Pharmacological and rAAV gene therapy rescue of visual functions in a blind mouse model of Leber congenital amaurosis

Abstract

Background: Leber congenital amaurosis (LCA), a heterogeneous early-onset retinal dystrophy, accounts for approximately 15% of inherited congenital blindness. One cause of LCA is loss of the enzyme lecithin:retinol acyl transferase (LRAT), which is required for regeneration of the visual photopigment in the retina.

Methods and findings: An animal model of LCA, the Lrat-/- mouse, recapitulates clinical features of the human disease. Here, we report that two interventions--intraocular gene therapy and oral pharmacologic treatment with novel retinoid compounds--each restore retinal function to Lrat-/- mice. Gene therapy using intraocular injection of recombinant adeno-associated virus carrying the Lrat gene successfully restored electroretinographic responses to approximately 50% of wild-type levels (p < 0.05 versus wild-type and knockout controls), and pupillary light responses (PLRs) of Lrat-/- mice increased approximately 2.5 log units (p < 0.05). Pharmacological intervention with orally administered pro-drugs 9-cis-retinyl acetate and 9-cis-retinyl succinate (which chemically bypass the LRAT-catalyzed step in chromophore regeneration) also caused long-lasting restoration of retinal function in LRAT-deficient mice and increased ERG response from approximately 5% of wild-type levels in Lrat-/- mice to approximately 50% of wild-type levels in treated Lrat-/- mice (p < 0.05 versus wild-type and knockout controls). The interventions produced markedly increased levels of visual pigment from undetectable levels to 600 pmoles per eye in retinoid treated mice, and approximately 1,000-fold improvements in PLR and electroretinogram sensitivity. The techniques were complementary when combined.

Conclusion: Intraocular gene therapy and pharmacologic bypass provide highly effective and complementary means for restoring retinal function in this animal model of human hereditary blindness. These complementary methods offer hope of developing treatment to restore vision in humans with certain forms of hereditary congenital blindness.

Figure 1. Retinoid Structures, Specificity of Retinoids in Regeneration of Visual Pigment, and Model of Absorption of 9-cis-R-Ac in Mammals

(A) Structures of retinoids used for gavage studies. (B) Levels of all-trans-RAL oximes, 9-cis-RAL oximes (corresponding to formation of visual pigment isorhodopsin), and all-trans-REs in Lrat ^−/− mouse eyes gavaged with 5 mg of each retinoid before 48–72 h dark adaptation (n ≥ 3, data shown with standard deviation [SD]). (C) Model of absorption of 9-cis-R-Ac in mammals.

Figure 2. Retinoids in the Liver and Eyes of Lrat ^−/− Mice after 9-cis-R-Ac Treatment

(A–F) Normal-phase HPLC analysis of nonpolar retinoids extracted from the tissues of dark-adapted Lrat ^+/+ or Lrat ^−/− mice following gavage with retinoids. Peaks marked * represent the solvent change artifact, and peaks labeled (1) indicate an unidentified non-specific compound with λ_max= 270 nm. In all experiments, mice were dark-adapted for 48 h after gavage. Shown are results from eyes of dark-adapted control Lrat ^+/+ (gray) and Lrat ^−/− mice (black) (A); eyes of dark-adapted Lrat ^−/− mouse gavaged with all-trans-R-Ac (B); eyes of dark-adapted Lrat ^−/− mouse gavaged with 9-cis-R-Ac (C); liver tissue from dark-adapted control Lrat ^+/+ (gray) and Lrat ^−/− (black) mice (D); liver tissue from dark-adapted Lrat ^−/− mouse gavaged with all-trans-R-Ac (E); and liver tissue from dark-adapted Lrat ^−/− mouse gavaged with 9-cis-R-Ac (F). (G–J) Time course of the levels of nonpolar and polar retinoids in the tissues of Lrat ^−/− mice following gavage with 9-cis-R-Ac measured by HPLC. After gavage, mice were dark adapted for indicated time before HPLC analysis (n ≥ 3, data shown with SD). The graphs depict: a short time course of 9-cis-RAL oxime levels detected in Lrat ^−/− mice eyes following a 20 μmol gavage of 9-cis-R-Ac (G); a longer time course of 9-cis-RAL oxime levels in Lrat ^−/− mice eyes following a 20 μmol gavage of 9-cis-R-Ac (H); time course of 9-cis-ROL blood levels in Lrat ^−/− mice following gavage with 20 μmol 9-cis-R-Ac (I); and time course of RE and RA levels in liver of Lrat ^−/− mice following a 20 μmol gavage with 9-cis-R-Ac (J).

