Advertisement

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

      Summary

      Current antiviral therapies, such as pegylated interferon-α and nucleos(t)ide analogues, effectively improve the quality of life of patients with chronic hepatitis B. However, they can only control the infection rather than curing infected hepatocytes. Complete HBV cure is hampered by the lack of therapies that can directly affect the viral minichromosome (in the form of covalently closed circular DNA [cccDNA]). Approaches currently under investigation in early clinical trials are aimed at achieving a functional cure, defined as the loss of HBsAg and undetectable HBV DNA levels in serum. However, achieving a complete HBV cure requires therapies that can directly target the cccDNA pool, either via degradation, lethal mutations or functional silencing. In this review, we discuss cutting-edge technologies that could lead to non-cytolytic direct cccDNA targeting and cure of infected hepatocytes.

      Keywords

      Introduction

      HBV can lead to acute and chronic infections, infecting more than 250 million people worldwide and causing over 780,000 deaths annually. Approved therapies include pegylated interferon-α (PEG-IFN) and nucleos(t)ide analogues (NAs). PEG-IFN can lead to sustained HBV suppression, though this was only observed in a limited number of patients, its mechanism of action is still unclear and prolonged therapy is poorly tolerated.
      • Isorce N.
      • Lucifora J.
      • Zoulim F.
      • Durantel D.
      Immune-modulators to combat hepatitis B virus infection: from IFN-α to novel investigational immunotherapeutic strategies.
      NAs improve clinical outcomes by controlling HBV replication and they are better tolerated than PEG-IFN.
      • Gish R.
      • Jia J.-D.
      • Locarnini S.
      • Zoulim F.
      Selection of chronic hepatitis B therapy with high barrier to resistance.
      However, neither of these treatments achieve a complete HBV cure (undetectable serum HBsAg and serum and intracellular HBV DNA, including covalently closed circular DNA [cccDNA] clearance) or a functional cure (undetectable serum HBV DNA and serum HBsAg, with or without seroconversion, accompanied or not by cccDNA silencing)
      • Cornberg M.
      • Lok A.S.-F.
      • Terrault N.A.
      • Zoulim F.
      2019 EASL-AASLD HBV treatment endpoints conference faculty. Guidance for design and endpoints of clinical trials in chronic hepatitis B - report from the 2019 EASL-AASLD HBV treatment endpoints conference‡.
      and in most cases, life-long treatments are required. Achieving a functional HBV cure after a finite course of treatment, leading to low rates of HBV reactivation and eliminating the need for life-long treatment, is the next goal for HBV therapies.
      The major challenge to achieving an HBV cure is the persistence of the cccDNA viral minichromosome, which is unaffected by current therapies.
      • Lebossé F.
      • Inchauspé A.
      • Locatelli M.
      • Miaglia C.
      • Diederichs A.
      • Fresquet J.
      • et al.
      Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
      • Lai C.-L.
      • Wong D.
      • Ip P.
      • Kopaniszen M.
      • Seto W.-K.
      • Fung J.
      • et al.
      Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.
      • Lai C.-L.
      • Wong D.K.-H.
      • Wong G.T.-Y.
      • Seto W.-K.
      • Fung J.
      • Yuen M.-F.
      Rebound of HBV DNA after cessation of nucleos/tide analogues in chronic hepatitis B patients with undetectable covalently closed circular DNA.
      • Nassal M.
      HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.
      cccDNA is HBV’s molecular reservoir and serves as the only transcriptional template for all viral RNAs, including pregenomic RNA (pgRNA). It contains 4 overlapping open reading frames (ORFs): S, C, P and X. These 4 overlapping ORFs encode 7 viral proteins: HBeAg (secreted protein), HBc (viral capsid protein), HBV POL/RT (polymerase reverse transcriptase), PreS1/PreS2/HBsAg (large, medium, and small surface envelope glycoproteins), and HBx (transcriptional activator). The pgRNA transcript serves as the substrate for reverse transcription into the new viral genome, the relaxed-circular DNA (rcDNA).
      • Hu J.
      • Seeger C.
      Hepadnavirus genome replication and persistence.
      NAs block pgRNA reverse transcription, preventing the formation of new rcDNA-containing virions. However, they do not prevent viral transcription from cccDNA and antigen production, which can be decreased to some extent by IFN-α. Just a few copies of cccDNA are sufficient for viral rebound after treatment cessation; thus, life-long treatments are needed to maintain control of the infection.
      • Lai C.-L.
      • Wong D.K.-H.
      • Wong G.T.-Y.
      • Seto W.-K.
      • Fung J.
      • Yuen M.-F.
      Rebound of HBV DNA after cessation of nucleos/tide analogues in chronic hepatitis B patients with undetectable covalently closed circular DNA.
      Although HBV DNA integration is not essential for the viral replication cycle, a byproduct generated during cccDNA formation, double-stranded linear DNA, can be integrated into the host genome.
      • Tu T.
      • Budzinska M.A.
      • Vondran F.W.R.
      • Shackel N.A.
      • Urban S.
      Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via sodium taurocholate cotransporting polypeptide-dependent uptake of enveloped virus particles.
      S and part of the HBx sequence can integrate into the host genome enabling the production of both HBsAg and a truncated, but potentially functional, HBx form.
      • Ali A.
      Hepatitis B virus, HBx mutants and their role in hepatocellular carcinoma.
      ,
      • Wooddell C.I.
      • Yuen M.-F.
      • Chan H.L.-Y.
      • Gish R.G.
      • Locarnini S.A.
      • Chavez D.
      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      Due to the fundamental role of cccDNA in the replication cycle of HBV, finding therapies that can directly target cccDNA is pivotal to develop a complete or functional cure for chronic hepatitis B (CHB), defined by cccDNA degradation, lethal mutations (e.g. early stop codons in main ORFs that lead to replication defective virus) or irreversible silencing. Herein, we discuss strategies to directly target cccDNA based on our current knowledge of its biology.
      The next achievable goal of current therapeutic research is achieving a functional cure, namely the suppression of circulating HBsAg, after a finite treatment.

      HBV cccDNA

      Biogenesis

      After internalisation via the cellular receptor sodium-taurocholate cotransporting polypeptide (NTCP),
      • Yan H.
      • Zhong G.
      • Xu G.
      • He W.
      • Jing Z.
      • Gao Z.
      • et al.
      Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
      HBV delivers its 3.2 kb rcDNA genome into the nuclei of the host hepatocyte. Since none of the DNA strands in rcDNA are covalently closed, it has to be “repaired” into the fully double-stranded cccDNA.
      The mechanism of rcDNA-to-cccDNA conversion is not fully understood, and its investigation has been hampered by the absence of robust methods to detect and quantify cccDNA using high-throughput approaches. Formation of cccDNA from rcDNA is a multistep process that begins with the removal of HBV POL leading to protein-free rcDNA (PF-rcDNA, aka deproteinated rcDNA (DP-rcDNA)) formation (Fig. 1). Initial studies suggested an essential role of tyrosyl-DNA phosphodiesterase 2 (TDP2) in this step.
      • Königer C.
      • Wingert I.
      • Marsmann M.
      • Rösler C.
      • Beck J.
      • Nassal M.
      Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses.
      However, other studies indicate that TDP2 may be dispensable; thus, the role of TDP-2 in rcDNA-to-cccDNA formation remains controversial.
      • Cui X.
      • McAllister R.
      • Boregowda R.
      • Sohn J.A.
      • Ledesma F.C.
      • Caldecott K.W.
      • et al.
      Does tyrosyl DNA phosphodiesterase-2 play a role in hepatitis B virus genome repair?.
      • Winer B.Y.
      • Huang T.S.
      • Pludwinski E.
      • Heller B.
      • Wojcik F.
      • Lipkowitz G.E.
      • et al.
      Long-term hepatitis B infection in a scalable hepatic co-culture system.
      • Cai D.
      • Yan R.
      • Xu J.Z.
      • Zhang H.
      • Shen S.
      • Mitra B.
      • et al.
      Characterization of the termini of cytoplasmic hepatitis B virus deproteinated relaxed circular DNA.
      Subsequently, the DNA flap and RNA primer are removed, leading to the final step of “repairing” the single-stranded DNA gap in the plus strand, which results in cccDNA. Several enzymes from the host repair system, including DNA pol κ and α,
      • Qi Y.
      • Gao Z.
      • Xu G.
      • Peng B.
      • Liu C.
      • Yan H.
      • et al.
      DNA polymerase κ is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B virus.
      ,
      • Tang L.
      • Sheraz M.
      • McGrane M.
      • Chang J.
      • Guo J.-T.
      DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA.
      DNA ligases 1 and 3
      • Long Q.
      • Yan R.
      • Hu J.
      • Cai D.
      • Mitra B.
      • Kim E.S.
      • et al.
      The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.
      and Flap endonuclease 1 (FEN-1),
      • Kitamura K.
      • Que L.
      • Shimadu M.
      • Koura M.
      • Ishihara Y.
      • Wakae K.
      • et al.
      Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus.
      provide all the activities required in this transformation. Notably, 5 cellular factors involved in DNA lagging strand synthesis are essential and sufficient for rcDNA-to-cccDNA conversion in vitro: proliferating cell nuclear antigen (PCNA), PCNA-loading replication factor complex (RFC), Pol δ, FEN-1 and DNA ligase 1.
      • Wei L.
      • Ploss A.
      Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.
      Interestingly, recent evidence suggests that differential repair of the minus and plus-strand of rcDNA requires different sets of human repair factors.
      • Wei L.
      • Ploss A.
      Hepatitis B virus cccDNA is formed through distinct repair processes of each strand.
      Nonetheless, cccDNA formation in vivo warrants further study, as it may be more complex and require additional host factors.
      HBV infection leads to the establishment of a pool of viral cccDNA in the nuclei of infected cells that is responsible for viral persistence.
      Figure thumbnail gr1
      Fig. 1cccDNA biogenesis.
      cccDNA biogenesis requires removal of the viral polymerase (POL) generating protein-free rcDNA (PF-rcDNA, aka DP-rcDNA). The DNA flap and RNA primer should be eliminated and the single-stranded DNA gap in the plus strand repaired to obtain the cccDNA molecule. A non-exhaustive list of the enzymes from the host-repair system potentially involved in cccDNA biogenesis are indicated in each step.
      cccDNA, covalently closed circular DNA; FEN-1, Flap endonuclease 1; rcDNA, relaxed circular DNA; TDP2, tyrosyl-DNA phosphodiesterase 2.
      NAs do not target the rcDNA-to-cccDNA conversion, thus reducing cccDNA levels using NA treatment would only be achieved if the precursor of cccDNA, rcDNA, is completely deleted. However, since NA treatment does not achieve complete suppression of reverse transcriptase activity, resulting in incomplete inhibition of rcDNA synthesis, the pool of cccDNA can still be replenished.
      • Boyd A.
      • Lacombe K.
      • Lavocat F.
      • Maylin S.
      • Miailhes P.
      • Lascoux-Combe C.
      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      Therefore, elucidating the complete list of host factors required for the conversion of rcDNA-to-cccDNA, as well as identifying antivirals that more profoundly inhibit viral DNA synthesis, are essential steps in the search for druggable viral targets that can be effectively targeted to prevent cccDNA pool replenishment via nuclear import of rcDNA from the cytoplasm, e.g. intracellular recycling, or via de novo infection of new hepatocytes.

      cccDNA chromatinisation and transcriptional regulation

      cccDNA is wrapped around nucleosomes containing core histones 3 (H3), H4, H2A and H2B, and is associated with viral core protein (HBc), HBx and host transcription factors in the nuclei of infected hepatocytes, forming a highly stable minichromosome.
      • Bock C.T.
      • Schranz P.
      • Schröder C.H.
      • Zentgraf H.
      Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell.
      • Bock C.T.
      • Schwinn S.
      • Locarnini S.
      • Fyfe J.
      • Manns M.P.
      • Trautwein C.
      • et al.
      Structural organization of the hepatitis B virus minichromosome.
      • Belloni L.
      • Pollicino T.
      • De Nicola F.
      • Guerrieri F.
      • Raffa G.
      • Fanciulli M.
      • et al.
      Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.
      • Tropberger P.
      • Mercier A.
      • Robinson M.
      • Zhong W.
      • Ganem D.E.
      • Holdorf M.
      Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.
      • Protzer U.
      Epigenetic control of HBV by HBx protein—releasing the break?.
      The mechanism of chromatin compaction, histone deposition and epigenetic regulation of cccDNA are still ill-defined. Like host genes, cccDNA is also subject to epigenetic regulation involving histone modifiers, chromatin remodellers and transcription factors.
      • Bernstein B.E.
      • Meissner A.
      • Lander E.S.
      The mammalian epigenome.
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      • Hong X.
      • Kim E.S.
      • Guo H.
      Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B: Hong, Kim, and Guo.
      Epigenetic regulation of HBV gene expression involves H3/H4-acetylation or methylation (e.g. H3 lysine4 trimethylation), which serve as activation marks, facilitating chromatin accessibility and enabling gene transcription. Conversely, histone hypoacetylation and/or methylation (e.g. H3 lysine27 trimethylation) leads to a more compact chromatin, silencing cccDNA transcription.
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      Accordingly, high viremia in patients with CHB correlates with hyperacetylation of cccDNA-associated H3/H4, potentially causing the opening of cccDNA chromatin, making it accessible to transcription factors and enabling efficient HBV transcription.
      • Hong X.
      • Kim E.S.
      • Guo H.
      Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B: Hong, Kim, and Guo.
      Viral proteins also play a role in the transcriptional activity of cccDNA. HBc is a structural component that increases cccDNA compaction,
      • Bock C.T.
      • Schwinn S.
      • Locarnini S.
      • Fyfe J.
      • Manns M.P.
      • Trautwein C.
      • et al.
      Structural organization of the hepatitis B virus minichromosome.
      yet de novo synthesis of HBc may not be required for cccDNA transcription and its role in cccDNA stability in vitro is still debated.
      • Tu T.
      • Zehnder B.
      • Qu B.
      • Urban S.
      De novo synthesis of Hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA.
      It is possible that HBc remains associated to rcDNA after nuclear uncoating, bypassing the need for de novo synthesis. cccDNA-associated HBx is essential for its transcriptional activity: mutants lacking HBx infect cells but its cccDNA is silenced.
      • Lucifora J.
      • Arzberger S.
      • Durantel D.
      • Belloni L.
      • Strubin M.
      • Levrero M.
      • et al.
      Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection.
      This silencing correlates with cccDNA-associated H3-hypoacetylation and a more compact/less accessible minichromosome. In the absence of HBx, the histone-lysine N-methyltransferase SET domain bifurcated histone-lysine N-methyltransferase 1 (SETDB1) is involved in cccDNA silencing, which can be overcome by reintroducing HBx.
      • Rivière L.
      • Gerossier L.
      • Ducroux A.
      • Dion S.
      • Deng Q.
      • Michel M.-L.
      • et al.
      HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.
      Additionally, cccDNA can be recognised and silenced by the structural maintenance of chromosomes complex 5/6 (Smc5/6). HBx counteracts the action of Smc5/6 on cccDNA by promoting its degradation via its binding partner, DNA damage-binding protein 1 (DDB1); HBx acts as a scaffold that enables the assembly of HBx-DDB1-CRL4 complexes, leading to ubiquitylation and degradation of the Smc5/6.
      • Brown J.S.
      • Jackson S.P.
      Ubiquitylation, neddylation and the DNA damage response.
      • Murphy C.M.
      • Xu Y.
      • Li F.
      • Nio K.
      • Reszka-Blanco N.
      • Li X.
      • et al.
      Hepatitis B virus X protein promotes degradation of SMC5/6 to enhance HBV replication.
      • Decorsière A.
      • Mueller H.
      • van Breugel P.C.
      • Abdul F.
      • Gerossier L.
      • Beran R.K.
      • et al.
      Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
      • Niu C.
      • Livingston C.M.
      • Li L.
      • Beran R.K.
      • Daffis S.
      • Ramakrishnan D.
      • et al.
      The Smc5/6 complex restricts HBV when localized to ND10 without inducing an innate immune response and is counteracted by the HBV X protein shortly after infection.
      It is unclear how the first molecules of HBx are produced; it is possible that early in infection, low cccDNA levels are not detected by Smc5/6, allowing for a first transcriptional wave to produce HBx, efficiently downregulating Smc5/6. Alternatively, HBx mRNA molecules found in Dane particles
      • Stadelmayer B.
      • Diederichs A.
      • Chapus F.
      • Rivoire M.
      • Neveu G.
      • Alam A.
      • et al.
      Full-length 5’RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum.
      could be functional, producing the first HBx molecules.
      Besides being regulated by associated histone modifications, cccDNA contains 2 or 3 CpG islands, depending on the HBV genotype, strategically placed in regulatory regions.
      • Kim J.-W.
      • Lee S.H.
      • Park Y.S.
      • Hwang J.-H.
      • Jeong S.-H.
      • Kim N.
      • et al.
      Replicative activity of hepatitis B virus is negatively associated with methylation of covalently closed circular DNA in advanced hepatitis B virus infection.
      While rcDNA is mostly unmethylated in serum and cytosol, nuclear HBV DNA methylation varies and could be associated with transcriptional repression. CpG2, located in the HBx promoter, is minimally methylated during active cccDNA transcription and methylation of CpG2 is associated with lower viremia and HBsAg production.
      • Zhang Y.
      • Mao R.
      • Yan R.
      • Cai D.
      • Zhang Y.
      • Zhu H.
      • et al.
      Transcription of hepatitis B virus covalently closed circular DNA is regulated by CpG methylation during chronic infection.
      Thus, DNA methylation could potentially control the transcriptional activity of cccDNA.

      cccDNA intracellular maintenance

      Newly synthesised HBc forms nucleocapsids that encapsidate pgRNA and allow the HBV reverse transcriptase to form rcDNA. Encapsidated rcDNA can be enveloped and released from cells as Dane particles that can infect new cells or be recycled back to the nucleus, where the rcDNA is transformed into cccDNA. Both phenomena lead to the replenishment of the nuclear cccDNA pool, either at the single cell level or by increasing the number of infected cells. It is accepted that nuclear levels of cccDNA are highly stable, however, single-molecule cccDNA maintenance remains controversial.
      A static cccDNA maintenance model suggests that as rcDNA nuclear import and rcDNA-to-cccDNA conversion decline, cccDNA levels are maintained by repressing de novo cccDNA formation/degradation (Fig. 2).
      • Lutgehetmann M.
      • Volz T.
      • Köpke A.
      • Broja T.
      • Tigges E.
      • Lohse A.W.
      • et al.
      In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice.
      • Zhu Y.
      • Yamamoto T.
      • Cullen J.
      • Saputelli J.
      • Aldrich C.E.
      • Miller D.S.
      • et al.
      Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.
      • Dandri M.
      • Burda M.R.
      • Will H.
      • Petersen J.
      Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir in vitro do not lead to reduction of the closed circular DNA.
      Consistent with this hypothesis, NAs indirectly affecting cccDNA levels by inhibiting de novo production of rcDNA, lead to minor effects on the nuclear pool of cccDNA. Translational studies indicate that intrahepatic cccDNA levels vary across CHB phases, with 1-10 copies/cell in HBeAg-positive patients and 1-2 logs less in HBeAg-negative patients.
      • Laras A.
      • Koskinas J.
      • Dimou E.
      • Kostamena A.
      • Hadziyannis S.J.
      Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      • Lebossé F.
      • Testoni B.
      • Fresquet J.
      • Facchetti F.
      • Galmozzi E.
      • Fournier M.
      • et al.
      Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.
      Observations from patients with CHB on NA treatment suggested that complete cccDNA clearance could take decades,
      • Lai C.-L.
      • Wong D.
      • Ip P.
      • Kopaniszen M.
      • Seto W.-K.
      • Fung J.
      • et al.
      Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.
      ,
      • Boyd A.
      • Lacombe K.
      • Lavocat F.
      • Maylin S.
      • Miailhes P.
      • Lascoux-Combe C.
      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      ,
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      ,
      • Gordon S.C.
      • Krastev Z.
      • Horban A.
      • Petersen J.
      • Sperl J.
      • Dinh P.
      • et al.
      Efficacy of tenofovir disoproxil fumarate at 240 weeks in patients with chronic hepatitis B with high baseline viral load: Hepatology.
      with most patients on long-term therapy (>3 years) still harbouring low levels of cccDNA in their livers (1-10 copies/103 cells).
      • Lebossé F.
      • Inchauspé A.
      • Locatelli M.
      • Miaglia C.
      • Diederichs A.
      • Fresquet J.
      • et al.
      Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
      ,
      • Lai C.-L.
      • Wong D.
      • Ip P.
      • Kopaniszen M.
      • Seto W.-K.
      • Fung J.
      • et al.
      Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.
      ,
      • Boyd A.
      • Lacombe K.
      • Lavocat F.
      • Maylin S.
      • Miailhes P.
      • Lascoux-Combe C.
      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      This suggests that nuclear import of de novo synthesised rcDNA plays a minor role in maintenance of the cccDNA pool and that nuclear cccDNA is highly stable.
      • Moraleda G.
      • Saputelli J.
      • Aldrich C.E.
      • Averett D.
      • Condreay L.
      • Mason W.S.
      Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.
      Figure thumbnail gr2
      Fig. 2cccDNA kinetics and stability.
      A dynamic model, summarised in the left panel assumes constant cccDNA degradation and formation. Both intracellular recycling of rcDNA to the nuclei of infected cells to form novel cccDNA and de novo infection could contribute to the maintenance of the cccDNA pool, while the mechanisms ruling its degradation are not currently known. On the right panel a static model of cccDNA maintenance is schemed in which nuclear cccDNA is stable. Thus, rcDNA recycling and de novo infections are not essential to maintain the cccDNA pool. In both models, cell division can lead to cccDNA dilution, thus reducing its levels in the infected liver. cccDNA, covalently closed circular DNA; NC, nucleocapsid; pgRNA, pregenomic RNA; rcDNA, relaxed circular DNA.
      Recent evidence defied the static cccDNA maintenance model and demonstrated persistence of residual levels of HBV replication during NA treatment, indicating that the cccDNA pool is maintained by renewal. Furthermore, serum from patients with CHB and low viremia (under NA treatment) is still infectious in chimeric mice, suggesting that dynamic turnover, i.e. constant cccDNA formation/degradation, could explain the maintenance of cccDNA levels (Fig. 2).
      • Zhu Y.
      • Yamamoto T.
      • Cullen J.
      • Saputelli J.
      • Aldrich C.E.
      • Miller D.S.
      • et al.
      Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.
      ,
      • Burdette D.
      • Cathcart A.
      • Shauf A.
      • Win R.
      • Zaboli S.
      • Hedskog C.
      • et al.
      PS-150-Evidence for the presence of infectious virus in the serum from chronic hepatitis B patients suppressed on nucleos (t)ide therapy with detectable but not quantifiable HBV DNA.
      ,
      • Huang Q.
      • Zhou B.
      • Cai D.
      • Zong Y.
      • Wu Y.
      • Liu S.
      • et al.
      Rapid turnover of hepatitis B virus covalently closed circular DNA indicated by monitoring emergence and reversion of signature-mutation in treated chronic hepatitis B patients.
      In this dynamic model, constant degradation and de novo synthesis of cccDNA maintains stable nuclear copy numbers.
      • Ko C.
      • Chakraborty A.
      • Chou W.-M.
      • Hasreiter J.
      • Wettengel J.M.
      • Stadler D.
      • et al.
      Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
      Supporting this model, retrospective studies on liver biopsies from patients treated with NAs estimated cccDNA turnover rates to be between 5-11 weeks in infected livers.
      • Huang Q.
      • Zhou B.
      • Cai D.
      • Zong Y.
      • Wu Y.
      • Liu S.
      • et al.
      Rapid turnover of hepatitis B virus covalently closed circular DNA indicated by monitoring emergence and reversion of signature-mutation in treated chronic hepatitis B patients.
      The underlying mechanism of cccDNA clearance and maintenance remains unclear: cccDNA destruction could occur dependently
      • Guo J.-T.
      • Zhou H.
      • Liu C.
      • Aldrich C.
      • Saputelli J.
      • Whitaker T.
      • et al.
      Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections.
      • Summers J.
      • Jilbert A.R.
      • Yang W.
      • Aldrich C.E.
      • Saputelli J.
      • Litwin S.
      • et al.
      Hepatocyte turnover during resolution of a transient hepadnaviral infection.
      • Mason W.S.
      • Jilbert A.R.
      • Summers J.
      Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection.
      or independently of infected hepatocyte death.
      • Guidotti L.G.
      Viral clearance without destruction of infected cells during acute HBV infection.
      • Wieland S.F.
      • Spangenberg H.C.
      • Thimme R.
      • Purcell R.H.
      • Chisari F.V.
      Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees.
      • Murray J.M.
      • Wieland S.F.
      • Purcell R.H.
      • Chisari F.V.
      Dynamics of hepatitis B virus clearance in chimpanzees.
      Given the constraints preventing single cell cccDNA quantification in vivo, these studies presented data as an average of cccDNA molecules per cell in the infected liver, instead of the actual cccDNA copy number per infected hepatocyte or its temporal distribution, thus hampering the assessment of single nuclei cccDNA stability. Therefore, it is still currently unknown if recycling of rcDNA to form cccDNA is an infrequent event in response to cccDNA loss, or if it is a permanent event counteracting constant cccDNA degradation, in vivo.
      cccDNA does not follow a semiconservative replication and is not tethered to chromosomes; thus, its fate after cell division remains controversial. Studies in HBV-infected liver-humanised mice, in the context of increased hepatocyte proliferation and efficient inhibition of viral reinfection, showed a reduction of the cccDNA pool, suggesting cccDNA loss upon cell division.
      • Allweiss L.
      • Volz T.
      • Giersch K.
      • Kah J.
      • Raffa G.
      • Petersen J.
      • et al.
      Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.
      cccDNA labelling by fluorescence in situ hybridisation in the presence of NAs suggested that cccDNA is asymmetrically distributed to daughter cells, instead of being lost or duplicated during cell division.
      • Li M.
      • Sohn J.A.
      • Seeger C.
      Distribution of hepatitis B virus nuclear DNA.
      Thus, in the presence of NAs, loss of cccDNA relies on the rate of infected hepatocyte death and further dilution by cell division. Accordingly, recent in vitro data in HepG2-NTCP cells suggested that cccDNA turnover is linked to the turnover of infected hepatocytes.
      • Tu T.
      • Zehnder B.
      • Qu B.
      • Urban S.
      De novo synthesis of Hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA.
      Elimination of cccDNA by agents targeting its biogenesis may thus be possible if the reduction in cccDNA levels is faster than its generation and if new cccDNA formation is effectively blocked. Thus, understanding the turnover time and fate of the cccDNA pool in infected hepatocytes is key in the design of antiviral strategies.
      Currently recommended therapies for HBV efficiently achieve viral suppression and improve patients’ quality of life; however, they are unable to eliminate the viral minichromosome and thus, to cure CHB.

