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The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure.
Current treatment of chronic hepatitis B can suppress HBV replication efficiently. However, a functional cure of infection defined by hepatitis B surface antigen (HBsAg) loss rarely occurs,
and thus long-term nucleos(t)ide analogue (NA) therapy is needed in the majority of patients. Because of the persistence of the viral minichromosome in the infected liver, i.e. covalently closed circular (ccc) DNA,
viral relapse is almost universal when treatment is stopped prior to HBsAg loss. The major barriers to HBV cure include the persistence of a reservoir of HBV-infected cells harbouring cccDNA and/or integrated viral sequences, and the impaired innate and adaptive immune responses against HBV.
Wang et al. make an important contribution to the HBV cure field and report on their attempts to identify small molecules to reduce cccDNA in chronically infected hepatocytes.
The episomal HBV cccDNA is located in the hepatocyte nucleus and serves as a transcriptional template for all HBV RNAs including pregenomic RNA (pgRNA) which is reverse transcribed into HBV DNA, and four messenger RNAs which are translated into viral proteins.
cccDNA is derived not only from incoming virions but also from intracellular recycling of nucleocapsids. The dual source of cccDNA and its long half-life explain why cccDNA levels are minimally decreased even after many years of NA therapy.
Permanent cccDNA elimination or silencing is the key objective to prevent any risk of viral reactivation, and thus cccDNA is considered as the ultimate target for HBV cure.
In that respect, efforts have been made to standardize the technologies for cccDNA quantification to monitor the effect of new strategies aimed at HBV cure.
HBV delivers its 3.2 kb relaxed cicurlar (rc) DNA genome into the nuclei of the host cell. rcDNA is then repaired into a fully double stranded cccDNA, through a multistep process that involves several nuclear enzymes
(Fig. 1). Since NAs do not target the rcDNA-to-cccDNA conversion, and do not induce a complete suppression of viral DNA synthesis, a low level replenishment of cccDNA through new rounds of infection and/or intracellular recycling may occur even during effective NA therapy.
Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.
It was also shown that the dynamics of cccDNA are affected by infected cell death and the resulting hepatocyte turnover which may lead to cccDNA clearance, provided that new virions are not produced or are neutralized by antibodies.
cccDNA is wrapped around nucleosomes containing core histones and is associated with viral capsid and HBx proteins and host transcription factors, forming a highly stable minichromosome
(Fig. 1). The mechanism and kinetics of cccDNA chromatin configuration, including histone deposition and recycling, and epigenetic regulation are not well defined. It was shown that rcDNA repair into cccDNA and histone deposition are coordinated, as is the case for host chromosomes.
Like cellular genes, cccDNA is subjected to the histone code thanks to the dynamic exchange between histone modifiers, chromatin remodelers and transcription factors.
Fig. 1Main steps of HBV cccDNA formation and chromatinization, and how ccc_RO8 might work on the already established cccDNA pool through the regulation of a host protein network.
Beside the elimination of the reservoir of infected cells, for instance by specific immune-mediated killing of infected hepatocytes, direct targeting of cccDNA remains a challenge.
Understanding the factors and mechanisms involved in cccDNA formation has revealed novel targets for inhibiting cccDNA biogenesis. It has been shown that cccDNA can be reduced by small molecule inhibitors that target various repair factors including: aphidicolin, a peptide derived from the cyclin-dependent kinase inhibitor p21, FEN-1 endonuclease inhibitor PTPD, topoisomerase inhibitors, DNA ligase inhibitors, and inhibitors of DNA checkpoint kinase ATR and CHK1.
Whether an approach targeting the host DNA repair pathway will be feasible to treat chronic HBV infections with a sufficient safety margin will require specific investigations. Moreover, it remains to be seen if blocking rcDNA to cccDNA conversion would lead to a significant reduction of the already established pool of cccDNA.
Targeting the pool of already established cccDNA for its degradation or silencing of its transcription/expression has thus been the subject of several investigations.
It was shown that interferon-alpha (IFN-α) and a lymphotoxin beta receptor agonist led to the non-cytolytic clearance of cccDNA via the upregulation of apolipoprotein B mRNA-editing enzyme 3 (APOBEC3) in HBV-infected HepaRG cells and primary human hepatocytes.
Indeed, APOBEC3 members recognize and deaminate foreign DNA, leading to destruction of HBV DNA, and potentially to cccDNA deamination. However, other studies showed that APOBECs preferentially target single-stranded DNA and rcDNA
Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.
. Studies in in vitro-infected hepatocytes showed that IFN-α treatment did not lead to cccDNA deamination by APOBECs, and instead suggested that G-A hypermutations in virions occurred independently of IFN-α.
Thus, while HBV suppression was clearly demonstrated, direct cccDNA targeting by APOBECs remains controversial. Advances in the gene editing field have given rise to a wide range of tools available for various applications including the targeting of the HBV genome and cccDNA.
Gene-editing approaches using meganucleases, Zinc-finger nucleases and TALENs (transcriptional activator-like effector nucleases) can disrupt the viral genome in a permanent manner.
Several challenges remain to be addressed for the development of CRISPR-Cas9 technologies to treat chronic HBV infections, including potential off target effects, double stranded breaks leading to chromosomal recombination, and efficient delivery of the ribonucleoprotein complex to the infected liver.
