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 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472bp 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 2.1kb 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.
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 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472bp 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 2.1kb 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.
Authors: Ekaterina S Lobanova; Rolf Herrmann; Stella Finkelstein; Boris Reidel; Nikolai P Skiba; Wen-Tao Deng; Rebecca Jo; Ellen R Weiss; William W Hauswirth; Vadim Y Arshavsky Journal: J Neurosci Date: 2010-05-19 Impact factor: 6.167
Authors: Katherine Mancuso; William W Hauswirth; Qiuhong Li; Thomas B Connor; James A Kuchenbecker; Matthew C Mauck; Jay Neitz; Maureen Neitz Journal: Nature Date: 2009-09-16 Impact factor: 49.962
Authors: Matthew C Mauck; Katherine Mancuso; James A Kuchenbecker; Thomas B Connor; William W Hauswirth; Jay Neitz; Maureen Neitz Journal: Vis Neurosci Date: 2008 May-Jun Impact factor: 3.241
Authors: A M Komáromy; J J Alexander; A E Cooper; V A Chiodo; L G Glushakova; G M Acland; W W Hauswirth; G D Aguirre Journal: Gene Ther Date: 2008-03-13 Impact factor: 5.250