      Modeling cccDNA kinetics in patients

      When a therapy intended to cure HBV is given to an individual, there is interest in determining the rate at which the cccDNA pool decreases, as this could provide some indication of when eradication can be achieved. This can be studied by modelling cccDNA levels as an exponential function of decay, from which we are able to estimate the half-life of cccDNA levels, i.e. the time taken to halve the cccDNA level at its initial value. Ideally, serial liver biopsies from individuals (or experimental animals) taken at several time points during treatment would be used to measure i) cccDNA quantity through PCR-based assays and ii) the total number of cells per sample. cccDNA copies/cells would be modelled as a function over time, which has been done previously in woodchucks
      • Dandri M.
      • Burda M.R.
      • Will H.
      • Petersen J.
      Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir in vitro do not lead to reduction of the closed circular DNA.
      and ducks.
      • Addison W.R.
      • Walters K.-A.
      • Wong W.W.S.
      • Wilson J.S.
      • Madej D.
      • Jewell L.D.
      • et al.
      Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo following inhibition of viral replication.
      If serial samples are unavailable, the copies/cells could be measured cross-sectionally at different time points and the same models applied. This was done in a previous study of HIV-HBV-coinfected individuals to estimate the half-life of cccDNA during treatment with tenofovir disoproxil fumarate.
      • Boyd A.
      • Lacombe K.
      • Lavocat F.
      • Maylin S.
      • Miailhes P.
      • Lascoux-Combe C.
      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      However, these models assume a constant exponential rate of cccDNA decline, which might not be the most appropriate function if levels stabilise at a certain point during treatment. These models are also unable to accommodate a number of other factors that might govern changes in cccDNA levels. For instance, impaired innate immunity,
      • Lebossé F.
      • Testoni B.
      • Fresquet J.
      • Facchetti F.
      • Galmozzi E.
      • Fournier M.
      • et al.
      Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.
      genome recycling during replication, secondary infections of neighbouring hepatocytes
      • Ko C.
      • Chakraborty A.
      • Chou W.-M.
      • Hasreiter J.
      • Wettengel J.M.
      • Stadler D.
      • et al.
      Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
      and clearance of infected hepatocytes
      • Tu T.
      • Zehnder B.
      • Qu B.
      • Urban S.
      De novo synthesis of Hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA.
      may all impact on the effectiveness of clearing cccDNA during therapy. There have been attempts to more thoroughly understand cccDNA half-life while simultaneously modelling the rates of these other “compartments”, namely cytotoxic T-lymphocytes (CTL) and non-CTL immune responses,
      • Murray J.M.
      • Goyal A.
      In silico single cell dynamics of hepatitis B virus infection and clearance.
      integration of HBV DNA,
      • Goyal A.
      • Chauhan R.
      The dynamics of integration, viral suppression and cell-cell transmission in the development of occult Hepatitis B virus infection.
      and cell-to-cell transmission of viral particles.
      • Goyal A.
      • Murray J.M.
      Modelling the impact of cell-to-cell transmission in hepatitis B virus.
      These models usually involve sets of ordinary differential equations,
      • Goyal A.
      • Liao L.E.
      • Perelson A.S.
      Within-host mathematical models of hepatitis B virus infection: past, present, and future.
      but have rarely extended to modelling cccDNA levels during therapy. These models could be used to establish potential phases of cccDNA decline given the different compartments leading to cccDNA persistence, and the combinations of direct and indirect therapies that could lead to faster cccDNA declines. Still, they are limited by the large quantities of inputs, missing inputs and the large numbers of assumptions required.
      Liver biopsy samples have become highly unpopular over the past decade, with non-invasive measures of liver fibrosis becoming the norm; thus, the ability to conduct a study with these ideal conditions is almost impossible. Alternative markers whose changes bear strong correlation with cccDNA changes could be used to shed light on cccDNA half-lives without the need for liver samples. Hepatitis B core-related antigen (HBcrAg) and circulating HBV RNAs are potential candidates.
      • Charre C.
      • Levrero M.
      • Zoulim F.
      • Scholtès C.
      Non-invasive biomarkers for chronic hepatitis B virus infection management.
      Interestingly, the proportion of lamivudine-resistant variants during lamivudine treatment, according to HBV RNAs, was used as a close proxy of the turnover rate of cccDNA pools, and hence cccDNA half-life in HBeAg-positive patients. The observed half-lives in that study ranged between 6.9–21.7 weeks, which is much shorter than expected.
      • Huang Q.
      • Zhou B.
      • Cai D.
      • Zong Y.
      • Wu Y.
      • Liu S.
      • et al.
      Rapid turnover of hepatitis B virus covalently closed circular DNA indicated by monitoring emergence and reversion of signature-mutation in treated chronic hepatitis B patients.
      These estimations came from individuals who likely had high transaminase levels, which could result in more rapid changes in cccDNA levels. Whether such a proxy could be used to estimate cccDNA levels during HBeAg-negative infection, especially when HBV-DNA levels are undetectable and transaminase levels are low, is uncertain. Despite improvements in these proxies, the potential for error remains significant.

      cccDNA targeting

      Targeting cccDNA formation

      cccDNA biosynthesis represents an attractive target to reduce the cccDNA pool in the nuclei of infected hepatocytes. However, understanding the complete list of host factors required in the rcDNA-to-cccDNA conversion is essential. One limitation for these studies is the functional redundancy among DNA repair components: residual levels of the targeted proteins, or their paralogue(s), may be sufficient to execute their function. Notwithstanding, functional redundancy exists because these factors are essential in cell homeostasis, so their complete knock-down is unachievable and they do not represent a potential therapeutic target in vivo. Hence, approaches that can specifically target the viral components or specific viral/host factor interactions, without affecting the essential functions of host proteins, are needed. Small molecules blocking rcDNA deproteination have been described, however their antiviral target and mechanism of action remains to be determined.
      • Cai D.
      • Mills C.
      • Yu W.
      • Yan R.
      • Aldrich C.E.
      • Saputelli J.R.
      • et al.
      Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation.
      ,
      • Liu C.
      • Cai D.
      • Zhang L.
      • Tang W.
      • Yan R.
      • Guo H.
      • et al.
      Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA.
      Full understanding of cccDNA biology remains essential to find potential druggable targets and to develop potentially curative HBV-targeted therapies.

      Targeting established cccDNA

      Direct cccDNA targeting

      Achieving complete or functional HBV cure requires direct cccDNA targeting; thus, strategies leading to its degradation, lethal mutations or irreversible silencing in a non-cytolytic manner are greatly needed. Recent efforts identified a novel small molecule that reduced cccDNA levels in HBV-infected primary human hepatocytes (PHHs) and in mouse liver after hydrodynamic injection with an HBV minicircle.
      • Wang L.
      • Zhu Q.
      • Zeng J.
      • Yan Z.
      • Feng A.
      • Young J.
      • et al.
      PS-074-A first-in-class orally available HBV cccDNA destabilizer ccc_R08 achieved sustainable HBsAg and HBV DNA suppression in the HBV circle mouse model through elimination of cccDNA-like molecules in the mouse liver.
      This data is encouraging, though the safety profile and mechanism of action of this molecule still need to be elucidated to determine if it directly targets cccDNA, or if cccDNA decline is an indirect effect of perturbed cellular processes.
      Gene-editing approaches can disrupt the viral genome in a permanent way (Fig. 3): Zinc-Finger nucleases (ZFNs)
      • Cradick T.J.
      • Keck K.
      • Bradshaw S.
      • Jamieson A.C.
      • McCaffrey A.P.
      Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs.
      and transcriptional activator-like effector nucleases (TALENs)
      • Bloom K.
      • Ely A.
      • Mussolino C.
      • Cathomen T.
      • Arbuthnot P.
      Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.
      target and cleave HBV DNA sequences. Target DNA analysis showed insertion-deletions (indels) in the target sequences, consistent with imprecise rejoining by the non-homologous end joining (NHEJ) repair system and a drop in the levels of viral parameters. Precision Bioscience’s meganuclease-HBV program (HBV-ARCUS) targets the viral POL leading to durable antigen loss in Hep3B cells containing integrated copies of the HBV genome and in HBV-infected PHHs (P-1057
      2020 ASGCT annual meeting abstracts.
      ). In vivo studies in an AAV-episomal mouse model showed robust editing in liver tissues. Being smaller than ZFNs and TALENs, meganucleases are easier to deliver. However, comprehensive screening of efficient target sequences using meganucleases is difficult given their labour-intensive production; thus, only a few selected target sequences were evaluated and more efficient target sites may have been missed.
      Figure thumbnail gr3
      Fig. 3Novel cccDNA targeting approaches and their expected mechanism of action.
      (A) Irreversible cccDNA silencing or degradation would lead to a sustained reduction of pgRNA and viral antigen production. (B) Permanent cccDNA alterations inducing lethal mutations leading to suppression of viral antigen production. In this scenario cccDNA levels will not be directly affected, unless high liver inflammation leads to increased cell division, “diluting” cccDNA. (C) Reversible cccDNA silencing leading to low viral antigen production intervals that could enable T-cell restoration. Once the HBV-specific CTL are re-established it is conceivable that they could control the infection, although it is possible that a CTL boost may be necessary. APOBEC, apolipoprotein B mRNA-editing enzyme; cccDNA, covalently closed circular DNA; CTL, cytotoxic T lymphocyte; pgRNA, pregenomic RNA; rcDNA, relaxed circular DNA.
      Since the discovery of CRISPR-Cas9 applications, gene-editing programmes have been advancing into clinics.
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
      • Ran F.A.
      • Hsu P.D.
      • Wright J.
      • Agarwala V.
      • Scott D.A.
      • Zhang F.
      Genome engineering using the CRISPR-Cas9 system.
      • Ledford H.
      • Callaway E.
      Pioneers of revolutionary CRISPR gene editing win chemistry Nobel.
      CRISPR-Cas9 can be redirected to DNA sequences by redesigning the guide RNAs (gRNAs) complementary to the desired target. Cas9 protein generates double stranded breaks (DSBs) that are often imperfectly repaired by the NHEJ, disrupting the target sequence. Several publications presented proof-of-concept that CRISPR-Cas9 can target HBV DNA.
      • Bloom K.
      • Maepa M.
      • Ely A.
      • Arbuthnot P.
      Gene therapy for chronic HBV—can we eliminate cccDNA?.
      However, different infection models lead to different outcomes, from HBV genome degradation to repair. Chromatin status in cccDNA, plasmids containing HBV genome and integrated HBV DNA can vary, leading to differential accessibilities that could account for the diverse outcomes observed after CRISPR-Cas9 editing.
      • Tropberger P.
      • Mercier A.
      • Robinson M.
      • Zhong W.
      • Ganem D.E.
      • Holdorf M.
      Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.
      CpG methylation can lead to recruitment of other factors, particularly in CpG islands, that could hinder Cas9 binding.
      • Verkuijl S.A.
      • Rots M.G.
      The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies.
      Nucleosomes strongly impair Cas9 binding to target sequences in cell-free assays. However, in vivo, nucleosomes are dynamic and sequences can regain accessibility due to nucleosome remodelling and breathing, where nucleosomal DNA is in equilibrium between wrapped and unwrapped states, allowing protein access to binding sites hidden by nucleosomes.
      • Isaac R.S.
      • Jiang F.
      • Doudna J.A.
      • Lim W.A.
      • Narlikar G.J.
      • Almeida R.
      Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.
      Despite the interest in designer nuclease approaches, several considerations should be addressed before their clinical application in CHB. Our group recently showed that CRISPR-Cas9 can target cccDNA in HBV-infected hepatocytes leading to cccDNA degradation and repair.
      • Martinez M.G.
      • Inchauspe A.
      • Delberghe E.
      • Chapus F.
      • Neveu G.
      • Alam A.
      • et al.
      SAT376 - targeting hepatitis B virus with CRISPR/Cas9 approach.
      However, HBV DNA variants were generated after dual gRNA targeting, highlighting the importance of understanding the fate of cccDNA after gene editing. 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.
      • Stone D.
      • Long K.R.
      • Loprieno M.A.
      • De Silva Feelixge H.S.
      • Kenkel E.J.
      • Liley R.M.
      • et al.
      CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice.
      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. However, pre-existing immunity to Cas9 could be a major obstacle and understanding the immunogenicity of Cas proteins in humans is essential: data on this topic are mixed, with reports of between 10% and 78% of tested individuals having pre-existing anti-SaCas9 antibodies (S.aureus), and between 2.5% and 58% having anti-SpCas9 antibodies (S.pyogenes).
      • Simhadri V.L.
      • McGill J.
      • McMahon S.
      • Wang J.
      • Jiang H.
      • Sauna Z.E.
      Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population.
      ,
      • Charlesworth C.T.
      • Deshpande P.S.
      • Dever D.P.
      • Camarena J.
      • Lemgart V.T.
      • Cromer M.K.
      • et al.
      Identification of preexisting adaptive immunity to Cas9 proteins in humans.
      Pre-existing immunity led to elimination of genome-edited cells in mice, however the outcome in humans is difficult to predict.
      • Li A.
      • Tanner M.R.
      • Lee C.M.
      • Hurley A.E.
      • De Giorgi M.
      • Jarrett K.E.
      • et al.
      AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9.
      Off-target effects are also a concern: the extent of these events is still unclear and better approaches to assess these events are needed. Another critical step is delivery: achieving long-lasting effects in the viral genome with minimal off-target effects will require efficient, and preferentially transient, delivery systems to target all infected hepatocytes.
      • Rouet R.
      • de Oñate L.
      • Li J.
      • Murthy N.
      • Wilson R.C.
      Engineering CRISPR-Cas9 RNA–protein complexes for improved function and delivery.
      The use of in vitro transcribed mRNAs, as opposed to viral vectors, to express the gene editors is associated with several advantages, including avoiding the risk of recombination with host DNA which exists with viral vectors.
      Nuclease approaches share a common disadvantage in relation to targeting the HBV genome: that is, they all lead to DSBs. Given the high sequence similarity between cccDNA, rcDNA and integrated HBV DNA, using designer-nuclease approaches will generate DSBs in the host genome, potentially risking genomic instability, chromosomal recombination and iatrogenesis.
      • Kosicki M.
      • Tomberg K.
      • Bradley A.
      Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.
      Conveniently, gene-editing tools are already showing a clear evolution as exemplified by base and prime editing. Both of these approaches lead to DNA rewriting without cleavage, allowing for control of the sequence outcome and reducing the risk of host genome rearrangements.
      • Rees H.A.
      • Liu D.R.
      Base editing: precision chemistry on the genome and transcriptome of living cells.
      ,
      • Anzalone A.V.
      • Koblan L.W.
      • Liu D.R.
      Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors.
      Yan et al. presented first proof-of-concept that cytosine base editors targeting the S gene sequence lead to a reduction of HBsAg and extracellular HBV DNA in HepG2.2.15 and HBV-infected HepG2-NTCP-C4 cells.
      • Yang Y.-C.
      • Chen Y.-H.
      • Kao J.-H.
      • Ching C.
      • Liu I.-J.
      • Wang C.-C.
      • et al.
      Permanent inactivation of HBV genomes by CRISPR/Cas9-Mediated non-cleavage base editing.
      These studies are encouraging as these approaches can potentially lead to the safe targeting of cccDNA and integrated HBV DNA (Fig. 3B). However, further studies evaluating the direct effect of base editors on cccDNA and its fate after editing are essential.
      • Yu Y.
      • Leete T.C.
      • Born D.A.
      • Young L.
      • Barrera L.A.
      • Lee S.-J.
      • et al.
      Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity.
      Assuming concerns regarding the use of gene/base editors to treat CHB are resolved, and given the extensive number of people chronically infected with HBV, careful evaluation of product development will be required to ensure unrestricted access to the treatment. In this regard, production of in vitro transcribed mRNA may provide an economic advantage, however development of mass-production technology at low cost will be crucial.
      Promising direct cccDNA targeting approaches aimed at reducing or silencing the viral minichromosome reservoir need to overcome delivery, safety and feasibility issues.

