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Vol 53(2019) N 2 p. 274-285; DOI 10.1134/S0026893319010072 Full Text

A.P. Kostyusheva1, D.S. Kostyushev1*, S.A. Brezgin1,2, D.N. Zarifyan1, E.V. Volchkova3, V.P. Chulanov1,3

Small Molecular Inhibitors of DNA Double Strand Break Repair Pathways Increase the ANTI-HBV Activity of CRISPR/Cas9

1Central Research Institute of Epidemiology, Federal Service for the Oversight of Consumer Protection and Welfare, Moscow, 111123 Russia
2Institute of Immunology of Federal Medical-Biological Agency, Moscow, 115478 Russia
3Sechenov Moscow State Medical University, Moscow, 119048 Russia

*dk@rcvh.ru
Received - 2018-09-05; Revised - 2018-09-20; Accepted - 2018-09-20

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.

CRISPR/Cas9, hepatitis B virus, covalently closed circular DNA, lentiviral transduction, HR, NHEJ, DNA double-strand breaks



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