonal H7N9 antibody heavy chain and an mRNA comprising an open reading frame encoding a monoclonal H7N9 antibody light chain. The mRNAs further each comprise: a 5' cap structure; a 5' UTR sequence; a 3' UTR sequence, and 4) a polyadenylate sequence. In the composition, the mass ratio of the mRNA comprising the open reading frame encoding the monoclonal H7N9 antibody heavy chain to the mRNA comprising the open reading frame encoding the monoclonal H7N9 antibody light chain is 1:1. The mRNA composition of the present invention can be directly expressed as a monoclonal antibody in vivo in a subject, with rapid onset of effect, and can be directly used for the treatment of H7N9 infection. Moreover, the effect is superior to that realized by direct injection of a monoclonal antibody.
Description
本发明属于免疫学和分子生物学领域，具体涉及一种编码抗禽流感H7N9病毒抗体的mRNA及其制法和应用。The invention belongs to the field of immunology and molecular biology, and specifically relates to mRNA encoding anti-avian influenza H7N9 virus antibodies and a preparation method and application thereof.
H7N9病毒是一种禽流感病毒，对传统的抗病毒药金刚烷胺(amantadine)和金刚烷乙胺（rimantadine）有耐药性，目前尚无有效治疗手段。2013年H7N9禽流感爆发，夺走40%感染病人的生命，上海等城市关闭活禽市场，扑杀至少17万只家禽，并连续多年流行。H7N9病毒在入侵细胞时需要依赖病毒自身表达的特定分子与人细胞上的受体结合，才能感染细胞，并进一步扩增。中和病毒的人源抗体是人B淋巴细胞产生的某些特异抗体，能够与病毒表面的抗原结合，从而阻止该病毒黏附靶细胞受体，防止病毒侵入细胞，能够高效防治H7N9流行性感冒。但是，抗体药物高昂的研发和生产成本以及复杂的卡脖子的技术要求限制了其在世界范围内的应用。治疗性单克隆抗体通常由细胞系产生，如中国仓鼠卵巢细胞，然后从细胞培养上清中大量纯化抗体和研制制药配方。抗体的生产有许多挑战，包括错误折叠或不正确的翻译后修饰可导致不良事件。每一种单克隆抗体的纯化都是特异性的，需要针对每一种抗体开发一种新方法，因此生产成本很高。The H7N9 virus is an avian influenza virus that is resistant to traditional antiviral drugs amantadine and rimantadine, and there is currently no effective treatment. The H7N9 avian influenza outbreak in 2013 killed 40% of infected patients. Cities such as Shanghai closed live poultry markets and culled at least 170,000 poultry, and the epidemic continued for many years. When invading cells, the H7N9 virus needs to rely on specific molecules expressed by the virus itself to bind to receptors on human cells in order to infect cells and further proliferate. Human antibodies that neutralize the virus are certain specific antibodies produced by human B lymphocytes, which can bind to antigens on the surface of the virus, thereby preventing the virus from adhering to target cell receptors and preventing the virus from invading cells. They can effectively prevent and treat H7N9 influenza. However, the high R&D and production costs of antibody drugs and the complex technical requirements that are bottlenecks limit their application worldwide. Therapeutic monoclonal antibodies are usually produced by cell lines, such as Chinese hamster ovary cells, and then the antibodies are purified in large quantities from the cell culture supernatant and pharmaceutical formulations are developed. There are many challenges in the production of antibodies, including misfolding or incorrect post-translational modifications that can lead to adverse events. The purification of each monoclonal antibody is specific, requiring the development of a new method for each antibody, making production costs high.
相比之下， mRNA的生产和纯化是简单、快速和经济有效的，因为它不需要复杂和昂贵的实验室基础设施，同样的方法可以用于所有的mRNA。新冠疫情的突然爆发使mRNA技术横空出世，得到了空前的关注。核酸编码的单克隆抗体，特别是基于mRNA的单克隆抗体，则为提高抗体治疗效果和广泛应用带来了巨大希望。治疗性mRNA疫苗研发周期短、安全性和有效性高、生产工艺简单、产能大，是当今最先进的生物医药技术。编码中和抗体的mRNA疫苗是治疗传染病药物研发的重要发展趋势：编码广谱中和HIV抗体VRC01的mRNA注射小鼠后体内成功产生了VRC01抗体，并保护人源小鼠免受HIV-1病毒感染；针对人类RSV，Tiwari将现有药物帕利维珠单抗开发成编码膜锚定中和抗体的mRNA，其效率高于帕利维珠单抗，并在转染7天后显著抑制RSV；编码中和基孔肯雅病毒抗体的mRNA在小鼠体内有效表达，并在接种2天后保护小鼠免受关节炎和肌肉骨骼组织感染，病毒血症降低到无法检测到的水平。抗禽流感病毒单克隆抗体能够预防和治疗禽流感病毒感染。但是抗体在细胞生产过程中，面临纯化和翻译后修饰的问题，而且研发和生产成本昂贵，因此，使用编码抗禽流感抗体的mRNA技术，以进行更高效、安全、低成本的单克隆抗体疗法，是预防和治疗禽流感的新方案。In contrast, the production and purification of mRNA is simple, rapid, and cost-effective because it does not require complex and expensive laboratory infrastructure, and the same method can be used for all mRNAs. The sudden outbreak of the COVID-19 pandemic has brought mRNA technology to the fore and has received unprecedented attention. Nucleic acid-encoded monoclonal antibodies, especially mRNA-based monoclonal antibodies, have brought great hope for improving the therapeutic effect of antibodies and their widespread application. Therapeutic mRNA vaccines have a short R&D cycle, high safety and effectiveness, simple production process, and large production capacity. They are the most advanced biomedical technology today. mRNA vaccines encoding neutralizing antibodies are an important development trend in the development of drugs for the treatment of infectious diseases: mRNA encoding VRC01, a broad-spectrum neutralizing HIV antibody, was successfully injected into mice to produce VRC01 antibodies in vivo and protect humanized mice from HIV-1 virus infection; for human RSV, Tiwari developed the existing drug palivizumab into mRNA encoding membrane-anchored neutralizing antibodies, which were more efficient than palivizumab and significantly inhibited RSV 7 days after transfection; mRNA encoding neutralizing antibodies against chikungunya virus was effectively expressed in mice and protected mice from arthritis and musculoskeletal tissue infection 2 days after inoculation, with viremia reduced to undetectable levels. Anti-avian influenza virus monoclonal antibodies can prevent and treat avian influenza virus infection. However, during the cell production process, antibodies face problems of purification and post-translational modification, and the development and production costs are expensive. Therefore, the use of mRNA technology encoding anti-avian influenza antibodies for more efficient, safe, and low-cost monoclonal antibody therapy is a new solution for the prevention and treatment of avian influenza.
抗体药物是治疗重大传染病的特效药，但其高昂的研发和生产成本以及复杂的卡脖子的技术要求限制了其在世界范围内的应用。mRNA药物研发周期短、安全性和有效性高、生产工艺简单、产能大，是当今最先进的生物技术。目前针对于还未见针对H7N9病毒感染的mRNA疫苗，且鲜有直接利用表达中和病毒的单克隆抗体mRNA作为治疗性mRNA疫苗用于抑制H7N9感染。Antibody drugs are effective drugs for treating major infectious diseases, but their high R&D and production costs and complex technical requirements limit their application worldwide. mRNA drugs have a short R&D cycle, high safety and effectiveness, simple production process, and large production capacity, and are the most advanced biotechnology today. Currently, there is no mRNA vaccine for H7N9 virus infection, and there are few mRNA vaccines that directly use monoclonal antibody mRNA that neutralizes the virus to inhibit H7N9 infection.
为解决上述问题，本发明的发明人利用前期研究中研发了的抗H7N9禽流感病毒的单克隆抗体（公告号:CN 109810189 B）序列。本发明将这个抗体基因转录为mRNA，并且包裹在LNP中，形成LNP-RNA疫苗，受试者接种后能够治疗其感染的H7N9病毒。To solve the above problems, the inventors of the present invention used the monoclonal antibody sequence against H7N9 avian influenza virus (Announcement No.: CN 109810189 B) developed in previous studies. The present invention transcribes this antibody gene into mRNA and encapsulates it in LNP to form an LNP-RNA vaccine, which can treat the H7N9 virus infected by the subject after vaccination.
本发明第一方面提供一种分离的mRNA组合物，其包括包含编码H7N9的单克隆抗体重链的mRNA和包含编码H7N9的单克隆抗体轻链的mRNA，The first aspect of the present invention provides an isolated mRNA composition, which includes mRNA encoding the heavy chain of a monoclonal antibody against H7N9 and mRNA encoding the light chain of a monoclonal antibody against H7N9.
所述H7N9的单克隆抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：The amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the H7N9 monoclonal antibody are shown below:
重链CDR1区：GFTFSSYA SEQ
ID NO.1；Heavy chain CDR1 region: GFTFSSYA SEQ
ID NO.1;
重链CDR2区：ISGSGGST SEQ
ID NO.2；Heavy chain CDR2 region: ISGSGGST SEQ
ID NO.2;
重链CDR3区：AKNRRGSMIVSFLAKSRAGMDV SEQ
ID NO.3；Heavy chain CDR3 region: AKNRRGSMIVSFLAKSRAGMDV SEQ
ID NO.3;
轻链CDR1区：QSISSY SEQ ID NO.4；Light chain CDR1 region: QSISSY SEQ ID NO.4;
轻链CDR2区：AAS ；Light chain CDR2 region: AAS;
轻链CDR3区：QQSYSTPWT SEQ ID NO.5；Light chain CDR3 region: QQSYSTPWT SEQ ID NO.5;
所述mRNA还分别包含：The mRNA further comprises:
1)5’帽结构；1) 5' cap structure;
2)5’UTR序列；2) 5’UTR sequence;
3)编码信号肽的序列3) Sequence encoding signal peptide
4) 终止密码子和酶切位点序列4) Stop codon and restriction site sequence
5)3’UTR序列；以及5) 3'UTR sequence; and
6)多聚腺苷酸序列，6) polyadenylation sequence,
其中，所述包含编码H7N9的单克隆抗体重链的mRNA或包含编码H7N9的单克隆抗体轻链的mRNA 按照5’→3’方向分别依次包括如下元件：5’帽结构，5’UTR 序列，编码信号肽的序列，H7N9的单克隆抗体重链或轻链的开放阅读框所对应的mRNA，终止密码子和酶切位点序列，3’UTR 序列和多聚腺苷酸序列。The mRNA encoding the heavy chain of the monoclonal antibody against H7N9 or the mRNA encoding the light chain of the monoclonal antibody against H7N9 respectively includes the following elements in sequence from 5' to 3' direction: a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, an mRNA corresponding to the open reading frame of the heavy chain or light chain of the monoclonal antibody against H7N9, a stop codon and a restriction site sequence, a 3' UTR sequence and a polyadenylation sequence.
进一步地，该抗体的重链可变区氨基酸序列如SEQ ID NO.6所示；Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.6;
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSS
SEQ
ID NO.6；和/或EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSS
SEQ
ID NO.6; and/or
该抗体的轻链可变区氨基酸序列如SEQ ID NO.7所示：The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.7:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK SEQ
ID NO.7。DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK SEQ
ID NO.7.
进一步地，该抗体的重链氨基酸序列如SEQ ID NO.8所示；Furthermore, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO.8;
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSSRSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8；和/或EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSSRSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8；和/or
该抗体的轻链氨基酸序列如SEQ ID NO.9所示：The light chain amino acid sequence of the antibody is shown in SEQ ID NO.9:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ
ID NO.9。DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ
ID NO.9.
进一步地，H7N9的单克隆抗体重链的开放阅读框所对应的RNA序列如SEQ ID NO.10所述的核酸序列所对应的RNA序列：Furthermore, the RNA sequence corresponding to the open reading frame of the heavy chain of the monoclonal antibody of H7N9 is the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.10:
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG SEQ
ID NO.10GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATG GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG SEQ
ID NO.10
H7N9的单克隆抗体轻链的开放阅读框所对应的RNA序列如SEQ ID NO.11所述的核酸序列所对应的RNA序列：The RNA sequence corresponding to the open reading frame of the monoclonal antibody light chain of H7N9 is the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.11:
gacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ
ID NO.11gacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGC TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ
ID NO.11
进一步地，所述5’帽结构选自m7GpppG、 m27,3′-OGpppG、m7Gppp(5′)N1或m7Gppp(m2′-O)N1中的至少一种。Furthermore, the 5' cap structure is selected from at least one of m7GpppG, m27,3'-OGpppG, m7Gppp(5')N1 or m7Gppp(m2'-O)N1.
