Multiplex gene editing suppresses random integration of hepatitis B virus DNA in chronically infected liver

Gene editing technologies have opened the possibility of directly targeting viral DNA for therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) serves as the master template for viral transcripts and gene products. In the present study, we evaluated anti-HBV multiplex gene editing with the CRISPR-Cas9 endonuclease from Staphylococcus aureus (SaCas9) using primary human hepatocytes (PHHs) and HBV mouse models. Nonviral delivery of SaCas9-encoding mRNA and a pair of HBV-targeting guide RNAs (gRNAs) substantially reduced viral biomarkers and intrahepatic HBV DNA copies in vitro and in vivo. Hybridization capture sequencing analyses showed that small insertions and deletions (indels) and structural variants including excisions and inversions of the viral sequences were introduced in the residual HBV DNA. These assays also demonstrated that transient expression of the HBV-targeting SaCas9 significantly suppressed random integration of HBV DNA into the host genome, while leading to no detectable increase in chromosomal translocations involving viral copies. Lastly, our gene editing approach blocked viral rebound after stopping treatment with a nucleos(t)ide analogue (NA), entecavir. Our results suggest that anti-HBV multiplex gene editing removes viral DNA from chronically infected hepatocytes, potentially reducing the risk of hepatocarcinogenesis associated with HBV DNA integration.