CN114317609A - 病毒载体及其应用 - Google Patents
病毒载体及其应用 Download PDFInfo
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- CN114317609A CN114317609A CN202111154043.1A CN202111154043A CN114317609A CN 114317609 A CN114317609 A CN 114317609A CN 202111154043 A CN202111154043 A CN 202111154043A CN 114317609 A CN114317609 A CN 114317609A
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明提出了一组病毒载体。该组病毒载体包括:第一病毒载体,所述第一病毒载体携带第一核酸分子,所述第一核酸分子编码包膜蛋白;第二病毒载体,所述第二病毒载体携带第二核酸分子,所述第二核酸分子编码融合蛋白,所述融合蛋白包括单链抗体和所述包膜蛋白的C端结构域,所述包膜蛋白的C端结构域包括包膜蛋白的跨膜区和胞内区,所述单链抗体的C端与所述包膜蛋白的C端结构域的N端相连,所述单链抗体靶向特异性抗原;所述第一核酸分子与所述第二核酸分子被设置为表达所述包膜蛋白与所述融合蛋白,并且所述包膜蛋白与所述融合蛋白呈非融合形式。该组病毒载体导入受体细胞后,可包装出高病毒滴度的病毒,且该病毒具有靶向感染性。
Description
技术领域
本发明涉及生物技术领域,具体地,本发明涉及病毒载体及其应用,更具体地,本发明涉及病毒载体、慢病毒、药物组合物及将目的基因导入受体细胞的方法。
背景技术
目前,慢病毒作为一种基因递送载体,被广泛的应用于各种疾病的基因治疗,且主要集中在ex vivo领域,如CAR-T制备、造血干细胞基因修饰等。慢病毒载体在in vivo领域的应用无法广泛开展,其主要原因之一是慢病毒转染缺乏靶向性。
科研工作者们一直都在致力于实现慢病毒的靶向转染。从已发表的工作来看,决定慢病毒转染宿主细胞类型的是包膜蛋白,科研工作者通过改变包膜蛋白的类型或结构,使慢病毒获得了对某类细胞的转染倾向性,但均存在一些缺陷。为使慢病毒获得转染倾向性或靶向性,科研工作者们主要在以下三方面进行改进:一是,将包膜蛋白替换为其他具有转染组织特异性的病毒的包膜蛋白,使慢病毒获得新的转染特性;但此类功能背景清晰、安全性有保障的包膜蛋白数量有限,且获得的重组慢病毒的滴度和稳定性下降。二是,将特异性结合某抗原的受体或抗体的scFv结构域融合表达至包膜蛋白的N端,或者以单独的膜蛋白形式组装到病毒载体上,使慢病毒可通过受体或scFv结合抗原,对表达该抗原的细胞具有转染倾向性,但是这种方式大大降低了慢病毒的生产滴度(降低约100倍)。三是,在包装病毒的同时共表达CD47蛋白,得到一种膜表面高表达CD47的慢病毒(CD47hi LV);CD47可同巨噬细胞表面受体相互作用,抑制巨噬细胞吞噬慢病毒,从而使CD47hi LV对肝细胞基因转移效率更高,急性炎症反应的激活减少;但是该方法是利用机体对病毒的代谢能力,被动增加了CD47hi LV对肝细胞基因转移效率,却也无法做到主动靶向感染肝细胞。
鉴于目前的技术仍无法在不显著降低慢病毒滴度的同时,解决慢病毒感染缺乏靶向性的问题,因此,需要开发新的技术来解决该问题。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本发明提出一种具有靶向感染力且病毒滴度得到显著提高的病毒载体。
在本发明的第一方面,本发明提出了一组病毒载体。根据本发明的实施例,所述病毒载体包括:第一病毒载体,所述第一病毒载体携带第一核酸分子,所述第一核酸分子编码包膜蛋白;至少一个第二病毒载体,所述第二病毒载体携带第二核酸分子,所述第二核酸分子编码至少一个融合蛋白,所述融合蛋白包括至少一个单链抗体和所述包膜蛋白的C端结构域,所述包膜蛋白的C端结构域包括包膜蛋白的跨膜区和胞内区,所述至少一个单链抗体的C端与所述包膜蛋白的C端结构域的N端相连,所述单链抗体靶向特异性抗原;所述第一核酸分子与所述第二核酸分子被设置为表达所述包膜蛋白与所述融合蛋白,并且所述包膜蛋白与所述融合蛋白呈非融合形式。根据本发明实施例的病毒载体导入受体细胞后,可包装出高病毒滴度的病毒,且该病毒具有靶向感染性。
根据本发明的实施例,上述病毒载体还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述病毒载体为反转录病毒、慢病毒和其他包膜病毒载体。
根据本发明的实施例,所述包膜病毒包括选自玻那病毒科(Bornaviridae)、尼亚马病毒科(Nyamaviridae)、沙粒病毒科(Arenaviridae)、丝状病毒科(Filoviridae)、汉坦病毒科(Hantaviridae)、内罗病毒科(Nairoviridae)、正粘病毒科(Orthomyxoviridae)、副黏病毒科(Paramyxoviridae)、布尼亚病毒科(Bunyaviridae)、白纤病毒科(Phenuiviridae)、弹状病毒科(Rhabdoviridae)、动脉炎病毒科(Arteriviridae)、冠状病毒科(Coronaviridae)、黄病毒科(Flaviviridae)、披膜病毒科(Togaviridae)、嗜肝DNA病毒科(Hepadnaviridae)、泡沫病毒(Spumavirus)、虹彩病毒科(Iridoviridae)、疱疹病毒科(Herpesviridae)、痘病毒科(Poxviridae)、丁型肝炎病毒科(Deltavirus)病毒的至少之一。
根据本发明的实施例,所述包膜蛋白为弹状病毒科水泡性口炎病毒的包膜G糖蛋白(VSV-G)或包膜G糖蛋白的突变体。水泡性口炎病毒的包膜G糖蛋白具有细胞膜吸附和融合能力,因此,根据本发明实施例的病毒载体所包装获得病毒具有细胞吸附和感染能力。
