Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. Approximately 30% of these patients progress to cirrhosis within 5 years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR-Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a "safe harbor," in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase minigene was precisely knocked in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be used as a new alternative in the development of a treatment for HCV-induced liver diseases.
Keywords: AAVS1; CRISPR/Cas9; NS5B RNA replicase; RNA aptamer; genome editing; hepatitis C virus.
© 2023 The Author(s).