Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness

Abstract

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Treatment Potential for LCA5-Associated Leber Congenital Amaurosis

“…One microliter of a solution containing AAV8-hLCA5 was injected into the subretinal space, delivering 1.0E+09 viral genomes (vg) per eye, an optimal dose based on previous expression and toxicity profiling studies. 18 Control injections were performed with excipient only. Pred-G ophthalmic ointment (Allergan) was applied to the eyes immediately after the procedure.…”

“… 33 , 34 Small ERGs were anticipated in Lca5 gt/gt mice, and thus the starting intensity for each of the series was set at ∼2 log units above the threshold response of wild-type mice. 18 Three mice developed corneal opacities after ERG testing and were excluded from subsequent pupillometry analyses. Transient pupillary light reflexes were recorded using a NeurOptics A-2000 (NeurOptics, Irvine, CA, USA) small animal pupillometry system as previously described.…”

“…Transient pupillary light reflexes were recorded using a NeurOptics A-2000 (NeurOptics, Irvine, CA, USA) small animal pupillometry system as previously described. 18 The subset of mice receiving bilateral injections (AAV-hLCA5 or excipient) were used for TPLR analysis, to avoid confounding of the treated eye causing a contralateral pupil constriction in the untreated eye. The amplitude of pupillary constriction was measured in response to a series of five 40-ms 1000 and 3000 lux flashes per eye in anesthetized, dark-adapted mice and was defined as the difference between the baseline pupil diameter and the diameter obtained 0.6 to 1.2 seconds after stimulus onset.…”

“… 7 , 16 , 17 In the Lca5 gt/gt mouse model of the disease, homozygous null Lca5 gene trap (gt) insertions cause a severe retinal degeneration within a few weeks of birth. 16 , 18 At postnatal day (P) 12, the outer nuclear layer (ONL) is normal in thickness, but the photoreceptor inner and outer segments (POS) are already shorter than normal. 16 By P28 the ONL thins to about one third of normal with only 3 or 4 rows of nuclei.…”

“… 19 , 20 We have previously shown that a recombinant adeno-associated viral vector (AAV7m8) containing human LCA5 cDNA can rescued ONL thickness, pupillary responses, and water maze performance when delivered to the subretinal space of neonatal Lca5 gt/gt mice. 16 , 18 Of note, these injections were performed in P4–P6 mice before retinal degeneration had started and thus is not translatable to the early phases of eventual human clinical trials. Patients with LCA5 -LCA typically show a moderate to severe retinal degeneration by the time they are diagnosed early in life.…”