Long Term Vision Restoration by Gene Therapy in the rd12 Mouse Model of RPE65 Leber Congenital Amaurosis

Abstract

Abstract: : Purpose: To test the duration of restored vision in rd12 mice by AAV–mediated gene therapy. Methods: 1 µl of AAV5–CBA–human RPE65 vector (3.9 x 10¹³ viral particles/ml) was injected subretinally into one eye of rd12 mice at postnatal day 14 (P14). The partner eye was untreated as the control. Eighteen months later, visual function was assessed with the electroretinogram (ERG) and vision–guided behavioral tests. Then both eyes of some mice were enucleated for histology and biochemical analysis for comparison with age–matched normal C57 BL/6 mice. Results: In AAV5–CBA–hRPE65 treated rd12 eyes 18 months after injection, dark–adapted ERG amplitudes are maintained at levels significantly improved over partner control eyes. Although amplitudes are reduced by 35% relative to the responses from the same treated eyes 11 months earlier, these changes were consistent with age–related changes. A comparison of ERG responses with control normal eyes suggests that treated rd12 eyes are about 75% of normal at all ages. ERGs are unrecordable in partner untreated eyes for the entire 18 month period. Vision–guided behavioral testing under dim light shows that rd12 mice treated in one eye have a performance statistically similar to normally sighted C57 BL/6 mice while untreated rd12 mice exhibit very poor performance. By light microscopy, untreated rd12 eyes show many clusters of lipid like droplets in their RPE cytoplasm and photoreceptor outer segments (OS) and outer nuclear layer (ONL) are disorganized and shortened. In contrast, treated eyes have well preserved OS and ONL morphology with few RPE droplets. Transmission electronic microscopy confirms these observations. In treated eyes, 11–cis retinal and rhodopsin levels are maintained at about 75% of normal compared to being undetectable in untreated partner eyes. Additionally, retinyl esters are significantly reduced in comparison with untreated eyes. Conclusions: AAV mediated gene therapy can restore a significant fraction of normal visual function to the rd12 mice and maintain it for at least 18 months. Although there is some deterioration in these parameters with aging, vision–guided behavioral testing shows that treated rd 12 mice maintain a nearly normal ability to visually locate objects.

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