定点切割和修饰HBV cccDNA抑制HBV复制的研究

Hepatitis B virus (HBV) infection is a major health problem worldwide, particularly in China. In the life cycle of the virus, the covalently closed circular DNA (cccDNA) plays a key role and permits the persistence of infection by serving as a template for the transcription of all viral transcripts, including the HBV pregenomic RNA. The cccDNA accumulates in hepatocyte nuclei as a stable minichromosome. Existing therapeutic regimens, including the modulation of patients' antiviral defenses and the inhibition of the viral reverse transcriptase, are unable to eradicate cccDNA, and thus they generally cannot cure the disease. Therapies that interfere with other HBV replication steps, particularly cccDNA reservoir, would be in huge demand for the enormous market of chronic HBV infection in China. Transcription activator-like effectors (TALEs) are bacterial effector proteins. Each TALE contains a DNA-binding domain consisting of 34 amino-acid tandem repeat modules where the twelfth and thirteenth residues specify the target DNA base. TALEs have been developed into a versatile platform for precise and efficient molecular genome modification tools applicable across a diverse range of biological systems. In this project, we propose to develop a novel therapeutic strategy to break and repress HBV cccDNA by harnessing the TALE DNA binding code. We hypothesize that couple the site-specific TALE domain with functional domains including nuclease, repressor and methylase will inhibit the activity of the cccDNA genes and suppress HBV replication, eventually depleting the cccDNA reservoir in HBV infected patients. We will synthesize a group of TALE nucleases (TALEN) by fusing HBV S or C gene binding domain to FokI nuclease domain; a group of TALE repressors (TALER) by fusing core promoter binding domain to SID repressor domain; a group of TALE methylases (TALEM) by fusing island 2 binding domain to DNMT3A or DNMT3B methylase domain. We will test whether the induced cccDNA cleavage, repression and hypermethylation will inhibit HBV replication in hepatocyte cell lines, and DHBV cccDNA cleavage and repression will inhibit DHBV replication in duck animal models.

乙型肝炎病毒（HBV）感染肝细胞后在核内形成的共价闭合环状DNA (cccDNA) 是HBV持续存在和复制的基础，cccDNA在肝细胞核内以稳定的微染色体形式存在，目前临床应用的所有抗HBV药物均不能清除cccDNA，如何有效抑制和清除cccDNA成为目前抗HBV治疗的难点和核心问题。本项目拟在我们前期对中国流行的HBV毒株基因组特点充分分析的基础上，构建HBV靶向性TALE，并分别与核酸酶、抑制子及甲基化酶功能域融合，体外细胞系研究对cccDNA上S和C结构基因的定点切割、C基因启动子的转录抑制、C基因启动子上游CpG岛2序列的甲基化修饰后，HBV复制、基因组转录及蛋白表达被抑制情况，同时用鸭乙肝动物模型做进一步的体内研究。本研究为乙型肝炎的治疗和HBV的清除，最终达到降低HBV相关肝病的发生和进展寻找一条全新的途径，研究结果具有非常重要的临床价值和意义。

乙型肝炎病毒（HBV）感染肝细胞后在核内形成的共价闭合环状DNA (cccDNA) 是HBV持续存在和复制的基础，如何有效抑制和清除cccDNA成为目前抗HBV治疗的难点和核心问题。本项目获资助后根据计划研究内容首先用TALEN基因编辑系统对HBV复制的抑制作用进行了研究，并根据国际上基因编辑技术的快速进展，利用CRISPR/Cas9 Sp基因编辑系统对HBV复制的抑制作用进行了研究，发现CRISPR/Cas9系统对HBV的切割抑制效率远高于TALEN系统，因此在研究TALEN系统抑制HBV复制的基础上，项目组及时调整研究策略，重点利用CRISPR/Cas9系统研究靶向切割抑制HBV复制的可行性。分别利用基因编辑工具CRISPR-CAS9 Sp和Sa系统，研究了其靶向切割抑制HBV复制的效率。首先利用SpCas9系统体外细胞系证实其抑制HBV复制的有效性，但该系统难以实现体内高效递送，其临床应用受限，因此课题组根据最新的SaCas9系统基因相对较小，可被AAV包装的优点，设计和筛选了针对SaCas9系统的HBV不同基因型靶位点，分别通过体外细胞系、高压尾静脉小鼠模型、慢性HBV感染小鼠模型，研究了新的SaCas9系统用于HBV基因组切割和复制抑制的高效性和可行性，尤其是通过高滴度AAV包装的SaCas9可在HBV慢性感染小鼠模型中抑制HBV复制，为目前如何利用Cas9系统靶向清除HBV的研究提供了新的临床前数据。