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

项目总结 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3,100多人引起 主要针对杆状感光细胞的80个基因的突变。在主要杆状死亡阶段之后，圆锥体 不管潜在的基因突变如何，死亡都会发生。目前，存在着一种知识鸿沟 了解光感受器中的有氧糖酵解如何影响视杆之间微妙的“代谢偶联” RP组视网膜色素上皮(RPE)。该项目的长期目标是开发一种治疗方法 将保留RP患者的视锥功能。这项提案的目标是研究新陈代谢如何 乳酸缺乏引起的调节失调导致RP的光感受器死亡，测试一种新的代谢物 重新编制战略，并满足提交研究前新药的重要安全要求 申请。假说是重新编程的杆状和锥体有氧酵解将促进锥体存活 RP不依赖于潜在的杆状特异基因突变。这一假设是基于以下假设提出的 申请者强劲的初步数据。这项拟议研究的基本原理是，通过针对新陈代谢 许多遗传异质性形式的RP共同的通路，锥体功能可能被保存下来 相当于人类10年或更长时间，这将对人类的生活产生非常积极的影响 RP患者。这一假设将通过追求三个具体目标来检验：1)调查 光感受器特异性消融含Pro羟基酶结构域蛋白(PHD)，这是一种代谢酶 常压下抑制有氧糖酵解，常染色体中通过增强有氧糖酵解来保存锥体 隐性RP小鼠模型的建立；2)评价黄连素增强视锥细胞存活和功能的有效性和可行性。 消融显性RP小鼠模型光感受器中的PHD2；3)建立治疗性PHD2的安全性 在WT和RP小鼠模型以及人类细胞中进行编辑。具体地说，目标1将决定 增强锥体光感受器的有氧糖酵解可以促进它们在一种新的遗传小鼠模型中的存活。 目标2将测试基因疗法通过促进有氧糖酵解来延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，因为这将是第一个细胞特异性的例子 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阶段试验中重新部署，不作任何修改。这项拟议的研究具有重要意义，因为它具有 有潜力大幅降低治疗成本，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.