CRISPR/Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Abstract Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and S. aureus (Sa)CRISPR/Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of 5 of 8 HBV-specific AAV-SaCas9 treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV positive human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.