进一步地，所述5’UTR序列选自SEQ ID NO.12所述的核酸序列所对应的RNA序列。Furthermore, the 5'UTR sequence is selected from the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.12.
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC SEQ ID NO.12。ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC SEQ ID NO.12.
在本发明的另一些实施方案中，所述3’UTR序列选自SEQ ID NO.13所述的核酸序列所对应的RNA序列。In other embodiments of the present invention, the 3'UTR sequence is selected from the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.13.
CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC
SEQ ID NO.13CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC
SEQ ID NO.13
进一步地，所述多聚腺苷酸序列包含25-400个腺苷酸的序列。Furthermore, the poly(A) sequence comprises a sequence of 25-400 adenylic acids.
进一步地，所述多聚腺苷酸序列包含50-400 个腺苷酸的序列。Furthermore, the poly(A) sequence comprises a sequence of 50-400 adenylic acids.
进一步地，所述多聚腺苷酸序列包含 50-300个腺苷酸的序列。Furthermore, the poly(A) sequence comprises a sequence of 50-300 adenylic acids.
进一步地，所述多聚腺苷酸序列包含50-250个腺苷酸的序列。Furthermore, the poly(A) sequence comprises a sequence of 50-250 adenylic acids.
进一步地，所述多聚腺苷酸序列包含80-120个腺苷酸的序列。Furthermore, the poly(A) sequence comprises a sequence of 80-120 adenylic acids.
进一步地，编码信号肽的序列选自SEQ ID NO.16所示序列对应的RNA序列。Furthermore, the sequence encoding the signal peptide is selected from the RNA sequence corresponding to the sequence shown in SEQ ID NO.16.
进一步地，酶切位点序列选自CTCGAG对应的RNA序列。Furthermore, the restriction site sequence is selected from the RNA sequence corresponding to CTCGAG.
本发明第二方面提供药物组合物，其包含第一方面所述的mRNA组合物，和任选的递送载体。The second aspect of the present invention provides a pharmaceutical composition comprising the mRNA composition described in the first aspect, and an optional delivery vector.
进一步地，所述递送载体为纳米颗粒。Furthermore, the delivery vehicle is a nanoparticle.
进一步地，所述递送载体为脂质纳米颗粒。Furthermore, the delivery vehicle is a lipid nanoparticle.
在本发明的另一些实施方案中，所述脂质纳米颗粒包括阳离子脂质和选自非阳离子脂质、固醇、PEG修饰脂质中的至少一种。In other embodiments of the present invention, the lipid nanoparticles include cationic lipids and at least one selected from non-cationic lipids, sterols, and PEG-modified lipids.
在本发明的一些实施方案中，所述脂质纳米颗粒为阳离子脂质、非阳离子脂质、固醇和PEG修饰脂质。In some embodiments of the present invention, the lipid nanoparticles are cationic lipids, non-cationic lipids, sterols and PEG-modified lipids.
在本发明的另一些实施方案中，所述阳离子脂质为可电离的阳离子脂质，选自以下一种或多种成分：2,2-二亚油基-4-二甲氨基乙基-[1,3]-二氧戊环、二亚油基-甲基-4-二甲氨基丁酸酯和9-((4-(二甲氨基)丁酰基)氧基)十七烷二酸二((Z)-壬-2-烯-1-基)酯，优选二亚油基-甲基-4-二甲氨基丁酸酯。在本发明的一些实施方案中，所述非阳离子脂质为中性脂质，选自二硬脂酰基磷脂酰胆碱(DSPC)、二油酰磷脂酰乙醇胺(DOPE)、二油酰基卵磷脂 (DOPC)和二油酰基磷脂酰丝氨酸(DOPS)中的至少一种。In other embodiments of the present invention, the cationic lipid is an ionizable cationic lipid selected from one or more of the following components: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, dilinoleyl-methyl-4-dimethylaminobutyrate and 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester, preferably dilinoleyl-methyl-4-dimethylaminobutyrate. In some embodiments of the present invention, the non-cationic lipid is a neutral lipid selected from at least one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS).
在本发明的另一些实施方案中，所述固醇为胆固醇。In other embodiments of the present invention, the sterol is cholesterol.
在本发明的一些实施方案中，所述PEG修饰脂质选自PEG-DMG、 PEG-DSG和PEG-DMPE中的至少一种。In some embodiments of the present invention, the PEG-modified lipid is selected from at least one of PEG-DMG, PEG-DSG and PEG-DMPE.
在本发明的另一些实施方案中，所述PEG修饰脂质的PEG长度为0.5-200KDa。In other embodiments of the present invention, the PEG length of the PEG-modified lipid is 0.5-200 KDa.
在本发明的一些实施方案中，所述药物组合物任选的含有佐剂。In some embodiments of the present invention, the pharmaceutical composition optionally contains an adjuvant.
本发明第三方面提供试剂盒，其包含本发明第一方面所述的mRNA组合物和/或本发明第二方面所述的药物组合物。The third aspect of the present invention provides a kit comprising the mRNA composition described in the first aspect of the present invention and/or the pharmaceutical composition described in the second aspect of the present invention.
本发明第四方面提供本发明第一方面所述的mRNA组合物，本发明第二方面所述的药物组合物，本发明第三方面所述的试剂盒在制备预防和/或治疗 H7N9病毒感染疾病的药物中的应用。The fourth aspect of the present invention provides the use of the mRNA composition described in the first aspect of the present invention, the pharmaceutical composition described in the second aspect of the present invention, and the kit described in the third aspect of the present invention in the preparation of drugs for preventing and/or treating H7N9 virus infection diseases.