根据本发明的实施例,所述包膜G糖蛋白的突变体具有K47Q和R354Q突变。具有K47Q和R354Q突变的包膜G糖蛋白突变体的细胞膜吸附能力减弱,但不影响其膜融合能力,因此,具有K47Q和R354Q突变的包膜G糖蛋白的突变体使得包装获得的病毒的非特异性吸附细胞的能力减弱,但不影响病毒感染细胞的能力。
根据本发明的实施例,所述包膜G糖蛋白的突变体具有SEQ ID NO:1所示的氨基酸序列。
KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEQELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:1)。
根据本发明的实施例,所述单链抗体靶向细胞特异性抗原。根据本发明实施例的单链抗体可选择设置为靶向任何特异性抗原,例如,靶向肿瘤细胞特异性抗原CD19或Siglec15,进而包装所获得的病毒在单链抗体与细胞特异性抗原靶向结合的介导下,实现了病毒对细胞(如:肿瘤细胞、免疫细胞)的特异性靶向结合,进而实现病毒对细胞的特异性感染。根据本发明的实施例,所述融合蛋白进一步包括第一连接肽。进而将单链抗体区与包膜蛋白C端区隔开,减少二者功能干扰。
根据本发明的实施例,所述第一连接肽具有SEQ ID NO:2所示的氨基酸序列。
AAATTT(SEQ ID NO:2)。
根据本发明的实施例,所述包膜蛋白的C端结构域具有SEQ ID NO:3所示的氨基酸序列。
FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI KLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:3)。
根据本发明的实施例,所述融合蛋白具有SEQ ID NO:4或SEQ ID NO:10所示的氨基酸序列。
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:4)。
DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVIQRPGQGLEWIGDIYCGSDTMHYNEKFKNKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARWWDYGSSYDYFDYWGQGTTLTVSSAAATTTFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:10)。
根据本发明的实施例,所述病毒载体进一步包括:第一启动子,所述第一启动子与所述第一核酸分子可操作地连接;以及第二启动子,所述第二启动子与所述第二核酸分子可操作地连接。进而第一核酸分子和第二核酸分子分别在第一启动子和第二启动子的调控下,实现对第一核酸分子和第二核酸分子的高效表达。
根据本发明的实施例,所述第一启动子和所述第二启动子分别独立地选自CMV、EF-1或RSV启动子。
根据本发明的实施例,所述第一核酸分子具有SEQ ID NO:5所示的核苷酸序列。
ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCCAAAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAACAGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAG(SEQ ID NO:5)。
根据本发明的实施例,所述第二核酸分子具有SEQ ID NO:6所示的核苷酸序列。
ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCGACATTCAGATGACTCAGACCACCTCCTCCCTGTCCGCCTCCCTGGGCGACCGCGTGACCATCTCATGCCGCGCCAGCCAGGACATCTCGAAGTACCTCAACTGGTACCAGCAGAAGCCCGACGGAACCGTGAAGCTCCTGATCTACCACACCTCCCGGCTGCACAGCGGAGTGCCGTCTAGATTCTCGGGTTCGGGGTCGGGAACTGACTACTCCCTTACTATTTCCAACCTGGAGCAGGAGGATATTGCCACCTACTTCTGCCAACAAGGAAACACCCTGCCGTACACTTTTGGCGGGGGAACCAAGCTGGAAATCACTGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGAAGTCAAGCTGCAGGAATCAGGACCTGGCCTGGTGGCCCCGAGCCAGTCACTGTCCGTGACTTGTACTGTGTCCGGAGTGTCGCTCCCGGATTACGGAGTGTCCTGGATCAGGCAGCCACCTCGGAAAGGATTGGAATGGCTCGGAGTCATCTGGGGTTCCGAAACCACCTATTACAACTCGGCACTGAAATCCAGGCTCACCATTATCAAGGATAACTCCAAGTCACAAGTGTTCCTGAAGATGAATAGCCTGCAGACTGACGACACGGCGATCTACTATTGCGCCAAGCACTACTACTACGGCGGATCCTACGCTATGGACTACTGGGGCCAGGGGACCAGCGTGACCGTGTCATCCGCGGCCGCAACTACCACCTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAG(SEQ ID NO:6)。
根据本发明的实施例,所述第一病毒载体和第二病毒载体为同一载体。
