Enhancing cone survival in retinitis pigmentosa through cell-specific therapeutic CRISPR editing of a roxadustat target

项目摘要 视网膜色素变性（RP）是最常见的遗传性视网膜营养不良（IRD），由超过3，100 80个基因的突变主要是针对视杆细胞感光器的。在主要视杆细胞死亡阶段之后， 无论潜在的基因突变如何，死亡都会发生。目前，存在知识差距， 了解光感受器中的有氧糖酵解如何影响杆之间微妙的“代谢耦合” RP组视网膜色素上皮（retinal pigment epithelium，RPE）的变化。该项目的长期目标是开发一种治疗方法， 将保留RP患者的视锥细胞功能。本提案的目的是研究代谢如何 由于乳酸缺乏导致的调节异常导致RP中的感光细胞死亡，测试一种新的代谢组 重新编程策略，并满足提交研究前新药的重要安全性要求 应用程序.假设重新编程视杆细胞和视锥细胞有氧糖酵解将促进视锥细胞的存活， RP独立于潜在的杆特异性基因突变。这一假设是根据以下事实提出的： 申请人的初步数据。这项研究的基本原理是，通过靶向代谢 途径共同的许多遗传异质性形式的RP，锥功能可能被保留， 相当于10年或更长的人类年，这将对人类的生活产生巨大的积极影响。 RP患者这一假设将通过追求三个具体目标来检验：1）调查是否 含脯氨酰羟化酶结构域蛋白（Phd）的光受体特异性消融，所述蛋白是一种代谢酶， 在常氧条件下抑制有氧糖酵解，通过增强常染色体中的有氧糖酵解来保护锥体。 隐性RP小鼠模型; 2）评估通过以下方法增强视锥细胞存活和功能的功效和可行性： 在显性RP小鼠模型的光感受器中消融PHD 2; 3）确定治疗性PHD 2的安全性 在WT和RP小鼠模型以及人类细胞中进行编辑。具体而言，目标1将确定是否 在一种新的遗传小鼠模型中，锥体光感受器中增强的有氧糖酵解可以促进它们的存活。 目的2将测试基因治疗通过增强有氧糖酵解来减缓光感受器退化的潜力 在不同的RP小鼠模型中。最后，目标3将定义有氧运动的药代动力学和安全性。 糖酵解重编程载体。这种方法是创新的，因为这将是细胞特异性的第一个例子。 CRISPR介导的精确代谢重编程，因为新的治疗编辑载体可以 在未来的I-IIA期试验中重新部署，无需修改。拟议的研究是重要的，因为它有 潜在的显着降低治疗成本，适用于分裂和非分裂细胞，形状 正在进行的CRISPR研究，并最终将有氧糖酵解定义为安全有效的治疗靶点。

PROJECT SUMMARY Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy (IRD), caused by more than 3,100 mutations in 80 genes that are primarily specific to rod photoreceptors. Following major rod death phase, cone death occurs regardless of the underlying gene mutations. Currently, there exists a knowledge gap in understanding how aerobic glycolysis in photoreceptors impact the delicate “metabolic coupling” between rods and the retinal pigment epithelium (RPE) in RP. The long-term goal of this project is to develop a therapy that will preserve cone function in patients with RP. The objectives of this proposal are to investigate how metabolic dysregulation due to lactate deficiency contributes to photoreceptor death in RP, test a novel metabolome reprogramming strategy, and fulfill important safety requirements for filing a Pre-Investigational New Drug application. The hypothesis is that reprogramming rod and cone aerobic glycolysis will promote cone survival in RP independent of the underlying rod-specific gene mutations. This hypothesis has been formulated based on the applicant’s strong preliminary data. The rationale for the proposed research is that by targeting a metabolic pathway common to many of the genetically heterogeneous forms of RP, cone function may be preserved for the equivalent of 10 or more human years, which would have a tremendously positive impact on the lives of patients with RP. This hypothesis will be tested by pursuing three specific aims: 1) Investigate whether photoreceptor-specific ablation of prolyl hydroxylase domain-containing protein (Phd), a metabolic enzyme that inhibits aerobic glycolysis under normoxia, preserves cones by enhancing aerobic glycolysis in an autosomal recessive RP mouse model; 2) Assess the efficacy and feasibility of enhancing cone survival and function by ablating PHD2 in photoreceptors of a dominant RP mouse model ; 3) Establish the safety of therapeutic Phd2 editing in a WT and RP mouse model as well as human cells. Specifically, Aim 1 will determine whether enhanced aerobic glycolysis in cone photoreceptors can promote their survival in a novel genetic mouse model. Aim 2 will test the potential of gene therapy to slow photoreceptor degeneration by enhancing aerobic glycolysis in a different mouse model of RP. Lastly, Aim 3 will define the pharmacokinetics and safety of the aerobic glycolysis reprogramming vector. The approach is innovative because this will be the first example of cell-specific CRISPR-mediated precision metabolic reprogramming and because the novel therapeutic-editing vectors can be redeployed in future Phase I-IIA trials without modification. The proposed research is significant as it has the potential to dramatically lower the cost of treatment, be applicable to dividing and nondividing cells, shape ongoing CRISPR research, and ultimately define aerobic glycolysis as a safe and effective therapeutic target.