Targeting Nr2e3 to prevent photoreceptor degeneration

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Project Summary Retinitis pigmentosa (RP) is the most common form of retinal dystrophy and can be caused by mutations in any one of dozens of rod-enriched genes. The genetic heterogeneity of RP represents a major challenge for the development of effective therapies. For this reason, gene-independent treatments for RP have become a long- sought goal in vision research. In this proposal, we will test the hypothesis that knockout of the rod-specific transcription factor Nr2e3 prevents photoreceptor degeneration in multiple mouse disease models. In Specific Aim 1, we will characterize the neuroprotective effects of developmental Nr2e3 knockout in multiple models of photoreceptor degeneration, including a light-damage model and four mechanistically diverse models of RP (Pde6brd10/rd10, RhoP23H/+, Rho-/- and Cngb1-/-). We will use a combination of molecular, cellular, physiological, and behavioral assays to evaluate the efficacy and versatility of this therapeutic approach. In Specific Aim 2, we will evaluate the therapeutic potential of acute, adeno-associated virus (AAV)-delivered, CRISPR-Cas9-mediated Nr2e3 knockout in the same four mouse RP models used in Aim 1. For each model, we will evaluate the ability of acute Nr2e3 knockout to protect photoreceptors at multiple stages of degeneration. We will also compare the effects of acute Nr2e3 knockout to those of Nr2e3 overexpression, which has also been suggested to prevent degeneration. Together, these studies will test the effectiveness of Nr2e3-based reprogramming as a gene- independent therapy for RP. Finally, in Specific Aim 3, we will determine the effects of acute Nr2e3 knockout in wild-type mouse rods and identify neuroprotective factors downstream of Nr2e3. We will first compare the effects of acute Nr2e3 knockout to those of developmental Nr2e3 knockout. We will then perform RNA-seq on Nr2e3- knockout rods to generate a list of Nr2e3-downstream candidate effector genes. We will knockout or overexpress selected candidate genes, singly and in combination, in four mouse models of RP to identity those that confer a neuroprotective effect. If successful, these studies will establish Nr2e3 knockout as a novel gene-independent therapy for RP, paving the way for future studies in large-animal models of photoreceptor degeneration and for clinical studies in human patients.