      Targeting cellular factors that can directly affect cccDNA

      Apolipoprotein B mRNA-editing enzyme 3 (APOBEC3) members recognise and deaminate foreign DNA, leading to C-to-T/G-to-A hypermutations and protecting against viral infection.
      • Love R.P.
      • Xu H.
      • Chelico L.
      Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A.
      Human APOBEC3s are activated by cytokines, such as IFNs, produced upon detection of foreign DNA. APOBEC 3A and 3B (A3A and A3B) overexpression leads to deamination and destruction of HBV DNA (Fig. 3),
      • Stenglein M.D.
      • Burns M.B.
      • Li M.
      • Lengyel J.
      • Harris R.S.
      APOBEC3 proteins mediate the clearance of foreign DNA from human cells.
      and was suggested to cause non-cytotoxic cccDNA deamination.
      • Lucifora J.
      • Xia Y.
      • Reisinger F.
      • Zhang K.
      • Stadler D.
      • Cheng X.
      • et al.
      Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.
      Accordingly, results in HBV-infected HepaRG cells and PHHs suggested that IFN and lymphotoxin beta receptor (LTBR) led to the non-cytolytic clearance of cccDNA via APOBEC upregulation.
      • Guidotti L.G.
      Viral clearance without destruction of infected cells during acute HBV infection.
      ,
      • Lucifora J.
      • Xia Y.
      • Reisinger F.
      • Zhang K.
      • Stadler D.
      • Cheng X.
      • et al.
      Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.
      A3A/B upregulation by HBV-specific CTL-associated cytokines leads to cccDNA minus strand editing, resulting in mismatch mutations and cccDNA degradation.
      • Nair S.
      • Zlotnick A.
      Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.
      CTLs can be engineered to exclusively target HBV-infected hepatocytes or hepatocytes producing antigens from HBV-DNA integrations.
      • Gehring A.J.
      • Xue S.-A.
      • Ho Z.Z.
      • Teoh D.
      • Ruedl C.
      • Chia A.
      • et al.
      Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.
      Given that a substantial number of hepatocytes could be infected during CHB, CTLs could trigger uncontrolled liver damage.
      • Bertoletti A.
      • Brunetto M.
      • Maini M.K.
      • Bonino F.
      • Qasim W.
      • Stauss H.
      T cell receptor-therapy in HBV-related hepatocellularcarcinoma.
      Engineered HBV-specific CTLs lacking cytolytic activity suppressed HBV replication via activation of the intracellular LTBR/APOBEC3 pathway.
      • Koh S.
      • Kah J.
      • Tham C.Y.L.
      • Yang N.
      • Ceccarello E.
      • Chia A.
      • et al.
      Nonlytic lymphocytes engineered to express virus-specific T-cell receptors limit HBV infection by activating APOBEC3.
      However, apoptosis and subsequent cell proliferation were observed upon treatment, supporting the possibility that the effect on cccDNA could be partly due to cell division leading to cccDNA dilution. Furthermore, APOBEC cytosine-deaminase activity preferentially targets single-stranded DNA; thus, replicative intermediates other than cccDNA would be more suitable substrates. The deamination pattern of A3A in HBV replicative intermediates suggested that rcDNA could be the preferential APOBEC substrate.
      • Nair S.
      • Zlotnick A.
      Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.
      Supporting this hypothesis, genomic data from patients showed that deamination occurred preferentially in the single-stranded region of the HBV genome.
      • Suspene R.
      • Guetard D.
      • Henry M.
      • Sommer P.
      • Wain-Hobson S.
      • Vartanian J.-P.
      Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo.
      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.
      • Seeger C.
      • Sohn J.A.
      Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA.
      Thus, while HBV suppression was clearly demonstrated, direct cccDNA targeting by APOBEC remains controversial.
      Strategies to regulate gene expression, such as CRISPR activation (CRISPRa), lead to specific APOBEC overexpression, providing a different tool to evaluate its effect on cccDNA (Fig. 3B-C). Briefly, the promoter of the gene of interest is targeted by a gRNA, locally recruiting a dead Cas9 (dCas9) protein fused to an activation domain.
      • Hilton I.B.
      • D’Ippolito A.M.
      • Vockley C.M.
      • Thakore P.I.
      • Crawford G.E.
      • Reddy T.E.
      • et al.
      Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.
      CRISPRa specifically activates A3A/3B expression, leading to deamination of foreign episomal DNA and the loss of foreign integrated DNA. However, evidence that activation of A3A/B (by CRISPRa) has a direct effect on cccDNA is still lacking. A major drawback of CRISPRa is that overexpression of intracellular deaminases can lead to mutagenesis of the host genome and cancer development,
      • Roberts S.A.
      • Lawrence M.S.
      • Klimczak L.J.
      • Grimm S.A.
      • Fargo D.
      • Stojanov P.
      • et al.
      An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers.
      which warrants careful evaluation.
      Therapies aimed at directly targeting cccDNA, either via degradation, lethal mutations or functional silencing are being actively explored.

