Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness

Abstract

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanism-based pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.

Figure 1

Retinoid and rhodopsin analysis in Rpe65 mice. (A) Chromatogram of retinoids extracted from eyes of Rpe65+/+, Rpe65+/−, and Rpe65−/− mice. The extraction procedures and derivatization with hydroxylamine to improve quantitative extraction of retinaldehydes were described previously (see ref. 13). Peaks are represented as follows: (1, 2) all-trans-retinyl and 11-cis-retinyl esters (truncated to fit in scale); (3,3′) anti- and syn- of 11-cis-retinal oximes; (4,4′) anti- and syn- of all-trans-retinal oximes; (5) 11-cis-retinol, and (6) all-trans-retinol. (B) Difference spectrum of bleached rhodopsin and rhodopsin extracted from eyes of Rpe65+/− and Rpe65−/− mice. The level of rhodopsin in Rpe65+/− mice is within the range of rhodopsin found in control Rpe65+/+ mice (●) and undetectable in Rpe65−/− mice. (C) Aberrant kinetics of retinoid and rhodopsin recovery in Rpe65+/− mice after a flash. Photolyzed rhodopsin releases all-trans-retinal (a), which is reduced to all-trans-retinol (b), and then transported to the RPE and esterified to retinyl esters (c). All-trans-retinol, or its derivative, is isomerized to 11-cis-retinol (d), which in turn, is oxidized to 11-cis-retinal (e). The rates of retinoid formations are compared to the rate obtained for Rpe65+/+ mice ().

Figure 2

Formation of isorhodopsin and kinetics of the retinoid flow in Rpe65−/− mice 48 h after 9-cis-retinal gavage. (A) Comparison spectra of rhodopsin from Rpe65+/+ mice and isorhodopsin from Rpe65−/− mice 48 h after 9-cis-retinal gavage (2.5 mg). Arrows denote differences in the absorption maximum for rhodopsin and isorhodopsin. (B) Isorhodopsin formation in Rpe65−/− mice at different time points after 0.5 mg or 2.5 mg of 9-cis-retinal gavage (n = 2). (C) Chromatograms illustrate the dark recovery of retinoids in Rpe65−/− mice 48 h after 9-cis-retinal gavage (2.5 mg) after a flash that bleached ≈45% of isorhodopsin. (C, Inset) isomeric composition of retinyl esters (8, 9-cis-retinol). (D) Dark recovery of isorhodopsin in Rpe65−/− mice 48 h after 9-cis-retinal gavage after a flash. For comparison, dark recovery of rhodopsin is shown for Rpe65+/+ (open hexagons) (7, 7′). anti- and syn- of 9-cis-Retinal oximes; all other peaks are as in the Fig. 1 legend.

Figure 3

Restoration of retinal function in Rpe65−/− mice 48 h after 9-cis-retinal gavage. (A and B) Serial ERG recordings in an Rpe65−/− mouse before and 48 h after 9-cis-retinal gavage compared to a representative Rpe65+/+ mouse. (A) Dark-adapted ERGs to increasing intensities of blue light stimuli (shown to the left of the traces) in an Rpe65−/− mouse show an elevated b-wave threshold compared to Rpe65+/+. The same stimuli after 9-cis-retinal treatment elicit ERGs at a lower threshold and with larger amplitude b-waves. (B) Leading edges (initial 4–15 ms depending on response) of dark-adapted ERG photoresponses (symbols) evoked by 3.6 and 2.2 log scot-cd⋅s⋅m⁻² flashes are fit with a model of phototransduction (smooth lines). The amplitude and sensitivity of the Rpe65−/− mouse photoresponses are reduced. After 9-cis-retinal treatment, photoresponses have larger amplitude and higher sensitivity. (C) Photoresponses in three Rpe65+/+ mice are compared to an untreated group of Rpe65−/− mice and a treated group of Rpe65−/− mice. Lines are the model of rod phototransduction activation fitted to a pair of photoresponses; only maximal responses are shown for clarity. (D) Maximum amplitude and sensitivity parameters of dark-adapted photoresponses in untreated and treated (48 h after 9-cis-retinal gavage) Rpe65−/− mice compared to the results in Rpe65+/+ mice. Untreated animals have significant differences (**, P < 0.05) in both parameters when compared to Rpe65+/+ or treated Rpe65−/− mice. Error bars represent 1 SEM and are smaller than symbols for some data.

Figure 4

Human phenotype of putative null mutation in RPE65. (A) Kinetic visual fields with a V-4e test target in the homozygote (black line) compared to normal (gray region); concentric circles are at 10^o intervals and meridians are at 15^o. (B) White stimulus thresholds of the homozygote (●) shows >6 log units of elevation under dark-adapted (DA) and >2 log units of elevation under light-adapted (2.7 log troland) conditions. Normal thresholds (gray symbols) on increasing background intensities (▿) and during the cone plateau following a full bleach (▵) are fit with empirical models of background adaptation (gray lines) to define the rod- and cone-mediated limbs. The normal cone adaptation model was shifted by 3.8 log units to the right and up to fit the patient data (solid line). (C) Cone flicker ERGs in homozygote are reduced ≈100-fold in amplitude and abnormally delayed in timing (arrows). Vertical gray lines mark stimulus times. A 29-Hz sinusoid was fitted to the homozygote data (dashed lines) to estimate amplitude and timing of the small signal. (D) Vertical OCT cross-sectional retinal images through the fovea (9^o inferior to 6^o superior retina) show central retinal structure is generally intact in the homozygote (compared to a heterozygous sibling) except for thinning of the outer retina-choroid complex, the red/white band toward bottom of panel. Optical reflectivity of retinal tissue is shown on a logarithmic pseudocolor scale: red and white are high reflectivity and blue and black are low reflectivity. Vitreous is toward the top and sclera is toward the bottom.