本发明第五方面提供本发明第一方面所述的mRNA组合物的制备方法，所述制备方法包括以下步骤：The fifth aspect of the present invention provides a method for preparing the mRNA composition according to the first aspect of the present invention, the preparation method comprising the following steps:
S1) 将H7N9单克隆抗体重链和轻链基因分别插入到质粒载体中，得到包含H7N9单克隆抗体重链或轻链基因的质粒载体；S1) inserting the H7N9 monoclonal antibody heavy chain and light chain genes into plasmid vectors respectively to obtain plasmid vectors containing the H7N9 monoclonal antibody heavy chain or light chain gene;
S2) 将步骤1）所得包含H7N9单克隆抗体重链和轻链基因的质粒载体分别转入宿主菌中培养并测序；S2) transferring the plasmid vectors containing the heavy chain and light chain genes of the H7N9 monoclonal antibody obtained in step 1) into host bacteria, culturing them and sequencing them;
S3) 将步骤2）中测序正确的单克隆进行扩大培养，并抽提质粒；S3) Expanding and culturing the single clone sequenced correctly in step 2) and extracting the plasmid;
S4) 以酶切质粒获得线性化质粒；S4) digesting the plasmid with enzymes to obtain a linearized plasmid;
S5) 将线性化质粒转录为RNA；S5) transcribe the linearized plasmid into RNA;
S6) 将步骤S5）所得RNA进行加帽获得mRNA，S6) capping the RNA obtained in step S5) to obtain mRNA,
S7) 将包含H7N9单克隆抗体重链和轻链基因开放阅读框的mRNA以1:1进行混合获得mRNA组合物。S7) Mixing mRNAs containing the H7N9 monoclonal antibody heavy chain and light chain gene open reading frames in a 1:1 ratio to obtain an mRNA composition.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明的H7N9 mRNA疫苗研发周期短，特别适合包括H7N9在内的新发突发传染病疫苗的开发；安全性高。本发明的mRNA组合物克服了现有技术中采用抗原mRNA疫苗治疗病毒感染疾病时可能出现的问题，例如在接种后不同受试者免疫反应程度不同，并不是所有受试者均能产生相应抗体，即使生成抗体，产抗体的时间也较为漫长，还可能出现产量不足以抑制病毒感染的问题。本发明的mRNA组合物能够直接在受试者体内表达为单克隆抗体，起效快，能够直接用于H7N9感染的治疗，且效果优于直接注射单克隆抗体。The H7N9 mRNA vaccine of the present invention has a short development cycle and is particularly suitable for the development of vaccines for emerging infectious diseases including H7N9; it has high safety. The mRNA composition of the present invention overcomes the problems that may occur when using antigen mRNA vaccines to treat viral infectious diseases in the prior art, such as different levels of immune response in different subjects after vaccination, not all subjects can produce corresponding antibodies, and even if antibodies are generated, the time for antibody production is relatively long, and there may be a problem that the production is not enough to inhibit viral infection. The mRNA composition of the present invention can be directly expressed as monoclonal antibodies in the subject, has a fast onset, can be directly used for the treatment of H7N9 infection, and has better effects than direct injection of monoclonal antibodies.
图1为质粒线性化凝胶电泳照片。Figure 1 is a photo of plasmid linearization gel electrophoresis.
图2为转录的mRNA凝胶电泳照片。Figure 2 is a photograph of gel electrophoresis of transcribed mRNA.
图3为ELISA验证LNP-mRNA在细胞中的抗体表达。FIG3 is an ELISA to verify the antibody expression of LNP-mRNA in cells.
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below, but it should not be understood as limiting the applicable scope of the present invention.
在本发明的一些实施方案中，所述mRNA为未经过化学改性或者经过化学改性，所述化学改性为对mRNA中的至少50％、至少60％、至少70％、至少80％、至少90％或 100％的尿嘧啶进行替换，将尿嘧啶替换为假尿苷、N1-甲基假尿苷、N1-乙基假尿苷、2-硫尿苷、4′-硫尿苷、5-甲基胞嘧啶、5-甲基尿苷、2-硫基-1-甲基-1- 去氮杂-假尿苷、2-硫基T-甲基-假尿苷、2-硫基-5-氮杂-尿苷、2-硫基-二氢假尿苷、2-硫基-二氢尿苷、2-硫基-假尿苷、4-甲氧基-2-硫基-假尿苷、4- 甲氧基-假尿苷、4-硫基-1-甲基-假尿苷、4-硫基-假尿苷、5-氮杂-尿苷、二氢假尿苷或5-甲氧基尿苷和2′-O-甲基尿苷中的至少一种，优选假尿苷或 N1-甲基假尿苷或N1-乙基假尿苷，进一步优选N1-甲基假尿苷；In some embodiments of the present invention, the mRNA is not chemically modified or is chemically modified, and the chemical modification is to replace at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil in the mRNA, and replace uracil with pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4- At least one of methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyluridine, preferably pseudouridine, N1-methylpseudouridine or N1-ethylpseudouridine, and more preferably N1-methylpseudouridine;
在本发明的一些实施方案中，所述5’帽结构选自m7GpppG、 m27,3′-OGpppG、m7Gppp(5′)N1或m7Gppp(m2′-O)N1中的至少一种。In some embodiments of the present invention, the 5' cap structure is selected from at least one of m7GpppG, m27,3'-OGpppG, m7Gppp(5')N1 or m7Gppp(m2'-O)N1.
根据不同mRNA的需求，可在mRNA 5’端灵活添加不同的5’帽结构。According to the requirements of different mRNAs, different 5’ cap structures can be flexibly added to the 5’ end of the mRNA.
“m7G”代表7-甲基鸟苷帽核苷，“ppp”代表帽核苷的5′碳和初级RNA 转录物的第一个核苷酸之间的三磷酸键，N1是最5′的核苷酸，“G”代表鸟嘌呤核苷，“m7”代表在鸟嘌呤的7-位上的甲基,“m2′-O”代表核苷酸2′-O 位上的甲基。"m7G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5' most nucleotide, "G" represents guanosine nucleoside, "m7" represents the methyl group at the 7-position of guanine, and "m2'-O" represents the methyl group at the 2'-O position of the nucleotide.
本发明中提供的纳米颗粒，可高效递送mRNA，具有如下特点和优势：例如，在包封mRNA时，酸性pH条件使可电离的阳离子脂质带有正电荷，压缩带负电荷的mRNA分子，进而获得较高的包封率；在生理pH条件下，可电离的脂质纳米颗粒具有电中性，不与带负电的细胞膜作用，生物相容性高；可电离的脂质纳米颗粒通过细胞内吞作用形成内涵体进入细胞后，内涵体中的酸性条件使纳米颗粒再次带上正电荷，与带有负电荷的内涵体膜发生静电相互作用，从而有利于mRNA的释放。The nanoparticles provided in the present invention can efficiently deliver mRNA and have the following characteristics and advantages: for example, when encapsulating mRNA, the acidic pH condition causes the ionizable cationic lipids to have a positive charge, compressing the negatively charged mRNA molecules, thereby obtaining a higher encapsulation rate; under physiological pH conditions, the ionizable lipid nanoparticles are electrically neutral and do not interact with the negatively charged cell membrane, and have high biocompatibility; after the ionizable lipid nanoparticles form endosomes and enter the cells through cellular endocytosis, the acidic conditions in the endosomes cause the nanoparticles to have a positive charge again, and electrostatically interact with the negatively charged endosomal membrane, thereby facilitating the release of mRNA.