      cccDNA epigenetic silencing

      cccDNA transcription is regulated by the host cell epigenetic machinery. IFN-α treatment leads to epigenetic changes in cccDNA-associated histones and recruitment of transcriptional repressors, which could at least partially explain the reduction in cccDNA transcriptional activity. Members of the tripartite motif (TRIM) family expressed upon IFN response, in particular TRIM22, inhibit HBV transcription and replication in a non-cytolytic manner.
      • Gao B.
      • Duan Z.
      • Xu W.
      • Xiong S.
      Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain.
      Pro-inflammatory cytokines had a direct effect on the transcriptional activity of cccDNA, without affecting its levels: IL-6 treatment reduced cccDNA-associated histone acetylation, affecting the transcriptional activity of cccDNA,
      • Palumbo G.A.
      • Scisciani C.
      • Pediconi N.
      • Lupacchini L.
      • Alfalate D.
      • Guerrieri F.
      • et al.
      IL6 inhibits HBV transcription by targeting the epigenetic control of the nuclear cccDNA minichromosome.
      and both IL-1β and IL-6 reduced HBV RNA levels more efficiently than IFN-α in vitro.
      • Isorce N.
      • Testoni B.
      • Locatelli M.
      • Fresquet J.
      • Rivoire M.
      • Luangsay S.
      • et al.
      Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.
      IL-4 and transforming growth factor-β1 affect HBV transcription and replication, however, direct evidence of their effect on cccDNA is lacking.
      • Lin S.-J.
      • Shu P.-Y.
      • Chang C.
      • Ng A.-K.
      • Hu C.
      IL-4 suppresses the expression and the replication of hepatitis B virus in the hepatocellular carcinoma cell line Hep3B.
      ,
      • Hong M.-H.
      • Chou Y.-C.
      • Wu Y.-C.
      • Tsai K.-N.
      • Hu C.
      • Jeng K.-S.
      • et al.
      Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression.
      Understanding the mechanisms and factors involved in the epigenetic regulation of cccDNA could be essential for the development of therapeutic strategies: though cccDNA levels would remain intact, complete transcriptional shutdown should stall HBV production leading to a functional cure.
      • Mitra B.
      • Thapa R.J.
      • Guo H.
      • Block T.M.
      Host functions used by hepatitis B virus to complete its life cycle: implications for developing host-targeting agents to treat chronic hepatitis B.
      cccDNA-associated histones can be directly targeted to silence cccDNA transcription. AGK2, an inhibitor of the histone deacetylase Sirtuin 2 (SIRT2), suppressed cccDNA transcription,
      • Yu H.-B.
      • Jiang H.
      • Cheng S.-T.
      • Hu Z.-W.
      • Ren J.-H.
      • Chen J.
      AGK2, A SIRT2 inhibitor, inhibits hepatitis B virus replication in vitro and in vivo.
      however integrated HBV DNA transcription was enhanced under the same conditions, suggesting different epigenetic regulation of integrated vs. episomal HBV DNA. A KDM5 (lysine demethylase 5) inhibitor led to a global increase in chromatin histone methylation, inhibiting viral RNAs and antigen production in PHHs.
      • Gilmore S.
      • Tam D.
      • Dick R.
      • Appleby T.
      • Birkus G.
      • Willkom M.
      • et al.
      Antiviral activity of GS-5801, a liver-targeted prodrug of a lysine demethylase 5 inhibitor, in a hepatitis B virus primary human hepatocyte infection model.
      PRMT5 (protein arginine methyltransferase 5) was suggested to preferentially regulate cccDNA vs. host chromatin, potentially by interacting directly with HBc.
      • Zhang W.
      • Chen J.
      • Wu M.
      • Zhang X.
      • Zhang M.
      • Yue L.
      • et al.
      PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.
      Treatment with the histone acetyltransferase p300/CBP inhibitor, C646, reduced HBV transcription.
      • Tropberger P.
      • Mercier A.
      • Robinson M.
      • Zhong W.
      • Ganem D.E.
      • Holdorf M.
      Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.
      Though epigenetic modifiers led to an interesting decrease in viral parameters, they could also silence tumour suppressor genes or other essential genes, contributing to hepatocarcinogenesis
      • Fernández-Barrena M.G.
      • Arechederra M.
      • Colyn L.
      • Berasain C.
      • Avila M.A.
      Epigenetics in hepatocellular carcinoma development and therapy: the tip of the iceberg.
      ; thus, their critical balance in cellular homeostasis should be carefully evaluated.
      The role of HBx on the transcriptional activity of cccDNA makes it an interesting therapeutic target.
      • Decorsière A.
      • Mueller H.
      • van Breugel P.C.
      • Abdul F.
      • Gerossier L.
      • Beran R.K.
      • et al.
      Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
      siRNAs targeting HBx mRNA transiently reduced HBx levels, leading to Smc5/6 complex reappeareance.
      • Kornyeyev D.
      • Ramakrishnan D.
      • Voitenleitner C.
      • Livingston C.M.
      • Xing W.
      • Hung M.
      • et al.
      Spatiotemporal analysis of hepatitis B virus X protein in primary human hepatocytes.
      Moreover, a recent study showed that silencing all viral transcripts using a combination of siRNAs and peg-IFNα substantially decreased HBx protein levels, leading to Smc6 rebound in vivo.
      • Allweiss L.
      • Giersch K.
      • Pirosu A.
      • Volz T.
      • Muench R.C.
      • Beran R.K.
      • et al.
      Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.
      Designer nucleases that introduce mutations into the HBx protein could provide a good strategy to achieve this goal. However, targeting integrated HBx DNA variants, which retain their ability to degrade Smc5/6, may be essential to achieve irreversible cccDNA silencing – thus non-DSB-led approaches should be considered.
      • Kornyeyev D.
      • Ramakrishnan D.
      • Voitenleitner C.
      • Livingston C.M.
      • Xing W.
      • Hung M.
      • et al.
      Spatiotemporal analysis of hepatitis B virus X protein in primary human hepatocytes.
      Epigenetic editors are also being explored to silence HBx expression: methylation of the HBx promoter using the catalytic domain of DNMT3a fused to ZF led to a reduction in HBV transcription.
      • Xirong L.
      • Rui L.
      • Xiaoli Y.
      • Qiuyan H.
      • Bikui T.
      • Sibo Z.
      • et al.
      Hepatitis B virus can be inhibited by DNA methyltransferase 3a via specific zinc-finger-induced methylation of the X promoter.
      Small molecules targeting the HBx-DDB1 interaction have been tested as potential antivirals for HBV. Data from HBV-infected PHHs treated with the FDA-approved small molecule nitazoxanide (NTZ) suggested a modest effect on Smc5 rebound and viral parameters and in vitro data suggests that NTZ targets the interphase of HBx-DDB1.
      • Sekiba K.
      • Otsuka M.
      • Ohno M.
      • Yamagami M.
      • Kishikawa T.
      • Suzuki T.
      • et al.
      Inhibition of HBV transcription from cccDNA with nitazoxanide by targeting the HBx–DDB1 interaction.
      Pevonedistat (MLN4924) is a small-molecule inhibitor of NEDD8-activating enzyme E1: NEDD8 is essential for cullin activation, blocking NEDD8 activation, which impairs the HBx-DDB1-ROC1 (CRL4) E3-ligase complex essential for HBx-dependent Smc5/6 degradation, thus blocking the transcriptional activity of cccDNA.
      • Sekiba K.
      • Otsuka M.
      • Ohno M.
      • Yamagami M.
      • Kishikawa T.
      • Seimiya T.
      • et al.
      Pevonedistat, a neuronal precursor cell-expressed developmentally down-regulated protein 8–activating enzyme inhibitor, is a potent inhibitor of hepatitis B virus.
      However, MLN4924 could directly affect HBx, contributing to reduced HBV replication. Evidently, epigenetic regulation could be exploited to control HBV infection (Fig. 3C) and repurposing FDA-approved compounds is an interesting strategy to identify novel antivirals. However, understanding the mechanisms by which these drugs could reduce viral parameters in the context of CHB remains essential.

      Perspectives

      Treatments that could non-cytopathically eradicate cccDNA have not yet reached clinical development, owing to their lack of specificity, inadequate and ineffective delivery approaches and to our incomplete understanding of off-target effects. Approaches aiming to achieve a functional cure, defined as serum viral DNA clearance and loss of HBsAg with or without anti-HBs seroconversion, are under development.
      • Fanning G.C.
      • Zoulim F.
      • Hou J.
      • Bertoletti A.
      Therapeutic strategies for hepatitis B virus infection: towards a cure.
      These approaches aim to evaluate therapeutic combinations that more effectively inhibit HBV replication and viral antigen production, and reinvigorate exhausted immune responses. These combinatorial strategies should result in decreased rcDNA supply and elimination of infected cells, thereby tipping the balance toward cccDNA loss.
      NA and capsid assembly modulator (CAM) combinations are being explored to achieve complete viral suppression: ongoing phase II results have shown continuing declines in serum HBV DNA and pgRNA levels.
      • Yuen M.-F.
      • Agarwal K.
      • Ma X.
      • Nguyen T.
      • Schiff E.R.
      • Hann H.-W.
      • et al.
      Antiviral activity and safety of the hepatitis B core inhibitor ABI-H0731 administered with a nucleos(t)ide reverse transcriptase inhibitor in patients with HBeAg-positive chronic hepatitis B infection in a long-term extension study.
      siRNAs and antisense oligonucleotides (ASOs) are also being explored in combination with NAs: siRNAs JNJ-3989,
      • Gane E.
      • Locarnini S.
      • Lim T.H.
      • Strasser S.
      • Sievert W.
      • Cheng W.
      • et al.
      Short-term treatment with RNA interference therapy, JNJ-3989, results in sustained hepatitis B surface antigen supression in patients with chronic hepatitis B receiving nucleos(t)ide analogue treatment.
      AB-729 or ASOs (GSK-836)
      • Yuen M.-F.
      • Heo J.
      • Jang J.W.
      • Yoon J.-H.
      • Kweon Y.O.
      • Park S.-J.
      • et al.
      Hepatitis B virus (HBV) surface antigen (HBsAg) inhibition with isis 505358 in chronic hepatitis B (CHB) patients on stable nucleos (t)ide analogue (NA) regimen and in NA-naive CHB patients: phase 2a, randomized, double-blind, placebo-controlled study.
      showed positive target engagement associated with decreasing HBsAg levels, which were sustained after treatment withdrawal in some patients. Results from longer term dual and triple therapy trials including a CAM
      • Yuen M.-F.
      • Locarnini S.
      • Given B.
      • Schluep T.
      • Hamilton J.
      • Biermer M.
      • et al.
      First clinical experience with RNA interference-based triple combination therapy in chronic hepatitis B: JNJ-3989, JNJ-6379 and a Nucleos (t) ide analogue.
      are eagerly awaited. The combination of the entry inhibitor bulevirtide with tenofovir and PEG-IFNα to treat chronic hepatitis B-Delta showed strong synergy and reduced HBsAg levels in a substantial number of patients,
      • Wedemeyer H.
      • Schöneweis K.
      • Bogomolov P.O.
      • Chulanov V.
      • Stepanova T.
      • Viacheslav M.
      • et al.
      48 weeks of high dose (10 mg) bulevirtide as monotherapy or with peginterferon alfa-2a in patients with chronic HBV/HDV co-infection.
      suggesting functional cure of HBV might be achievable. Similar results were obtained by triple combination of NAs, peg-IFNa and nucleic acid polymers,
      • Bazinet M.
      • Pântea V.
      • Placinta G.
      • Moscalu I.
      • Cebotarescu V.
      • Cojuhari L.
      • et al.
      Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naïve to nucleos(t)ide therapy.
      however studies in larger cohorts are essential to evaluate the safety and efficacy of these combinations. Increasing evidence suggests that decreasing HBsAg levels is not enough to restore anti-HBV immune responses; therefore, combinations of NAs and innate immunity boosters,
      • Gane E.
      • Dunbar P.R.
      • Brooks A.
      • Zhao Y.
      • Tan S.
      • Lau A.
      • et al.
      Efficacy and safety of 24 weeks treatment with oral TLR8 agonist, selgantolimod, in virally-suppressed adult patients with chronic hepatitis B: a phase 2 study.
      NAs and therapeutic vaccines,
      • Michler T.
      • Kosinska A.D.
      • Festag J.
      • Bunse T.
      • Su J.
      • Ringelhan M.
      • et al.
      Knockdown of virus antigen expression increases therapeutic vaccine efficacy in high-titer hepatitis B virus carrier mice.
      or NAs in combination with checkpoint inhibitors and therapeutic vaccines
      • Gane E.
      • Verdon D.J.
      • Brooks A.E.
      • Gaggar A.
      • Nguyen A.H.
      • Subramanian G.M.
      • et al.
      Anti-PD-1 blockade with nivolumab with and without therapeutic vaccination for virally suppressed chronic hepatitis B: a pilot study.
      are currently under evaluation. In a scenario where persistence of transcriptionally active cccDNA remains the mainstay, sustained restoration of immune control would be needed. It may rely on complex combinations of viral replication and antigen expression inhibitors together with immune invigoration and stimulation by therapeutic vaccines.
      • Maini M.K.
      • Pallett L.J.
      Defective T-cell immunity in hepatitis B virus infection: why therapeutic vaccination needs a helping hand.
      Exciting approaches to directly target cccDNA include gene-editing,
      • Bloom K.
      • Maepa M.
      • Ely A.
      • Arbuthnot P.
      Gene therapy for chronic HBV—can we eliminate cccDNA?.
      ,
      • Martinez M.G.
      • Inchauspe A.
      • Delberghe E.
      • Chapus F.
      • Neveu G.
      • Alam A.
      • et al.
      SAT376 - targeting hepatitis B virus with CRISPR/Cas9 approach.
      epigenetic suppression of cccDNA transcription
      • Dandri M.
      Epigenetic modulation in chronic hepatitis B virus infection.
      and stimulation of hepatocyte innate immune signalling which could lead to lethal mutations in cccDNA via APOBEC nucleotide deamination.
      • Revill P.A.
      • Chisari F.V.
      • Block J.M.
      • Dandri M.
      • Gehring A.J.
      • Guo H.
      • et al.
      A global scientific strategy to cure hepatitis B.
      Envisioning the use of engineer nucleases as a therapy for HBV requires an in-depth study of their off-target effects, the impact of DSBs when targeting integrated HBV DNA, efficient and preferentially specific delivery to infected hepatocytes, and broad spectrum targeting of different viral strains. Editing and inactivation of the HBV genome through innate immune pathway stimulation – that could be accomplished through oral administration of small molecules – is promising, but achieving robust and safe anti-HBV immune activation requires further investigation. Epigenetic modulators could obviate the challenges of eliminating cccDNA, but lack of virus over host chromatin specificity still represents a major drawback. Furthermore, if cccDNA silencing is the aim of HBx targeting, both cccDNA and integrated HBx should be disrupted to prevent HBx expression and lead to sustained Smc5/6 reappearance.
      Basic studies of cccDNA biology are required to better understand its formation, maintenance and kinetics, as well as its interplay with host chromatin, and to guide the careful experimental evaluation of novel targeted therapies prior to proceeding to clinical evaluations. Novel approaches will also have to show an optimal safety profile and efficiency to be considered for clinical development. In particular, possible combination approaches with NAs that alleviate the load of replicative intermediates, could lead to more efficient cccDNA targeting. Though evidence on the synergistic effects and potential undesirable effects of any such combinations will have to be carefully evaluated.

      Abbreviations

      A3A, APOBEC3A; A3B, APOBEC3B; APOBEC3, apolipoprotein B mRNA-editing enzyme 3; ASOs, antisense oligonucleotides; CAM, capsid assembly modulator; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; CTL, cytotoxic T-lymphocyte; DDB1, DNA damage-binding protein 1D; SB, double strand breaks; FEN-1, Flap endonuclease 1; gRNA, guide RNAs; LTBR, lymphotoxin beta receptor; NAs, nucleos(t)ide analogues; NHEJ, non-homologous end joining; NTZ, nitazoxanide; ORF, open reading frame; PEG-IFN, pegylated interferon-α; pgRNA, pregenomic RNA; PHHs, primary human hepatocytes; rcDNA, relaxed circular, TALENs, transcriptional activator-like effector nucleases; TDP2, tyrosyl-DNA phosphodiesterase 2; TRIM, tripartite motif; ZFN, Zinc-Finger nucleases.

