mirGE vs miR-30 reads

Gene knock-down using miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knock-down through optimizing the structure of miRNA mimics. Knock-down of two target genes was analyzed: CCR5 and GFP. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs, positioning of the targeting strand on the 5' side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knock-down with an efficiency of over 90% upon single copy transduction. Importantly, single copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knock-down efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV-resistant through single copy vector transduction, rendering this approach more compatible with clinical applications.