Long-term effect of gene therapy on Leber's congenital amaurosis

Abstract

Background: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited.

Methods: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings.

Results: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG.

Conclusions: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Figure 1. Retinal Sensitivity Measured by Means of Dark-Adapted Perimetry

Retinal sensitivity in the dark-adapted state was evaluated with the use of a pointwise linear regression for each of the 76 locations tested. The sum of loci with a significant positive slope (P<0.05) is plotted against time for the study eye (solid line) and contralateral control eye (broken line) for each of the 12 participants. Participants 3, 5, and 6 had a clear improvement in retinal sensitivity by approximately month 6, followed by a decline in sensitivity, which returned to near-baseline levels by 3 years after therapy. Participants 8, 10, and 12 also had some improvement. The term vg denotes vector genomes.

Figure 2. Vision-Guided Ambulatory Navigation

The participants’ abilities to navigate a course at a range of illuminances 6 months after vector administration were assessed by measurement of the number of navigational errors made when the study eye and control contralateral eye were used independently. The dark blue bars indicate, for each participant tested, the difference between the study eye and control eye in the number of errors made. Bars that extend upward from 0 indicate fewer errors for the study eye than for the control eye for each participant. The gray-shaded areas indicate the test–retest variability, determined by one-way analysis of variance with the use of multiple baseline measurements. The four rows of data are results for each of the lighting conditions; only Participants 5 through 12 were tested at 2 lux. Participants 5, 8, and 10 had better performance with their study eyes at lower illuminances. Participants 1 and 9 had better performance with their untreated control eye at the highest illuminance. NP denotes that the participant was not able to perform the test, and NT denotes that the participant was not tested.

Figure 3. Dose-Dependent Responses to Gene-Augmentation Therapy in Dogs

Panel A shows the results of an immunohistochemical analysis demonstrating levels of RPE65 (in red) after administration of the indicated doses of vector (subpanels a–d). RPE65 signals are similar to wild-type levels (subpanel e) only at the highest dose. No signal is detected in negative control samples without primary antibody (subpanel f). Autofluorescent signal (in green) in the subretinal space, shown in subpanels on the left, has been removed in the subpanels on the right to facilitate visualization of RPE65. DAPI denotes 4′,6-diamidino-2-phenylindole, and GAPDH glyceraldehyde 3-phosphate dehydrogenase. Panel B shows the results of a Western-blot analysis indicating levels of recombinant RPE65 that are appreciable but lower than wild-type levels at the highest dose of injected vector. Panel C shows that the highest level of 11-cis retinal production corresponds to the highest dose of vector. The term vg denotes vector genomes. In Panel D, electroretinographic findings (left graph) show substantial restoration of the b-wave only at the highest dose of vector, whereas vision-guided behavior (right graph) shows dose dependency, with improved function evident even at lower doses of vector.