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M. Hernandez, S. E. Pearce-Kelling, D. Rodriguez, A. Rivas, G. Aguirre, E. Vecino; Recovery of Molecular Marker Expression in RPE65 Mutant Dog Retinas After Gene Therapy using Adeno-Associated Virus. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4616.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the expression of molecular markers specific for different retinal cell types in RPE65 mutant dogs before and after retinal gene therapy, and to establish the therapeutic time window whereby treatment restores expression.
Retinas from wt, RPE 65 -/- affected and treated dogs were analyzed. The treatment consisted in one single subretinal injection of rAAV-mediated RPE65. The dog retinas were analyzed at 17 months of age in affected and treated animals. In the treated animals, the injection of rAAV was performed 5 months before the analysis. The retinas then processed for immunocytochemistry, and the different cell types were examined using antibodies against various molecular markers of the outer retina (M/Lopsin, S opsin, hCAR and rod opsin), inner retina (Goα or PKC, Calretinin, GABA, TH, BDNF) and glial cells (GFAP and vimentin). RPE65 labeling was used as a marker to identify treated areas.
In affected retinas we found a decrease in the expression of photoreceptor and amacrine cell markers, increase in the expression of glial markers, and changes in the synaptic processes of amacrine and bipolar cells. After treatment, we found a restoration in the expression of the photoreceptor, glial and amacrine markers. However, 5 moths after treatment, the synaptic changes in the inner retina are still present.
Subretinally delivered recombinant adeno-associated virus with wild-type RPE65 cDNA can restore a normal structure of the photoreceptor cells in RP65 -/- affecteddog retinas. This restoration has a partial effect in the inner retinal layers.
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