WO2024098498A1 - 一种编码抗禽流感H7N9病毒抗体的mRNA及其制法和应用 - Google Patents
一种编码抗禽流感H7N9病毒抗体的mRNA及其制法和应用 Download PDFInfo
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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_110746503_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 GYIFTSYD SEQ ID NO.1;
- Heavy chain CDR2 region MNPDSGDT SEQ ID NO.2;
- Heavy chain CDR3 region ATGNADCSGGSCYNWFDP SEQ ID NO.3;
- Light chain CDR1 region RLRSYY SEQ ID NO.4;
- 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 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 endocytosis, the acidic conditions in the endosomes cause the nanoparticles to have a positive charge again, and electrostatic interaction occurs 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:
- the 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 358.1 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 721 ng/mL, and the yield was 2.095 mg.
- 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.
- LNP encapsulating 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 0.11, and the particle size of the nanoparticles is 92.84 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 8 mice in each group, and PBS, LNP-mRNA (1.4 mg/kg) and antibody (20 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
本发明涉及一种编码抗禽流感H7N9病毒抗体的mRNA及其制法和应用。具体地,提供了一种分离的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的单克隆抗体的mRNA,所述mRNA还分别包含: 5'帽结构;5'UTR序列; 3'UTR序列以及4)多聚腺苷酸序列,所述组合物中包含编码H7N9的单克隆抗体重链的开放阅读框的mRNA和包含编码H7N9的单克隆抗体轻链的开放阅读框的mRNA的质量比为1:1。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。
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。
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。
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。
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。
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_110746503_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。
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,
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:
重链CDR1区:GYIFTSYD SEQ
ID NO.1;
重链CDR2区:MNPDSGDT SEQ ID NO.2;
重链CDR3区:ATGNADCSGGSCYNWFDP SEQ ID NO.3;
轻链CDR1区:RLRSYY SEQ ID NO.4;
轻链CDR2区:GKN ;
轻链CDR3区:NSRDTSGYHLV SEQ
ID NO.5;
所述mRNA还分别包含:
1)5’帽结构;
2)5’UTR序列;
3)编码信号肽的序列
4) 终止密码子和酶切位点序列
5)3’UTR序列;以及
6)多聚腺苷酸序列,
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。
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;
QVQLVESGAEVKKPGASVKVSCKASGYIFTSYDINWVRQATGQGLEWMGWMNPDSGDTGFAQKFQGRVTMTRNTSITTAYMELSSLTSEDTAVYYCATGNADCSGGSCYNWFDPWGQGTLVTVSS
SEQ
ID NO.6;和/或
该抗体的轻链可变区氨基酸序列如SEQ ID NO.7所示:
SSELTQDPAVSVALGQTVRITCQGDRLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDTSGYHLVFGGGTKLTVLV 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;
QVQLVESGAEVKKPGASVKVSCKASGYIFTSYDINWVRQATGQGLEWMGWMNPDSGDTGFAQKFQGRVTMTRNTSITTAYMELSSLTSEDTAVYYCATGNADCSGGSCYNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8;和/或
该抗体的轻链氨基酸序列如SEQ ID NO.9所示:
SSELTQDPAVSVALGQTVRITCQGDRLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDTSGYHLVFGGGTKLTVLVTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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:
caagtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacatattcaccagttatgatatcaactgggtgcgacaggccactggccaagggcttgagtggatgggatggatgaaccctgacagtggtgacacaggctttgcacagaagttccagggcagagtcaccatgaccaggaacacctccataaccacagcctacatggagctgagcagcctgacttctgaggacacggccgtgtattactgtgcgacaggaaatgcggattgtagtggtggtagctgctacaattggttcgacccctggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga 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:
tcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagactcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacaccagtggttaccatctggtgttcggcggagggaccaagctgaccgtcctagtaaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggcaccgcctccgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggttacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgctag 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。
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。
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。
CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC
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。
进一步地,所述多聚腺苷酸序列包含50-400 个腺苷酸的序列。
进一步地,所述多聚腺苷酸序列包含 50-300个腺苷酸的序列。
进一步地,所述多聚腺苷酸序列包含50-250个腺苷酸的序列。
进一步地,所述多聚腺苷酸序列包含80-120个腺苷酸的序列。
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。
进一步地,酶切位点序列选自CTCGAG对应的RNA序列。
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。
进一步地,所述递送载体为纳米颗粒。
进一步地,所述递送载体为脂质纳米颗粒。
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。
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。
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。
在本发明的另一些实施方案中,所述固醇为胆固醇。
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。
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。
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。
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。
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。
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:
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;
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;
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;
S4) 以酶切质粒获得线性化质粒;
S5) 将线性化质粒转录为RNA;
