Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CB^SB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CB^SB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21–24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 × 10¹⁰ vector genomes in 150 μl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30–90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p < 0.001); whereas, for control eyes, sensitivity changes were not significant (p = 0.99). Comparisons are drawn between the present work and two other studies of ocular gene therapy for RPE65-LCA that were carried out contemporaneously and reported.

The RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65 -LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with <1 h in normal eyes. Cone-sensitivity recovery time was rapid. These results demonstrate dramatic, albeit imperfect, recovery of rod- and cone-photoreceptor-based vision after RPE65 gene therapy.

To determine the safety and efficacy of subretinal gene therapy in the RPE65 form of Leber congenital amaurosis using recombinant adeno-associated virus 2 (rAAV2) carrying the RPE65 gene. Open-label, dose-escalation phase I study of 15 patients (range, 11-30 years of age) evaluated after subretinal injection of the rAAV2- RPE65 vector into the worse-functioning eye. Five cohorts represented 4 dose levels and 2 different injection strategies. Primary outcomes were systemic and ocular safety. Secondary outcomes assayed visual function with dark-adapted full-field sensitivity testing and visual acuity with Early Treatment Diabetic Retinopathy Study charts. Further assays included immune responses to the vector, static visual fields, pupillometry, mobility performance, and optical coherence tomography. No systemic toxicity was detected; ocular adverse events were related to surgery. Visual function improved in all patients to different degrees; improvements were localized to treated areas. Cone and rod sensitivities increased significantly in the study eyes but not in the control eyes. Minor acuity improvements were recorded in many study and control eyes. Major acuity improvements occurred in study eyes with the lowest entry acuities and parafoveal fixation loci treated with subretinal injections. Other patients with better foveal structure lost retinal thickness and acuity after subfoveal injections. Gene therapy for Leber congenital amaurosis caused by RPE65 mutations is sufficiently safe and substantially efficacious in the extrafoveal retina. There is no benefit and some risk in treating the fovea. No evidence of age-dependent effects was found. Our results point to specific treatment strategies for subsequent phases. Gene therapy for inherited retinal disease has the potential to become a future part of clinical practice. clinicaltrials.gov Identifier: NCT00481546.

Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Here we show that mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptors.