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.

Fig. 1.

Schematic of the retina, retinoid cycle, and localized delivery of gene therapy in three patients with inherited blindness. (A) Light enters the eye and is absorbed by visual pigments (rhodopsin) in photoreceptors of the retina (red), the complex brain-like neural layer. (B) The retinoid cycle and its isomerase, RPE65, converts all-trans- to 11-cis-retinal to regenerate the visual pigment. (C) Series of retinal cross-sections from patient 2 rendered in three dimensions and overlaid onto the ocular fundus view illustrate the superior retinal site of subretinal vector injection. Fovea and optic nerve head locations are shown. (D) Photoreceptor cell layer (i.e., ONL) thickness topography (pseudocolor scale) in a normal subject and pretreatment in study eyes of patients 1, 2, and 3 (P1, P2, and P3). Site of injection (syringe tip) and the estimate of the bleb formed by the injection (dashed circle) are shown. Data in C and D are shown in equivalent left retina representation for clarity.

Fig. 2.

Biological activity resulting from localized gene therapy in three patients with inherited blindness. (A) Clinical kinetic visual field maps (represented as left retina for clarity and comparability) in control (blue) and study eyes at 1 month after treatment (red) are shown. Light sensitivity measures in study eyes along vertical (patients 1 and 2) and horizontal (patient 3) meridians at 1, 2, and 3 months after treatment compared with before treatment are also shown. The increased superior (patient 1), inferior (patient 2), and temporal (patient 3) retinal extent of vision boundary on visual field maps of study eyes corresponds to the region of vector injection. These regions show increases in light sensitivity after treatment compared with before treatment. (B) Retinal loci demonstrating significant change (stars) in light sensitivity at 1, 2, and 3 months after treatment compared with before treatment. All significant changes were increases in sensitivity (ranging from 1 to 3 log units) and correspond to regions of study eyes that received gene therapy; control eyes of the three patients did not show significant changes. Test-retest variability estimated from five additional patients with RPE65-LCA along the vertical meridian was smaller than the magnitude of sensitivity changes observed in study eyes. Biological activity in the study eye of patient 3 is assumed to cover a large contiguous region even though significance could be mathematically determined only at a subset of far temporal loci where pretreatment sensitivities were available. The baseline for each patient corresponds to the mean of two visits within 20 months of treatment. F, fovea.

Fig. 3.

Rod- and cone-photoreceptor-mediated visual function across the retinal region of study eyes showing biological activity. (A) The magnitude of the biological activity can be improved by allowing for a period of extended dark adaptation (Ext DA) before testing. Changes in the shapes of the light sensitivity profiles in patient 1 and patient 2 between standard dark adaptation (StdDA) and extended dark adaptation conditions suggest local differences in adaptation rate. (B) Dark adaptation kinetics measured with chromatic stimuli after a 7 log scot-td.s yellow adapting flash (presented at time 0) in patient 2 (at 3.6 and 7.2 mm inferior loci) and patient 3 (at 17 mm temporal locus). Also shown are detailed results from one normal (N) subject at 3.6 mm inferior (Left) and mean results from normal subjects at each location (gray lines). Cone adaptation kinetics (red symbols) are fast and do not show a difference from healthy cones. Rod adaptation kinetics (blue symbols) are extremely slow compared with healthy rods, and in patient 2 there is evidence for a large intraretinal difference in recovery rate. Additionally, absolute thresholds of both rod and cone systems are abnormally elevated. Note, the vertical threshold scale is inverted compared to the sensitivity scale in A to be consistent with traditional presentations of such data sets. (C) Maximal improvement in rod-mediated function (blue) across the region of treated retina ranged from 2.3 log in patient 1, 4.8 log in patient 2, and 4.5 log in patient 3; cone vision improvements (red) range from 1.7 log in patient 2 to 1.2 log in patient 3. Note that pretreatment estimates for rod- or cone-mediated vision (dashed lines) are best-case extrapolations from achromatic sensitivity measures. Only in patient 2 were pretreatment chromatic sensitivities measurable at 1–3 mm superior retina, and these measures were consistent with cone mediation. For patient 1, after treatment, specialized testing with a 4° diameter stimulus centered at 4.8 mm inferior retina suggested rod mediation for the blue stimulus. Pretreatment (Pre) results are from ≈18 to ≈3 months before treatment; posttreatment (Post) results are from 1–3 months after treatment.

Fig. 4.

Pathophysiology of complex visual loss resulting from photoreceptor degeneration and biochemical blockade, and the effect of gene therapy. (A) Retinal cross-sectional images at the region of maximal biological activity show the photoreceptor layer (ONL, brackets) to be abnormally thinned; greater degeneration of photoreceptors is evident in patient 1 compared with patient 2. Normal retina and patient 2 are at ≈5 mm superior retina, patient 1 is at ≈5 mm inferior retina. Schematics of photoreceptors and RPE are overlaid (yellow) and to the right of the actual optical images; these are derived from cynomolgus monkey histology (3, 28). INL, inner nuclear layer; PR-IS and PR-OS, photoreceptor inner and outer segments. (B) The relationship between photoreceptor layer thickness and rod sensitivity loss in patients 1 and 2 before and after therapy compared with normal subjects and patients with retinitis pigmentosa without mutations in the RPE65 gene. Normal variability is described by the ellipse encircling the 95% C.I. of a bivariate Gaussian distribution. Dotted lines define the idealized model of the relationship between structure and function in pure photoreceptor degenerations and the region of uncertainty that results from translating the normal variability along the idealized model. Gene therapy (arrows) results in partial (patient 1) or a nearly complete (patient 2) amelioration of the biochemical blockade transforming the complex RPE65-LCA disease phenotype into a simpler retinal degeneration phenotype.