Gene Therapy restores Vision in a Natural Model of RPE65 Leber Congenital Amaurosis: the rd12 mouse

Abstract

Abstract: : Purpose: To test whether AAV–mediated gene delivery can restore the vision in a mouse model with a spontaneous RPE65 mutation. Methods: 1 µl of AAV5–CBA–human RPE65 vector suspension (3.9 x 10¹³ viral particles/ml) was injected subretinally to one eye of rd12 mice at postnatal day (P) 14. The partner eye (untreated eye) was either not injected or similarly injected with control AAV5–CBA–GFP vector suspension. ERGs were recorded regularly. Seven months later, both eyes were enucleated for histology, immunostaining and retinoid analysis for comparison with age–matched normal C57 BL/6 mice. Results: In AAV5–CBA–hRPE65 treated rd12 eyes, dark adapted ERG waveforms were restored as soon as 1 week after treatment. These restored rod ERGs became stable in amplitude at 3 weeks post–treatment and remained so for 7 months after treatment, the latest time checked. Restored b–wave amplitudes of rod ERGs were 60–80% of normal. There were no recordable rod ERGs in untreated eyes. Treated eyes also maintained normal cone ERGs while they were delayed and exhibited progressive loss in amplitude in untreated eyes. By light microscopy, photoreceptor outer segments became shortened, disorganized and contained many voids in untreated rd 12 mice; the outer nuclear layer (ONL) was about 30% thinner than normal. By transmission electronic microscopy, frequent large lipid–like droplets and vacules accumulated in the RPE cytoplasm. In contrast, treated eyes had nearly normal outer segment morphology and showed only an occassional, small lipid–like droplet in RPE cells. Immunostaining with anti–RPE65 antibody showed strong RPE65 expression in the RPE cells with expression in the ONL of treated rd12 eyes, but no expression in untreated eyes. RPE65 expression was observed only in the RPE cells of normal mice. In treated eyes, the 11–cis retinal and rhodopsin were restored to approximately 60% of normal. Additionally, retinyl esters were reduced in comparison with untreated eyes . Reduction in retinyl ester levels paralleled the loss of RPE lipid–like droplets.Conclusions: AAV mediated gene therapy can restore a significant fraction of the visual function in this naturally occurring mouse lacking RPE65. This work also shows that a heterologous RPE65 cDNA, the human version, is therapeutic in a nonhuman RPE65 LCA model. Behavioral testing is underway.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • retinal pigment epithelium