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N.J. Sund, X. Yang, A. Kuroki, N. Mirza, J. Bennett, A. Auricchio, M.J. Tolentino; Heat-inducible AAV-mediated Gene Delivery in RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):454.
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Purpose: Tightly-controlling gene delivery of therapeutic proteins to the retina will be important in the treatment of retinal pathology. The purpose of this study is to develop a heat-inducible viral delivery system using an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) under the control of the heat shock protein 70 (hsp70) promoter. Methods: To observe gene activation under different temperatures, recombinant AAV-hsp70-GFP was used to infect human retinal pigment epithelial cell line D407. The cells were then heated between 43 and 47 degrees celsius at different time intervals. Reporter GFP expression was evaluated 24 hours later by fluorescence microscopy and quantified using Image Pro Plus software. Results: AAV-hsp70-GFP is highly-induced in RPE cells when heated compared to non-heat shocked controls. We report the optimal temperature and conditions for maximum transgene expression from this recombinant vector, which is comparable to AAV-GFP under the control of a CMV constitutive promoter. Conclusions: The AAV-hsp70 promoter is active in human RPE cells in vitro and is highly inducible under optimal conditions. This study suggests that an AAV-mediated delivery system based on the hsp70 promoter can deliver therapeutic proteins in a space- and time- specific manner. This may prove useful in developing novel therapies for many types of retinal pathologies.
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