The Stability of covalently closed circular DNA during mitosis of Hepatitis B Virus infected cells

Abstract

Chronic HBV infection is typically lifelong due to the persistence of the viral episomal covalently closed circular (ccc)DNA, from which all viral products can be produced. HBV cccDNA is a highly stable, long-lived transcriptional template for HBV. Elimination of this form of HBV DNA would represent a cure for chronic HBV infection. Determining the true fate of cccDNA in mitosis may in addition result in developing a novel treatment for cccDNA elimination.

To this end, we have chosen to explore two methods of approaching elimination of cccDNA. Direct degradation utilising novel CRISPR-cas9 therapy and elucidating the role of cell mitosis in cccDNA loss both in vitro and in vivo.

Using a dual guide RNA-based approach, one guide pair transiently reduced HBeAg and HBsAg expression respectively up to 36% and 42% with expression returning to baseline 9 days post transfection.

In vitro studies showed that cccDNA is lost after cell mitosis, resulting in uninfected daughter cells, however alse revealed he cccDNA retaining cells expressing β-galactosidase, suggesting mitotic arrest. RNA sequencing of this population revealed upregulation of pathways associated with cell cycle arrest. A panel of drugs targeting mitotic arrest pathways was screened and candidates identified which effectively induced up to a 12-fold reduction in cccDNA levels.

We tested these drugs in a liver turnover mouse model, with a green fluorescent protein (GFP) adeno-associated virus vector as HBV cccDNA surrogate. We found a subset of hepatocytes which retained GFP and expressed p53. Treatment of these mice with drug candidates resulted in a ~3-fold reduction in these cells.

Overall, we have illuminated a new aspect of cccDNA persistence which has previously only been speculated on and have demonstrated the existence of these cells and a potential pathway by which we can treat them. Targeting these reservoir cells may be crucial for developing novel cure therapies for chronic hepatitis B.