RPE65 Gene Delivery Effectively Restores the Retinol Isomerase Activity in the Rpe65–/– Mouse

Abstract

Abstract: : Purpose: RPE 65 is essential for isomerization of all–trans retinol to 11–cis isomers. In homozygous RPE65 knockout (Rpe65^–/–) mice, no 11–cis retinoids were formed. Although previous studies have shown that RPE65 gene delivery restores some retinal functions, the isomerase activity after the gene delivery in the RPE has not been reported. The purpose of this study is to investigate if the RPE65 gene delivery can restore isomerase activity in Rpe65^–/–mice. Methods: Adenovirus expressing RPE65 and its mutants were injected into the subretinal space of Rpe65^–/–mice. Immunohistochemistry was applied to demonstrate the location of RPE65. Western blot analysis was performed to measure the expression of RPE65. Retinol isomerase activities in the mouse eyecup homogenate from injected mice and wilt–type (wt) C57BL/6 and sv129 mice were measured using all–trans [³H] retinol as a substrate. The generation of 11–cis retinol was analyzed by HPLC. Results: RPE65 gene delivery resulted in expression of RPE65 in a large area of the RPE in Rpe65^–/– mice. The expression levels of RPE65 were comparable to that in wt C57BL/6 mice. RPE65 gene delivery generated robust retinol isomerase activities in the Rpe65^–/–eyecup. The isomerase activities in Rpe65^–/–mice injected with the RPE65 mutants, R91W and Y368H did not display any detectable retinol isomerase activity. Conclusions: Adenovirus mediated RPE65 gene delivery results in efficient RPE65 expression and restores retinol isomerase activity in the RPE of Rpe65^–/–mice to the level of wt animals.

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