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B. Zangerl, K. E. Guziewicz, A. M. Komaromy, S. J. Lindauer, J. J. Alexander, L. G. Glushakova, W. W. Hauswirth, G. M. Acland, G. D. Aguirre; Human Bestrophin Promoter Drives Rpe-Specific Gfp Expression in the Canine Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1701.
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Canine multifocal retinophathy (cmr), caused by mutations in the bestrophin gene, is a large animal model for human Best disease. To begin the development of a gene therapy approach to treat cmr, a recombinant Adeno-Associated virus (rAAV) was made to express GFP under control of the human bestrophin promoter (hVMD2-GFP). Normal canine eyes were used to determine the level and specificity of expression.
The VMD2 promoter was a gift from D. Zak. Normal dogs at 4 months of age had one eye injected in either the subretinal or sub-RPE space with 3.35 x1013 vg of hVMD2-GFP AAV2/1; the contralateral eye was either injected with saline or remained uninjected. Fundus exams were performed 2 and 4 weeks after injection, and eyes were collected after 4 weeks, fixed in 4% paraformaldehyde, and embedded in OCT. GFP expression was evaluated on 7µm sections with and without specific antibody.
The fundus exam revealed a small scar at the site of injection, but eyes were otherwise normal. After 4 weeks, ophthalmoscopy with a blue light source showed green fluorescence only with subretinally injected hVMD2-GFP. In sections, GFP expression was evident in the area corresponding to the hVMD2-GFP subretinal bleb by direct GFP fluorescence and antibody labeling. Sub-RPE injection of hVMD2-GFP yielded a significantly lower number of GFP expressing cells, and expression could only be detected by antibody labeling.
The human bestrophin promoter drives stable and specific GFP expression in canine RPE cells after in vivo transduction with rAAV. Currently, we are evaluating rAAV mediated expression of wild-type bestrophin in place of GFP in the same construct. These results provide a basis for further therapeutic studies in the cmr dog which are of petential significance for therapy in human patients with Best disease.
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