title: Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction.
abstract: Gene knockdown using micro RNA (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 knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp), 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. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC); incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown 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 knockdown 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.
authors.affiliation: 1] Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland [2] Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland [3] Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
authors.forename: Renier
authors.initials: R
authors.lastname: Myburgh
authors.affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Ophélie
authors.initials: O
authors.lastname: Cherpin
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Erika
authors.initials: E
authors.lastname: Schlaepfer
authors.affiliation: Functional Genomics Center, University of Zurich, Zurich, Switzerland.
authors.forename: Hubert
authors.initials: H
authors.lastname: Rehrauer
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Roberto F
authors.initials: RF
authors.lastname: Speck
authors.affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Krause
authors.affiliation: Department of Neurosciences, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Salmon