Generation of hepatitis C virus–resistant liver cells by genome editing–mediated stable expression of RNA aptamer

• Published in: Molecular therapy. Methods & clinical development Vol. 31; p. 101151
• Main Authors: Kim, Tae Hyeong, Lee, Seong-Wook
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 14.12.2023; Elsevier
• Subjects: AAVS1; CRISPR/Cas9; genome editing; hepatitis C virus; NS5B RNA replicase; RNA aptamer; RNA aptamer; hepatitis C virus; genome editing; CRISPR/Cas9; AAVS1; NS5B RNA replicase
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2023.101151

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. [Display omitted] Lee and colleagues generated hepatitis C virus (HCV)–resistant liver cells by specifically inserting an HCV NS5B replicase-specific RNA aptamer into AAVS1 using the CRISPR/Cas9 system. The cells were safely and effectively protected from HCV replication using an RNA aptamer as a decoy with minimal off-target effects. The authors proposed therapeutics to overcome HCV recurrence after liver transplantation.