利用锁核酸化学偶联FokⅠ核酸酶靶向切割HBV基因的体外实验

doi:10.16288/j.yczz.15-435

遗传 ›› 2016, Vol. 38 ›› Issue (4): 350-359.doi: 10.16288/j.yczz.15-435

利用锁核酸化学偶联FokⅠ核酸酶靶向切割HBV基因的体外实验

马丽¹, 陈红岩¹, 朱化星², 李威³, 卢大儒¹

1. 复旦大学生命科学学院,遗传工程国家重点实验室,上海 200433;
2. 上海近岸科技有限公司,上海 201203;
3. 生工生物工程(上海)股份有限公司,上海 201611

收稿日期:2015-10-17 修回日期:2015-11-19 出版日期:2016-04-20 发布日期:2016-04-20
通讯作者: 卢大儒,博士,教授,研究方向：人类医学分子遗传学。E-mail: drlu@fudan.edu.cn
作者简介:马丽,硕士在读,专业方向：生物工程。Tel：021-51630621;E-mail: maryguoji@gmail.com陈红岩,博士,副教授,研究方向：人类医学分子遗传学。E-mail: chenhy@fudan.edu.cn马丽和陈红岩为并列第一作者。

Locked nucleic acid couples with FokⅠnucleases to target and cleave hepatitis B virus’s gene in vitro

Li Ma¹, Hongyan Chen¹, Huaxing Zhu², Wei Li³, Daru Lu¹

1. State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China;
2. Shanghai Novoprotein Technology Co., Ltd., Shanghai 201203, China;
3. Sangon Biotech (Shanghai) Co., Ltd., Shanghai 201611, China

Received:2015-10-17 Revised:2015-11-19 Online:2016-04-20 Published:2016-04-20

摘要/Abstract

摘要： 乙肝病毒(HBV)是有缺口的双链DNA病毒,侵入人体肝细胞后形成共价闭合环状DNA(cccDNA)持续复制,并在逆转录过程中随机整合入宿主基因组。慢性乙型肝炎感染者平均每个细胞中含有33个cccDNA拷贝,半衰期长达35~57 d,很难从体内彻底清除。利用锁核酸抑制HBV转录,是乙肝治疗的新策略。此外,利用基因组编辑技术靶向切割HBV基因组,有望从源头治愈乙型肝炎。基于锁核酸与双链DNA形成三股螺旋的能力、抵御核酸酶及聚合酶的稳定性以及对单碱基错配的敏感性,本研究以靶向切割乙型肝炎病毒为例,设计构建锁核酸修饰的寡核苷酸作为DNA结合域,有效增强对靶基因的特异性识别;同时利用FokⅠ核酸酶分子量小、二聚化时才具有酶活等特点,设计构建FokⅠ切割域二聚体重组蛋白作为DNA切割域;进而通过双功能交联剂GMBS,建立了5′端氨基(-NH₂)修饰的锁核酸与N端巯基(-SH)修饰的FokⅠ核酸酶定向化学偶联的方法,并在体外验证了新型工具对HBV基因的靶向切割。该方法为此后在体内进行高特异性、无整合风险的抗病毒基因治疗提供了全新的技术思路。

关键词: 乙肝病毒, 基因编辑, 锁核酸, FokⅠ, 化学偶联

Abstract: Hepatitis B virus (HBV) is a dented double-stranded DNA virus. After infecting human hepatic cells, it forms cccDNA that replicates persistently and integrates randomly into the host’s genome during the process of reserve transcription. On average, in each cell with chronic HBV infection, there are about 33 copies of cccDNA with a half of 35-57 days, which can be difficult to eradicate. A new strategy is to inhibit HBV transcription by using locked nucleic acid (LNA). Besides, cleaving HBV genome by targeted genome editing technologies could potentially cure patients. In this study, we explored new genome editing tools for HBV treatment. Based on LNA’s ability to form triple helix by binding to duplex DNA, its stability towards nuclease and polymerase, and its sensitivity to single base mismatches, we designed LNA-modified oligonucleotides as DNA binding domain to effectively increase the specificity of target gene recognition. Meanwhile, by utilizing the small molecular weight and dimerization dependent activity of nuclease FokⅠ, we used FokⅠrecombinant dimer protein as DNA cleavage domain. Here, we established a method by chemical coupling of LNA-oligonucleotide with FokⅠcleavage domain, and also validated the targeted cleavage of HBV genes with our new tools in vitro. These results provide new possibilities for future in vivo anti-virus gene therapy with high specificity and no integration risk.

Key words: hepatitis B virus, gene editing, locked nucleic acid, FokⅠ, chemical coupling

马丽, 陈红岩, 朱化星, 李威, 卢大儒. 利用锁核酸化学偶联FokⅠ核酸酶靶向切割HBV基因的体外实验[J]. 遗传, 2016, 38(4): 350-359.

Li Ma, Hongyan Chen, Huaxing Zhu, Wei Li, Daru Lu. Locked nucleic acid couples with FokⅠnucleases to target and cleave hepatitis B virus’s gene in vitro[J]. HEREDITAS(Beijing), 2016, 38(4): 350-359.

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利用锁核酸化学偶联FokⅠ核酸酶靶向切割HBV基因的体外实验

Locked nucleic acid couples with FokⅠnucleases to target and cleave hepatitis B virus’s gene in vitro

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