title: Regulated and multiple miRNA and siRNA delivery into primary cells by a lentiviral platform.
abstract: RNA interference (RNAi) has tremendous potential for investigating gene function and developing new therapies. However, the design and validation of proficient vehicles for stable and safe microRNA (miR) and small interfering RNA (siRNA) delivery into relevant target cells remains an active area of investigation. Here, we developed a lentiviral platform to efficiently coexpress one or more natural/artificial miR together with a gene of interest from constitutive or regulated polymerase-II (Pol-II) promoters. By swapping the stem-loop (sl) sequence of a selected primary transcript (pri-miR) with that of other miR or replacing the stem with an siRNA of choice, we consistently obtained robust expression of the chimeric/artificial miR in several cell types. We validated our platform transducing a panel of engineered cells stably expressing sensitive reporters for miR activity and on a natural target. This approach allowed us to quantitatively assess at steady state the target suppression activity and expression level of each delivered miR and to compare it to those of endogenous miR. Exogenous/artificial miR reached the concentration and activity typical of highly expressed natural miR without perturbing endogenous miR maturation or regulation. Finally, we demonstrate the robust performance of the platform reversing the anergic/suppressive phenotype of human primary regulatory T cells (Treg) by knocking-down their master gene Forkhead Transcription Factor P3 (FOXP3).
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Amendola
authors.forename: Laura
authors.initials: L
authors.lastname: Passerini
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Pucci
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gentner
authors.forename: Rosa
authors.initials: R
authors.lastname: Bacchetta
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
mesh_terms: D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D035683:MicroRNAs; D034741:RNA, Small Interfering