Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial

Abstract

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-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21-24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 x 10(10) vector genomes in 150 microl 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 < 0.001); whereas, for control eyes, sensitivity changes were not significant (p = 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.

FIG. 1.

Fundus images with near-infrared illumination of a normal subject and of P1, P2, and P3 with RPE65-LCA. The macula is defined by a circle on the normal image; the fovea (F) and the optic nerve head are indicated. Dotted circles on the images of individual patients represent the estimated areas of retinal detachment from drawings at time of surgery. White “syringe” indicates the site of the retinotomy that produced the detachment. All images depicted as left eyes to enable comparisons.

FIG. 2.

Immune assays before surgery (baseline) and at 14 and 90 days after surgery. (A) Humoral immune response to AAV serotype 2 (AAV2) assayed by determining circulating serum antibody titers against AAV2 capsid in each study subject before and after surgery. Arrow along vertical axis indicates mean levels from a normal reference population (n = 79). (B) Antigen-specific lymphocyte proliferation response (ASR) assayed in peripheral blood lymphocytes incubated in the presence versus in the absence of AAV2 capsid antigen. The stimulation index (the ratio of [³H]thymidine uptake in the presence of antigen to the uptake in its absence) in each study subject after surgery is compared with baseline values. (C) T cell immune response to AAV2 capsid in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from each study subject before and after surgery were stimulated with three AAV2 capsid peptide pools (2A, 2B, and 2C) and assayed for IFN-γ secretion by ELISpot. The number of spot-forming cells (SFC) per 10⁶ PBMCs before and after surgery is compared with a negative control (medium alone); responses to a CEF pool served as reference.

FIG. 3.

Central vision and retinal structure in the RPE65-LCA patients. (A) Visual acuity as a function of time before (baselines) and after the day of surgery (0) in the study eyes of P1, P2, and P3. (B) OCT scans along the horizontal meridian before surgery and at 90 days after surgery in study eyes. (C) Visual acuity and (D) OCT scans in control eyes for comparison. Arrows, epiretinal membranes (detectable in all scans).

FIG. 4.

Dark-adapted full-field sensitivity tests (FSTs) before (Pre) and 30 to 90 days after (Post) surgery in study and control eyes of the three patients. (A) Mean FST sensitivity and its variability (error bars, SD) at four visits presurgery compared with three visits postsurgery for the study and control eyes. Normal range for the FST is shown along the horizontal axis (Roman et al., ; Aguirre et al., 2007). (B) Changes in sensitivity from mean baseline postsurgery show all positive numbers in study eyes compared with positive and negative changes in control eyes or pretreatment. Groups of three bars shown for each patient postsurgery refer to days 30 (top), 60 (middle), and 90 (bottom). p values refer to t test statistics between the pooled pre- and postsurgery results shown.