      Financial support

      This work was funded by the University of Lyon “Excellence laboratories” grant (Labex DevWeCan n° ANR-10-LABX-61) and Agence Nationale de Recherches sur le SIDA et les hepatites virales (ANRS) to FZ and BT paid to their institution. AB received grants from the ANRS and Sidaction , paid to his institution.

      Authors’ contributions

      MGM, AB, EC and BT wrote the manuscript. FZ defined the outline and extensively revised the manuscript.

      Conflict of interest

      BT and FZ declare a patent licensed by Hoffmann-La Roche. FZ received grants from Beam Therapeutics and Evotec paid to his institution. FZ received consulting fees from Aligos, Antios, Assembly Bioscience and Enochian. FZ received honoraria from Gilead and MYR Pharma as a consultant.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

      References

        • Isorce N.
        • Lucifora J.
        • Zoulim F.
        • Durantel D.
        Immune-modulators to combat hepatitis B virus infection: from IFN-α to novel investigational immunotherapeutic strategies.
        Antiviral Res. 2015; 122: 69-81https://doi.org/10.1016/j.antiviral.2015.08.008
        • Gish R.
        • Jia J.-D.
        • Locarnini S.
        • Zoulim F.
        Selection of chronic hepatitis B therapy with high barrier to resistance.
        Lancet Infect Dis. 2012; 12: 341-353https://doi.org/10.1016/S1473-3099(11)70314-0
        • Cornberg M.
        • Lok A.S.-F.
        • Terrault N.A.
        • Zoulim F.
        2019 EASL-AASLD HBV treatment endpoints conference faculty. Guidance for design and endpoints of clinical trials in chronic hepatitis B - report from the 2019 EASL-AASLD HBV treatment endpoints conference‡.
        J Hepatol. 2020; 72: 539-557https://doi.org/10.1016/j.jhep.2019.11.003
        • Lebossé F.
        • Inchauspé A.
        • Locatelli M.
        • Miaglia C.
        • Diederichs A.
        • Fresquet J.
        • et al.
        Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
        Sci Rep. 2020; 10: 21097https://doi.org/10.1038/s41598-020-78001-1
        • Lai C.-L.
        • Wong D.
        • Ip P.
        • Kopaniszen M.
        • Seto W.-K.
        • Fung J.
        • et al.
        Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.
        J Hepatol. 2017; 66: 275-281https://doi.org/10.1016/j.jhep.2016.08.022
        • Lai C.-L.
        • Wong D.K.-H.
        • Wong G.T.-Y.
        • Seto W.-K.
        • Fung J.
        • Yuen M.-F.
        Rebound of HBV DNA after cessation of nucleos/tide analogues in chronic hepatitis B patients with undetectable covalently closed circular DNA.
        JHEP Rep. 2020; 2: 100112https://doi.org/10.1016/j.jhepr.2020.100112
        • Nassal M.
        HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.
        Gut. 2015; 64: 1972-1984https://doi.org/10.1136/gutjnl-2015-309809
        • Hu J.
        • Seeger C.
        Hepadnavirus genome replication and persistence.
        Cold Spring Harb Perspect Med. 2015; 5: a021386https://doi.org/10.1101/cshperspect.a021386
        • Tu T.
        • Budzinska M.A.
        • Vondran F.W.R.
        • Shackel N.A.
        • Urban S.
        Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via sodium taurocholate cotransporting polypeptide-dependent uptake of enveloped virus particles.
        J Virol. 2018; 92: e02007-e02017https://doi.org/10.1128/JVI.02007-17
        • Ali A.
        Hepatitis B virus, HBx mutants and their role in hepatocellular carcinoma.
        WJG. 2014; 20: 10238https://doi.org/10.3748/wjg.v20.i30.10238
        • Wooddell C.I.
        • Yuen M.-F.
        • Chan H.L.-Y.
        • Gish R.G.
        • Locarnini S.A.
        • Chavez D.
        • et al.
        RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
        Sci Transl Med. 2017; 9https://doi.org/10.1126/scitranslmed.aan0241
        • Yan H.
        • Zhong G.
        • Xu G.
        • He W.
        • Jing Z.
        • Gao Z.
        • et al.
        Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
        ELife. 2012; 1https://doi.org/10.7554/eLife.00049
        • Königer C.
        • Wingert I.
        • Marsmann M.
        • Rösler C.
        • Beck J.
        • Nassal M.
        Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses.
        Proc Natl Acad Sci USA. 2014; 111: E4244-E4253https://doi.org/10.1073/pnas.1409986111
        • Cui X.
        • McAllister R.
        • Boregowda R.
        • Sohn J.A.
        • Ledesma F.C.
        • Caldecott K.W.
        • et al.
        Does tyrosyl DNA phosphodiesterase-2 play a role in hepatitis B virus genome repair?.
        PloS One. 2015; 10e0128401https://doi.org/10.1371/journal.pone.0128401
        • Winer B.Y.
        • Huang T.S.
        • Pludwinski E.
        • Heller B.
        • Wojcik F.
        • Lipkowitz G.E.
        • et al.
        Long-term hepatitis B infection in a scalable hepatic co-culture system.
        Nat Commun. 2017; 8: 125https://doi.org/10.1038/s41467-017-00200-8
        • Cai D.
        • Yan R.
        • Xu J.Z.
        • Zhang H.
        • Shen S.
        • Mitra B.
        • et al.
        Characterization of the termini of cytoplasmic hepatitis B virus deproteinated relaxed circular DNA.
        J Virol. 2020; 95 (e00922-20, /jvi/95/1/JVI.00922-20.atom)https://doi.org/10.1128/JVI.00922-20
        • Qi Y.
        • Gao Z.
        • Xu G.
        • Peng B.
        • Liu C.
        • Yan H.
        • et al.
        DNA polymerase κ is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B virus.
        Plos Pathog. 2016; 12e1005893https://doi.org/10.1371/journal.ppat.1005893
        • Tang L.
        • Sheraz M.
        • McGrane M.
        • Chang J.
        • Guo J.-T.
        DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA.
        Plos Pathog. 2019; 15e1007742https://doi.org/10.1371/journal.ppat.1007742
        • Long Q.
        • Yan R.
        • Hu J.
        • Cai D.
        • Mitra B.
        • Kim E.S.
        • et al.
        The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.
        Plos Pathog. 2017; 13e1006784https://doi.org/10.1371/journal.ppat.1006784
        • Kitamura K.
        • Que L.
        • Shimadu M.
        • Koura M.
        • Ishihara Y.
        • Wakae K.
        • et al.
        Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus.
        Plos Pathog. 2018; 14e1007124https://doi.org/10.1371/journal.ppat.1007124
        • Wei L.
        • Ploss A.
        Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.
        Nat Microbiol. 2020; 5: 715-726https://doi.org/10.1038/s41564-020-0678-0
        • Wei L.
        • Ploss A.
        Hepatitis B virus cccDNA is formed through distinct repair processes of each strand.
        Nat Commun. 2021; 12: 1591https://doi.org/10.1038/s41467-021-21850-9
        • Boyd A.
        • Lacombe K.
        • Lavocat F.
        • Maylin S.
        • Miailhes P.
        • Lascoux-Combe C.
        • et al.
        Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
        J Hepatol. 2016; 65: 683-691https://doi.org/10.1016/j.jhep.2016.05.014
        • Bock C.T.
        • Schranz P.
        • Schröder C.H.
        • Zentgraf H.
        Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell.
        Virus Genes. 1994; 8: 215-229
        • Bock C.T.
        • Schwinn S.
        • Locarnini S.
        • Fyfe J.
        • Manns M.P.
        • Trautwein C.
        • et al.
        Structural organization of the hepatitis B virus minichromosome.
        J Mol Biol. 2001; 307: 183-196https://doi.org/10.1006/jmbi.2000.4481
        • Belloni L.
        • Pollicino T.
        • De Nicola F.
        • Guerrieri F.
        • Raffa G.
        • Fanciulli M.
        • et al.
        Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.
        Proc Natl Acad Sci USA. 2009; 106: 19975-19979https://doi.org/10.1073/pnas.0908365106
        • Tropberger P.
        • Mercier A.
        • Robinson M.
        • Zhong W.
        • Ganem D.E.
        • Holdorf M.
        Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.
        Proc Natl Acad Sci USA. 2015; 112: E5715-E5724https://doi.org/10.1073/pnas.1518090112
        • Protzer U.
        Epigenetic control of HBV by HBx protein—releasing the break?.
        Nat Rev Gastroenterol Hepatol. 2015; 12: 558-559https://doi.org/10.1038/nrgastro.2015.152
        • Bernstein B.E.
        • Meissner A.
        • Lander E.S.
        The mammalian epigenome.
        Cell. 2007; 128: 669-681https://doi.org/10.1016/j.cell.2007.01.033
        • Pollicino T.
        • Belloni L.
        • Raffa G.
        • Pediconi N.
        • Squadrito G.
        • Raimondo G.
        • et al.
        Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
        Gastroenterology. 2006; 130: 823-837https://doi.org/10.1053/j.gastro.2006.01.001
        • Hong X.
        • Kim E.S.
        • Guo H.
        Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B: Hong, Kim, and Guo.
        Hepatology. 2017; 66: 2066-2077https://doi.org/10.1002/hep.29479
        • Tu T.
        • Zehnder B.
        • Qu B.
        • Urban S.
        De novo synthesis of Hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA.
        JHEP Rep. 2020; : 100195https://doi.org/10.1016/j.jhepr.2020.100195
        • Lucifora J.
        • Arzberger S.
        • Durantel D.
        • Belloni L.
        • Strubin M.
        • Levrero M.
        • et al.
        Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection.
        J Hepatol. 2011; 55: 996-1003https://doi.org/10.1016/j.jhep.2011.02.015
        • Rivière L.
        • Gerossier L.
        • Ducroux A.
        • Dion S.
        • Deng Q.
        • Michel M.-L.
        • et al.
        HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.
        J Hepatol. 2015; 63: 1093-1102https://doi.org/10.1016/j.jhep.2015.06.023
        • Brown J.S.
        • Jackson S.P.
        Ubiquitylation, neddylation and the DNA damage response.
        Open Biol. 2015; 5: 150018https://doi.org/10.1098/rsob.150018
        • Murphy C.M.
        • Xu Y.
        • Li F.
        • Nio K.
        • Reszka-Blanco N.
        • Li X.
        • et al.
        Hepatitis B virus X protein promotes degradation of SMC5/6 to enhance HBV replication.
        Cell Rep. 2016; 16: 2846-2854https://doi.org/10.1016/j.celrep.2016.08.026
        • Decorsière A.
        • Mueller H.
        • van Breugel P.C.
        • Abdul F.
        • Gerossier L.
        • Beran R.K.
        • et al.
        Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
        Nature. 2016; 531: 386-389https://doi.org/10.1038/nature17170
        • Niu C.
        • Livingston C.M.
        • Li L.
        • Beran R.K.
        • Daffis S.
        • Ramakrishnan D.
        • et al.
        The Smc5/6 complex restricts HBV when localized to ND10 without inducing an innate immune response and is counteracted by the HBV X protein shortly after infection.
        PloS One. 2017; 12e0169648https://doi.org/10.1371/journal.pone.0169648
        • Stadelmayer B.
        • Diederichs A.
        • Chapus F.
        • Rivoire M.
        • Neveu G.
        • Alam A.
        • et al.
        Full-length 5’RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum.
        J Hepatol. 2020; 73: 40-51https://doi.org/10.1016/j.jhep.2020.01.028
        • Kim J.-W.
        • Lee S.H.
        • Park Y.S.
        • Hwang J.-H.
        • Jeong S.-H.
        • Kim N.
        • et al.
        Replicative activity of hepatitis B virus is negatively associated with methylation of covalently closed circular DNA in advanced hepatitis B virus infection.
        Intervirology. 2011; 54: 316-325https://doi.org/10.1159/000321450
        • Zhang Y.
        • Mao R.
        • Yan R.
        • Cai D.
        • Zhang Y.
        • Zhu H.
        • et al.
        Transcription of hepatitis B virus covalently closed circular DNA is regulated by CpG methylation during chronic infection.
        PloS One. 2014; 9e110442https://doi.org/10.1371/journal.pone.0110442
        • Lutgehetmann M.
        • Volz T.
        • Köpke A.
        • Broja T.
        • Tigges E.
        • Lohse A.W.
        • et al.
        In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice.
        Hepatology. 2010; 52: 16-24https://doi.org/10.1002/hep.23611
        • Zhu Y.
        • Yamamoto T.
        • Cullen J.
        • Saputelli J.
        • Aldrich C.E.
        • Miller D.S.
        • et al.
        Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.
        J Virol. 2001; 75: 311-322https://doi.org/10.1128/JVI.75.1.311-322.2001
        • Dandri M.
        • Burda M.R.
        • Will H.
        • Petersen J.
        Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir in vitro do not lead to reduction of the closed circular DNA.
        Hepatology. 2000; 32: 139-146https://doi.org/10.1053/jhep.2000.8701
        • Laras A.
        • Koskinas J.
        • Dimou E.
        • Kostamena A.
        • Hadziyannis S.J.
        Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.
        Hepatology. 2006; 44: 694-702https://doi.org/10.1002/hep.21299
        • Volz T.
        • Lutgehetmann M.
        • Wachtler P.
        • Jacob A.
        • Quaas A.
        • Murray J.M.
        • et al.
        Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
        Gastroenterology. 2007; 133: 843-852https://doi.org/10.1053/j.gastro.2007.06.057
        • Werle-Lapostolle B.
        • Bowden S.
        • Locarnini S.
        • Wursthorn K.
        • Petersen J.
        • Lau G.
        • et al.
        Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
        Gastroenterology. 2004; 126: 1750-1758
        • Lebossé F.
        • Testoni B.
        • Fresquet J.
        • Facchetti F.
        • Galmozzi E.
        • Fournier M.
        • et al.
        Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.
        J Hepatol. 2017; 66: 897-909https://doi.org/10.1016/j.jhep.2016.12.024
        • Gordon S.C.
        • Krastev Z.
        • Horban A.
        • Petersen J.
        • Sperl J.
        • Dinh P.
        • et al.
        Efficacy of tenofovir disoproxil fumarate at 240 weeks in patients with chronic hepatitis B with high baseline viral load: Hepatology.
        Hepatology. 2013; 58: 505-513https://doi.org/10.1002/hep.26277
        • Moraleda G.
        • Saputelli J.
        • Aldrich C.E.
        • Averett D.
        • Condreay L.
        • Mason W.S.
        Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.
        J Virol. 1997; 71: 9392-9399https://doi.org/10.1128/JVI.71.12.9392-9399.1997
        • Burdette D.
        • Cathcart A.
        • Shauf A.
        • Win R.
        • Zaboli S.
        • Hedskog C.
        • et al.
        PS-150-Evidence for the presence of infectious virus in the serum from chronic hepatitis B patients suppressed on nucleos (t)ide therapy with detectable but not quantifiable HBV DNA.
        J Hepatol. 2019; 70: e95https://doi.org/10.1016/S0618-8278(19)30168-9
        • Huang Q.
        • Zhou B.
        • Cai D.
        • Zong Y.
        • Wu Y.
        • Liu S.
        • et al.
        Rapid turnover of hepatitis B virus covalently closed circular DNA indicated by monitoring emergence and reversion of signature-mutation in treated chronic hepatitis B patients.
        Hepatology. 2021; 73: 41-52https://doi.org/10.1002/hep.31240
        • Ko C.
        • Chakraborty A.
        • Chou W.-M.
        • Hasreiter J.
        • Wettengel J.M.
        • Stadler D.
        • et al.
        Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
        J Hepatol. 2018; 69: 1231-1241https://doi.org/10.1016/j.jhep.2018.08.012
        • Guo J.-T.
        • Zhou H.
        • Liu C.
        • Aldrich C.
        • Saputelli J.
        • Whitaker T.
        • et al.
        Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections.
        J Virol. 2000; 74: 1495-1505https://doi.org/10.1128/JVI.74.3.1495-1505.2000
        • Summers J.
        • Jilbert A.R.
        • Yang W.
        • Aldrich C.E.
        • Saputelli J.
        • Litwin S.
        • et al.
        Hepatocyte turnover during resolution of a transient hepadnaviral infection.
        Proc Natl Acad Sci. 2003; 100: 11652-11659https://doi.org/10.1073/pnas.1635109100
        • Mason W.S.
        • Jilbert A.R.
        • Summers J.
        Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection.
        Proc Natl Acad Sci. 2005; 102: 1139-1144https://doi.org/10.1073/pnas.0409332102
        • Guidotti L.G.
        Viral clearance without destruction of infected cells during acute HBV infection.
        Science. 1999; 284: 825-829https://doi.org/10.1126/science.284.5415.825
        • Wieland S.F.
        • Spangenberg H.C.
        • Thimme R.
        • Purcell R.H.
        • Chisari F.V.
        Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees.
        Proc Natl Acad Sci USA. 2004; 101: 2129-2134https://doi.org/10.1073/pnas.0308478100
        • Murray J.M.
        • Wieland S.F.
        • Purcell R.H.
        • Chisari F.V.
        Dynamics of hepatitis B virus clearance in chimpanzees.
        Proc Natl Acad Sci. 2005; 102: 17780-17785https://doi.org/10.1073/pnas.0508913102
        • Allweiss L.
        • Volz T.
        • Giersch K.
        • Kah J.
        • Raffa G.
        • Petersen J.
        • et al.
        Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.
        Gut. 2018; 67: 542-552https://doi.org/10.1136/gutjnl-2016-312162
        • Li M.
        • Sohn J.A.
        • Seeger C.
        Distribution of hepatitis B virus nuclear DNA.
        J Virol. 2017; 92 (e01391-17)https://doi.org/10.1128/JVI.01391-17
        • Addison W.R.
        • Walters K.-A.
        • Wong W.W.S.
        • Wilson J.S.
        • Madej D.
        • Jewell L.D.
        • et al.
        Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo following inhibition of viral replication.
        J Virol. 2002; 76: 6356-6363https://doi.org/10.1128/jvi.76.12.6356-6363.2002
        • Murray J.M.
        • Goyal A.
        In silico single cell dynamics of hepatitis B virus infection and clearance.
        J Theor Biol. 2015; 366: 91-102https://doi.org/10.1016/j.jtbi.2014.11.020
        • Goyal A.
        • Chauhan R.
        The dynamics of integration, viral suppression and cell-cell transmission in the development of occult Hepatitis B virus infection.
        J Theor Biol. 2018; 455: 269-280https://doi.org/10.1016/j.jtbi.2018.06.020
        • Goyal A.
        • Murray J.M.
        Modelling the impact of cell-to-cell transmission in hepatitis B virus.
        PloS One. 2016; 11e0161978https://doi.org/10.1371/journal.pone.0161978
        • Goyal A.
        • Liao L.E.
        • Perelson A.S.
        Within-host mathematical models of hepatitis B virus infection: past, present, and future.
        Curr Opin Syst Biol. 2019; 18: 27-35https://doi.org/10.1016/j.coisb.2019.10.003
        • Charre C.
        • Levrero M.
        • Zoulim F.
        • Scholtès C.
        Non-invasive biomarkers for chronic hepatitis B virus infection management.
        Antivir Res. 2019; 169: 104553https://doi.org/10.1016/j.antiviral.2019.104553
        • Cai D.
        • Mills C.
        • Yu W.
        • Yan R.
        • Aldrich C.E.
        • Saputelli J.R.
        • et al.
        Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation.
        Antimicrob Agents Chemother. 2012; 56: 4277-4288https://doi.org/10.1128/AAC.00473-12
        • Liu C.
        • Cai D.
        • Zhang L.
        • Tang W.
        • Yan R.
        • Guo H.
        • et al.
        Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA.
        Antiviral Res. 2016; 134: 97-107https://doi.org/10.1016/j.antiviral.2016.08.026
        • Wang L.
        • Zhu Q.
        • Zeng J.
        • Yan Z.
        • Feng A.
        • Young J.
        • et al.
        PS-074-A first-in-class orally available HBV cccDNA destabilizer ccc_R08 achieved sustainable HBsAg and HBV DNA suppression in the HBV circle mouse model through elimination of cccDNA-like molecules in the mouse liver.
        J Hepatol. 2019; 70: e48https://doi.org/10.1016/S0618-8278(19)30086-6
        • Cradick T.J.
        • Keck K.
        • Bradshaw S.
        • Jamieson A.C.
        • McCaffrey A.P.
        Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs.
        Mol Ther. 2010; 18: 947-954https://doi.org/10.1038/mt.2010.20
        • Bloom K.
        • Ely A.
        • Mussolino C.
        • Cathomen T.
        • Arbuthnot P.
        Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.
        Mol Ther. 2013; 21: 1889-1897https://doi.org/10.1038/mt.2013.170
      1. 2020 ASGCT annual meeting abstracts.
        Mol Ther. 2020; 28: 1-592https://doi.org/10.1016/j.ymthe.2020.04.019
        • Jinek M.
        • Chylinski K.
        • Fonfara I.
        • Hauer M.
        • Doudna J.A.
        • Charpentier E.
        A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
        Science. 2012; 337: 816-821https://doi.org/10.1126/science.1225829
        • Ran F.A.
        • Hsu P.D.
        • Wright J.
        • Agarwala V.
        • Scott D.A.
        • Zhang F.
        Genome engineering using the CRISPR-Cas9 system.
        Nat Protoc. 2013; 8: 2281-2308https://doi.org/10.1038/nprot.2013.143
        • Ledford H.
        • Callaway E.
        Pioneers of revolutionary CRISPR gene editing win chemistry Nobel.
        Nature. 2020; 586: 346-347https://doi.org/10.1038/d41586-020-02765-9
        • Bloom K.
        • Maepa M.
        • Ely A.
        • Arbuthnot P.
        Gene therapy for chronic HBV—can we eliminate cccDNA?.
        Genes. 2018; 9: 207https://doi.org/10.3390/genes9040207
        • Verkuijl S.A.
        • Rots M.G.
        The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies.
        Curr Opin Biotechnol. 2019; 55: 68-73https://doi.org/10.1016/j.copbio.2018.07.005
        • Isaac R.S.
        • Jiang F.
        • Doudna J.A.
        • Lim W.A.
        • Narlikar G.J.
        • Almeida R.
        Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.
        ELife. 2016; 5e13450https://doi.org/10.7554/eLife.13450
        • Martinez M.G.
        • Inchauspe A.
        • Delberghe E.
        • Chapus F.
        • Neveu G.
        • Alam A.
        • et al.
        SAT376 - targeting hepatitis B virus with CRISPR/Cas9 approach.
        J Hepatol. 2020; 73: S841-S842https://doi.org/10.1016/S0168-8278(20)32126-7
        • Stone D.
        • Long K.R.
        • Loprieno M.A.
        • De Silva Feelixge H.S.
        • Kenkel E.J.
        • Liley R.M.
        • et al.
        CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice.
        Mol Ther - Methods Clin Dev. 2021; 20: 258-275https://doi.org/10.1016/j.omtm.2020.11.014
        • Simhadri V.L.
        • McGill J.
        • McMahon S.
        • Wang J.
        • Jiang H.
        • Sauna Z.E.
        Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population.
        Mol Ther - Methods Clin Dev. 2018; 10: 105-112https://doi.org/10.1016/j.omtm.2018.06.006
        • Charlesworth C.T.
        • Deshpande P.S.
        • Dever D.P.
        • Camarena J.
        • Lemgart V.T.
        • Cromer M.K.
        • et al.
        Identification of preexisting adaptive immunity to Cas9 proteins in humans.
        Nat Med. 2019; 25: 249-254https://doi.org/10.1038/s41591-018-0326-x
        • Li A.
        • Tanner M.R.
        • Lee C.M.
        • Hurley A.E.
        • De Giorgi M.
        • Jarrett K.E.
        • et al.
        AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9.
        Mol Ther. 2020; 28: 1432-1441https://doi.org/10.1016/j.ymthe.2020.04.017
        • Rouet R.
        • de Oñate L.
        • Li J.
        • Murthy N.
        • Wilson R.C.
        Engineering CRISPR-Cas9 RNA–protein complexes for improved function and delivery.
        CRISPR J. 2018; 1: 367-378https://doi.org/10.1089/crispr.2018.0037
        • Kosicki M.
        • Tomberg K.
        • Bradley A.
        Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.
        Nat Biotechnol. 2018; 36: 765-771https://doi.org/10.1038/nbt.4192
        • Rees H.A.
        • Liu D.R.
        Base editing: precision chemistry on the genome and transcriptome of living cells.
        Nat Rev Genet. 2018; 19: 770-788https://doi.org/10.1038/s41576-018-0059-1
        • Anzalone A.V.
        • Koblan L.W.
        • Liu D.R.
        Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors.
        Nat Biotechnol. 2020; 38: 824-844https://doi.org/10.1038/s41587-020-0561-9
        • Yang Y.-C.
        • Chen Y.-H.
        • Kao J.-H.
        • Ching C.
        • Liu I.-J.
        • Wang C.-C.
        • et al.
        Permanent inactivation of HBV genomes by CRISPR/Cas9-Mediated non-cleavage base editing.
        Mol Ther - Nucleic Acids. 2020; 20: 480-490https://doi.org/10.1016/j.omtn.2020.03.005
        • Yu Y.
