Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

Abstract 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.