根据本发明的实施例,所述第一病毒载体和第二病毒载体为同一载体,所述载体进一步包括:内部核糖体进入位点序列IRES,所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间。内部核糖体进入位点前后的两个蛋白的表达通常是成比例的。内部核糖体进入位点序列的引入使得第一核酸分子和第二核酸分子分别独立的翻译表达,得到的包膜蛋白和融合蛋白呈非融合形式。内部核糖体进入位点序列的引入有效保证了包膜蛋白和融合蛋白的生物学作用,使得包装所获得的病毒的特异性结合吸附和感染能力显著,且病毒的滴度高。
根据本发明的实施例,所述第一病毒载体和第二病毒载体为同一载体,所述载体进一步包括:第三核酸分子,设置在所述第一核酸分子与所述第二核酸分子之间,并且所述第三核酸分子编码第二连接肽,所述第二连接肽能够被切割。第三核酸分子的引入使得包膜蛋白和融合蛋白成非融合状态的表达,从而保证了包膜蛋白和融合蛋白的生物学作用,使得包装所获得的病毒的特异性结合吸附和感染能力显著,且病毒的滴度高。
根据本发明的实施例,所述第一病毒载体和第二病毒载体为同一载体,所述第一核酸分子和第二核酸分子的拷贝数之比为1:1~4:1。需要说明的是,本申请所述的“第一核酸分子和第二核酸分子的拷贝数之比”是指当第一病毒载体和第二病毒载体为同一载体,即第一核酸分子和第二核酸分子在同一载体时,第一核酸分子和第二核酸分子在载体上的携带数量比,以尽可能保证第一核酸分子和第二核酸分子的蛋白表达量之比约为同比。发明人发现,当载体上携带所述第一核酸分子和第二核酸分子的数量比为1:1~4:1时,病毒滴度和病毒的感染效率均较高。
根据本发明的实施例,所述第一核酸分子和第二核酸分子的拷贝数之比为2:1~4:1。根据本发明的具体实施例,当载体上携带所述第一核酸分子和第二核酸分子的数量比为2:1~4:1时,病毒滴度和病毒的感染效率得到进一步提高。
根据本发明的实施例,所述第一核酸分子和第二核酸分子的拷贝数之比为2:1。发明人发现,当载体上携带所述第一核酸分子和第二核酸分子的数量比为2:1时,病毒滴度和病毒的感染效率会达到一个最佳的平衡。
根据本发明的实施例,所述第一病毒载体和第二病毒载体为pMD2.G、pCMV、pMD2.G突变体或pCMV的突变体。根据本发明实施例的第一病毒载体和第二病毒载体的种类不受特别限制,可以表达VSV-G的载体或可表达VSV-G或VSV-G突变体的载体的突变体均可使用。
根据本发明的实施例,进一步包括第三病毒载体和第四病毒载体,所述第三病毒载体携带目的基因,所述第四病毒载体携带病毒结构蛋白基因和病毒包装酶基因以及任选的调节因子rev基因。
根据本发明的实施例,所述结构蛋白基因、病毒包装酶基因和调节因子rev基因设置于同一第四病毒载体或不同第四病毒载体上。例如,慢病毒载体psPAX2表达产物有结构蛋白gag、包装酶pol(包括逆转录酶、蛋白酶和整合酶)、调节因子rev,其中rev可一定程度提高产物滴度,但对慢病毒包装是非必需的。可将rev(pRSV-rev)和gag-pol(pMDLg-pRRE)分成两个质粒进行表达;或将rev(pRSV-rev),gag(pCMV-gag)、pol(pCMV-gag)分为三质粒进行表达。
根据本发明的实施例,所述病毒包装酶包括逆转录酶、蛋白酶和整合酶的至少之一。
根据本发明的实施例,所述第三病毒载体为转移载体。
根据本发明的实施例,所述转移载体包含慢病毒包装信号。
根据本发明的实施例,所述慢病毒包装信号包括:Ψ。
根据本发明的实施例,所述转移载体为pLV载体。
根据本发明的实施例,所述第四病毒载体为psPAX2。
根据本发明的实施例,所述病毒载体是非致病性病毒。
在本发明的第二方面,本发明提出了一种获得慢病毒的方法。根据本发明的实施例,所述方法包括将前面所述的病毒载体导入第一受体细胞;将导入病毒载体的第一受体细胞进行培养,以便获得所述病毒。根据本发明实施例的方法所获得的慢病毒滴度高,且具有靶向结合和感染细胞的能力显著提高。
根据本发明的实施例,上述方法还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述病毒为慢病毒,所述第一病毒载体和第二病毒载体不为同一载体,所述第三病毒载体、第四病毒载体、第一病毒载体和第二病毒载体的质量比为2:1:1:0.25~2:1:1:1。根据本发明实施例所述的病毒载体的比例,慢病毒滴度和慢病毒感染效率均较高。
根据本发明的实施例,所述第三病毒载体、第四病毒载体、第一病毒载体和第二病毒载体的质量比为2:1:1:0.5。发明人发现,当所述第三病毒载体、第四病毒载体、第一病毒载体和第二病毒载体的质量比为2:1:1:0.5时,慢病毒滴度和慢病毒感染效率会达到一个最佳的平衡。
根据本发明的实施例,所述第一受体细胞为293T。
在本发明的第三方面,本发明提出了一种慢病毒。根据本发明的实施例,所述慢病毒是通过前面所述的方法包装获得的。根据本发明实施例的慢病毒滴度高,且具有靶向结合和感染细胞的能力。
在本发明的第四方面,本发明提出了一种慢病毒。根据本发明的实施例,所述慢病毒表达包膜蛋白以及融合蛋白,所述融合蛋白包括单链抗体和所述包膜蛋白的C端结构域,所述包膜蛋白的C端结构域包括包膜蛋白的跨膜区和胞内区,所述单链抗体的C端与所述包膜蛋白的C端结构域的N端相连。根据本发明实施例的慢病毒滴度高,且具有靶向结合和感染细胞的能力。
根据本发明的实施例,所述包膜蛋白为水泡性口炎病毒的包膜G糖蛋白或包膜G糖蛋白的突变体。
在本发明的第五方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物包括前面所述的病毒载体或前面所述的慢病毒,所述病毒载体或慢病毒携带目的基因。需要说明的是,本申请所述的“目的基因”是指病毒载体或慢病毒需要携带的外源基因,该外源基因随着病毒感染细胞后,进入靶向细胞内,在靶向细胞内实现目的基因的表达。当所述目的基因在靶向细胞的表达能够实现直接或间接的治疗作用时,所述病毒载体或慢病毒所形成的药物组合物实现了将目的基因靶向带入靶向细胞的作用,实现了对疾病的靶向治疗。
