Cone-specific expression using a human red opsin promoter in recombinant AAV

Purpose: To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector. Methods: An rAAV vector was constructed that contains a 2.1 kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472 bp mouse rod opsin promoter, previously shown to drive photoreceptor-specific expression, was also used. Each recombinant virus was injected into the subretinal space of rat, ferret or guinea pig eyes. GFP expression was analyzed 4-6 weeks after injection microscopically. Result: The human 2.1 kb cone opsin gene upstream sequence targeted GFP expression only to a subset of photoreceptors. Cone-specific expression was shown by co-localization of GFP fluorescence and cone-specific opsin antibody staining. Additionally, in rats, expression was specific for L/M-cones whereas no S-cones exhibited GFP fluorescence. The efficiency of rAAV mediated cone transduction surrounding the injection site was high since every L/M-cone antibody-staining cone was also positive for GFP expression. Conclusion: The human red/green opsin gene promoter used in this study is sufficient to direct efficient cone-specific gene expression in several mammalian species, suggesting that key cell-type specific regulatory elements must be broadly conserved in mammals. These observations have significance in devising gene therapy strategies for retinal dystrophies that primarily affect cones and point toward a way to functionally dissect the cone opsin promoter in vivo. (c) 2008 Published by Elsevier Ltd.