Generation of combinatorial lentiviral vectors expressing multiple anti hepatitis C virus shRNAs and their validation on a novel HCV replicon double reporter cell line.

Despite the introduction of the direct antiviral agents (DAA), there is still space for the development of novel therapeutic strategies to efficiently tackle hepatitis C virus (HCV). Indeed, drug costs, patient compliance and viral resistance to treatment might compromise the success of DAA. Here we describe the generation and characterization of an array of lentiviral vectors expressing single double or triple cassettes expressing different combinations of short hairpin (sh)RNAs, targeting both viral and cellular factors crucial for HCV replication. Upon transduction of a hepatocyte derived cellular carcinoma cell line, the developed combinatorial platforms led to shRNA expression without significant cytopatic effects. Finally, their antiviral effect was tested on a novel HCV replicon double reporter cell line that allowed a rapid and efficient quantification of the viral replication capacity in the presence of the shRNA. All vectors were effective in reducing HCV replication, with the ones encoding shRNAs targeting viral sequences acting earlier upon transduction. The developed combinatorial platforms as well as the double reporter cell lines 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.