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L.G. Glushakova, A.M. Timmers, T.D. Doyle, Q. Li, V. Chiodo, W.W. Hauswirth; AAV Transfers Human Blue Cone Opsin Promoter Targeted Reporter Gene Expression to Rat S-cone Photoreceptors with High Efficiency . Invest. Ophthalmol. Vis. Sci. 2003;44(13):440.
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Purpose:To evaluate the specificity and efficiency of the transgene expression driven by a human blue opsin (BH) promoter in the rodent retina via AAV serotype 5 vector and to test the feasibility of this vector as a tool to develop an S-cone gene delivery system. Methods:Upstream sequences of the BH gene were amplified from blood, cloned into a rAAV2 cassette upstream of a GFP reporter gene, and then packaged into rAAV5 capsids by routine procedures. Two µl of purified viral suspension was injected subretinally into male Sprague-Dawley rats at PN 40-48. Animals were euthanized at the PN 100-130 and imminocytochemistry used to analyze patterns and efficiency of GFP reporter expression. Results: Subretinal injections of rAAV5.BH.GFP resulted in photoreceptor-exclusive GFP expression. Photoreceptor transduction appeared to be on average 15-20 - fold more efficient than via an analogue, serotype 2 vector, rAAV2.BH.GFP. Dual GFP / S-cone, GFP / M-cone immunostaning and GFP / cone specific PNA labeling revealed that S-cones constituted 37.5 ± 8% of the GFP-expressing photoreceptor cells while M -cones and rods constituted 13.5±3% and 49 ±5% respectively. Since the prevalence of S-cones vs. M-cones vs. rods in the rat is 1:16:1983 (Szel and Rolich, Exp Eye Res 1992;55:47-52) the relative transduction efficiency of rAAV5.BH.GFP is 1500:34:1 for S-cones vs.M-cones vs. rods respectively. Conclusions: 1. rAAV5 BH.GFP virus injected into subretinal space of rats targeted gene expression to photoreceptor cells with an efficiency approximately 15-20 - fold higher than rAAV2.BH.GFP. 2. In the context of the BH promoter, S-cones transduction is extremely specific: it’s efficiency is approximately 1,500 and 34 - fold higher than transduction to rod and M-cone photoreceptors respectively, suggesting that this system may be an effective way to transduce S-cones in a therapeutic paradigm. Supported by: EY 07864, EY 11123, EY 11596, EY 413101, NS 36302, FFB, MVRF, RPB. CR: P
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