Targeting Nr2e3 to prevent photoreceptor degeneration

项目摘要 色素性视网膜炎（RP）是视网膜营养不良的最常见形式，并且可以由任何基因突变引起。 几十种杆状基因之一RP的遗传异质性是研究的主要挑战。 开发有效的治疗方法。因此，RP的基因非依赖性治疗已成为一个长期的， 在视觉研究中寻求目标。在这个建议中，我们将测试的假设，敲除杆特异性 转录因子Nr 2 e3在多种小鼠疾病模型中预防光感受器变性。在特定 目的1，我们将描述发育性Nr 2 e3基因敲除在多种模型中的神经保护作用。 感光细胞变性，包括光损伤模型和四种不同机制的RP模型 （Pde 6 brd 10/rd 10、RhoP 23 H/+、Rho-/-和Cngb 1-/-）。我们将结合分子、细胞、生理和 行为测定以评估该治疗方法的功效和通用性。在第二阶段，我们将 评估急性、腺相关病毒（AAV）递送、CRISPR-Cas9介导的 在Aim 1中使用的相同四种小鼠RP模型中的Nr 2 e3敲除。对于每个模型，我们将评估 急性Nr 2 e3敲除，以保护光感受器在多个阶段的退化。我们还将比较 急性Nr 2 e3敲除对Nr 2 e3过表达的影响，这也被认为可以防止 退化总之，这些研究将测试基于Nr 2 e3的重编程作为基因的有效性， RP的独立治疗。最后，在具体目标3中，我们将确定急性Nr 2 e3敲除对 野生型小鼠视杆细胞并鉴定Nr 2 e3下游的神经保护因子。我们先来对比一下 急性Nr 2 e3基因敲除与发育性Nr 2 e3基因敲除之间的差异。然后我们将对Nr 2 e3进行RNA-seq- 敲除棒以产生Nr 2 e3下游候选效应基因的列表。我们将淘汰或过度表达 在四种RP小鼠模型中，单独和组合地选择候选基因，以鉴定赋予 神经保护作用如果成功，这些研究将建立Nr 2 e3敲除作为一种新的基因独立的基因敲除。 治疗RP，为未来在感光细胞变性的大型动物模型中的研究和 人类患者的临床研究。

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.