Elucidating the molecular and cellular mechanisms underlying cone survival in the peripheral retina in mouse models of Retinitis Pigmentosa

项目摘要 视网膜色素变性(RP)是一种遗传性视网膜疾病，全世界每4,000人中就有1人患有此病。这种病 最初的进展是由于杆状光感受器退化引起的杆状特异基因突变，尽管 在这种疾病中，不同基因的不同突变汇聚成相同的杆状变性表型。 然而，正是随后的视锥感光细胞退化导致了日光颜色视力和 最终，大多数患者的生活质量会下降。而用基因治疗来取代突变的基因 功能复制已经成功，考虑到突变和基因的异质性，很难治疗所有的RP 通过瞄准棒子来处理案件。相反，在视杆细胞退化的情况下保存视锥细胞的通用疗法可能会导致 更全面的治疗方案。尽管取得了进展，但这种继发性疾病的分子机制 视锥细胞退行性变仍不清楚。在RP小鼠模型中，视杆细胞摘除后，视网膜中央的视锥细胞变性 死亡，但有趣的是，外围视网膜中的视锥细胞可以长期存活。这项拟议研究的目标是 目的是了解小鼠外周锥体存活的分子和细胞机制。 反相。在这笔赠款的指导阶段(K99阶段)，可能足够(目标1)和/或必要的因素 (目标2)视锥细胞存活的机制将通过细胞类型特异性RNA测序、体内视网膜电穿孔、 和时间调节的基因缺失。在本赠款的独立阶段(R00阶段)，原因是 将探讨血-视网膜-屏障破坏和视锥细胞变性之间的关系(目标3)。完成 这些被提议的目标将导致在RP小鼠模型中识别锥体存活的关键调控因素。 此外，我们可能首次确定BRB击穿和次级锥体之间的因果关系 死亡，打开新的细胞靶点以防止RP患者的视锥细胞丢失。从长远来看，概述的方法 在这笔赠款中可以成为回答有关神经元和其他细胞如何 在整个中枢神经系统的神经退行性疾病中，支持细胞退化。被指导者 这一阶段的赠款是在康斯坦斯·切普科博士的指导下进行的，他的实验室开发了许多 多年来在体内对视网膜进行基因操作并发现基因调控网络的技术 潜在的视网膜细胞类型规范。哈佛大学切普科实验室周围的科学环境 医学院提供了宝贵的职业发展机会，有助于为 独立研究视网膜变性的分子机制。

Project Summary Retinitis Pigmentosa (RP) is an inherited retinal disease afflicting 1 in 4,000 people worldwide. The disease progresses initially by rod photoreceptor degeneration caused by mutations in rod-specific genes, although different mutations in different genes converge upon the same rod degeneration phenotype in this disease. However, it is the subsequent cone photoreceptor degeneration that causes loss in daylight color vision and ultimately, diminishing quality of life for most patients. While gene therapy to replace a mutated gene with a functional copy has been successful, given the heterogeneity in mutations and genes, it is difficult to treat all RP cases by targeting the rods. Instead, a generic therapy to preserve the cones upon rod degeneration may lead to a more comprehensive therapeutic option. Despite progress, the molecular mechanism for this secondary cone degeneration remains unclear. In mouse models of RP, the cones in the central retina degenerate after rod death, but interestingly, the cones in the peripheral retina survive long-term. The goal of this proposed research is to understand the molecular and cellular mechanisms underlying peripheral cone survival in mouse models of RP. During the mentored phase of this grant (K99 phase), factors that may be sufficient (Aim 1) and/or necessary (Aim 2) for cone survival will be elucidated by cell type specific RNA sequencing, in vivo retinal electroporation, and temporally-regulated gene deletion. During the independent phase of this grant (R00 phase), the causal relationship between blood-retina-barrier breakdown and cone degeneration will be explored (Aim 3). Completion of the proposed aims will lead to identification of key regulators of cone survival in mouse models of RP. Moreover, we may identify, for the first time, a causal relationship between BRB breakdown and secondary cone death, opening new cellular targets to prevent cone loss in patients with RP. Long-term, the approaches outlined in this grant can become the cornerstone for answering questions regarding how, in general, neurons and other supporting cells degenerate in neurodegenerative disorders across the central nervous system. The mentored phase of this grant is conducted under the guidance of Dr. Constance Cepko, whose lab has developed many techniques over the years to genetically manipulate the retina in vivo and discovered gene regulatory networks underlying retinal cell type specification. The scientific environment that surrounds the Cepko Lab at Harvard Medical School offers valuable opportunities for career development, helping to build a strong foundation for an independent career investigating the molecular mechanisms of retinal degeneration.