在本发明的第六方面,本发明提出了一种表达目的基因的方法。根据本发明的实施例,所述方法包括:将整合有目的基因的病毒载体或慢病毒导入第二受体细胞;将导入病毒载体或慢病毒的第二受体细胞进行培养,以便表达目的基因。根据本发明实施例的方法,有效实现了目的基因在受体细胞的表达。例如在本发明的实施例中,mCherry或BFP作为一种可携带的目的基因,为了验证根据本发明实施例的靶向载体平台的可行性,发明人采用mcherry基因或BFP基因作为一种标签基因,以表达荧光蛋白,进而在受体细胞中表征病毒感染阳性率。
根据本发明的实施例,上述方法还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述导入第二受体细胞是通过电转、转染或感染的方式进行的。需要说明的是,所述“电转”或“转染”是将病毒载体导入受体细胞的方法,所述“感染”是指病毒主动结合和融合细胞膜进而进入细胞的过程。其中,“电转”是指通过电刺激的方式,将病毒包装用载体导入受体细胞的方法,所述“转染”是指通过化学介导物,如脂质体,将病毒包装用载体导入受体细胞的方法。
根据本发明的实施例,所述第二受体细胞为体细胞。
根据本发明的实施例,所述第二受体细胞为肿瘤细胞。当所述目的基因在肿瘤细胞的表达能够实现直接或间接的治疗作用时,根据本发明实施例的方法实现了肿瘤细胞的特异性杀伤,实现了对肿瘤的特异性治疗。
根据本发明的实施例,所述“pMD2.G突变体”或“pMD2.G-Mut”均是指包含K47Q\R354Q突变位点的表达慢病毒包膜蛋白的质粒(VSV-G-K47Q\R354Q)。
附图说明
图1是根据本发明实施例的靶向CD19抗原的包膜融合蛋白(antiCD19-G)结构模式图,其中,Anti-CD19 scFv表示CD19单链抗体的可变区序列,VSV-G C-terminal表示慢病毒包膜蛋白的C端结构域;
图2是根据本发明实施例的pMD2.G突变体(pMD2.G-Mut)的图谱;
图3是根据本发明实施例的不同比例的pMD2.G-Mut和pMD2.antiCD19-G包装获得的靶向CD19的慢病毒的滴度结果图,其中,横坐标表示慢病毒的类别,每种慢病毒包含载体种类及比例如表1所示,纵坐标表示慢病毒感染HEK-293T-CD19细胞所测得的慢病毒滴度;
图4是根据本发明实施例的包装获得的靶向CD19慢病毒的滴度结果图,其中,横坐标表示慢病毒的类别,每种慢病毒包含载体种类及比例如表2所示,纵坐标表示慢病毒感染HEK-293T细胞所测得的慢病毒滴度;
图5是根据本发明实施例的包装获得的靶向CD19慢病毒感染293T的流式分析图,其中CD19ta是能够与CD19结合的scFv;
图6是根据本发明实施例的包装获得的靶向CD19慢病毒靶向感染293T的能力分析图,其中,横坐标表示慢病毒的类别,每种慢病毒包含载体种类及比例如表2所示,纵坐标表示慢病毒感染HEK-293T-CD19与HEK-293T细胞混合体系后,HEK-293T-CD19细胞与HEK-293T细胞的mCherry阳性率的比值,CD19ta是能够与CD19结合的scFv;
图7是根据本发明实施例的包装获得的靶向Siglec15慢病毒的靶向感染293T细胞的能力分析图,其中,横坐标表示慢病毒的类别,每种慢病毒包含载体种类及比例如表3所示,纵坐标表示慢病毒感染HEK-293T-HS15与HEK-293T细胞混合体系后,HEK-293T-HS15细胞与HEK-293T细胞的BFP阳性率的比值,HEK-293T-HS15表示稳定表达人源Siglec15蛋白的HEK 293T细胞,5G12表示能够与Siglec15结合的scFv。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。
根据本发明的具体实施例,本发明提供了一组新型具有靶向转染力的慢病毒载体,该组载体,一个携带包膜蛋白VSV-G或VSV-G突变体的编码区,以及一个携带由单链抗体scFv和VSV-G的C端结构域通过连接肽相连得到的融合蛋白的编码区。
根据本发明实施例的慢病毒载体具有如下特点:①VSV-G突变体为减弱VSV-G吸附靶细胞能力的突变体,但是保留了细胞膜融合能力;②scFv可为一个或多个串联;③VSV-G的C端结构域中至少包括VSV-G的胞内区和跨膜区;④一个病毒载体上可含有一种或多种融合蛋白。
此研究的优势:①scFv与VSV-G的C端结构域形成的融合蛋白,具有同VSV-G相同的跨膜区和胞内区,使得该融合蛋白仍维持同基质蛋白的相互作用,使高效的组装到慢病毒颗粒包膜上,并且不干扰病毒颗粒的出芽,从而不对病毒滴度造成显著影响。②慢病毒颗粒包膜上仍含有完整的VSV-G或其突变体,可以维持病毒颗粒的稳定性。③通过scFv同相应抗原的结合,使得慢病毒颗粒主动靶向感染表达相应抗原的靶细胞,并且在相同感染复数条件下提高对靶细胞的感染效率。④scFv功能背景清晰、安全性有保障,而且任何抗原都可筛选到与之特异性结合的scFv,使得此类慢病毒载体所包装的病毒可普遍适用于各类细胞的靶向感染。⑤VSV-G替换为吸附靶细胞能力减弱的突变体,使得scFv同相应抗原之间的特异性结合力在病毒颗粒吸附过程中占据主导地位,进一步提高重组慢病毒的靶向性。
根据本发明的具体实施例,本发明还提供了一类具有靶向转染基因能力的新型慢病毒载体的构建和使用方法,包括以下步骤(以靶向转染CD19+细胞的慢病毒载体为例):
1.设计靶向CD19的单链抗体和VSV-G C端结构域形成的融合蛋白(CD19 scFv-VSV-G-CT(简称antiCD19-G)),结构模式如图1所示。其中,VSV-G信号肽位于VSV-G前体蛋白的氨基末端,是VSV-G蛋白的膜定位端肽,在帮助VSV-G蛋白定位到内质网上,蛋白成熟后就水解脱离,病毒颗粒上的VSV-G蛋白并不含有该信号肽。
信号肽的代表氨基酸序列如SEQ ID NO:7所示。
MKCLLYLAFLFIGVNC(SEQ ID NO:7)。
anti-CD19 scFv的氨基酸序列如SEQ ID NO:8所示。
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:8)。
第一连接肽(Linker)的氨基酸序列如SEQ ID NO:2所示。
