Viral vectors expressing a single microRNA-based short-hairpin RNA result in potent gene silencing in vitro and in vivo

• Development of a generic cloning platform to generate knockdown viral vectors.
• Evaluation of miR-based viral vectors encoding up to six miR-hairpin repeats.
• Knockdown potency was evaluated in cell culture and in vivo for different targets.
• Potent knockdown was achieved with single miR-based short-hairpin RNA viral vectors.

The characterization of RNA interference and the accompanying microRNAs (miRs), together with the exogenous expression of artificial miR-like elements, has led to the development of strategies for specific and potent gene silencing. In turn, this allows manipulation of gene expression levels for target validation purposes in cell culture or for the generation of animal models. In this study we determined the optimal strategy to achieve the most potent knockdown using miR-based viral vectors. We studied polycistronic miRs in a viral vector context and evaluated knockdown potency of multiple-miRs targeting the same seed sequence in parallel with miRs targeting different seed sequences, both for a reporter and endogenous mRNA targets. We demonstrate that potent knockdown can be obtained in vitro and in vivo using viral vectors that encode a single miR-based short-hairpin RNA and report a generic and effective cloning platform for artificial miR30-based short-hairpin RNAs to generate potent knockdown viral vectors.