Abstract
Purpose: :
To evaluate adeno–associated virus (AAV)–mediated green fluorescent protein (GFP) expression in canine cones using the human red and blue cone opsin promoters.
Methods: :
AAV vectors with serotype 5 capsids containing GFP cDNA under control of either the human red (PR2.1) or blue (HB570) cone opsin promoters were injected into the subretinal space of young, normal dogs. A total of 12 eyes of 6 dogs were injected with 150 µl with 1.2–1.6x1012 vector genomes. Retinal sections were examined for GFP expression after 4 and 8 weeks under fluorescence microscopy. Immunocytochemistry using blue and red/green opsin antibodies helped confirm the targeting specificity of GFP expression.
Results: :
No adverse effects of the viral vectors or the GFP expression were noted clinically and histologically. The PR2.1 promoter led to specific GFP expression in the red/green cones, evident by GFP fluorescence co–localizing with red/green opsin staining. The entire cells were brightly fluorescent. Alternatively, the HB570 promoter led to the expression of GFP in a subgroup of red/green cones, rods, and in the RPE.
Conclusions: :
The human cone opsin promoters are effective in directing expression of transferred genes in canine cone photoreceptor cells. While the red cone opsin promoter (PR2.1) showed a high degree of efficiency and specificity in expressing GFP in red/green cones, the blue cone opsin promoter (HB570) was less robust and specific as evidenced by GFP expression in some rod photoreceptors and RPE cells. As red/green cones outnumber the blue cones in mammalian retinas, our results indicate that cone directed gene replacement therapy should be possible with the PR2.1 promoter.
Keywords: gene transfer/gene therapy • photoreceptors • retina