Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication

作者：Jinyu Zhang, Yi Zhang, Sushant Khanal, Dechao Cao, Juan Zhao, Xindi Dang, Lam Ngoc Thao Nguyen, Madison Schank, Xiao Y. Wu, Yong Jiang, Shunbin Ning, Ling Wang, Mohamed El Gazzar, Jonathan P. Moorman, Haitao Guo, Zhi Q. Yao · 发表于：Journal of Medical Virology · 年份：2023 · DOI：10.1002/jmv.28952 · 被引用次数：21 · 研究领域：Hepatitis B Virus Studies、Vibrio bacteria research studies、RNA Interference and Gene Delivery

The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed...