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K. E. Guziewicz, A. M. Komaromy, W. W. Hauswirth, S. L. Boye, V. A. Chiodo, J. Slavik, G. M. Acland, G. D. Aguirre, B. Zangerl; Targeting Gene Expression to the RPE With AAV2/1 and AAV2/2 Vectors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3132.
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It has been shown in dogs and humans that primary disorders of the RPE can be treated by gene therapy with the AAV2 vector. Canine multifocal retinopathy (cmr), caused by mutations in the VMD2 gene, has been established as a model for human Best disease, and therapeutic approaches are being developed using AAV vectors. The AAV1 serotype is thought to be highly specific in targeting RPE cells, and has greater transduction efficiency than AAV2. We have compared the two vector serotypes carrying hGFP reporter gene under control of the human VMD2 promoter. We determined onset, efficiency, and stability of gene expression as well as potential adverse effects of subretinal injection with both AAV1 and AAV2 in dogs.
Dogs 9-10 months of age were subretinally injected with either AAV1 (1.75x1012 vg/ml) or AAV2 (6.07x1011 vg/ml) carrying a construct encoding humanized green fluorescent protein (hGFP) under the control of the human VMD2 promoter (courtesy of D. Zack). Each injected eye was monitored clinically following the injection, and collected at 2, 4, or 6 weeks after injection, respectively. Tissues were fixed in 4% paraformaldehyde, and OCT embedded. hGFP expression was evaluated on 7µm sections with and without the use of a GFP specific antibody.
Fundus exams revealed a small scar at the site of the injection, but eyes were otherwise normal. No significant difference in the onset and expression level of hGFP was observed using AAV1 or AAV2. No adverse effects were noted with either vector secondary to the subretinal injection. At 2 weeks native hGFP expression was not present in the RPE, but was detected with antibody labeling. Specific signal was present 4 weeks after injection, and was stable and comparable to the results at 6 weeks after injection. hGFP expression by either direct hGFP fluorescence or antibody labeling was only present in areas corresponding to the subretinal bleb.
Our results suggest that gene therapy targeting the RPE using the human VMD2 promoter is specific, stable, and safe with either AAV1 or AAV2. Experiments with the same viral vectors expressing canine bestrophin are now ongoing to determine optimal dosage and time for gene therapy in the canine best disease model.
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