AAV-mediated Rescue Of Rod Photoreceptors In The Rd3 Mouse Model Of Leber Congenital Amaurosis Type 12 (LCA12)

Abstract

Purpose: : Mutations in RD3, a 23 kDa protein required for the stable expression and trafficking of guanylate cyclases (GC1 and GC2) in photoreceptor cells, are responsible for retinal degeneration in LCA12 patients, the rd3 mouse, and the rcd2 collie. The purpose of this study was to determine if AAV-mediated delivery of the mouse Rd3 gene can prevent photoreceptor degeneration in the rd3 mouse.

Methods: : AAV8(Y733F) containing the mouse Rd3 cDNA under control of the rhodopsin kinase promoter (hGRK1) was injected into the subretinal space of the right eyes of P14 rd3 mice; the uninjected left eyes served as controls. Eyes were evaluated at various times post-injection by electroretinography (ERGs), optical coherence tomography (OCT), histology, immunocytochemistry and SDS gels.

Results: : GC1 and GC2 expression was observed in rods and cones of the Rd3-AAV treated eye 7 days post-injection. The level of expression increased with time, and at 1 month post-injection GC1 and GC2 expression was observed in the outer segment layer which persisted for at least 4 months. The outer nuclear layer (ONL) thickness in the treated eye stabilized at about 60% that of wild-type mice, although a gradual loss in cones was evident. In the untreated eye, both rods and cones degenerated rapidly with essentially complete loss in the ONL by 4 months. The scotopic ERGs were maintained in the treated eyes, but rapidly disappeared in the untreated eyes. Photopic ERGs were absent in both the treated and untreated eye at all times.

Conclusions: : Rd3-AAV gene replacement therapy restored GC1 and GC2 expression and outer segment localization in rod cells of the rd3 mouse. GC expression resulted in sustained scotopic ERGs and rod cell survival. Although GC1 was detected in cones after treatment, the photopic ERG remained undetectable and progressive cone cell degeneration was observed.

Keywords: retinal degenerations: hereditary • gene transfer/gene therapy • photoreceptors