May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
AAV Transfers Human Blue Cone Opsin Promoter Targeted Reporter Gene Expression to Rat S-cone Photoreceptors with High Efficiency
L.G. Glushakova; A.M. Timmers; T.D. Doyle; Q. Li; V. Chiodo; W.W. Hauswirth
Author Affiliations & Notes
  • L.G. Glushakova
    Ophthalmology, University of Florida, Gainesville, FL, United States
  • A.M. Timmers
    Ophthalmology@ Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
  • T.D. Doyle
    Molecular Genetics, University of Florida, Gainesville, FL, United States
  • Q. Li
    Molecular Genetics, University of Florida, Gainesville, FL, United States
  • V. Chiodo
    Molecular Genetics, University of Florida, Gainesville, FL, United States
  • W.W. Hauswirth
    Ophthalmology@Molecular Genetics@Powell gene Therapy Center, University of Florida, Gainesville, FL, United States
  • Footnotes
    Commercial Relationships  L.G. Glushakova, None; A.M. Timmers, None; T.D. Doyle, None; Q. Li, None; V. Chiodo, None; W.W. Hauswirth, None.
  • Footnotes
    Support  EY 07864, EY 11123, EY 11596, NS36302, FFB, MVRF, RPB
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 440. doi:
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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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Abstract

Abstract: : 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

Keywords: gene transfer/gene therapy • photoreceptors • animal model 
© 2003, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2024 Association for Research in Vision and Ophthalmology.
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