The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery.
Bibliographical noteFunding Information:
This material is based upon work supported in part by the US Army Research Laboratory and the US Army Research Office under grant number W911NF-07-R-0003-4. R.A.L. and A.M.P. are supported by the NYU-MSSM Mechanisms of Virus–Host Interactions National Institutes of Health T32 training grant (no. AI007647-09). B.R.tO is supported in part by the Pew Charitable Trust and the Burroughs Wellcome Fund. We would additionally like to thank Alexander Tarakhovsky (Rockefeller University) and Donal O'Carroll (EMBL, Monterotondo, Italy) for Dicer1 −/− fibroblasts, and the members of the MSSM Microbiology department for helpful discussions.
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