AAATTT(SEQ ID NO:2)。
VSV-G C-Terminal包含VSV-G蛋白的第405-495位(共91个)氨基酸,其氨基酸序列如SEQ ID NO:3所示。
FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI KLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:3)。
2.构建CG融合膜蛋白表达质粒
以VSV-G信号肽-scFv-Linker-(VSV-G-CT)模式设计DNA序列委托基因合成公司,并以pMD2.G质粒(VSV-G蛋白表达质粒)为载体构建融合蛋白表达质粒pMD2.antiCD19-G。
3.构建VSV-G弱吸附力突变体(参考文献Structural basis for therecognition of LDL-receptor family members by VSV glycoprotein.NatureCommunications.2018)
设计K47Q和R354Q双点突变体的DNA序列委托基因合成公司,以VSV-G表达质粒pMD2.G为载体,构建VSV-G-K47Q\R354Q表达质粒pMD2.G-Mut,研究发现,K47Q和R354Q突变只会降低VSV-G的膜吸附能力,不会影响VSV-G的膜融合能力。
4.慢病毒包装、收获
①将慢病毒载体包装质粒共转染293T细胞。
②转染48-72h后收集含病毒的培养上清;用0.45μm滤器过滤,分装后,冻存于超低温冰箱内。
5.慢病毒载体靶向转染能力检测和分析
①用慢病毒载体对CD19+和CD19-混合培养的细胞进行感染,对比两类细胞感染阳性率。
②相同感染条件下,感染CD19+细胞阳性率/感染CD19-细胞的阳性率,可用于评估慢病毒靶向感染CD19+细胞的能力(简称靶向感染力)。当靶向感染力≤1时,该慢病毒无靶向感染CD19+细胞的能力;当靶向转染力>1时,该慢病毒有靶向感染CD19+细胞的能力,且数值越大,靶向感染CD19+细胞的能力越强。
下面将更详细地描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
需要说明的是,以下实施中所述的“质粒”与“载体”具有相同的意义,可互换使用。
实施例1
1.1包装一系列不同antiCD19-G含量的靶向感染CD19+细胞的慢病毒
1.1.1慢病毒包装信息
按表1中所示质粒及质量比(维持每组间pLV-mCherry和psPAX2质粒质量一致)共转染293T细胞,包装慢病毒。
表1:
慢病毒 | 慢病毒载体名称及比例 |
LVm-CD19ta(1:0.25) | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:0.25 |
LVm-CD19ta(1:0.5) | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:0.5 |
LVm-CD19ta(1:1) | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:1 |
LVm-CD19ta(1:2) | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:2 |
LVm-CD19ta(1:4) | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:4 |
LVm-CD19ta(1:8) | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:8 |
其中,pLV-mCherry为携带mCherry序列的转移质粒,携带目的基因为mCherry序列,psPAX2为表达慢病毒结构蛋白gag和pol(逆转录酶、蛋白酶、整合酶,包装到病毒颗粒内,非结构蛋白)的质粒,pMD2.G-Mut为表达慢病毒膜蛋白突变体(VSV-G-K47Q\R354Q)的质粒,pMD2.antiCD19-G为表达包含靶向CD19的scFv与VSV-G蛋白C端结构域的嵌合蛋白的质粒,其中,本实施例所用pMD2.G突变体的图谱如图2所示。
1.1.2慢病毒载体产量评价
1)评价细胞系构建:
构建稳定共表达CD19和GFP的293T细胞系(HEK-293T-CD19),表达GFP的目的是用GFP阳性指示CD19阳性细胞。
2)慢病毒包装、收获及产量评价
按表1设置的实验组,分别共转染293T细胞48-72h,然后收集各组病毒液,将各组病毒液经0.45μm无菌过滤器过滤。将200μL HEK-293T-CD19细胞铺入24孔板孔内。将过滤后的慢病毒液进行100倍稀释,按200μL/孔加入对应细胞孔内,并轻轻充分混匀,于37℃,5%CO2条件下静置培养3-4天。采用流式细胞术检测被感染细胞中mCherry+细胞百分比。
3)结果及分析
表达VSV-G-K47Q\R354Q蛋白和antiCD19-G融合蛋白的质粒质量比为1:0.25~1:1(即4:1~1:1)时,病毒滴度较高,其中以1:0.5(即2:1)最高(结果如图3所示)。病毒产量=病毒滴度×病毒液体积,在收获病毒液体积相同的情况下,病毒滴度高低即代表病毒产量高低,即表达VSV-G-K47Q\R354Q蛋白和antiCD19-G融合蛋白的质粒质量比为1:0.25~1.1(即4:1~1:1)时,病毒产量较高,其中以1:0.5最高。
1.2包装一系列靶向感染CD19+细胞的慢病毒
1.2.1慢病毒包装信息
按下表2中的质粒及比例(维持每组间pLV-mCherry和psPAX2质粒量一致),共转染293T细胞,包装慢病毒。
表2:
慢病毒 | 慢病毒载体名称及比例 |
Negative Control(NC) | pLV-mCherry:psPAX2=2:1 |
LV(Lentivirus) | pLV-mCherry:psPAX2:pMD2.G=2:1:1 |