实施例1制备mRNAExample 1 Preparation of mRNA
（1）中和禽流感病毒抗体基因插入到pUC57-kana载体中：用限制性酶EcoR1和Hind3（购自Thermo）分别双酶切H7N9抗体重链基因、H7N9抗体轻链基因和pUC57载体（本实验室保存），并用T4连接酶（Thermo）分别连接抗体重链基因和抗体轻链基因相连；获得连接有中和禽流感病毒抗体重链基因的载体和连接有中和禽流感病毒抗体轻链基因的载体。(1) Inserting the neutralizing avian influenza virus antibody gene into the pUC57-kana vector: Use restriction enzymes EcoR1 and Hind3 (purchased from Thermo) to double-digest the H7N9 antibody heavy chain gene, H7N9 antibody light chain gene and pUC57 vector (stored in this laboratory), and use T4 ligase (Thermo) to connect the antibody heavy chain gene and the antibody light chain gene, respectively; obtain a vector connected with the neutralizing avian influenza virus antibody heavy chain gene and a vector connected with the neutralizing avian influenza virus antibody light chain gene.
H7N9抗体重链基因如SEQ ID NO.10所示The H7N9 antibody heavy chain gene is shown in SEQ ID NO.10
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG
SEQ
ID NO.10。GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTG TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG
SEQ
ID NO.10.
在编码H7N9抗体重链基因序列前还包含了信号肽序列SEQ ID NO.16；ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC
SEQ ID NO.16。信号肽表达后可以引导新合成的蛋白质向分泌通路转移，本发明所用信号肽为现有技术中已知的信号肽序列，也可以替换为功能相同的其他信号肽序列。A signal peptide sequence SEQ ID NO.16 is also included before the gene sequence encoding the H7N9 antibody heavy chain; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC
SEQ ID NO.16. After the signal peptide is expressed, it can guide the newly synthesized protein to transfer to the secretory pathway. The signal peptide used in the present invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
也可以使用能够编码H7N9抗体重链SEQ ID NO.8的其他基因序列。H7N9抗体重链序列如SEQ ID NO.8所示：Other gene sequences that can encode the H7N9 antibody heavy chain SEQ ID NO.8 can also be used. The H7N9 antibody heavy chain sequence is shown in SEQ ID NO.8:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSSRSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8。EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSSRSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8.
H7N9抗体轻链基因如SEQ ID NO.11所示：The H7N9 antibody light chain gene is shown in SEQ ID NO.11:
gacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO.11。gacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGG TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO.11.
在编码H7N9抗体轻链基因序列前还包含了信号肽序列SEQ ID NO.16；ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC
SEQ ID NO.16。信号肽表达后可以引导新合成的蛋白质向分泌通路转移，本发明所用信号肽为现有技术中已知的信号肽序列，也可以替换为功能相同的其他信号肽序列。A signal peptide sequence SEQ ID NO.16 is also included before the gene sequence encoding the light chain of the H7N9 antibody; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC
SEQ ID NO.16. After the signal peptide is expressed, it can guide the newly synthesized protein to transfer to the secretory pathway. The signal peptide used in the present invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
也可以使用能够编码H7N9抗体轻链SEQ ID NO.9的其他基因序列。H7N9抗体轻链序列如SEQ ID NO.9所示：Other gene sequences that can encode the H7N9 antibody light chain SEQ ID NO.9 can also be used. The H7N9 antibody light chain sequence is shown in SEQ ID NO.9:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.9。DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.9.
（2）载体转化XL1-blue菌株：取一管感受态细胞，加入1μL（50ng）的步骤（1）所得含轻链基因的质粒或含重链基因的质粒，混匀；冰浴5分钟，热激90秒，冰上放置5分钟；加入500μL的2YT液体培养基，37℃，200 rpm，45分钟；吸50μL菌液涂板，培养过夜；挑单克隆送到金唯智公司测序；测序结果如SEQ ID NO.14和SEQ ID NO.15所示：ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO.14；(2) Vector transformation of XL1-blue strain: Take a tube of competent cells, add 1 μL (50 ng) of the plasmid containing the light chain gene or the plasmid containing the heavy chain gene obtained in step (1), mix well; ice bath for 5 minutes, heat shock for 90 seconds, and place on ice for 5 minutes; add 500 μL of 2YT liquid culture medium, incubate at 37°C, 200 rpm, for 45 minutes; aspirate 50 μL of bacterial solution and spread on the plate, culture overnight; select a single clone and send it to Genewise for sequencing; the sequencing results are shown in SEQ ID NO.14 and SEQ ID As shown in No.15: ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGTGAGGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGG AAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGG GACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO.14；
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCgacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO.15；ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCgacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCA GTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCA CCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO.15;
（3）单克隆扩大培养，质粒抽提：将测序正确的单克隆培养在400ml的2YT培养基中，37℃培养过夜。利用CWBIO公司的质粒抽提试剂盒抽提质粒，最终获得6.4mg质粒，测序正确；(3) Monoclonal expansion and plasmid extraction: The correctly sequenced monoclonal was cultured in 400 ml of 2YT medium at 37°C overnight. The plasmid was extracted using CWBIO's plasmid extraction kit, and 6.4 mg of plasmid was finally obtained, which was sequenced correctly;
