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.

Significance: The first retinal gene therapy in human blindness from RPE65 mutations has focused on safety and efficacy, as defined by improved vision. The disease component not studied, however, has been the fate of photoreceptors in this progressive retinal degeneration. We show that gene therapy improves vision for at least 3 y, but photoreceptor degeneration progresses unabated in humans. In the canine model, the same result occurs when treatment is at the disease stage equivalent to humans. The study shows the need for combinatorial therapy to improve vision in the short term but also slow retinal degeneration in the long term.

Retinal gene therapy for Leber’s congenital amaurosis, an autosomal recessive childhood blindness, has been widely considered to be safe and efficacious. Three years after therapy, improvement in vision was maintained, but the rate of loss of photoreceptors in the treated retina was the same as that in the untreated retina. Here we describe long-term follow-up data from three treated patients. Topographic maps of visual sensitivity in treated regions, nearly 6 years after therapy for two of the patients and 4.5 years after therapy for the third patient, indicate progressive diminution of the areas of improved vision. (Funded by the National Eye Institute; ClinicalTrials.gov number, NCT00481546.)

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.

Mutations in ABCA4, which encodes a photoreceptor specific ATP-binding cassette transporter (ABCR), cause autosomal recessive forms of human blindness due to retinal degeneration (RD) including Stargardt disease. The exact disease sequence leading to photoreceptor and vision loss in ABCA4-RD is not known. Extrapolation from murine and in vitro studies predicts that two of the earliest pathophysiological features resulting from disturbed ABCR function in man would be slowed kinetics of the retinoid cycle and accelerated deposition of lipofuscin in the retinal pigment epithelium (RPE). To determine the human pathogenetic sequence, we studied surrogate measures of retinoid cycle kinetics, lipofuscin accumulation, and rod and cone photoreceptor and RPE loss in ABCA4-RD patients with a wide spectrum of disease severities. There were different extents of photoreceptor/RPE loss and lipofuscin accumulation in different regions of the retina. Slowing of retinoid cycle kinetics was not present in all patients; when present, it was not homogeneous across the retina; and the extent of slowing correlated well with the degree of degeneration. The orderly relationship between these phenotypic features permitted the development of a model of disease sequence in ABCA4-RD. The model predicted lipofuscin accumulation as a key and early component of the disease expression in man, as in mice. In man, however, abnormal slowing of the rod and cone retinoid cycle occurs at later stages of the disease sequence. Knowledge of the human ABCA4 disease sequence will be critical for defining rates of progression, selecting appropriate patients and retinal locations for future therapy, and choosing appropriate treatment outcomes.

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.