S6) 将步骤S5)所得RNA进行加帽获得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。
本发明的有益效果为:
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。
图1为质粒凝胶电泳照片。
图2为转录的mRNA凝胶电泳照片。
图3为ELISA验证LNP-mRNA在细胞中的抗体表达。
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。
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;
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。
根据不同mRNA的需求,可在mRNA 5’端灵活添加不同的5’帽结构。
“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。
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。
实施例1制备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。
H7N9抗体重链基因如SEQ ID NO.10所示
caagtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacatattcaccagttatgatatcaactgggtgcgacaggccactggccaagggcttgagtggatgggatggatgaaccctgacagtggtgacacaggctttgcacagaagttccagggcagagtcaccatgaccaggaacacctccataaccacagcctacatggagctgagcagcctgacttctgaggacacggccgtgtattactgtgcgacaggaaatgcggattgtagtggtggtagctgctacaattggttcgacccctggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
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。
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:
QVQLVESGAEVKKPGASVKVSCKASGYIFTSYDINWVRQATGQGLEWMGWMNPDSGDTGFAQKFQGRVTMTRNTSITTAYMELSSLTSEDTAVYYCATGNADCSGGSCYNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8。
H7N9抗体轻链基因如SEQ ID NO.11所示:
tcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagactcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacaccagtggttaccatctggtgttcggcggagggaccaagctgaccgtcctagtaaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggcaccgcctccgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggttacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgctag
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。
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:
SSELTQDPAVSVALGQTVRITCQGDRLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDTSGYHLVFGGGTKLTVLVTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
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:ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCcaagtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacatattcaccagttatgatatcaactgggtgcgacaggccactggccaagggcttgagtggatgggatggatgaaccctgacagtggtgacacaggctttgcacagaagttccagggcagagtcaccatgaccaggaacacctccataaccacagcctacatggagctgagcagcctgacttctgaggacacggccgtgtattactgtgcgacaggaaatgcggattgtagtggtggtagctgctacaattggttcgacccctggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO.14;
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCtcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagactcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacaccagtggttaccatctggtgttcggcggagggaccaagctgaccgtcctagtaaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggcaccgcctccgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggttacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgctagTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
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5.7mg质粒,测序正确;
(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358.1ng/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5101 ng/μL RNA,一共7.22 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721ng/mL,产量2.095 mg。
(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。
实施例2制备LNP包封的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0.11,纳米颗粒粒径为92.84 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。
结果如图3所示:转染LNP-mRNA的细胞表达目的抗体在上清中。
实施例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8只小鼠,分别尾静脉接种PBS、LNP-mRNA(1.4 mg/kg)和抗体(20mg/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
结果显示:PBS组动物全部死亡,LNP-mRNA组的小鼠存活率87.5%,抗体治疗组的小鼠存活率为75%。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。
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,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:重链CDR1区:GYIFTSYD SEQ ID NO.1;重链CDR2区:MNPDSGDT SEQ ID NO.2;重链CDR3区:ATGNADCSGGSCYNWFDP SEQ ID NO.3;轻链CDR1区:RLRSYY SEQ ID NO.4;轻链CDR2区:GKN ;轻链CDR3区:NSRDTSGYHLV 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的mRNA还分别包含:1)5’帽结构;2)5’UTR序列;3)编码信号肽的序列4) 终止密码子和酶切位点序列5)3’UTR序列;以及6)多聚腺苷酸序列,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;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;优选地,所述多聚腺苷酸序列包含25-400个腺苷酸的序列;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。
- 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: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/或所述H7N9的单克隆抗体的轻链氨基酸序列如SEQ ID NO.9所示。
- 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;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。
- 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。
- 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。
- 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;优选地,所述递送载体为纳米颗粒;优选地,所述递送载体为脂质纳米颗粒。
- 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。
- 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。
- 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。
- 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: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;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;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;S4) 以酶切质粒获得线性化质粒;S5) 将线性化质粒转录为RNA;S6) 将步骤S5)所得RNA进行加帽获得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。
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