Japan Digestive Disease Week 2021
International Session (Workshop)1 (JSGE, JSH)
November 4, 15:00–17:00, Room 11 (Portopia Hotel South Wing Topaz)
IS-W1-4_H
Novel anti-HBV combination treatment of CRISPR/Cas9 and suppression of NHEJ-mediated DNA repair Young Award
- 1
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine
Background and aim: Current anti-Hepatitis B virus (HBV) therapies have little effect on covalently closed circular DNA (cccDNA). HBV uses host DNA repair machinery for cccDNA biosynthesis. We investigated efficacy of CRISPR/Cas9 targeting HBV genome in combination with suppression of DNA repair machinery.
Methods: We designed gRNAs targeting HBV genome (HBV-gRNA). We lentivirally transduced tandem Cas9 and HBV-gRNA vector (LV-gRNA/Cas9) into HBV genome-integrated cells (HepG2.2.15), and single HBV-gRNA vector into HBV-infected cells with inducible Cas9 expression (HepG2-hNTCP-iCas9). We examined anti-viral effect of the CRISPR/Cas9 treatment with or without suppression of DNA repair machinery by siRNA.
Results: Transduction of LV-gRNA/Cas9 induced indel formation in HBV genome in HepG2.2.15. CRISPR/Cas9 treatment targeting HBV genome reduced the levels of HBV DNA, HBs and HBe antigen in the supernatant and pregenomic RNA levels in HepG2.2.15 and HepG2-hNTCP-iCas9 cells, and cccDNA levels in HepG2-hNTCP-iCas9 cells. Suppression of BRCA or RAD51, essential to homologous recombination, did not alter the efficacy of CRISPR/Cas9 treatment in HepG2-hNTCP-iCas9 cells. Whereas, suppression of PARP2 or HPF1, important for the initial step of DNA repair, or LIG4, essential to non-homologous end joining (NHEJ), significantly enhanced anti-viral effect of CRISPR/Cas9 treatment.
Conclusion: Suppression of NHEJ-mediated DNA repair machinery enhances the efficacy of CRISPR/Cas9 treatment targeting HBV genome.
Methods: We designed gRNAs targeting HBV genome (HBV-gRNA). We lentivirally transduced tandem Cas9 and HBV-gRNA vector (LV-gRNA/Cas9) into HBV genome-integrated cells (HepG2.2.15), and single HBV-gRNA vector into HBV-infected cells with inducible Cas9 expression (HepG2-hNTCP-iCas9). We examined anti-viral effect of the CRISPR/Cas9 treatment with or without suppression of DNA repair machinery by siRNA.
Results: Transduction of LV-gRNA/Cas9 induced indel formation in HBV genome in HepG2.2.15. CRISPR/Cas9 treatment targeting HBV genome reduced the levels of HBV DNA, HBs and HBe antigen in the supernatant and pregenomic RNA levels in HepG2.2.15 and HepG2-hNTCP-iCas9 cells, and cccDNA levels in HepG2-hNTCP-iCas9 cells. Suppression of BRCA or RAD51, essential to homologous recombination, did not alter the efficacy of CRISPR/Cas9 treatment in HepG2-hNTCP-iCas9 cells. Whereas, suppression of PARP2 or HPF1, important for the initial step of DNA repair, or LIG4, essential to non-homologous end joining (NHEJ), significantly enhanced anti-viral effect of CRISPR/Cas9 treatment.
Conclusion: Suppression of NHEJ-mediated DNA repair machinery enhances the efficacy of CRISPR/Cas9 treatment targeting HBV genome.
- Index Term 1: CRISPR/Cas9
- Index Term 2: cccDNA