Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.

由于病毒游离 DNA (cccDNA) 的持续存在，慢性乙型肝炎病毒 (HBV) 感染构成了一个全球健康问题，目前治疗效果有限。CRISPR/Cas9 系统是一种新开发的强大的基因组编辑和潜在基因治疗工具，需要有效传递 CRISPR 组件才能成功应用。在这里，我们研究了慢病毒或腺相关病毒 2 (AAV2) 载体介导的从 16 个 gRNA 中选择的 3 个引导 (g)RNA/Cas9 递送的影响。这些显着抑制了细胞中的 HBV 复制，WJ11/Cas9 表现出最高的功效并被选择用于体内学习。AAV2/WJ11-Cas9 还显着抑制了 HBV 复制并显着减少了测试细胞中的 cccDNA。此外，当联合使用时，AAV2/WJ11-Cas9 增强了恩替卡韦的作用，表明了不同的作用模式。值得注意的是，在人源化嵌合小鼠中，AAV2/WJ11-Cas9 显着抑制肝组织中的 HBcAg、HBsAg 和 HBV DNA 以及 cccDNA，而没有显着的细胞毒性；因此，下一代测序数据显示没有显着的基因组突变。据我们所知，这是首次使用 HBV 自然感染模式对 CRISPR/Cas9 系统进行评估。因此，由相对安全的AAV2载体递送的WJ11/Cas9可能为消除HBV感染提供新的治疗策略，并作为治愈慢性HBV感染的有效平台。