Gene Therapy in the Three Month–Old rd12 Mouse, a Model for Mid–Course RPE65 Leber Congenital Amaurosis, Leads to Long–Term Vision Restoration

Abstract

Purpose: : To test if long–term restoration of retinal structure and function can be obtained by AAV–mediated gene therapy in the rd12 mouse model of Leber’s Congenital Amaurosis.

Methods: : 1 µl of AAV5–CBA–human RPE65 vector (3.9 x 10¹⁰ vector genome particles) was injected subretinally into one eye of five 3 month–old rd12 mice. Partner control eyes were injected with either AAV5–CBA–GFP or remained uninjected. Retinal function was monitored by scotopic ERG from 3 weeks to 18 months following injection. At 18 months, results of histological and biochemical analysis were compared with age–matched normal C57 BL/6 mice.

Results: : In RPE65 treated rd12 eyes, significant dark–adapted ERG restoration was obtained as early as 3 weeks and maintained for 18 months following injection. At 18 months, ERG amplitudes in treated eyes were about half of that in age–matched normals. A comparison of ERG responses between 3 weeks and 18 months following injection suggests that about 1/3 of the recovered ERG amplitude at 3 weeks was lost at 18 months similar to the age–related ERG loss seen in normals. ERGs were barely recordable in partner control rd12 eyes for the entire 18–month period. By funduscopic examination, retinas in treated eyes appear nearly normal whereas small yellow–white spots devoid of pigment were dispersed throughout the fundus of control eyes. By light microscopy, control rd12 eyes showed many clusters of lipid–like droplets in their RPE cytoplasm, photoreceptor outer segments (OS) were disorganized and shortened and the outer nuclear layer (ONL) was thinned. In contrast, treated eyes had well preserved OS and relatively preserved ONL morphology with few RPE droplets. Transmission EM confirms these observations. In treated eyes, 11–cis retinal and rhodopsin levels were maintained at about half of normal whereas they were both undetectable in control partner eyes. Additionally, retinyl esters were significantly reduced in treated eyes.

Conclusions: : AAV mediated gene therapy applied to 3 month–old rd12 mice can restore a significant fraction of normal retinal function and maintain it for at least 18 months. Although the level of rescue is not as great as that reported when 14 day–old rd12 mice were treated, our data suggest that vector–mediated therapy is still successful and can last as long as 18 months even when treatment is initiated after the start of retinal degeneration.

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