Institut for Biomedicin

Jacob Giehm Mikkelsen

AAV-delivered polycistronic microRNA-clusters for knockdown of VEGF in vivo

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BACKGROUND: Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases including cancer, rheumatoid arthritis, and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors. METHODS: Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retina pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative RT-PCR, Western blotting and immunostaining analysis. In vivo efficacy was tested by injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR. RESULTS: Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNAi effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF. CONCLUSIONS: We have developed miRNA clusters encoding anti-VEGF effectors and shown in a mouse model that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters. Copyright © 2012 John Wiley & Sons, Ltd.
TidsskriftThe Journal of Gene Medicine
StatusUdgivet - 2012

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