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

PurposeTo determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector.MethodsAn 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.ResultThe 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.ConclusionThe 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.