Analysis of RPE65 Promoter Activity Using Lentiviral Vectors for Gene Transfer in Leber Congenital Amaurosis Mouse Model

Abstract

Abstract: : Purpose: Gene transfer is a promising approach for single gene defect in several retinal degeneration disorders. Among them, 10–15% of Leber congenital amaurosis (LCA) cases bear a mutation in the RPE65 gene, the function of which is essential for the visual cycle. AAV–mediated gene therapy was already described in dog and mouse models of LCA using CMV or composite promoters. We evaluated the efficiency of gene transfer in rodent models of LCA using a lentiviral vector containing a RPE65 promoter fragment driving the expression of the therapeutic RPE65 cDNA. Methods: An HIV–1–derived lentiviral vector expressing either the GFP or the RPE65 mouse cDNA under the control of a 0.8 kb fragment of the human RPE65 promoter (R0.8) was produced using transient transfection in 293T cells. Concentrated preparations were injected into RPE65 knockout or RPE65–/– GNAT1–/– double knockout adult and newborn mice. Transgene expressions were analysed using direct fluorescence and immunohistochemistry 7 days post–injection. Results: Both RPE65 and GFP transgenes were expressed in RPE cells of both adult and newborn injected mice. However, in newborn injected mice, GFP was also detected in rare photoreceptors and at the external layer of the corneal epithelium. Conclusions: This preliminary study suggests that the R0.8 promoter activity is suitable for gene transfer in RPE of adult injected mice. However the tropism of the vector which is different between adults and newborn may lead to targeting of other cell types, thus complementary studies on R0.8 activity during development are needed.

Keywords: gene transfer/gene therapy • transgenics/knock-outs • retinal degenerations: hereditary