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 CRISPR- Staphylococcus aureus ( Sa )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 five of eight HBV-specific AAV- Sa Cas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of Sa Cas9 delivery to HBV + human hepatocytes than those without gene editing. HBV-specific AAV- Sa Cas9 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 it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.

The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer

“…HBV-specific AAV-Sa Cas9 therapy significantly improves the survival of human hepatocytes, shows a trend toward decreasing the total liver HBV DNA and cccDNA, and is well tolerated. 64 These results are hopeful in terms of curing chronic HBV infection.…”

Covalently closed circular DNA: The ultimate therapeutic target for curing HBV infections

“…CRISPR-Cas9 treatment led to HBV DNA editing in 5/8 chronically infected liver-humanised mice undergoing NA treatment: though there was a trend towards reduced cccDNA levels, there was no change in viremia after treatment, indicating that a large cccDNA fraction remained unaffected, leading to viral rebound and dampening any therapeutic effect. 81 Several factors will be instrumental in the clinical application of gene-editing approaches for HBV therapy. Maximising gene-editing efficiency, which is connected to high Cas9 expression levels in target cells, is essential.…”

Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

“…Simultaneously, the CRISPR/Cas9 system was also applied to target HBsAg or HBV X protein (HBx) in cell culture and in animal experiments, respectively, and demonstrated that the expression levels of HBsAg in cell culture supernatant and mouse serum were both decreased [ 45 ]. Recently, a large number of studies and similar data have been reported on using the CRISPR/Cas9 system to target HBV genes [ 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ], implying that CRISPR/Cas9-based gene editing may be a potential therapeutic method for HBV infection.…”