LV-CD19ta | pLV-mCherry:psPAX2:pMD2.G:pMD2.antiCD19-G=2:1:1:0.5 |
LVm | pLV-mCherry:psPAX2:pMD2.G-Mut=2:1:1 |
LVm-CD19ta | pLV-mCherry:psPAX2:pMD2.G-Mut:pMD2.antiCD19-G=2:1:1:0.5 |
其中,pLV-mCherry为携带mCherry序列的转移质粒,携带目的基因为mCherry序列,psPAX2为表达慢病毒结构蛋白gag和pol(逆转录酶、蛋白酶、整合酶,包装到病毒颗粒内,非结构蛋白)的质粒,pMD2.G为表达慢病毒包膜蛋白的质粒(表达VSV-G),pMD2.G-Mut为表达慢病毒膜蛋白突变体(VSV-G-K47Q\R354Q)的质粒,pMD2.antiCD19-G为表达包含靶向CD19的scFv与VSV-G蛋白C端结构域的嵌合蛋白的质粒,其中,本实施例所用pMD2.G突变体的图谱如图2所示。
1.2.2靶向CD19+细胞的慢病毒载体靶向性评价
1)评价细胞系构建:
构建稳定共表达CD19和GFP的293T细胞系(HEK-293T-CD19),表达GFP的目的是用GFP阳性指示CD19阳性细胞。
2)慢病毒包装、收获、滴度测定及靶细胞转染
按表2设置实验组,分别共转染293T 48-72h后收集各组病毒液,分装冻存于超低温冰箱(<-75℃)内。用293T细胞进行滴度测定。
将293T同HEK-293T-CD19细胞按1:1比例混合铺入24孔板孔内。将冻存的慢病毒液进行100倍稀释,按200μL/孔加入对应细胞孔内,并轻轻充分混匀。(两种细胞混合转染的目的有两个:a.保证两种细胞的转染条件完全一致;b.模拟in vivo基因治疗中,靶细胞和非靶细胞共存时转染条件。)
将24孔板置于37℃,5%CO2条件下静置培养3-4天。流式细胞术检测被感染细胞中mCherry+细胞百分比和GFP阳性(GFP+)细胞百分比。
3)结果及分析
表达antiCD19-G融合蛋白不会引起慢病毒生产滴度的显著下降(结果如图4所示),病毒滴度可达107以上,显著优于现有技术靶向性病毒的生产滴度,且可增强慢病毒对CD19+细胞的感染能力(结果如图5和图6所示)。
突变体包膜蛋白(VSV-G-K47Q\R354Q)的表达降低了慢病毒的感染能力(结果可参考图5D和图5B),antiCD19-G融合蛋白的表达特异性增强了慢病毒对CD19+细胞的感染能力(结果如图5E所示),使得LVm-CD19ta比LV-CD19ta对CD19+细胞有更强的靶向感染能力(结果如图6所示)。
实施例2:
2.1包装一系列靶向感染人Siglec15+细胞的慢病毒
按下表3中的质粒及比例(维持每组间pLV-BFP和psPAX2质粒量一致),共转染HEK293T细胞,包装慢病毒。
表3:
慢病毒 | 慢病毒载体名称及比例 |
LV-BFP | pLV-BFP:psPAX2:pMD2.G=2:1:1 |
LV-BFP-5G12 | pLV-BFP:psPAX2:pMD2.G:pMD2.antiSiglec15-G=2:1:1:0.5 |
LVm-BFP | pLV-BFP:psPAX2:pMD2.G-Mut=2:1:1 |
LVm-BFP-5G12 | pLV-BFP:psPAX2:pMD2.G-Mut:pMD2.antiSiglec15-G=2:1:1:0.5 |
其中,pLV-BFP为携带BFP(Blue Fluorescent Protein,蓝色荧光蛋白)序列的转移质粒,携带目的基因为BFP序列,psPAX2为表达慢病毒结构蛋白gag和pol(逆转录酶、蛋白酶、整合酶,包装到病毒颗粒内,非结构蛋白)的质粒,pMD2.G为表达慢病毒包膜蛋白(VSV-G)的质粒,pMD2.G-Mut为表达慢病毒膜蛋白突变体(VSV-G-K47Q\R354Q)的质粒,pMD2.antiSiglec15-G为表达包含靶向Siglec15的scFv与VSV-G蛋白C端结构域的嵌合蛋白的质粒,其中,本实施例所用pMD2.G突变体的图谱如图2所示。
anti-Siglec15 scFv的氨基酸序列如SEQ ID NO:9所示。
DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVIQRPGQGLEWIGDIYCGSDTMHYNEKFKNKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARWWDYGSSYDYFDYWGQGTTLTVSS(SEQ ID NO:9)。
2.2靶向Siglec15+细胞的慢病毒载体靶向性评价
1)评价细胞系构建:
构建稳定共表达Siglec15和BFP的293T细胞系(293T-Siglec15+)。
2)慢病毒包装、收获、滴度测定及靶细胞转染
按表2设置实验组,分别共转染293T 48-72h后收集各组病毒液,分装冻存于超低温冰箱(<-75℃)内。用293T细胞进行滴度测定。
将293T细胞同293T-Siglec15+细胞按1:1比例混合铺入24孔板孔内。将冻存的慢病毒液进行100倍稀释,按200μL/孔加入对应细胞孔内,并轻轻充分混匀。
将混合转染的两种细胞于37℃,5%CO2条件下静置培养3-4天。流式细胞术检测被感染细胞中BFP+细胞百分比和Siglec15阳性(Siglec15+)细胞百分比。
4)结果及分析
具体实验结果如图7所示,antiSiglec15-G融合蛋白的表达特异性增强了慢病毒对Siglec15+细胞的感染能力,使得LV-BFP-5G12比LV-BFP有更强的靶向能力,LVm-BFP-5G12比LVm-BFP有更强的靶向能力。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 广东东阳光药业有限公司
<120> 病毒载体及其应用
<130> SI4210293
