Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity

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Abstract

Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million of people die annually due to consequences of chronic hepatitis B: liver cirrhosis and hepatocellular carcinoma. The key factor of hepatitis B virus persistency is a special form of viral genome called circular covalently closed DNA. Current therapeutics suppress viral replication but have no effect on circular covalently closed DNA as it exists in the nuclei of hepatocytes as a minichromosome and is not accessible for therapeutics. Commonly, viral reactivation occurs after cessation of treatment. Therefore, duration of antiviral treatment is supposed to be indefinitely long. One of the most promising approaches to target circular covalently closed DNA is the technology of site-specific nucleases CRISPR/Cas9 from Streptococcus pyogenes. A short guide RNA recruits an SpCas9 protein to the viral genome and induces generation of DNA double strand breaks. However, there are several limitations of CRISPR/Cas9 hampering translation of this technology into the clinic. First, efficacy of CRISPR/Cas9 needs to be improved. Second, CRISPR/Cas9-mediated off-target mutagenesis represents a menacing problem which has to be addressed. To overcome these limitations, several approaches have been devised to improve CRISPR/Cas9 activity (modification of guide RNAs) and reduce off-target mutagenesis (a Cas9 protein with enhanced specificity, eSpCas9). In this study, we compared antiviral activity of a classic SpCas9 with an eSpCas9 system as well as analyzed effects of gRNAs modification on anti-HBV effects. Here, we demonstrated that SpCas9 has the highest antiviral potency, reducing transcription and replication of HBV over 90%. Hepatitis B virus covalently closed circular DNA declined over 90% post CRISPR/Cas9 transfection. Although it was previously shown that modified guide RNAs increase nucleolytic activity of CRISPR/Cas9, our results indicated that this modification impairs antiviral activity of CRISPR/Cas9. To conclude, CRISPR/Cas9 effectively suppress viral replication and transcription per se. Described modifications do not potentiate antiviral activity of CRISPR/Cas9 system and should not be used for development of future therapeutics. The best strategy to improve CRISPR/Cas9 efficacy is to design new highly effective guide RNAs. 

About the authors

A. P. Kostyusheva

Central Research Institute of Epidemiology

Author for correspondence.
Email: ak@rcvh.ru
ORCID iD: 0000-0002-2335-6582

Junior Researcher, Laboratory of Viral Hepatitis

Contacts: Anastasiya P. Kostyusheva 111123, Russian Federation, Moscow, Novogireevskaja str., 3A, Central Research Institute of Epidemiology. Phone: +7 (925) 310-91-24

Russian Federation

S. A. Brezgin

Central Research Institute of Epidemiology;
Institute of Immunology FMBA

Email: sb@rcvh.ru
ORCID iD: 0000-0003-4792-0739
Junior Researcher, Laboratory of Viral Hepatitis, Central Research Institute of Epidemiology,; PhD Student, Laboratory No. 73 of Clinical Pharmacology, Institute of Immunology FMBA

D. N. Zarifyan

Central Research Institute of Epidemiology

Email: dz@rcvh.ru
Research Technician, Laboratory of Viral Hepatitis

D. S. Chistyakov

I.M. Sechenov Moscow State Medical University

Email: dchistakoff@gmail.com
Technician, Laboratory of Viral Hepatitis

V. I. Gegechkory

I.M. Sechenov Moscow State Medical University

Email: mr.faul@mail.ru
PhD (Pharmaceutical Sciences), Senior Faculty Member, Assistant Professor, A.P. Arzamastsev Department of Pharmaceutical and Toxicological Chemistry

E O. Bayurova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products

Email: 79153645941@ya.ru
Junior Researcher, Laboratory of Immunobiological Processes Modeling with the Experimental Clinic of the Marmosets of the Department of Innovative Biotechnological Preparations

E. A. Volchkova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products

Email: antononina@rambler.ru
ORCID iD: 0000-0003-4581-4510
Junior Researcher, Laboratory of Immunobiological Processes Modeling with the Experimental Clinic of the Marmosets of the Department of Innovative Biotechnological Preparations

D. S. Kostyushev

Central Research Institute of Epidemiology

Email: ak@rcvh.ru
ORCID iD: 0000-0002-1851-7441
Junior Researcher, Laboratory of Viral Hepatitis

V. P. Chulanov

Central Research Institute of Epidemiology;
I.M. Sechenov Moscow State Medical University

Email: vladimir.chulanov@rcvh.ru
ORCID iD: 0000-0001-6303-9293
PhD, MD (Medicine), Head of the Laboratory of Viral Hepatitis, Central Research Institute of Epidemiology; Professor, Department of Infectious Diseases, Preventive Medicine Department, I.M. Sechenov Moscow State Medical University

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Copyright (c) 2019 Kostyusheva A.P., Brezgin S.A., Zarifyan D.N., Chistyakov D.S., Gegechkory V.I., Bayurova E.O., Volchkova E.A., Kostyushev D.S., Chulanov V.P.

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