An Animal Model for Inherited Human Retinal Dystrophy Caused by the R91W Mutation in Rpe65

Abstract

Purpose:: RPE65 is essential for the regeneration of 11-cis retinal - the chromophore of both cone and rod visual pigments. More than 80 disease-associated mutations have been identified in the Rpe65 gene. The most frequent mutation found in patients causes an amino acid substitution at position 91 (R91W). Patients with this mutation have useful cone-mediated vision in the first decade of life suggesting that the mutant RPE65 protein is at least partially functional. All currently available animal models for visual defects associated with RPE65 are functional knock-outs. We generated an R91W knock-in mouse model to understand the mechanism of retinal degeneration caused by aberrant RPE65 function.

Methods:: The animal strains used were: single mutant (R91W, R91W/wt, Rpe65-/-, Rho-/-), double mutant (R91W;Rho-/-, Rpe65-/-;Rho-/-) and control wt (129S6) mice. The analyses were: RT-PCR, Western blotting and immunohistochemistry for gene/protein screening, HPLC for retinoid composition; spectrophotometry for rhodopsin content; light microscopy for morphology; and ERG for function.

Results:: The R91W mutation caused: 1) reduced RPE65 protein levels, 2) reduced levels of 11-cis retinal, 3) disturbed rhodopsin regeneration, 4) progressive loss of photoreceptors, 5) severely reduced retinal function. After selective ablation of the rod opsin remnant function, it turned out that residual vision is mediated by cones in young R91W mice. The phenotype of R91W knock-in mice was distinctive from that of the Rpe65 knock-out mouse as evidenced by: 1) presence of 11-cis retinal and rhodopsin, 2) slower degeneration 3) prolonged survival of cones, 4) preserved cone function, which is lost early postnatally in Rpe65-/- mice.

Conclusions:: The R91W knock-in mouse mimics many aspects of the human pathology caused by this mutation and complements the Rpe65 knock-out mouse as model for pre-clinical investigations. Particularly, the relative longevity of cones in the R91W knock-in mice may have dramatic impact on upcoming clinical trials with regard to the therapeutic window in the corresponding patients.

Keywords: retinal degenerations: hereditary • retinal pigment epithelium • retina