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atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60
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gacatcacct tcttctcaga ggacggagag ctatcatccc tgggaaagga gggcacaggg 660
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ttcgaacatc ctcacattca agacgctgct tcgcaacttc ctgatgatga gagtttattt 1320
tttggtgata ctgggctatc caaaaatcca atcgagcttg tagaaggttg gttcagtagt 1380
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aagctcctga tctaccacac ctcccggctg cacagcggag tgccgtctag attctcgggt 240
tcggggtcgg gaactgacta ctcccttact atttccaacc tggagcagga ggatattgcc 300
acctacttct gccaacaagg aaacaccctg ccgtacactt ttggcggggg aaccaagctg 360
gaaatcactg ggggcggagg atccggtgga ggcggaagcg ggggtggagg atccgaagtc 420
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aaaggattgg aatggctcgg agtcatctgg ggttccgaaa ccacctatta caactcggca 600
ctgaaatcca ggctcaccat tatcaaggat aactccaagt cacaagtgtt cctgaagatg 660
aatagcctgc agactgacga cacggcgatc tactattgcg ccaagcacta ctactacggc 720
ggatcctacg ctatggacta ctggggccag gggaccagcg tgaccgtgtc atccgcggcc 780
gcaactacca ccttcgaaca tcctcacatt caagacgctg cttcgcaact tcctgatgat 840
gagagtttat tttttggtga tactgggcta tccaaaaatc caatcgagct tgtagaaggt 900
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ggactattct tggttctccg agttggtatc catctttgca ttaaattaaa gcacaccaag 1020
aaaagacaga tttatacaga catagagatg aaccgacttg gaaag 1065
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Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys
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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
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Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
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Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
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Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 9
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Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly
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Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr
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Claims (22)
1.一组病毒载体,其特征在于,包括:
第一病毒载体,所述第一病毒载体携带第一核酸分子,所述第一核酸分子编码包膜蛋白;
至少一个第二病毒载体,所述第二病毒载体携带第二核酸分子,所述第二核酸分子编码至少一个融合蛋白,所述融合蛋白包括至少一个单链抗体和所述包膜蛋白的C端结构域,所述包膜蛋白的C端结构域包括包膜蛋白的跨膜区和胞内区,所述至少一个单链抗体的C端与所述包膜蛋白的C端结构域的N端相连,所述单链抗体靶向特异性抗原;
所述第一核酸分子与所述第二核酸分子被设置为表达所述包膜蛋白与所述融合蛋白,并且所述包膜蛋白与所述融合蛋白呈非融合形式。
2.根据权利要求1所述的病毒载体,其特征在于,所述病毒载体为反转录病毒、慢病毒和其他包膜病毒载体。
