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K Narfstrom, R Bragadòttir, TM Redmond, ML Katz, B Lei, CM Lai, EP Rakoczy; Gene Therapy in 6 Dogs With RPE65 Null Mutation Improves Visual Function: A Short-term Study Using Clinical Observations, Electrophysiology and Morphology . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4601.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To obtain evidence that gene therapy using an AAV.RPE65 gene construct restores functional vision in dogs affected with an RPE65 null mutation, a model for Leber´s congenital amaurosis. Methods: The normal RPE65 dog cDNA was subcloned into an rAAV vector under the control of a CMV promoter. A control vector with GFP cDNA in place of the RPE65 cDNA was also constructed. Batches of rAAV.GFP and rAAV.RPE65 were produced, the latter with a titer of 6x1013 particles/ml. Base-line behavioural vision tests, funduscopic examination and bilateral full-field electroretinography (ERG) were performed in six affected 4-mo-old dogs. At ages 4 mo (3 dogs), 8 mo (2 dogs) and 11 mo (1 dog), the dogs underwent subretinal injections, using a two-port entry through the pars plana. 30 to 100 ml of the rAAV.RPE65 construct was injected subretinally into one eye and, in 4 dogs, the same volume of rAAV.GFP contralaterally. Multiple follow-up ERGs were performed. An 8-mo-old dog was euthanized 3 mo post-operatively and both eyes used for morphologic studies. Results: All dogs were initially functionally blind, had nystagmus, abnormal pupillary responses but normal fundus with unrecordable darkadapted ERG. Distinct low-amplitude ERG responses were recorded when using photopic flicker stimuli. After treatment all six dogs showed limited vision, proven by behavioural studies, the earliest 1 mo after surgery. Follow-up ERGs showed that the scotopic ERG b-wave amplitudes recovered to an average of 21% of normal dogs, and the photopic b-wave responses to 17% of normals. LM and EM of the AAV.RPE65 treated eye showed a lack in the treated area of the lipoid-like inclusions found in eyes of affected animals. Conclusion: Successful AAV.RPE65 gene transfer results in useful clinical vision and correlates with limited but definite retinal functional and structural improvement, including prevention/reversal of a major phenotypic change in the RPE. Whether there is long-term structural saving of residual photoreceptors of the mutant dog remains to be elucidated.
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