Shutdown of HBV transcripts using siRNA followed by entry inhibition induces sustained silencing of the cccDNA in vivo

Question There is an urgent need for therapeutic strategies repressing the hepatitis B virus (HBV) nuclear template, the covalently closed circular DNA (cccDNA), since silencing of the viral reservoir may promote functional cure of chronic HBV infection. The structural maintenance of chromosomes 5/6 complex (SMC5/6) is a host factor with the potential to suppress cccDNA activity. However, SMC5/6 is antagonized by the viral regulatory HBx protein, which triggers its degradation. We aimed to investigate the impact of an siRNA that targets all HBV transcripts for degradation on HBx and SMC5/6. In particular, we assessed whether siRNA and other interventions able to lower HBV transcripts can achieve silencing of cccDNA transcription in vivo.

Methods HBV-infected human liver chimeric mice were treated with siRNA for 4 weeks. Virological and host changes were analyzed at the end of treatment and during follow-up by qRT-PCR, ELISA, immunoblotting and chromatin immunoprecipitation (ChIP). RNA in situ hybridization was combined with immunofluorescence to detect SMC6 and HBV RNAs at the single cell level. The entry inhibitor Myrcludex-B was used during the follow-up phase to avoid new infection events. In addition, some mice received pegylated interferon alpha (peg-IFNα) or beta (peg-IFNβ) treatment, which have been shown to affect cccDNA activity.

Results siRNA strongly reduced all HBV transcripts and viral markers in the serum and liver except cccDNA. Interestingly, HBx protein levels were also reduced, thus enabling the reappearance of SMC5/6 in the liver of treated mice. RNA-ISH+IF analysis revealed that the reappearance of the SMC5/6 complex was restricted to hepatocytes negative for HBV transcripts and SMC5/6 was found associated with the cccDNA in ChIP assays. Also peg-IFNα and beta peg-IFNb reduced HBx proteins, promoted SMC5/6 reappearance and achieved cccDNA silencing. However, the antiviral effects did not persist off treatment and SMC5/6 was degraded again regardless of the treatment used. Remarkably, blockade of viral entry during follow-up hindered renewed degradation of SMC5/6 and maintained cccDNA transcriptional silencing.

Conclusions Our results reveal that interventions abrogating HBx promote SMC5/6-mediated silencing of the cccDNA minichromosome, while strategies shielding the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Publication History

Article published online:
04 January 2021