3.根据权利要求1所述的病毒载体,其特征在于,所述包膜病毒包括选自玻那病毒科(Bornaviridae)、尼亚马病毒科(Nyamaviridae)、沙粒病毒科(Arenaviridae)、丝状病毒科(Filoviridae)、汉坦病毒科(Hantaviridae)、内罗病毒科(Nairoviridae)、正粘病毒科(Orthomyxoviridae)、副黏病毒科(Paramyxoviridae)、布尼亚病毒科(Bunyaviridae)、白纤病毒科(Phenuiviridae)、弹状病毒科(Rhabdoviridae)、动脉炎病毒科(Arteriviridae)、冠状病毒科(Coronaviridae)、黄病毒科(Flaviviridae)、披膜病毒科(Togaviridae)、嗜肝DNA病毒科(Hepadnaviridae)、泡沫病毒(Spumavirus)、虹彩病毒科(Iridoviridae)、疱疹病毒科(Herpesviridae)、痘病毒科(Poxviridae)、丁型肝炎病毒科(Deltavirus)病毒的至少之一;
任选地,所述包膜蛋白为弹状病毒科水泡性口炎病毒的包膜G糖蛋白或包膜G糖蛋白的突变体。
4.根据权利要求3所述的病毒载体,其特征在于,所述包膜G糖蛋白的突变体具有K47Q和R354Q突变;
任选地,所述包膜G糖蛋白的突变体具有SEQ ID NO:1所示的氨基酸序列。
5.根据权利要求1所述的病毒载体,其特征在于,所述单链抗体靶向细胞特异性抗原。
6.根据权利要求3~5任一项所述的病毒载体,其特征在于,所述融合蛋白进一步包括第一连接肽;
任选地,所述第一连接肽具有SEQ ID NO:2所示的氨基酸序列;
任选地,所述包膜蛋白的C端结构域具有SEQ ID NO:3所示的氨基酸序列;
任选地,所述融合蛋白具有SEQ ID NO:4或SEQ ID NO:10所示的氨基酸序列。
7.根据权利要求1所述的病毒载体,其特征在于,进一步包括:
第一启动子,所述第一启动子与所述第一核酸分子可操作地连接;以及
第二启动子,所述第二启动子与所述第二核酸分子可操作地连接。
8.根据权利要求7所述的病毒载体,其特征在于,所述第一启动子和所述第二启动子分别独立地选自CMV、EF-1或RSV启动子。
9.根据权利要求1所述的病毒载体,其特征在于,所述第一核酸分子具有SEQ ID NO:5所示的核苷酸序列;
任选地,所述第二核酸分子具有SEQ ID NO:6所示的核苷酸序列。
10.根据权利要求1所述的病毒载体,其特征在于,所述第一病毒载体和第二病毒载体为同一载体。
11.根据权利要求10所述的病毒载体,其特征在于,进一步包括:
内部核糖体进入位点序列,所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间。
12.根据权利要求10所述的病毒载体,其特征在于,进一步包括:
第三核酸分子,设置在所述第一核酸分子与所述第二核酸分子之间,并且所述第三核酸分子编码第二连接肽,所述第二连接肽能够被切割。
13.根据权利要求10所述的病毒载体,其特征在于,所述第一核酸分子和第二核酸分子的拷贝数之比为1:1~4:1,
任选地,所述第一核酸分子和第二核酸分子的拷贝数之比为2:1~4:1,
优选地,所述第一核酸分子和第二核酸分子的拷贝数之比为2:1。
14.根据权利要求1所述的病毒载体,其特征在于,所述第一病毒载体和第二病毒载体为pMD2.G、pCMV、pMD2.G突变体或pCMV的突变体。
15.根据权利要求1所述的病毒载体,其特征在于,进一步包括第三病毒载体和第四病毒载体,所述第三病毒载体携带目的基因,所述第四病毒载体携带病毒结构蛋白基因和病毒包装酶基因以及任选的调节因子rev基因;
任选地,所述结构蛋白基因、病毒包装酶基因和调节因子rev基因设置于同一第四病毒载体或不同第四病毒载体上;
任选地,所述病毒包装酶包括逆转录酶、蛋白酶和整合酶的至少之一。
16.根据权利要求15所述的病毒载体,其特征在于,所述第三病毒载体为转移载体,所述转移载体含有慢病毒包装信号,
任选地,所述慢病毒包装信号包括:Ψ;
任选地,所述转移载体为pLV;
任选地,所述第四病毒载体为psPAX2。
17.一种获得慢病毒的方法,其特征在于,将权利要求1~16任一项所述的病毒载体导入第一受体细胞;将导入病毒载体的第一受体细胞进行培养,以便获得所述病毒。
18.根据权利要求17所述的方法,其特征在于,所述病毒为慢病毒,所述第一病毒载体和第二病毒载体不为同一载体,所述第三病毒载体、第四病毒载体、第一病毒载体和第二病毒载体的质量比为2:1:1:0.25~2:1:1:1,
优选地,所述第三病毒载体、第四病毒载体、第一病毒载体和第二病毒载体的质量比为2:1:1:0.5;
任选地,所述第一受体细胞为293T。
19.一种慢病毒,其特征在于,是通过权利要求17或18所述的方法包装获得的。
20.一种慢病毒,其特征在于,表达包膜蛋白以及融合蛋白,所述融合蛋白包括单链抗体和所述包膜蛋白的C端结构域,所述包膜蛋白的C端结构域包括包膜蛋白的跨膜区和胞内区,所述单链抗体的C端与所述包膜蛋白的C端结构域的N端相连,
任选地,所述包膜蛋白为水泡性口炎病毒的包膜G糖蛋白或包膜G糖蛋白的突变体。
21.一种药物组合物,其特征在于,包括权利要求1~16任一项所述的病毒载体或权利要求19或20所述的慢病毒,所述病毒载体或慢病毒携带目的基因。
22.一种表达目的基因的方法,其特征在于,包括:
将整合有目的基因的病毒载体或慢病毒导入第二受体细胞;
将导入病毒载体或慢病毒的第二受体细胞进行培养,以便表达目的基因;
任选地,所述导入第二受体细胞是通过电转、转染或感染的方式进行的;
任选地,所述第二受体细胞为体细胞;
任选地,所述第二受体细胞为肿瘤细胞。
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WO2024067870A1 (zh) * | 2022-09-30 | 2024-04-04 | 深圳市济因生物科技有限公司 | 靶向载体及其制备方法与用途 |
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