Gamma-retrovirus design for efficacious microRNA-mediated gene knock-down and protein co-expression without
Romain Vuillefroy de Silly1, Bili Seijo1, Jimmy Maillard1
1Ludwig Institute for Cancer Research, University of Lausanne and Department of Oncology, Lausanne University Hospital (CHUV), CH-1011 Lausanne, Switzerland.
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RNA interference (RNAi) is a powerful tool for controlling gene expression. Although stable gene down-regulation in cells can be achieved with viral vectors expressing short hairpin RNAs (shRNAs), incorporating shRNAs into a microRNA (miR) backbone can improve both the efficacy and safety of RNAi by exploiting the endogenous miR-processing machinery. For gamma-retroviral vectors there are two main strategies, LTR (Pol II)- and U6 (Pol III)-driven expression of the miR-based shRNA, but both have limitations in viral titers generated, knock-down efficacy, and/or protein/reporter gene co-expression levels. Here, we sought to evaluate the effectiveness of inserting miR-30a-based shRNA within the psi/packaging-containing intron of the murine stem cell virus (MSCV)-based splice-gag vector (MSGV) for which transcription is LTR-driven. Systematic comparison to previously described designs within the same viral backbone comprising a range of different shRNAs targets revealed maximal viral titer, knock-down and level of protein co-expression upon intronic insertion of miR-based shRNA. In addition, we demonstrated that the strategy can be extended to gene knock-down by wild-type miRs, as demonstrated for miR-30c1 and let-7a. Taken together, we have developed a gamma-retroviral vector design facilitating efficacious miR-mediated gene knock-down and maintaining strong viral titers.