The development of new base and prime editing technologies has enabled DNA rewriting without cleavage, allowing for more precise DNA sequence modification and reducing the risk of host genome rearrangements. Yang et al. recently showed that cytosine base editors targeting the S open reading frame led to a reduction of HBV replication in cultured hepatocytes.
Epigenetic silencing of cccDNA to shut down its transcription is another potential approach to functional cure. cccDNA transcription is regulated by the host cell epigenetic machinery. It was shown that IFN-α treatment leads to changes in post-translational modifications of cccDNA-associated histones and recruitment of transcriptional repressors, which could lead to a reduction in its transcriptional activity. Pro-inflammatory cytokines such as IL-6 and IL-1β also have a direct effect on the transcriptional activity of cccDNA, without affecting its levels.
cccDNA-associated histones can be directly targeted to silence cccDNA transcription. Drugs interfering with histone acetylation or methylation were shown to suppress cccDNA transcription (reviewed in
). Though epigenetic modifiers led to a decrease in viral parameters, a major caveat is that they could also interfere with host gene regulation. The discovery of the role of HBx in cccDNA transcription and the initiation of infection has opened new perspectives on the search for virus-specific targets to silence cccDNA.
Moreover, it was shown that silencing all viral transcripts using a combination of siRNAs and peg-IFNα substantially decreased HBx protein levels, leading to Smc6 rebound in vivo.
Small molecules targeting the HBx-DDB1 interaction, nitazoxanide and pevonedistat (MLN4924), have been tested in cultured hepatocytes and were shown to induce a rebound of Smc5/6 associated with a decrease in both cccDNA transcriptional activity and viral parameters.
Pevonedistat, a neuronal precursor cell-expressed developmentally down-regulated protein 8-activating enzyme inhibitor, is a potent inhibitor of hepatitis B virus.
The in vivo confirmation of these findings is warranted.
Given the complexity of cccDNA biology and its key role in viral persistence, the discovery of orally available antiviral agents that could deplete the pool of intrahepatic cccDNA would change the HBV cure paradigm. Wang et al.
report the discovery of a small molecule, ccc_R08, that decreases the HBV cccDNA reservoir in highly relevant study models, including a high-throughput screening in HBV-infected primary human hepatocytes and HepDES19 cells, and experimental validation in the HBV circle mouse model and the uPA-SCID humanized liver mouse model. The compound ccc_R08 demonstrated its ability to reduce HBsAg, HBeAg, HBV DNA, and pgRNA in the blood circulation and to decrease the cccDNA reservoir in the liver. It is the first time that a small molecule has been shown to act on the already established cccDNA pool.
Although the authors called their compound a cccDNA ‘inhibitor’, the exact mode of action of the compound remains to be determined as it may be ascribed to cellular processes involved in cccDNA degradation or instability (Fig. 1). Interestingly, their findings showed that ccc_R08 administration was associated with a strong reduction in viral transcripts, i.e. pre-C/pregenomic and X transcripts, as well as an impact on host gene expression. In their study models, where integration is thought to be negligeable, HBsAg is mainly produced by transcriptionally active cccDNA. Therefore, the observation that cccDNA levels were reduced to a lesser extent than HBsAg levels suggests that the compound might preferentially destabilize the transcriptionally active cccDNA species. Thus, it will be interesting to study the chromatin configuration of residual cccDNA to determine if compacted cccDNA species would be less sensitive to ccc_R08. The integrated analysis of host transcriptomics and viral RNA expression and replication suggested that the anti-HBV effect of ccc_R08 is most likely mediated by the regulation of a host gene regulatory network. To overcome the difficulties encountered with knockdown and knockout approaches, the authors used a clever forward and reverse pharmacology approach and identified two gene lists that are likely involved in the pharmacological effect of ccc_R08. This enabled them to identify already reported putative targets of ccc_R08 including CHEK1, CHEK2, TOP2A, and ATM,
but also novel candidates to be explored by future studies, such as transcription factors, druggable proteins, and signal transduction proteins. Further functional investigations of these candidates may reveal new avenues for the identification of molecular targets involved in cccDNA depletion.
not only provides new hope in the search for drugs to eliminate cccDNA, but also provides the ground to generate new knowledge on the cellular networks involved in the persistence and regulation of the HBV minichromosome.
Financial support
FZ and BT have received public grants overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program (reference: ANR-17-RHUS-0003) and by the European Union (grant EU H2020-847939-IP-cure-B).
Conflict of interest
FZ received consulting fees from Aicuris, Aligos, Antios, Assembly, Blue Jay, Evotec, Gilead, GSK, Zhimeng. FZ and BT received research grants from Assembly, Beam, Janssen, Viravaxx.
Please refer to the accompanying ICMJE disclosure forms for further details.
Authors’ contributions
FZ wrote the manuscript, BT prepared the figure and revised the manuscript.
Supplementary data
The following are the supplementary data to this article:
Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.
Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.
Pevonedistat, a neuronal precursor cell-expressed developmentally down-regulated protein 8-activating enzyme inhibitor, is a potent inhibitor of hepatitis B virus.
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