Reprogramming the metabolome to overcome the genetic heterogeneity in retinitis pigmentosa

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，由&gt；71突变引起 主要导致杆状感光细胞死亡。圆锥体的死亡总是伴随着杆子 死亡，大调结束后开始 杆状细胞死亡阶段与潜在的杆状细胞特异性基因突变无关。积累的证据表明 RP中的锥体死亡是由于葡萄糖饥饿所致。在理解细胞方面存在着知识鸿沟 视锥内代谢及视锥与RPE之间的“代谢偶联”。我们的长期目标是防止 视锥细胞死亡致双眼失明。我们的总体目标是定义视锥细胞和RPE是如何代谢的 并测试旨在恢复这种关系的RP的潜在疗法。我们的中央 假设重新编程视锥细胞和RPE代谢可以促进RP患者视锥细胞存活 与潜在的杆状特异基因突变无关。为了检验这一假设，我们提出了三个假设 明确的目标。在目标1中，我们将使用锥体特异的条件等位基因来增强糖酵解或OXPHOS 然后在RP小鼠模型中确定对视锥细胞存活和功能的影响。在目标2中，我们将 使用RPE特有的条件等位基因来增强或抑制RPE中的OXPHOS，并确定其对 RP小鼠模型的光感受器功能。目标1和目标2的结果将阐明糖酵解的作用。 RP模型小鼠视锥细胞和RPE细胞中的OXPOHS。在目标3中，我们将使用病毒基因疗法来测试 重编程视锥和RPE代谢在两种RP临床前模型中的治疗价值。影响：它将 解决视锥细胞代谢和OXPHOS在退行性RPE生物学中的作用的这一知识缺口 视网膜，这将极大地促进我们对视锥细胞和RPE之间代谢偶联的理解 并可能为临床前基因治疗干预提供机会，以防止锥体死亡或延迟 光感受器通过细胞代谢的重新编程而退化。这个建议很有新意 因为它：1)将使用创新的感应式单元特定CRE驱动器来解决之前未回答的问题 需要精确控制视锥细胞和RPE中基因操作时机的问题；2)将使用创新的 条件过表达敲入小鼠；3)将第一个测试重新编程的创新假说 视锥细胞和RPE代谢可以促进视锥细胞在RP中的存活，而不依赖于潜在的视杆细胞特异性基因 突变；4)将使用独特的RP敲入小鼠模型；以及5)将使用 AAV：：CONE特异性CRISPR/Cas9靶向基因以重新编程细胞代谢。

Project Summary Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, caused by >71 mutations that primarily cause rod photoreceptor death. Cone death always follows rod death and starts after the end of major rod death phase regardless of the underlying rod specific gene mutations. Accumulation evidence have shown that cone death in RP is due to glucose starvation. There is a knowledge gap in understanding cellular metabolism in cone and “metabolic coupling” between cone and RPE in RP. Our long-term goal is to prevent blindness in RP due to cone death. Our overall objective is to define how cones and RPE are metabolically coupled and to test potential therapies for RP that are designed to restore this relationship. Our central hypothesis is that reprogramming cone and RPE metabolism can promote cone survival in RP independently of the underlying rod-specific gene mutations. To test this hypothesis, we propose three specific aims. In Aim 1, we will use a cone-specific conditional allele to enhance glycolysis or OXPHOS in cone cells and then determine the effects on cone survival and function in RP mouse models. In Aim 2, we will use an RPE-specific conditional allele to enhance or suppress OXPHOS in RPE and determine the effects on photoreceptor function in RP mouse models. The results from Aim 1 & 2 will elucidate the role of glycolysis and OXPOHS in Cone and RPE cells in mouse model of RP. In Aim 3, we will use virus gene therapy to test therapeutic value of reprogramming cone and RPE metabolisms in 2 preclinical models of RP. Impact: it will address this knowledge gap in cone metabolism and the role of OXPHOS in RPE biology in degenerated retina, which will greatly advance our understanding of the metabolic coupling between cones and the RPE and may provide opportunities for preclinical gene-therapy interventions to prevent cone death or delay photoreceptor degeneration through reprogramming of cellular metabolism. This proposal is innovative because it: 1) will use innovative inducible cell-specific Cre drivers to address previously unanswered questions that require precise control of timing of gene manipulation in cones and RPE; 2) will use innovative conditional overexpress knock-in mouse ; 3)will be the first to test innovative hypothesis that reprogramming cone and RPE metabolism can promote cone survival in RP independently of the underlying rod specific gene mutations; 4) will use unique knock-in mouse model of RP; and 5) will test preclinical animal trial using AAV::Cone specific CRISPR/Cas9 to target a gene to reprogram cellular metabolism.