Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector

Lucifora, Julie; Salvetti, Anna; Marniquet, Xavier; Mailly, Laurent; Testoni, Barbara; Fusil, Floriane; Inchauspé, Aurore; Michelet, Maud; Michel, Marie‐Louise; Levrero, Massimo; Cortez, Pierre; Baumert, Thomas F.; Cosset, François‐Loïc; Challier, Cécile; Zoulim, Fabien; Durantel, David

doi:10.1016/j.antiviral.2017.07.006

Cited by 52 publications

(39 citation statements)

References 39 publications

(39 reference statements)

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“…This LNA-SSO demonstrated superior antiviral efficacy as compared to its unconjugated counterpart, as judged by HBsAg reduction both in natural infection assay, in which both HBV replication and expression are driven by viral cccDNA, and in the AAV-HBV mouse model, where HBV expression originates from both episomal AAV-HBV vector and HBV cccDNA. 39 Most importantly, the effect of this oligonucleotide was highly dependent upon an exact sequence match to its target viral RNA sequence, and it was not associated with non-specific cytokine, complement, or other generalized toxicity induction.…”

Section: Discussionmentioning

confidence: 95%

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo

Javanbakht

Mueller

Walther

et al. 2018

Molecular Therapy - Nucleic Acids

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Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

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Section: Discussionmentioning

confidence: 95%

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo

Javanbakht

Mueller

Walther

et al. 2018

Molecular Therapy - Nucleic Acids

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“…HBsAg and HBeAg secretion were quantified by chemiluminescence immunoassay (Autobio) following the manufacturer's instructions. Southern blot detection of HBV cccDNA was performed using digoxigenin (DIG)-labeled (Roche) specific probes as described 62 . Total DNA from HBV-infected cells was extracted using the Hirt method as described 63 .…”

Section: Methodsmentioning

confidence: 99%

A genome-wide gain-of-function screen identifies CDKN2C as a HBV host factor

et al. 2020

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Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle.

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“…After total DNA isolation and T5 exonuclease digestion (NEB, Fance) digestion for 1 h, cccDNA was quantified as previously described 30 . Alternatively, cccDNA was analyzed by Southern blot after Hirt extraction as described previously 31 .…”

Section: Methodsmentioning

confidence: 99%

Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes

Lucifora¹,

Bonnin²,

Aillot³

et al. 2018

Sci Rep

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Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or “adjuvant” potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.

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Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector

Cited by 52 publications

References 39 publications

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo

A genome-wide gain-of-function screen identifies CDKN2C as a HBV host factor

Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes

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