Identifying photoreceptors in blind eyes caused by RPE65 mutations: Prerequisite for human gene therapy success

Abstract

Mutations in RPE65, a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65-mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65-mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65(-/-) mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.

Figures

Fig. 1.

RPE65-mutant human retinas with normal thickness topography or localized retinal thinning. Topographical maps from high-resolution depth imaging of the central retina in a normal subject, age 22 (A), and five patients with RPE65 mutations (ages 20-41) (B-F). (Insets Upper Right) En face images. (A Inset Lower Right) The lower limit (mean - 2 SD) of normal. (B-F Insets Lower Right) A difference map (subtracted from normal lower limit) showing regions of thinned retina (gray). All images are depicted as left eyes. ON, optic nerve. F, fovea. N, nasal, T, temporal, S, superior, I, inferior.

Fig. 2.

Photoreceptor nuclear layer in RPE65-mutant retinas. (A-D) Cross-sectional retinal images along the horizontal (Left) and vertical (Right) meridia through the fovea in a normal subject (A) and three patients with RPE65 mutations (B-D). ONL is indicated to the left of the images. (E) ONL thickness across horizontal (Left) and vertical (Right) meridia in normal subjects and patients with RPE65 mutations. Normal ONL thickness mean (thin line) and ±2 SD (gray) are indicated.

Fig. 3.

RPE65-mutant human retinas can have more photoreceptor nuclear layer than predicted from vision. (A) Foveal ONL thickness as a function of dark-adapted cone-mediated sensitivity (650 nm). (B and C) ONL thickness as a function of dark-adapted sensitivity (500 nm) at 3.6 mm in temporal (B) and superior (C) retina. Rod, rod-mediated sensitivity; Cone, cone-mediated sensitivity; Pts, patients without RPE65 mutations. Normal variability is described by the ellipses encircling the 95% confidence interval 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 by translating the normal variability along the idealized model. The region encompassing data with greater than expected ONL thickness is marked as treatment (Tx) potential. (Inset) Retinal location (white arrow on fundus image) of colocalized measures of structure and function. Overlaid onto the fundus image are cone density (a) and rod density (b and c) along horizontal and vertical meridia (26).

Fig. 4.

Gene therapy in Rpe65-/- mice at advanced disease stages leads to limited visual restoration. (A) Comparison of ERGs in young (4 mo) and old (>15 mo) WT (Left) and Rpe65-/- mice (Center and Right). (Center) Saline-injected control eyes of Rpe65-/- mice illustrate the severe ERG abnormality. (Right) The AAV2/1-CMV-hRPE65-treated eyes show visual restoration. Stimulus onset is at trace onset; stimulus luminance is at left of traces. (B) Summary of treatment results. ERG thresholds in >15-mo-old Rpe65-/- mice after treatment with AAV2/1-CMV-hRPE65 (Tx1) or oral 9-cis retinal (Tx2) are compared with saline-injected (Ctrl) eyes. Unfilled symbols (treatment success) are results falling beyond the 99% confidence interval limit (upper boundary of gray) determined from uninjected, age-matched, Rpe65-/- mice. Frequency of treatment success by ERG and retinoid biochemistry for Tx1 and Tx2 in young (empty bars) versus old (filled bars) Rpe65-/- mice is shown. (C) ONL thickness (1 mm superior to optic nerve) in 3-mo-old vs. 24-mo-old Rpe65-/- mice (•) is compared with age-matched WT (○) mice. ONL thickness data (mean ± 2 SD) for young WT mice (25) is shown. (D) Retinal histological sections (1 mm superior to optic nerve) from an old WT mouse (Left) are compared with two old Rpe65-/- mice treated with gene therapy: one with ERG success (Center) and one with failure (Right). The ONL and the inner nuclear layer (INL) are indicated by brackets.