CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Daniel Stone; Kelly R Long; Michelle A Loprieno; Harshana S De Silva Feelixge; Elizabeth J Kenkel; R Matt Liley; Stephen Rapp; Pavitra Roychoudhury; Thuy Nguyen; Laurence Stensland; Rossana Colón-Thillet; Lindsay M Klouser; Nicholas D Weber; Connie Le; Jessica Wagoner; Erin A Goecker; Alvason Zhenhua Li; Karsten Eichholz; Lawrence Corey; D Lorne Tyrrell; Alexander L Greninger; Meei-Li Huang; Stephen J Polyak; Martine Aubert; John E Sagartz; Keith R Jerome

doi:10.1016/j.omtm.2020.11.014

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Mol Ther Methods Clin Dev. 2020 Nov 26:20:258-275. doi: 10.1016/j.omtm.2020.11.014. eCollection 2021 Mar 12.

Authors

Daniel Stone¹, Kelly R Long², Michelle A Loprieno¹, Harshana S De Silva Feelixge¹, Elizabeth J Kenkel³, R Matt Liley², Stephen Rapp², Pavitra Roychoudhury³, Thuy Nguyen³, Laurence Stensland³, Rossana Colón-Thillet¹, Lindsay M Klouser¹, Nicholas D Weber¹, Connie Le⁴, Jessica Wagoner³, Erin A Goecker³, Alvason Zhenhua Li¹, Karsten Eichholz¹, Lawrence Corey^{1

3}, D Lorne Tyrrell⁴, Alexander L Greninger³, Meei-Li Huang³, Stephen J Polyak³, Martine Aubert¹, John E Sagartz², Keith R Jerome^{1

3}

Affiliations

¹ Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
² Seventh Wave Laboratories, LLC, 19 Worthington Access Drive, Maryland Heights, MO 63043, USA.
³ Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.
⁴ Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2E1, Canada.

Abstract

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV⁺ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.

Keywords: AAV; CRISPR/Cas9; Gene editing; HBV; Humanized mouse.

Abstract

Grants and funding