| International Session（Panel Discussion）1(JSH・JSGE・JSGCS) |
| Fri. November 6th 9:40 - 12:00 Room 8: Portopia Hotel Main Building Kairaku 1+2 |
| Efficacy of novel anti-HBV combination therapy with CRISPR/Cas9 targeting HBV genome and inhibitors of host DNA synthesis/repair pathways. | |||
| Kazuhiro Murai1, Takahiro Kodama1, Tetsuo Takehara1 | |||
| 1Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine | |||
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| Background and aim: Current anti-Hepatitis B virus (HBV) therapies have little effect on cccDNA and fail to eliminate HBV. HBV uses host cellular DNA repair machinery for cccDNA biosynthesis. We investigated efficacy of novel therapeutics targeting the HBV genome using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 in combination with inhibitors of DNA synthesis/repair pathway. 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 susceptible cells with inducible Cas9 expression (HepG2-hNTCP-iCas9). Cas9 expression was induced two days after HBV infection in HepG2-hNTCP-iCas9 cells. We examined the anti-viral effect of CRISPR/Cas9 in the presence or absence of DNA synthesis inhibitor (CD437) or repair inhibitor (olaparib). Results: Transduction of LV-gRNA/Cas9 induced indel formation in HBV genome in HepG2.2.15. CRISPR/Cas9 therapy targeting HBV genome reduced the levels of HBV DNA, HBs and HBe antigen in the supernatant and pregenomic RNA (pgRNA) levels in HepG2.2.15 and HepG2-hNTCP-iCas9 cells, and cccDNA levels in HepG2-hNTCP-iCas9 cells. While neither the PARP inhibitor olaparib nor the DNA polymerase alpha inhibitor CD437 showed anti-viral effect, both inhibitors enhanced the anti-viral effect of CRISPR/Cas9 in HepG2-hNTCP-iCas9 cells. Conclusion: Efficacy of anti-HBV therapy targeting HBV genome by CRISPR/Cas9 was enhanced by modulating host DNA synthesis/repair pathways. |
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Index Term 1: CRISPR/Cas9 Index Term 2: cccDNA |
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