Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca 2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration , were challenging to interpret because of negative effects on synaptic transmission (45). We therefore altered our approach to limit ablation of Sirt6 to rod photoreceptors with an inducible gene disruption strategy. Using this model, we tested whether upregulation of glycolytic flux through Sirt6 knockout can preserve both rod and cone photoreceptors in a preclinical, Pde6-associated RP model. The Journal of Clinical Investigation R E S E A R C H A R T I C L E ResultsGeneration of experimental and control groups. The third most common cause of autosomal recessive RP is deficiency in the PDE6 enzyme, which controls the depolarization state of rods by regulating cGMP levels (9, 46-48). An established preclinical model for RP involves a homozygous point mutation (H620Q) in the gene months by increasing glucose uptake and utilization for NADPH production in 4 different mouse models of RP (11,12). In our report, we propose a similar strategy that improves both survival and function of degenerating rods and cones. We hypothesized that Pde6-associated RP provokes a metabolic aberration in the rod cells that forces them to succumb to the consequences of elevated cGMP and Ca 2+ via cyclic nucleotide-gated (CNG) channels and Na + /Ca 2+ -K + exchangers (36-39). The histone deacetylase sirtuin 6 (SIRT6) is a transcriptional repressor of glycolytic enzymes that has been extensively studied in the context of metabolism and cancer biology (40). Normally, S...

Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.

As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest.

Retinal degenerations present a unique challenge as disease progression is irreversible and the retina has little regenerative potential. No current treatments for inherited retinal disease have the ability to reverse blindness, and current dietary supplement recommendations only delay disease progression with varied results. However, the retina is anatomically accessible and capable of being monitored at high resolution in vivo. This, in addition to the immune-privileged status of the eye, has put ocular disease at the forefront of advances in gene- and cell-based therapies. This review provides an update on gene therapies and randomized control trials for inherited retinal disease, including Leber congenital amaurosis, choroideremia, retinitis pigmentosa, Usher syndrome, X-linked retinoschisis, Leber hereditary optic neuropathy, and achromatopsia. New gene-modifying and cell-based strategies are also discussed. © 2016 Wiley Periodicals, Inc.