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> 71个突变引起， 主要导致视杆细胞死亡。锥死亡总是跟随杆 死亡和开始后，结束主要 视杆细胞死亡阶段，而不管潜在的视杆细胞特异性基因突变。积累的证据表明 RP中视锥细胞死亡是由于葡萄糖饥饿。在理解细胞生物学方面， RP视锥细胞的代谢和视锥细胞与RPE之间的"代谢偶联"。我们的长期目标是防止 视锥细胞死亡导致RP失明。我们的总体目标是确定视锥细胞和RPE是如何代谢的， 并测试旨在恢复这种关系的RP潜在疗法。我们的中央 假设重新编程视锥和RPE代谢可以促进RP中视锥存活 独立于潜在的杆特异性基因突变。为了验证这一假设，我们提出了三个 具体目标。在目标1中，我们将使用视锥细胞特异性条件等位基因来增强糖酵解或OXPHOS， 视锥细胞，然后确定对RP小鼠模型中视锥细胞存活和功能的影响。在目标2中，我们将 使用RPE特异性条件等位基因来增强或抑制RPE中的OXPHOS，并确定对 RP小鼠模型中的光感受器功能。目标1和2的结果将阐明糖酵解的作用 以及RP小鼠模型中视锥细胞和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 在RP的2个临床前模型中重编程锥和RPE代谢的治疗价值。影响：将 解决视锥细胞代谢和OXPHOS在视网膜色素上皮生物学中的作用的知识缺口， 视网膜，这将大大推进我们对视锥细胞和RPE之间的代谢耦合的理解 并可能为临床前基因治疗干预提供机会，以防止视锥细胞死亡或延迟 光感受器通过细胞代谢重编程而退化。这一建议具有创新性 因为它：1）将使用创新的诱导型细胞特异性Cre驱动程序来解决以前未回答的问题 需要精确控制视锥细胞和RPE中基因操作的时间的问题; 2）将使用创新的 条件过表达敲入小鼠; 3）将是第一个测试创新假设，即重编程 视锥细胞和视网膜色素上皮的代谢可以促进视锥细胞的存活，而与视杆细胞特异性基因无关 突变; 4）将使用RP的独特敲入小鼠模型;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.