Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line

Viruses

Khaled Thabet¹  ClaudiaDel Del Vecchio²  Francesco Ciccarese²  Adriana Vitiello²  Eleonora Ponterio²  Giorgio Palù²  Cristiano Salata²  Arianna Calistri²  Cristina Parolin²  Elisa Franchin²  Gualtiero Alvisi²  Hanieh Ghassabian²  VeronicaDi Di Antonio²  Mekky M. Abouzied³  Hossein M. Elbadawy³  Mohi I. Mohammed Abdul³  Naif Aljuhani³  Heba M. Eltahir³  Ahmed M. Shehata³  Saleh Bahashwan³  Mohamed A. Shaker⁴ 

[1] Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61519, Egypt;Department of Molecular Medicine, University of Padua, 35121 Padua, Italy;Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia;Pharmaceutics and Pharmaceutical Technology Department, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia;

关键词: hepatitis C virus;    reporter cell line;    antivirals;    gene therapy;    siRNA;   

DOI  :  10.3390/v12091044

来源: DOAJ

【 摘 要 】

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

【 授权许可】

Unknown