Figure 3. Levels of 9-cis-RAL Oximes in the Eyes of Lrat ^−/− Mice after a Single or Multiple Dose of 9-cis-R-Ac

(A) The level of 9-cis-RAL in Lrat ^−/− mouse eyes after a varying dose of 9-cis-R-Ac. (B) The level of 9-cis-RAL in Lrat ^−/− mouse eyes after a varying size and number of doses of 9-cis-R-Ac. The solid gray line represents a maximal level of isorhodopsin as measured by the level of 9-cis-retinal oximes in Lrat ^−/− mouse eyes after ten gavages; dashed gray line indicates the SD. The maximal level of isorhodopsin is comparable to the level of rhodopsin in WT mice (blue dotted line, shown as pmol of 11-cis-retinal/eye). (C) The level of 9-cis-RAL in Lrat ^−/− mouse eyes after 9-cis-R-Ac treatment and light exposure or after exposure to light and re-gavage (n ≥ 3, data shown with SD). (D and E) Changes in the RPE-ROS interface in control Lrat ^−/− mice and Lrat ^−/− mice treated with 9-cis-R-Ac. Treated Lrat ^−/− mice were gavaged with 9-cis-R-Ac (10 μmol per gavage) six times, 3 d apart, and analyzed (D). Control retina from age-matched (8 wk old) untreated Lrat ^−/− mice (E). Considerably improved RPE-ROS processes were observed in all treated mice. RPE, retinal pigment epithelium; ROS, rod outer segments; IS, inner segments. Scale bar, 10 μm.

Figure 4. Rescue of Visual Responses Measured by Single-Cell Recording and ERG Responses of single Lrat ^+/+ and Lrat ^−/− Rods

(A–D) Flash families measured for a Lrat ^+/+ rod (A), a control Lrat ^−/− rod (B), a Lrat ^−/− rod after a single gavage with 9-cis-R-Ac (C), and a Lrat ^−/− rod after three gavages with 9-cis-R-Ac (D). Each panel superimposes average responses to five to 20 repeats of a flash, with the flash strength increasing by a factor of two for each successively larger response. (E) Stimulus-response relations for the same cells in (A–D). Maximal response amplitudes are plotted against the flash strength. Fits are saturating exponential functions, used to estimate the strength of the flash producing a half-maximal response (Lrat ^+/+, 26 photons/μm²; Lrat ^−/−, 43,000 photons/μm²; singly treated Lrat ^−/−, 590 photons/μm²; and multiply treated Lrat ^−/−, 69 photons/μm²). (F and G) Comparison of WT mice scotopic single flash ERG to Lrat ^−/− 9-cis-R-Ac gavaged mice and Lrat ^−/− and Lrat ^+/+ control mice. Lrat ^−/− mice were gavaged nine times with 5 μmol 9-cis-R-Ac over a 1-mo time period (n ≥ 3, data shown with SD).

Figure 5. Immunocytochemistry and ERG of rAAV-Lrat-Treated Lrat ^−/− Mice

(A) Immunolocalization of LRAT (green) in rAAV-Lrat treated Lrat ^−/− mouse eye. Anti-LRAT antibody was directly labeled by Alexa 488. Expression of LRAT is locally restricted (arrowheads) in the eye. Nuclei are stained by Hoechst 33342 (blue). (B) Higher-power magnification image of (A). LRAT (green) is observed specifically in the RPE cell layer. (C) Subcellular localization of LRAT in rAAV-Lrat treated Lrat ^−/− mouse eye. RPE cells were labeled by anti-LRAT and detected by Cy3-labeled secondary antibody (red). Nuclei are stained by Hoechst 33342 (blue). RPE cell layer was mounted flat on a coverslip and imaged. LRAT is localized in the internal membrane of the RPE cells. Similar localization was observed for WT mouse RPE cells [12]. (D) Control flat-mounted RPE cell layer of untreated Lrat ^−/− mouse. Anti-LRAT antibody does not show any non-specific labeling. Bars indicate 100 μm. (E) Comparison of scotopic single flash ERG of rAAV-Lrat treated, Lrat ^+/+, and Lrat ^−/− control mice as measured by a-wave amplitudes, (n ≥ 16, data shown with SD). (F) A plot of a-wave amplitudes at 2.8 cd·s·m⁻² intensity as a function of post-treatment time for rAAV-Lrat treated mice.

Figure 6. Light-Induced Pupillary Constriction of Lrat ^−/− Mice Before and After Treatment with 9-cis-R-Ac or rAAV-Lrat

(A–H) 470 nm light (4.79 × 10¹³ photons·cm⁻²·sec⁻¹) was used to stimulate pupillary constriction. Untreated Lrat ^−/− pupil before (A) and during (B) light exposure. Same mouse as in (A and B) subsequent to treatment with 9-cis-R-Ac, before (C) and during (D) light exposure. Control, untreated pupil of Lrat ^−/− mouse before (E) and during (F) light exposure. Contralateral eye of mouse shown in (E and F) treated with rAAV-Lrat, before (G) and during (H) light exposure. (I) Irradiance-response relations for PLR to 470 nm light.