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F Rolling, H Conrath, M Weber, D Briot, Y Cherel, D Schmidt-Morand, P Moullier; Kinetics of Recombinant Adeno-Associated Virus Mediated Gene Transfer into Dog Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4607.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The goals were to realize i) a precise and reproducible rAAV-2.CMVgfp subretinal injection, ii) efficient rAAV-mediated transfer, and iii) accurate noninvasive quantification of retinal transgene expression over time in the dog. Methods: Subretinal injection in the dog retina was optimized by designing a new automated and controlled injection device. GFP protein expression in live dogs was monitored by fluorescent retinal imaging using a retinal camera connected to a digital imaging system. Digital images taken at various timepoints postinjection were analyzed using Matrox Inspector software. We extracted and counted the green pixels within the transduced area overtime and the kinetics of expression were calculated. Additionally, viral tropism was determined by histology. Results: Subretinal injection of rAAV-2.CMVgfp was performed via a transvitreal route within the tapetal retina of dogs. Quantitative results obtained over a four-month monitoring period showed that gfp expression was detected as early as 4 days post-injection and gradually increased before reaching maximal levels at 60-70 days postinjection. Additionally, subretinal readministration of rAAV allowed efficient gene transfer and preexistence of serotype-specific neutralizing antibody did not impact on efficient transduction and transgene expression pattern. Ninety to 100% of RPE cells and photoreceptors were homogeneously transduced within the targeted area. Conclusions: Our results showed accurate subretinal injection in defined sites and efficient rAAV mediated gene transfer with rapid onset of transgene expression which are desirable features for clinical application. Both RPE and photoreceptor cells were efficiently transduced suggesting that rAAV holds promise as gene therapy vector for retinal degenerative disease originating in RPE and photoreceptors.
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