Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics

Abstract

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...

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

“…11,12 In contrast, in clinical studies in which smaller amounts of AAV vectors were introduced into immunoprivileged body compartments, limited or no immune responses to the AAV capsid were observed. [13][14][15][16] These data support that the route of administration and the total antigen load (that is total vector capsid dose) determine the kinetics and the nature of the immune response against the AAV capsid. Increasing the transgene expression efficiency, vector transduction efficiency, and vector purity, with the objective to minimize viral antigen dose and minimize potentially antigenic moieties, are important approaches to reduce the potential for deleterious host immune responses after vector administration.…”

Optogenetic therapy for retinitis pigmentosa

“…Progress in developing gene therapy for a form of Leber congenital amaurosis 7 caused by defects in the retinal pigment epithelium-specific gene, RPE65, has not only offered hope for patients with this disease, but has also boosted general confidence in gene therapy strategies for retinal disorders by demonstrating the safety and efficacy of adeno-associated viral (AAV) vectors, following intraocular administration in humans. [8][9][10][11] As gene supplementation can only be applied when the cell type expressing the gene is still alive, early diagnosis and gene therapy in childhood may be necessary in the case of the most common rod-specific genes. Gene supplementation is more complicated in dominant forms of RP, 12 where the mutation induces a toxic pathway or in recessive RP, if the gene exceeds the packaging capacity of viral vectors that are suitable for use in humans.…”

Gene supplementation therapy for recessive forms of inherited retinal dystrophies

“…49,50 Reports of the results of these trials to date indicate that despite differences in administered viral titres, injection volumes and assessment methods, RPE65 gene augmentation can improve retinal light sensitivity and vision. [48][49][50][51][52] Where assessments allowed for distinction between rod photoreceptor-mediated and cone-mediated responses, in most subjects the improvements were detected in rod photoreceptor sensitivity, although in some cases improvements in the cone-mediated visual function have been detected. 52 Despite robust improvements in psychophysical assessments of vision, improvements in electroretinographic responses have yet to be reported.…”

“…[48][49][50][51][52] Where assessments allowed for distinction between rod photoreceptor-mediated and cone-mediated responses, in most subjects the improvements were detected in rod photoreceptor sensitivity, although in some cases improvements in the cone-mediated visual function have been detected. 52 Despite robust improvements in psychophysical assessments of vision, improvements in electroretinographic responses have yet to be reported. 48,50,51 This contrasts with the results of the preclinical studies in which substantial electrophysiological improvements are predictably recorded in the mice and the dogs following vector administration.…”