Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9

Dmitry Kostyushev; Anastasiya Kostyusheva; Sergey Brezgin; Natalia Ponomareva; Natalia F Zakirova; Aleksandra Egorshina; Dmitry V Yanvarev; Ekaterina Bayurova; Anna Sudina; Irina Goptar; Anastasiya Nikiforova; Elena Dunaeva; Tatiana Lisitsa; Ivan Abramov; Anastasiia Frolova; Alexander Lukashev; Ilya Gordeychuk; Andrey A Zamyatnin Jr; Alexander Ivanov; Vladimir Chulanov

doi:10.1016/j.omtn.2023.02.001

Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9

Mol Ther Nucleic Acids. 2023 Feb 4:31:482-493. doi: 10.1016/j.omtn.2023.02.001. eCollection 2023 Mar 14.

Authors

Dmitry Kostyushev^{1

2}, Anastasiya Kostyusheva¹, Sergey Brezgin^{1

2}, Natalia Ponomareva^{1

2

3}, Natalia F Zakirova⁴, Aleksandra Egorshina¹, Dmitry V Yanvarev⁴, Ekaterina Bayurova⁵, Anna Sudina⁶, Irina Goptar⁷, Anastasiya Nikiforova⁷, Elena Dunaeva⁸, Tatiana Lisitsa⁶, Ivan Abramov⁶, Anastasiia Frolova⁹, Alexander Lukashev¹, Ilya Gordeychuk^{5

10}, Andrey A Zamyatnin Jr^{2

9

11

12}, Alexander Ivanov⁴, Vladimir Chulanov^{2

10

13

14}

Affiliations

¹ Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, Moscow 119991, Russia.
² Scientific Center for Genetics and Life Sciences, Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia.
³ Department of Pharmaceutical and Toxicological Chemistry, Sechenov First Moscow State Medical University, Moscow 119146, Russia.
⁴ Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Science, Moscow 119991, Russia.
⁵ Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, Moscow 108819, Russia.
⁶ Federal State Budgetary Institution Centre for Strategic Planning and Management of Biomedical Health Risks of the Federal Medical Biological Agency, Moscow 119435, Russia.
⁷ Izmerov Research Institute of Occupational Health, Moscow 105275, Russia.
⁸ Central Research Institute of Epidemiology, Moscow 111123, Russia.
⁹ Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow 119991, Russia.
¹⁰ Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Moscow 127994, Russia.
¹¹ Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
¹² Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, UK.
¹³ Department of Infectious Diseases, Sechenov First Moscow State Medical University, Moscow 119146, Russia.
¹⁴ National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, Moscow 127994, Russia.

Abstract

CRISPR-Cas9 systems can directly target the hepatitis B virus (HBV) major genomic form, covalently closed circular DNA (cccDNA), for decay and demonstrate remarkable anti-HBV activity. Here, we demonstrate that CRISPR-Cas9-mediated inactivation of HBV cccDNA, frequently regarded as the "holy grail" of viral persistence, is not sufficient for curing infection. Instead, HBV replication rapidly rebounds because of de novo formation of HBV cccDNA from its precursor, HBV relaxed circular DNA (rcDNA). However, depleting HBV rcDNA before CRISPR-Cas9 ribonucleoprotein (RNP) delivery prevents viral rebound and promotes resolution of HBV infection. These findings provide the groundwork for developing approaches for a virological cure of HBV infection by a single dose of short-lived CRISPR-Cas9 RNPs. Blocking cccDNA replenishment and re-establishment from rcDNA conversion is critical for completely clearing the virus from infected cells by site-specific nucleases. The latter can be achieved by widely used reverse transcriptase inhibitors.

Keywords: Cas9 RNPs; DNA methylation; MT: RNA/DNA Editing; antiviral therapy; cccDNA elimination; chronic hepatitis B; cure; epigenetics; reverse transcription; viral rebound.