(RLH) system and suppresses HBV replication during HBV/HCV co-infection and that HCV elimination by DAA therapy decreases RLH system induction and enhances HBV replication.
Current anti–hepatitis B virus (HBV) therapies have little effect on covalently closed circular DNA (cccDNA) and fail to eliminate HBV. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been reported to directly target cccDNA and exert antiviral effects. In this study, we hypothesized that the inhibition of the DNA repair machinery, which is important for the repair of CRISPR‐induced double‐strand breaks, may enhance the effect of CRISPR targeting cccDNA, and we investigated the antiviral effect of potential combination therapy. The antiviral effect of CRISPR targeting cccDNA (HBV‐CRISPR) was evaluated in HBV‐susceptible HepG2‐hNTCP‐C4 cells expressing Cas9 (HepG2‐hNTCP‐C4‐iCas9) or primary human hepatocytes (PHHs) expressing Cas9. Following HBV infection, HBV‐CRISPR reduced cccDNA levels, accompanied by decreases in pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen and hepatitis B e antigen levels in HepG2‐hNTCP‐C4‐iCas9 cells, and PHHs. HBV‐CRISPR induced indel formation in cccDNA and up‐regulated poly(adenosine diphosphate ribose) polymerase (PARP) activity in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells. The suppression of PARP2‐Histone PARylation factor 1 (HPF1) (involved in the initial step of DNA repair) with small interfering RNA (siRNA) targeting either PARP2 or HPF1 increased the reduction in pgRNA and cccDNA by HBV‐CRISPR in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells. The suppression of DNA Ligase 4 (LIG4) (essential for nonhomologous end joining [NHEJ]) but not breast cancer susceptibility gene (BRCA) (essential for homologous recombination) enhanced the antiviral effect of HBV‐CRISPR in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells. Finally, the clinically available PARP inhibitor olaparib increased the reductions in pgRNA and cccDNA levels induced by HBV‐CRISPR in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells and PHHs.
Conclusion
: The suppression of the NHEJ‐mediated DNA repair machinery enhances the effect of CRISPR targeting cccDNA. The combination of CRISPR and olaparib may represent a therapy for HBV elimination.
Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen‐associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV‐infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41‐4109 (BAY41), to HBV‐infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)–stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN‐α in HBV‐infected PHHs but did not change ISG induction by IFN‐α in uninfected PHHs. Compared with BAY41 or IFN‐α alone, coadministration of BAY41 and IFN‐α significantly suppressed extracellular HBV‐DNA levels. HBV‐infected human liver–chimeric mice were treated with vehicle, BAY41, pegylated IFN‐α (pegIFN‐α), or BAY41 and pegIFN‐α together. Compared with the vehicle control, pegIFN‐α highly up‐regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN‐α further enhanced intrahepatic antiviral ISG expression, which was up‐regulated by pegIFNα. The serum HBV‐DNA levels in mice treated with the combination of BAY41 and pegIFN‐α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN‐α, likely due to increased cytoplasmic extracapsid pgRNA.
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