（4）质粒线性化：利用Bsa I酶酶切质粒，在离心管中依次加入质粒（2887μL）、酶（333μL）、缓冲液（1665μL）和DEPC水（11765μL），37℃水浴过夜。用100K超滤管换液回收，最终获得线性化质粒浓度387.5ng/μL；(4) Plasmid linearization: Use Bsa I enzyme to digest the plasmid, add plasmid (2887 μL), enzyme (333 μL), buffer (1665 μL) and DEPC water (11765 μL) in a centrifuge tube in sequence, and incubate in a 37°C water bath overnight. Use a 100K ultrafiltration tube to replace the liquid and recover, and finally obtain a linearized plasmid concentration of 387.5 ng/μL;
（5）转录：在离心管中加入412 μL质粒，320 μL缓冲液，64 μL ATP，64 μL GTP，64 μL CTP，64 μL pUTP，160 μL RNase抑制剂，3.2 μL 无机焦磷酸酶，160 μL T7 RNA聚合酶，1888.8 μL DEPC水。37℃反应3小时。加入160 μL DNase I
酶消化15分钟；加入4800 μL LiCl沉淀液和4800 μL DEPC水，混匀后置于-20℃冰箱静置30分钟；12000 rpm离心15分钟，70%乙醇清洗2次，尽量去除离心管中残留的乙醇；加入1500 μL 水溶解RNA。最终获得5037 ng/μL RNA，一共7.56 mg；(5) Transcription: Add 412 μL plasmid, 320 μL buffer, 64 μL ATP, 64 μL GTP, 64 μL CTP, 64 μL pUTP, 160 μL RNase inhibitor, 3.2 μL inorganic pyrophosphatase, 160 μL T7 RNA polymerase, and 1888.8 μL DEPC water to a centrifuge tube. React at 37°C for 3 hours. Add 160 μL DNase I
enzyme digestion for 15 minutes; add 4800 μL LiCl precipitation solution and 4800 μL DEPC water, mix well and place in a -20°C refrigerator for 30 minutes; centrifuge at 12000 rpm for 15 minutes, wash twice with 70% ethanol to remove as much ethanol as possible from the centrifuge tube; add 1500 μL water to dissolve the RNA. Finally, 5037 ng/μL RNA was obtained, a total of 7.56 mg;
（6）加帽m7Gppp(5′)N1：离心管中加入596 μL RNA，3424 μL水，65℃变性5分钟；加入600 μL缓冲液，600 μL GTP，150 μL SAM，150 μL RNase抑制剂，240 μL加帽酶；240 μL甲基转移酶，37℃反应1h；加入9mL LiCl沉淀液和9mLDEPC水，混匀后置于-20℃冰箱静置30min；12000rpm离心15分钟，70%乙醇清洗2次，尽量去除离心管中残留的乙醇；开盖置于超净台中晾干，加入3000μL水溶解RNA。Nanodrop检测浓度为715ng/mL，产量2.145 mg，得率71.5%。(6) Capping m7Gppp(5′)N1: Add 596 μL RNA and 3424 μL water to a centrifuge tube, denature at 65°C for 5 minutes; add 600 μL buffer, 600 μL GTP, 150 μL SAM, 150 μL RNase inhibitor, 240 μL capping enzyme; 240 μL methyltransferase, react at 37°C for 1 hour; add 9 mL LiCl precipitation solution and 9 mL DEPC water, mix well and place in a -20°C refrigerator for 30 minutes; centrifuge at 12000 rpm for 15 minutes, wash twice with 70% ethanol to remove as much ethanol as possible from the centrifuge tube; open the tube and place in a clean bench to dry, add 3000 μL water to dissolve the RNA. The Nanodrop detection concentration was 715 ng/mL, the yield was 2.145 mg, and the yield was 71.5%.
(7)制备mRNA混合物：将分别将包含H7N9单克隆抗体重链和轻链基因开放阅读框的mRNA以质量比1:1进行混合，获得mRNA组合物。(7) Preparation of an mRNA mixture: mRNAs containing the open reading frames of the heavy chain and light chain genes of the H7N9 monoclonal antibody were mixed at a mass ratio of 1:1 to obtain an mRNA composition.
实施例2制备LNP包封的mRNAExample 2 Preparation of LNP-encapsulated mRNA
LNP包封mRNA的实验由上海近岸科技有限公司完成，LNP-mRNA外观是乳白色液体，包封率>98%，分散系数是0.103，纳米颗粒粒径为90.35 nm。The experiment of LNP encapsulation of mRNA was completed by Shanghai Jinan Technology Co., Ltd. The appearance of LNP-mRNA is milky white liquid, the encapsulation rate is >98%, the dispersion coefficient is 0.103, and the particle size of the nanoparticles is 90.35 nm.
LNP-mRNA利用LIPO3000（Invitrogen）转染293T细胞，48小时后收集上清和细胞裂解液，分别用PBS稀释20倍后100μL/孔包被ELISA板子，37℃孵育2小时，PBS洗涤2次，5%脱脂奶粉封闭1小时，PBS洗涤2次，每孔加入100 μL抗his标签抗体，37℃孵育1小时，PBST洗涤3次，每孔加入100 μL显色液显示10分钟，最后加入100μL终止液，在分光光度计记录OD450的数据。LNP-mRNA was transfected into 293T cells using LIPO3000 (Invitrogen). After 48 hours, the supernatant and cell lysate were collected and diluted 20 times with PBS, and 100 μL/well was coated on the ELISA plate. The plates were incubated at 37°C for 2 hours, washed twice with PBS, blocked with 5% skim milk powder for 1 hour, washed twice with PBS, and 100 μL anti-his tag antibody was added to each well. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and 100 μL of color development solution was added to each well for 10 minutes. Finally, 100 μL of stop solution was added, and the OD450 data was recorded on a spectrophotometer.
结果如图3所示：转染LNP-mRNA的细胞表达了目的抗体。The results are shown in Figure 3: the cells transfected with LNP-mRNA expressed the target antibody.
实施例3Example 3
用生理盐水配制终浓度为1%的戊巴比妥钠溶液，0.22μm滤器过滤，4℃低温保存。称量小鼠体重，以150μl/20g的剂量通过腹腔给药麻醉小鼠，滴鼻感染20 μL含10×LD50的H7N9病毒。12小时后，将小鼠随机分为PBS组、LNP-mRNA实验组和抗体实验组，每组6只小鼠，分别尾静脉接种PBS、LNP-mRNA（1.4 mg/kg）和抗体（30mg/kg）。滴鼻的日期为小鼠实验第0天。自感染之日起，记录小鼠存活数，直至感染后2周Use physiological saline to prepare a sodium pentobarbital solution with a final concentration of 1%, filter it with a 0.22μm filter, and store it at 4°C. Weigh the mice, anesthetize them by intraperitoneal administration of 150μl/20g, and infect them with 20 μL of H7N9 virus containing 10×LD50 by intranasal drip. After 12 hours, the mice were randomly divided into PBS group, LNP-mRNA experimental group and antibody experimental group, with 6 mice in each group, and PBS, LNP-mRNA (1.4 mg/kg) and antibody (30 mg/kg) were inoculated through the tail vein. The date of intranasal drip was day 0 of the mouse experiment. From the date of infection, the number of surviving mice was recorded until 2 weeks after infection.