        • Leete T.C.
        • Born D.A.
        • Young L.
        • Barrera L.A.
        • Lee S.-J.
        • et al.
        Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity.
        Nat Commun. 2020; 11: 2052https://doi.org/10.1038/s41467-020-15887-5
        • Love R.P.
        • Xu H.
        • Chelico L.
        Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A.
        J Biol Chem. 2012; 287: 30812-30822https://doi.org/10.1074/jbc.M112.393181
        • Stenglein M.D.
        • Burns M.B.
        • Li M.
        • Lengyel J.
        • Harris R.S.
        APOBEC3 proteins mediate the clearance of foreign DNA from human cells.
        Nat Struct Mol Biol. 2010; 17: 222-229https://doi.org/10.1038/nsmb.1744
        • Lucifora J.
        • Xia Y.
        • Reisinger F.
        • Zhang K.
        • Stadler D.
        • Cheng X.
        • et al.
        Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.
        Science. 2014; 343: 1221-1228https://doi.org/10.1126/science.1243462
        • Nair S.
        • Zlotnick A.
        Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.
        J Virol. 2018; 92 (e02190-17, /jvi/92/10/e02190-17.atom)https://doi.org/10.1128/JVI.02190-17
        • Gehring A.J.
        • Xue S.-A.
        • Ho Z.Z.
        • Teoh D.
        • Ruedl C.
        • Chia A.
        • et al.
        Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.
        J Hepatol. 2011; 55: 103-110https://doi.org/10.1016/j.jhep.2010.10.025
        • Bertoletti A.
        • Brunetto M.
        • Maini M.K.
        • Bonino F.
        • Qasim W.
        • Stauss H.
        T cell receptor-therapy in HBV-related hepatocellularcarcinoma.
        OncoImmunology. 2015; 4e1008354https://doi.org/10.1080/2162402X.2015.1008354
        • Koh S.
        • Kah J.
        • Tham C.Y.L.
        • Yang N.
        • Ceccarello E.
        • Chia A.
        • et al.
        Nonlytic lymphocytes engineered to express virus-specific T-cell receptors limit HBV infection by activating APOBEC3.
        Gastroenterology. 2018; 155 (180-193.e6)https://doi.org/10.1053/j.gastro.2018.03.027
        • Suspene R.
        • Guetard D.
        • Henry M.
        • Sommer P.
        • Wain-Hobson S.
        • Vartanian J.-P.
        Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo.
        Proc Natl Acad Sci. 2005; 102: 8321-8326https://doi.org/10.1073/pnas.0408223102
        • Seeger C.
        • Sohn J.A.
        Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA.
        Mol Ther. 2016; 24: 1258-1266https://doi.org/10.1038/mt.2016.94
        • Hilton I.B.
        • D’Ippolito A.M.
        • Vockley C.M.
        • Thakore P.I.
        • Crawford G.E.
        • Reddy T.E.
        • et al.
        Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.
        Nat Biotechnol. 2015; 33: 510-517https://doi.org/10.1038/nbt.3199
        • Roberts S.A.
        • Lawrence M.S.
        • Klimczak L.J.
        • Grimm S.A.
        • Fargo D.
        • Stojanov P.
        • et al.
        An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers.
        Nat Genet. 2013; 45: 970-976https://doi.org/10.1038/ng.2702
        • Gao B.
        • Duan Z.
        • Xu W.
        • Xiong S.
        Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain.
        Hepatology. 2009; 50: 424-433https://doi.org/10.1002/hep.23011
        • Palumbo G.A.
        • Scisciani C.
        • Pediconi N.
        • Lupacchini L.
        • Alfalate D.
        • Guerrieri F.
        • et al.
        IL6 inhibits HBV transcription by targeting the epigenetic control of the nuclear cccDNA minichromosome.
        PloS One. 2015; 10e0142599https://doi.org/10.1371/journal.pone.0142599
        • Isorce N.
        • Testoni B.
        • Locatelli M.
        • Fresquet J.
        • Rivoire M.
        • Luangsay S.
        • et al.
        Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.
        Antiviral Res. 2016; 130: 36-45https://doi.org/10.1016/j.antiviral.2016.03.008
        • Lin S.-J.
        • Shu P.-Y.
        • Chang C.
        • Ng A.-K.
        • Hu C.
        IL-4 suppresses the expression and the replication of hepatitis B virus in the hepatocellular carcinoma cell line Hep3B.
        J Immunol. 2003; 171: 4708-4716https://doi.org/10.4049/jimmunol.171.9.4708
        • Hong M.-H.
        • Chou Y.-C.
        • Wu Y.-C.
        • Tsai K.-N.
        • Hu C.
        • Jeng K.-S.
        • et al.
        Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression.
        PloS One. 2012; 7e30360https://doi.org/10.1371/journal.pone.0030360
        • Mitra B.
        • Thapa R.J.
        • Guo H.
        • Block T.M.
        Host functions used by hepatitis B virus to complete its life cycle: implications for developing host-targeting agents to treat chronic hepatitis B.
        Antiviral Res. 2018; 158: 185-198https://doi.org/10.1016/j.antiviral.2018.08.014
        • Yu H.-B.
        • Jiang H.
        • Cheng S.-T.
        • Hu Z.-W.
        • Ren J.-H.
        • Chen J.
        AGK2, A SIRT2 inhibitor, inhibits hepatitis B virus replication in vitro and in vivo.
        Int J Med Sci. 2018; 15: 1356-1364https://doi.org/10.7150/ijms.26125
        • Gilmore S.
        • Tam D.
        • Dick R.
        • Appleby T.
        • Birkus G.
        • Willkom M.
        • et al.
        Antiviral activity of GS-5801, a liver-targeted prodrug of a lysine demethylase 5 inhibitor, in a hepatitis B virus primary human hepatocyte infection model.
        J Hepatol. 2017; 66: S690-S691https://doi.org/10.1016/S0168-8278(17)31855-X
        • Zhang W.
        • Chen J.
        • Wu M.
        • Zhang X.
        • Zhang M.
        • Yue L.
        • et al.
        PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.
        Hepatology. 2017; 66: 398-415https://doi.org/10.1002/hep.29133
        • Fernández-Barrena M.G.
        • Arechederra M.
        • Colyn L.
        • Berasain C.
        • Avila M.A.
        Epigenetics in hepatocellular carcinoma development and therapy: the tip of the iceberg.
        JHEP Rep. 2020; 2: 100167https://doi.org/10.1016/j.jhepr.2020.100167
        • Kornyeyev D.
        • Ramakrishnan D.
        • Voitenleitner C.
        • Livingston C.M.
        • Xing W.
        • Hung M.
        • et al.
        Spatiotemporal analysis of hepatitis B virus X protein in primary human hepatocytes.
        J Virol. 2019; 93 (e00248-19, /jvi/93/16/JVI.00248-19.atom)https://doi.org/10.1128/JVI.00248-19
        • Allweiss L.
        • Giersch K.
        • Pirosu A.
        • Volz T.
        • Muench R.C.
        • Beran R.K.
        • et al.
        Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.
        Gut. 2021; (gutjnl-2020-322571)https://doi.org/10.1136/gutjnl-2020-322571
        • Xirong L.
        • Rui L.
        • Xiaoli Y.
        • Qiuyan H.
        • Bikui T.
        • Sibo Z.
        • et al.
        Hepatitis B virus can be inhibited by DNA methyltransferase 3a via specific zinc-finger-induced methylation of the X promoter.
        Biochem Mosc. 2014; 79: 111-123https://doi.org/10.1134/S0006297914020047
        • Sekiba K.
        • Otsuka M.
        • Ohno M.
        • Yamagami M.
        • Kishikawa T.
        • Suzuki T.
        • et al.
        Inhibition of HBV transcription from cccDNA with nitazoxanide by targeting the HBx–DDB1 interaction.
        Cell Mol Gastroenterol Hepatol. 2019; 7: 297-312https://doi.org/10.1016/j.jcmgh.2018.10.010
        • Sekiba K.
        • Otsuka M.
        • Ohno M.
        • Yamagami M.
        • Kishikawa T.
        • Seimiya T.
        • et al.
        Pevonedistat, a neuronal precursor cell-expressed developmentally down-regulated protein 8–activating enzyme inhibitor, is a potent inhibitor of hepatitis B virus.
        Hepatology. 2019; 69: 1903-1915https://doi.org/10.1002/hep.30491
        • Fanning G.C.
        • Zoulim F.
        • Hou J.
        • Bertoletti A.
        Therapeutic strategies for hepatitis B virus infection: towards a cure.
        Nat Rev Drug Discov. 2019; 18: 827-844https://doi.org/10.1038/s41573-019-0037-0
        • Yuen M.-F.
        • Agarwal K.
        • Ma X.
        • Nguyen T.
        • Schiff E.R.
        • Hann H.-W.
        • et al.
        Antiviral activity and safety of the hepatitis B core inhibitor ABI-H0731 administered with a nucleos(t)ide reverse transcriptase inhibitor in patients with HBeAg-positive chronic hepatitis B infection in a long-term extension study.
        J Hepatol. 2020; 73: S140https://doi.org/10.1016/S0168-8278(20)30790-X
        • Gane E.
        • Locarnini S.
        • Lim T.H.
        • Strasser S.
        • Sievert W.
        • Cheng W.
        • et al.
        Short-term treatment with RNA interference therapy, JNJ-3989, results in sustained hepatitis B surface antigen supression in patients with chronic hepatitis B receiving nucleos(t)ide analogue treatment.
        J Hepatol. 2020; 73: S20https://doi.org/10.1016/S0168-8278(20)30597-3
      2. Oral abstracts.
        Hepatology. 2020; 72: 1A-130Ahttps://doi.org/10.1002/hep.31578
        • Yuen M.-F.
        • Heo J.
        • Jang J.W.
        • Yoon J.-H.
        • Kweon Y.O.
        • Park S.-J.
        • et al.
        Hepatitis B virus (HBV) surface antigen (HBsAg) inhibition with isis 505358 in chronic hepatitis B (CHB) patients on stable nucleos (t)ide analogue (NA) regimen and in NA-naive CHB patients: phase 2a, randomized, double-blind, placebo-controlled study.
        J Hepatol. 2020; 73: S49-S50https://doi.org/10.1016/S0168-8278(20)30646-2
        • Yuen M.-F.
        • Locarnini S.
        • Given B.
        • Schluep T.
        • Hamilton J.
        • Biermer M.
        • et al.
        First clinical experience with RNA interference-based triple combination therapy in chronic hepatitis B: JNJ-3989, JNJ-6379 and a Nucleos (t) ide analogue.
        Hepatology. 2019; 70: 1489A
        • Wedemeyer H.
        • Schöneweis K.
        • Bogomolov P.O.
        • Chulanov V.
        • Stepanova T.
        • Viacheslav M.
        • et al.
        48 weeks of high dose (10 mg) bulevirtide as monotherapy or with peginterferon alfa-2a in patients with chronic HBV/HDV co-infection.
        J Hepatol. 2020; 73: S52-S53https://doi.org/10.1016/S0168-8278(20)30651-6
        • Bazinet M.
        • Pântea V.
        • Placinta G.
        • Moscalu I.
        • Cebotarescu V.
        • Cojuhari L.
        • et al.
        Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naïve to nucleos(t)ide therapy.
        Gastroenterology. 2020; 158: 2180-2194https://doi.org/10.1053/j.gastro.2020.02.058
        • Gane E.
        • Dunbar P.R.
        • Brooks A.
        • Zhao Y.
        • Tan S.
        • Lau A.
        • et al.
        Efficacy and safety of 24 weeks treatment with oral TLR8 agonist, selgantolimod, in virally-suppressed adult patients with chronic hepatitis B: a phase 2 study.
        J Hepatol. 2020; 73: S52https://doi.org/10.1016/S0168-8278(20)30650-4
        • Michler T.
        • Kosinska A.D.
        • Festag J.
        • Bunse T.
        • Su J.
        • Ringelhan M.
        • et al.
        Knockdown of virus antigen expression increases therapeutic vaccine efficacy in high-titer hepatitis B virus carrier mice.
        Gastroenterology. 2020; 158 (1762-1775.e9)https://doi.org/10.1053/j.gastro.2020.01.032
        • Gane E.
        • Verdon D.J.
        • Brooks A.E.
        • Gaggar A.
        • Nguyen A.H.
        • Subramanian G.M.
        • et al.
        Anti-PD-1 blockade with nivolumab with and without therapeutic vaccination for virally suppressed chronic hepatitis B: a pilot study.
        J Hepatol. 2019; 71: 900-907https://doi.org/10.1016/j.jhep.2019.06.028
        • Maini M.K.
        • Pallett L.J.
        Defective T-cell immunity in hepatitis B virus infection: why therapeutic vaccination needs a helping hand.
        Lancet Gastroenterol Hepatol. 2018; 3: 192-202https://doi.org/10.1016/S2468-1253(18)30007-4
        • Dandri M.
        Epigenetic modulation in chronic hepatitis B virus infection.
        Semin Immunopathol. 2020; 42: 173-185https://doi.org/10.1007/s00281-020-00780-6
        • Revill P.A.
        • Chisari F.V.
        • Block J.M.
        • Dandri M.
        • Gehring A.J.
        • Guo H.
        • et al.
        A global scientific strategy to cure hepatitis B.
        Lancet Gastroenterol Hepatol. 2019; 4: 545-558https://doi.org/10.1016/S2468-1253(19)30119-0