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. dark adaptation ͉ photoreceptor ͉ retinal degeneration ͉ retinoid cycle T he enzymatic pathway in the human eye that regenerates light-altered vitamin A molecules is known as the retinoid cycle of vision. Molecular defects in retinoid cycle genes can lead to inherited retinal diseases in man (1). The severity of visual disturbance in these diseases is thought to be related to how the mutation alters the biochemical activity and whether there is redundancy at the multiple biochemical steps of the cycle. A severe form of incurable childhood blindness, Leber congenital amaurosis (LCA), is caused by mutations in RPE65 (retinal pigment epithelium-specific protein, 65 kDa), the gene in the retinal pigment epithelium (RPE) that encodes the isomerase. This is the only known enzyme that catalyzes isomerization of all-trans-retinyl esters to 11-cis-vitamin A. In RPE65 deficiency, photoreceptor cells do not regenerate their visual pigment and vision is not sustained. Retinal anatomy also degenerates, but not entirely (2, 3).RPE65-deficient animals have been characterized, and proofof-principle studies using recombinant adeno-associated virus (AAV) vector delivery of RPE65 to RPE cells have described restoration of vision (2,(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). These studies provided the impetus for human safety studies of RPE65 gene replacement (trials NCT00481546, NCT00643747, NCT00516477, and NCT00422721, www.clinicaltri...

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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 ϫ 10 10 vector genomes in 150 l 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 Ͻ 0.001); whereas, for control eyes, sensitivity changes were not significant (p ϭ 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. 979

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Autosomal dominant retinitis pigmentosa (ADRP) is a hereditary form of retinitis pigmentosa which accounts for about 15% of all types of the latter disease. Recently, close to 50 mutations, mostly point mutations, have been identified in the rhodopsin gene in ADRP patients. We have introduced these mutations in the synthetic bovine rhodopsin gene and herein report on the expression of the mutant genes in COS-1 cells and studies in vitro of the properties of the expressed opsins. The mutant phenotypes fall into three classes: Class I mutants are expressed in COS-1 cells at wild-type levels, form the normal rhodopsin chromophore with 11-cis-retinal, and are transported to the cell surface. However, on illumination, they activate transducin inefficiently. Class II mutants remain in the endoplasmic reticulum and do not bind 11-cis-retinal to form the chromophore. Class III mutants are expressed at low levels and form rhodopsin chromophore only poorly. They also remain in the endoplasmic reticulum and, as expected, show high mannose glycosylation. Nearly all of the mutants studied show abnormal sensitivity to light compared to the wild type, and they activate transducin less efficiently. We conclude that the majority of the ADRP mutants have folding defects.

Human gene therapy with rAAV2-vector was performed for the RPE65 form of childhood blindness called Leber congenital amaurosis. In three contemporaneous studies by independent groups, the procedure was deemed safe and there was evidence of visual gain in the short term. At 12 months after treatment, our young adult subjects remained healthy and without vector-related serious adverse events. Results of immunological assays to identify reaction to AAV serotype 2 capsid were unchanged from baseline measurements. Results of clinical eye examinations of study and control eyes, including visual acuities and central retinal structure by in vivo microscopy, were not different from those at the 3-month time point. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months. The retinal extent and magnitude of rod and cone components of the visual sensitivity between 3 and 12 months were also the same. The safety and efficacy of human retinal gene transfer with rAAV2-RPE65 vector extends to at least 1 year posttreatment.

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Rhodopsin, the dim light photoreceptor ofthe rod cell, is an integral membrane protein that is glycosylated at Asn-2 and Asn-15. Here we report experiments on the role of the glycosylation in rhodopsin folding and function. Nonglycosylated opsin was prepared by expression of a wild-type bovine opsin gene in COS-1 cells in the presence of tunicamycin, an inhibitor of asparagine-linked glycosylation. The nonglycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (Aa, 500 nm) with 11-cis-retinal. However, the nonglycosylated rhodopsin showed strikingly low light-dependent activation of GT at concentration levels comparable with those of glycosylated rhodopsin. Amino acid replacements at positions 2 and 15 and the cognate tripeptide consensus sequence [Asn-2 -* Gln, Gly-3 -+ Cys (Pro), Thr-4 -+ Lys, Asn-15 Ala (Cys, Glu, Lys, Gln, Arg), Lys-16 -* Cys (Arg), Thr-17 Met (Val)] showed that the substitutions at Asn-2, Gly-3, and Thr-4 had no sgnificant effect on the folding, cellular transport, and/or function of rhodopsin, whereas those at Asn-15 and Lys-16 caused poor folding and were defective in transport to the cell surface.Further, mutant pigments with amino acid replacements at Asn-15 and Thr-17 activated Gr very poorly. We conclude that Asn-15 glycosylation is important in signal tanduction.Asparagine-linked (N-linked) glycosylation of membrane and secreted proteins is observed frequently, although the role that glycosylation may serve is not always evident (1-3). Bovine rhodopsin is glycosylated at Asn residues 2 and 15 (Fig. 1) by the hexasaccharide sequence Man3GlcNac3 (4, 5). We have now examined the role of N-linked glycosylation in rhodopsin folding and function. We expressed the wild-type bovine opsin gene (6) in the presence of the glycosylation inhibitor tunicamycin (TM). The resulting nonglycosylated opsin was normally palmitoylated, was transported to the cell surface, and formed the characteristic rhodopsin chromophore with 11-cisretinal. However, the nonglycosylated rhodopsin showed strikingly diminished light-dependent activation of transducin (GT) when compared with glycosylated rhodopsin. We next studied opsin mutants that contained amino acid replacements in the regions ofthe two glycosylation sites (Fig. 1). Mutations at or near Asn-2 had little effect on cell-surface expression, chromophore formation, and/or GT activation. In contrast, mutations at Asn-15 and Lys-16 resulted in opsins that were defective in cellular transport and formed little or no chromophore with il-cis-retinal. The mutations at Asn-15 and Thr-17 resulted in pigments that were defective in signal transduction. These results show that while glycosylation of rhodopsin is not required for its folding to an apparently correct ground-state structure, it is necessary for full activity in signal transduction.