结果显示：PBS组动物全部死亡，LNP-mRNA组的小鼠存活率83%，抗体治疗组的小鼠存活率为66%。相比抗体治疗组，更低剂量下编码该抗体的LNP-mRNA能够更显著保护小鼠免受H7N9病毒的致死攻击。编码抗禽流感抗体的LNP-mRNA能够直接在体内表达抗体，并显著保护小鼠免受H7N9病毒的致死攻击。The results showed that all animals in the PBS group died, the survival rate of mice in the LNP-mRNA group was 83%, and the survival rate of mice in the antibody treatment group was 66%. Compared with the antibody treatment group, the LNP-mRNA encoding the antibody at a lower dose can more significantly protect mice from the lethal attack of the H7N9 virus. The LNP-mRNA encoding anti-avian influenza antibodies can directly express antibodies in vivo and significantly protect mice from the lethal attack of the H7N9 virus.
Claims (10)
- 一种分离的mRNA组合物，其特征在于，其包括包含编码H7N9的单克隆抗体重链的mRNA和包含编码H7N9的单克隆抗体轻链的mRNA，An isolated mRNA composition, characterized in that it comprises mRNA encoding the heavy chain of a monoclonal antibody against H7N9 and mRNA encoding the light chain of a monoclonal antibody against H7N9,所述H7N9的单克隆抗体的重链和轻链具有如下所示CDR1、CDR2及CDR3区：The heavy chain and light chain of the monoclonal antibody of H7N9 have the following CDR1, CDR2 and CDR3 regions:重链CDR1区：GFTFSSYA SEQ ID NO.1；Heavy chain CDR1 region: GFTFSSYA SEQ ID NO.1;重链CDR2区：ISGSGGST SEQ ID NO.2；Heavy chain CDR2 region: ISGSGGST SEQ ID NO.2;重链CDR3区：AKNRRGSMIVSFLAKSRAGMDV SEQ ID NO.3；Heavy chain CDR3 region: AKNRRGSMIVSFLAKSRAGMDV SEQ ID NO.3;轻链CDR1区：QSISSY SEQ ID NO.4；Light chain CDR1 region: QSISSY SEQ ID NO.4;轻链CDR2区：AAS ；Light chain CDR2 region: AAS ;轻链CDR3区：QQSYSTPWT SEQ ID NO.5；Light chain CDR3 region: QQSYSTPWT SEQ ID NO.5;所述包含编码H7N9的单克隆抗体重链的mRNA或包含编码H7N9的单克隆抗体轻链的mRNA还分别包含：The mRNA encoding the heavy chain of the monoclonal antibody against H7N9 or the mRNA encoding the light chain of the monoclonal antibody against H7N9 further comprises:1)5’帽结构；1) 5' cap structure;2)5’UTR序列；2) 5’UTR sequence;3)编码信号肽的序列3) Sequence encoding signal peptide4)终止密码子和酶切位点序列4) Stop codon and restriction site sequence5)3’UTR序列；以及5) 3'UTR sequence; and6)多聚腺苷酸序列，6) polyadenylation sequence,其中，所述包含编码H7N9的单克隆抗体重链的mRNA或包含编码H7N9的单克隆抗体轻链的mRNA按照5’→3’方向依次包括如下元件：5’帽结构，5’UTR 序列，编码信号肽的序列，编码H7N9的单克隆抗体重链或轻链的开放阅读框所对应的RNA序列，终止密码子和酶切位点序列，3’UTR 序列和多聚腺苷酸序列；The mRNA encoding the heavy chain of the monoclonal antibody against H7N9 or the mRNA encoding the light chain of the monoclonal antibody against H7N9 includes the following elements in order from 5' to 3': a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, an RNA sequence corresponding to an open reading frame encoding the heavy chain or light chain of the monoclonal antibody against H7N9, a stop codon and a restriction site sequence, a 3' UTR sequence and a polyadenylation sequence;优选地，所述5’帽结构选自m7GpppG、 m27,3′-OGpppG、m7Gppp(5′)N1或m7Gppp(m2′-O)N1中的至少一种；Preferably, the 5' cap structure is selected from at least one of m7GpppG, m27,3'-OGpppG, m7Gppp(5')N1 or m7Gppp(m2'-O)N1;优选地，所述多聚腺苷酸序列包含25-400个腺苷酸的序列；Preferably, the poly(A) sequence comprises a sequence of 25-400 adenylic acids;优选地，所述组合物中包含编码H7N9的单克隆抗体重链的mRNA和包含编码H7N9的单克隆抗体轻链的mRNA的质量比为1:1。Preferably, the mass ratio of mRNA encoding the heavy chain of the monoclonal antibody against H7N9 to mRNA encoding the light chain of the monoclonal antibody against H7N9 in the composition is 1:1.
- 根据权利要求1所述的分离的mRNA组合物，其特征在于，所述H7N9的单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.6所示；和/或The isolated mRNA composition according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody against H7N9 is as shown in SEQ ID NO.6; and/or所述H7N9的单克隆抗体的抗体的轻链可变区氨基酸序列如SEQ ID NO.7所示：The amino acid sequence of the light chain variable region of the monoclonal antibody against H7N9 is shown in SEQ ID NO.7:优选地，所述H7N9的单克隆抗体的重链氨基酸序列如SEQ ID NO.8所示；和/或Preferably, the heavy chain amino acid sequence of the monoclonal antibody against H7N9 is as shown in SEQ ID NO.8; and/or所述H7N9的单克隆抗体的轻链氨基酸序列如SEQ ID NO.9所示。The light chain amino acid sequence of the monoclonal antibody against H7N9 is shown in SEQ ID NO.9.
- 根据权利要求1-2任一项所述的分离的mRNA组合物，其特征在于，编码H7N9的单克隆抗体重链的开放阅读框核酸序列如SEQ ID NO.10 所示；The isolated mRNA composition according to any one of claims 1 to 2, characterized in that the open reading frame nucleic acid sequence encoding the heavy chain of the monoclonal antibody against H7N9 is as shown in SEQ ID NO.10;编码H7N9的单克隆抗体轻链的开放阅读框核酸序列如SEQ ID NO.11 所示。The open reading frame nucleic acid sequence encoding the light chain of the monoclonal antibody against H7N9 is shown in SEQ ID NO.11.
- 根据权利要求1-2任一项所述的分离的mRNA组合物，其特征在于，所述5’UTR序列选自SEQ ID NO.12所述的核酸序列所对应的RNA序列。The isolated mRNA composition according to any one of claims 1-2, characterized in that the 5'UTR sequence is selected from the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.12.
- 根据权利要求1-3任一项所述的分离的mRNA组合物，其特征在于，所述3’UTR序列选自SEQ ID NO.13所述的核酸序列所对应的RNA序列。The isolated mRNA composition according to any one of claims 1 to 3, characterized in that the 3'UTR sequence is selected from the RNA sequence corresponding to the nucleic acid sequence described in SEQ ID NO.13.
- 一种药物组合物，其特征在于，其包含权利要求1-5任一项所述的mRNA组合物，和任选的递送载体；A pharmaceutical composition, characterized in that it comprises the mRNA composition according to any one of claims 1 to 5, and an optional delivery vector;优选地，所述递送载体为纳米颗粒；Preferably, the delivery vehicle is a nanoparticle;优选地，所述递送载体为脂质纳米颗粒。Preferably, the delivery vehicle is a lipid nanoparticle.
- 根据权利要求6所述的药物组合物，其特征在于，所述药物组合物任选的含有佐剂。The pharmaceutical composition according to claim 6, characterized in that the pharmaceutical composition optionally contains an adjuvant.
- 一种试剂盒，其特征在于，其包含权利要求1-5任一项所述的mRNA组合物和/或权利要求6或7所述的药物组合物。A kit, characterized in that it comprises the mRNA composition according to any one of claims 1 to 5 and/or the pharmaceutical composition according to claim 6 or 7.
- 权利要求1-5任一项所述的mRNA组合物所述的mRNA组合物，权利要求6或7所述的药物组合物，权利要求8所述的试剂盒在制备预防和/或治疗 H7N9病毒感染疾病的药物中的应用。Use of the mRNA composition described in any one of claims 1 to 5, the pharmaceutical composition described in claim 6 or 7, and the kit described in claim 8 in the preparation of drugs for preventing and/or treating H7N9 virus infection diseases.
- 权利要求1-5任一项所述的mRNA组合物的制备方法，其特征在于，所述制备方法包括以下步骤：The method for preparing the mRNA composition according to any one of claims 1 to 5, characterized in that the preparation method comprises the following steps:S1) 将H7N9单克隆抗体重链和轻链基因分别插入到质粒载体中，得到包含H7N9单克隆抗体重链或轻链基因的质粒载体；S1) inserting the H7N9 monoclonal antibody heavy chain and light chain genes into plasmid vectors respectively to obtain plasmid vectors containing the H7N9 monoclonal antibody heavy chain or light chain gene;S2) 将步骤1）所得包含H7N9单克隆抗体重链和轻链基因的质粒载体分别转入宿主菌中培养并测序；S2) transferring the plasmid vectors containing the heavy chain and light chain genes of the H7N9 monoclonal antibody obtained in step 1) into host bacteria, culturing them and sequencing them;S3) 将步骤2）中测序正确的单克隆进行扩大培养，并抽提质粒；S3) Expanding and culturing the single clone sequenced correctly in step 2) and extracting the plasmid;S4) 以酶切质粒获得线性化质粒；S4) digesting the plasmid with enzymes to obtain a linearized plasmid;S5) 将线性化质粒转录为RNA；S5) transcribe the linearized plasmid into RNA;S6) 将步骤S5）所得RNA进行加帽获得mRNA；S6) capping the RNA obtained in step S5) to obtain mRNA;S7) 将包含H7N9单克隆抗体重链和轻链基因开放阅读框的mRNA以1:1进行混合获得mRNA组合物。S7) Mixing mRNAs containing the H7N9 monoclonal antibody heavy chain and light chain gene open reading frames in a 1:1 ratio to obtain an mRNA composition.
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| PCT/CN2022/137375 WO2024098491A1 (en) | 2022-11-11 | 2022-12-07 | Mrna for encoding anti-avian influenza h7n9 virus antibody, preparation method therefor, and use thereof |
Country Status (2)
| Country | Link |
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| CN (1) | CN118021957A (en) |
| WO (1) | WO2024098491A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109810189A (en) * | 2017-11-21 | 2019-05-28 | 中国科学院深圳先进技术研究院 | The full human monoclonal antibody 4L3 of anti-H7N9 and its preparation method and application |
| CN113151312A (en) * | 2020-03-02 | 2021-07-23 | 中国科学院微生物研究所 | Novel coronavirus SARS-CoV-2mRNA vaccine and its preparation method and application |
| CN113736801A (en) * | 2020-05-28 | 2021-12-03 | 上海蓝鹊生物医药有限公司 | mRNA and novel coronavirus mRNA vaccine containing same |
| CN114081943A (en) * | 2021-11-08 | 2022-02-25 | 中国医学科学院医学生物学研究所 | Varicella-zoster mRNA vaccine composition and preparation method and application thereof |
| CA3194325A1 (en) * | 2020-11-06 | 2022-05-12 | Danilo Casimiro | Lipid nanoparticles for delivering mrna vaccines |
-
2022
- 2022-11-11 CN CN202211410073.9A patent/CN118021957A/en active Pending
- 2022-12-07 WO PCT/CN2022/137375 patent/WO2024098491A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109810189A (en) * | 2017-11-21 | 2019-05-28 | 中国科学院深圳先进技术研究院 | The full human monoclonal antibody 4L3 of anti-H7N9 and its preparation method and application |
| CN113151312A (en) * | 2020-03-02 | 2021-07-23 | 中国科学院微生物研究所 | Novel coronavirus SARS-CoV-2mRNA vaccine and its preparation method and application |
| CN113736801A (en) * | 2020-05-28 | 2021-12-03 | 上海蓝鹊生物医药有限公司 | mRNA and novel coronavirus mRNA vaccine containing same |
| CA3194325A1 (en) * | 2020-11-06 | 2022-05-12 | Danilo Casimiro | Lipid nanoparticles for delivering mrna vaccines |
| CN114081943A (en) * | 2021-11-08 | 2022-02-25 | 中国医学科学院医学生物学研究所 | Varicella-zoster mRNA vaccine composition and preparation method and application thereof |
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| CN118021957A (en) | 2024-05-14 |
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