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

项目概要 色素性视网膜炎 (RP) 是最常见的遗传性视网膜营养不良 (IRD)，由超过 3,100 种疾病引起 主要针对杆状光感受器的 80 个基因发生突变。在主要杆死亡阶段之后，锥体 无论潜在的基因突变如何，都会发生死亡。目前，在这方面存在知识差距 了解光感受器中的有氧糖酵解如何影响视杆细胞之间微妙的“代谢耦合” 以及 RP 中的视网膜色素上皮 (RPE)。该项目的长期目标是开发一种疗法 将保留 RP 患者的锥体功能。该提案的目的是研究代谢如何 乳酸缺乏导致的失调导致 RP 中光感受器死亡，测试一种新的代谢组 重编程策略，并满足提交研究前新药的重要安全要求 应用程序。假设重新编程视杆细胞和视锥细胞的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。这一假设的提出是基于 申请人强有力的初步数据。拟议研究的基本原理是通过针对代谢 许多遗传异质形式的 RP 所共有的途径，锥体功能可能会被保留 相当于人类10年或更长的时间，这将对人类的生活产生巨大的积极影响 RP 患者。该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的蛋白（Phd）的光感受器特异性消融，这是一种代谢酶， 抑制常氧条件下的有氧糖酵解，通过增强常染色体中的有氧糖酵解来保护视锥细胞 隐性RP小鼠模型； 2) 评估增强视锥细胞存活和功能的功效和可行性 消除显性 RP 小鼠模型光感受器中的 PHD2； 3) 确定治疗性 Phd2 的安全性 在 WT 和 RP 小鼠模型以及人类细胞中进行编辑。具体来说，目标 1 将确定是否 视锥光感受器中增强的有氧糖酵解可以促进其在新型遗传小鼠模型中的存活。 目标 2 将测试基因疗法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。该方法具有创新性，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，并且因为新型治疗编辑载体可以 无需修改即可在未来的 I-IIA 期试验中重新部署。拟议的研究意义重大，因为它具有 显着降低治疗成本的潜力，适用于分裂和非分裂细胞，形状 正在进行的 CRISPR 研究，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.