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Silencing HBV transcription with SMC5/6: has a path been found?
  1. John Tavis,
  2. Ranjit Chauhan
  1. Molecular Microbiology and Immunology, Saint Louis University, Saint Louis, Missouri, USA
  1. Correspondence to Dr John Tavis, Molecular Microbiology and Immunology, Saint Louis University, Saint Louis, MO 63104, USA; john.tavis{at}health.slu.edu

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Chronic hepatitis B virus (HBV) infection is maintained by the viral nuclear covalently closed circular DNA (cccDNA) that is the transcriptional template for HBV’s mRNAs. The cccDNA is durable during current therapies, but a functional cure for HBV infections will require stable, off-treatment silencing of any cccDNA remaining in the body after treatment cessation.1 The cccDNA is silenced naturally during infection by binding of the structural maintenance of chromosome 5/6 (SMC5/6) complex to the cccDNA, and HBV antagonises this silencing with the HBx protein that binds to SMC5/6 and triggers its proteasomal degradation.2 In Gut, Allweiss and Giersch et al3 explored what happens to HBV mRNA transcription during and after suppression of viral mRNA and antigen levels in HBV-infected chimeric mice carrying humanised livers. Suppressing all HBV mRNAs with a small interfering RNA (siRNA) or with pegylated interferon α (Peg-IFNα) increased SMC5/6 binding to the cccDNA, suppressed HBV transcription and reduced HBV protein production. Withdrawal of siRNA and Peg-IFNα led to degradation of SMC5/6 and rebound of viral transcription and protein levels. Finally, treating the mice with the potent HBV entry inhibitor myrcludex B (bulevirtide, Hepcludex) after cessation of siRNA or Peg-IFNα treatment greatly increased durability of SMC5/6-mediated transcriptional repression. This is a well-done study using the best available animal model of HBV infection.

These results have two major implications. First, they strongly emphasise that restoring SMC5/6 function will be essential to achieve the durable repression of cccDNA transcription needed for a functional cure. This in turn raises the importance of developing agents that reduce HBx production (as is done by both the siRNA and Peg-IFNα treatments), disrupt the HBx:DDB1:SMC5/6 binding interfaces4 or induce HBx degradation. Mechanistically, sustained SMC5/6 repression of transcription from the cccDNA could underlie the unexpectedly robust HBV surface antigen (HBsAg) suppression months after termination of siRNA treatment, as has been observed for JNJ-3989.5

The second implication stems from the exciting finding that administering the entry inhibitor myrcludex B upon siRNA or Peg-IFNα withdrawal increases durability of SMC5/6-mediated cccDNA repression. This indicates that interaction of extracellular HBV virions with hepatocytes plays a larger role in rebound of functional cccDNA, at least after siRNA and Peg-IFNα treatment, than our current understanding of infection would predict. Myrcludex B presumably acts by inhibiting de novo infection of cells that would create new cccDNA molecules and new HBx protein to antagonise SMC5/6, but it is also possible that HBV binding to or entry into cells could trigger a signalling cascade that prolongs SMC5/6-mediated repression.

The effect of myrcludex B in delaying viral rebound also adds to the growing evidence that HBV infections are more dynamic than has long been believed. Myrcludex B monotherapy can suppress HBV titers by >1 log10 after 12 or 24 weeks of treatment,6 indicating that de novo infection during chronic infection contributes to cccDNA maintenance. Preventing formation of new capsids in cultured cells after HBV infection can block cccDNA amplification,7 indicating that replenishment of cccDNA in this culture system primarily occurs by infection of cells. A more dynamic state for HBV infections is also supported by the rate of reversion of nucleoside analogue resistance that occurs via cccDNA turnover. cccDNA was recently measured to have a half-life of 16–28 weeks using genetic reversion analyses, and HBV mRNAs had fully reverted to the wild-type sequence within 24–48 weeks following nucleos(t)ide analogue withdrawal.8 This is substantially faster than predicted by the 8.6 (HBeAg positive) to 26.2 (HBeAg negative) month half-life recently measured for cccDNA during nucleos(t)ide analogue treatment.9 However, HBV DNA decay kinetics analysed in this study also revealed that tenofovir did not completely block reverse transcription. Ongoing viral replication likely lengthened the measured cccDNA half-lives due to new cccDNA formation by infection of cells and/or intracellular replenishment, further supporting dynamic maintenance of HBV in the liver. A more dynamic state for the cccDNA pool bodes well for the development of curative combination therapies because it reveals that natural turnover of the cccDNA will play a greater role in clearing the infection than the research community had anticipated.

Limitations to the Allweiss et al’s study stem primarily from constraints of the chimeric mouse model for HBV. These mice are fragile and extremely expensive, limiting the number of mice used in their studies. More significantly, the mice lack an adaptive immune response, and intercell signalling in some cases is impaired due to mismatches between the mouse tissues and human hepatocytes in the chimeric liver. It will be extremely interesting to extend these studies into humans where the immunological limitations of the chimeric mice are absent.

As with all good science, the Allweiss et al’s study raises many more questions than it answers. Mechanistically these include: Does SMC5/6-mediated suppression of cccDNA transcription underlie the persistence of HBV in patients who appear to have cleared HBV yet rebound on immunosuppression? Could HBsAg suppression expose the anti-HBsAg antibodies present in some patients, possibly mimicking myrcludex B’s effects? Could variable levels of endogenous HBsAg antibodies help explain differences in the rate of rebound in patients treated with antigen-suppressing therapies? Does HBx rebound in some cells on treatment withdrawal despite the presence of SMC5/6, or is all of the rebound due to de novo infection plus resurgence in cells that never fully established SMC5/6 repression? What role would expression of HBx from chromosomal HBV integrants play in restoring transcription from the cccDNA? Practical questions raised by this study include: Does altering the dose, timing or duration of siRNA, Peg-IFNα and myrcludex B treatment improve durability of cccDNA silencing? Would a triple combination of siRNA, Peg-IFNα and myrcludex B lead to a higher fraction of cells in which HBV becomes durably suppressed? Are there other drug combinations that could exploit this suppressive effect? Answering these questions will significantly advance our understanding of cccDNA regulation within hepatocytes and may well lead to improved treatment for chronic hepatitis B patients.

References

Footnotes

  • Contributors Both authors wrote the manuscript jointly.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Commissioned; internally peer reviewed.

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