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A. Bemelmans, C. Kostic, D. Hornfeld, W.W. Hauswirth, J. Lem, D.F. Schorderet, F.L. Munier, M. Seeliger, A. Wenzel, Y. Arsenijevic; Lentiviral Gene Transfer of RPE65 cDNA in Two Knock–Out Mouse Models of Leber Congenital Amaurosis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5214.
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Purpose: In 10 to 15 % of Leber congenital amaurosis (LCA) cases, a severe retinal degeneration of early childhood, the disease is due to mutations in RPE65, a gene expressed in the retinal pigment epithelium (RPE). It has been shown that RPE65 gene transfer is able to restore, at least partially, vision in several animal models of LCA. To explore the effect of RPE65 restoration on the different photoreceptor subtypes, we studied its effect in two models of LCA, the RPE65 and the RPE65/GNAT1 knock–out mice. In the latter, deletion of the rod transducin alpha–subunit gene prevent the functioning of rod photoreceptors. Methods: RPE65 gene transfer was achieved using a lentiviral vector in which the human RPE65 promoter drives the expression of the RPE65 mouse cDNA (LV–RPE65). Control eyes received a GFP vector (LV–GFP) or vehicle alone (sham). We assessed for retinal function by electroretinogram recordings (ERG) at different times after vector injection. Following the last ERG, animals were sacrificed for analysis of GFP, RPE65 expression and rhodopsin content. Results: Using LV–GFP, we first ensured that our lentiviral construct led to a strong transgene expression in the RPE of injected eyes. We next explored the therapeutic benefit of RPE65 gene transfer into three to six months old RPE65 knock–out mice, an age at which almost no ERG activity is normally detected. ERG provided evidence that RPE65 gene transfer partially restores the photoreceptors function in RPE65 knock–out mice. Nevertheless, we observed a great variability in the response among animals. In the double knock–out model, no improvement was seen in the ERG after RPE65 gene transfer. Conclusions: We report the first lentiviral gene transfer of RPE65 leading to a partial rescue of the phenotype in RPE65 knock–out mouse. Nevertheless, differences in ERG restoration between single and double knock–out mice suggest that RPE65 gene transfer is able to restore function of rod photoreceptors but not cones. Further histological analysis will correlate ERG amplitudes with the size of the retinal area expressing RPE65 and with rhodopsin content.
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