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(Circulation Research. 2009;105:604.)
© 2009 American Heart Association, Inc.

Molecular Medicine

Efficient Regulation of VEGF Expression by Promoter-Targeted Lentiviral shRNAs Based on Epigenetic Mechanism

A Novel Example of Epigenetherapy

Mikko P. Turunen, Tiia Lehtola, Suvi E. Heinonen, Genet S. Assefa, Petra Korpisalo, Roseanne Girnary, Christopher K. Glass, Sami Väisänen, Seppo Ylä-Herttuala

From Ark Therapeutics Oy (M.P.T., G.S.A., R.G.), Kuopio, Finland; the Department of Biosciences (T.L., S.V.) and the Department Biotechnology and Molecular Medicine and Department of Medicine (S.E.H., S.Y.-H.), University of Kuopio, Finland; the Gene Therapy Unit (S.Y.-H.), Kuopio University Hospital, Finland; and the Department of Cellular & Molecular Medicine (C.K.G.), School of Medicine, University of California, San Diego.

Correspondence to Seppo Ylä-Herttuala, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail seppo.ylaherttuala{at}uku.fi

Rationale: We studied a possibility that shRNAs can lead to transcriptional gene activation at the promoter level via epigenetic mechanism.

Objective: The purpose of this study was to test the effects on vascular endothelial growth factor (VEGF-A) expression by promoter targeted small hairpin RNAs (shRNAs) in vitro and in experimental animals in vivo using stable local lentiviral gene transfer.

Methods and Results: One shRNA was identified which strongly increased VEGF-A expression in C166 endothelial cells at mRNA and protein level whereas another shRNA decreased VEGF-A expression. Quantitative chromatin immunoprecipitation analysis revealed that the repressing shRNA caused epigenetic changes, which increased nucleosome density within the promoter and transcription start site and led to repression of VEGF-A expression. Epigenetic changes caused by the activating shRNA were opposite to those caused by the repressing shRNA. These results were confirmed in vivo in an ischemic mouse hindlimb model after local gene transfer where VEGF-A upregulation achieved by promoter-targeted shRNA increased vascularity and blood flow.

Conclusions: We show that lentivirus-mediated delivery of shRNA molecules targeted to specific regions in the mVEGF-A promoter either induce or repress VEGF-A expression via epigenetic modulation. Thus, we describe a new approach of gene therapy, epigenetherapy, based on an epigenetic mechanism at the promoter level. Controlling transcription through manipulation of specific epigenetic marks provides a novel approach for the treatment of several diseases.

Key Words: endothelial cells • gene regulation • gene therapy • transcription factors • transcriptional regulation • vascular endothelial growth factor