Role of OPA1 in Retinal Ganglion Cell Differentiation and the Pathogenesis of Dominant Optic Atrophy

项目摘要 常染色体显性视神经萎缩（DOA）是最常见的诊断遗传性视神经病变。 OPA 1基因的突变，它编码一种线粒体动力蛋白，如GT3，占所有突变的60- 70 死亡案例虽然OPA 1在全身表达，继发于线粒体功能障碍， 具有DOA相关OPA 1突变的患者特异性地表现出视网膜神经节细胞（RGC）的损失。尽管 深入的研究和DOA小鼠模型的可用性，关于OPA 1突变如何 导致DOA患者中人RGC特异性丧失的研究仍然没有答案，目前没有 治疗这种情况。人类RGC模型将极大地促进疾病机制的研究， 以及药物发现的努力。然而，从DOA患者样品获得RGC是不可行的，这是由于 DOA供体眼睛的稀有性，人视网膜中RGC的稀疏性，以及分离后RGC的活力差。的 拟议的研究将通过开发和详细描述三种人类 DOA的多能干细胞（hPSC）模型，其跟踪从干细胞分化到RGC的疾病进展 退化我们的干细胞模型的一个重要特征是，它们利用产生 大量高度纯化的RGC显示出长期存活，对生物化学重要的特征， 功能、形态和转录组学分析。我们将该方案与CRISPR/Cas9 基因组编辑以模拟OPA 1单倍不足，并开发了诱导型CRISPR推理（CRISPRi） DOA模型控制OPA 1功能丧失的时间。我们建议使用这两个互补的 模型与来源于患者iPSC的RGC一起研究OPA 1在RGC分化中的作用， 退化在未来，这些特征良好的干细胞模型可用于大规模功能性 基因组学研究和神经保护和再生剂的高通量筛选。

PROJECT SUMMARY Autosomal dominant optic atrophy (DOA) is the most commonly diagnosed inherited optic neuropathy. Mutations in the OPA1 gene, which encodes a mitochondrial dynamin like GTPase, account for 60-70% of all DOA cases. Although OPA1 is expressed throughout the body, secondary to dysfunctional mitochondria, patients with DOA associated OPA1 mutations exhibit loss of retinal ganglion cells (RGCs) specifically. Despite intensive study and the availability of mouse models of DOA, critical questions regarding how OPA1 mutations lead to specific loss of human RGCs in DOA patients remain unanswered and there are currently no treatments for this condition. A human RGC model would greatly facilitate the study of disease mechanisms as well as drug discovery efforts. Obtaining RGCs from DOA patient samples is not feasible, however, due to the rarity of DOA donor eyes, the sparsity of RGCs in the human retina, and poor RGC viability upon isolation. The proposed studies will address this unmet need by developing and characterizing in detail three human pluripotent stem cell (hPSC) models of DOA that track disease progress from stem cell differentiation to RGC degeneration. An important feature of our stem cell models is that they make use of techniques that produce large quantities of highly purified RGCs that display long term survival, features important for biochemical, functional, morphological, and transcriptomic analyses. We combined this protocol with CRISPR/Cas9 genome-editing to model OPA1 haploinsufficiency and developed an inducible CRISPR inference (CRISPRi) DOA model to control the timing of OPA1 loss of function. We propose to use these two complementary models together with RGCs derived from patient iPSCs to study the role of OPA1 in RGC differentiation and degeneration. In the future, these well-characterized stem cell models could be used for large-sale functional genomics studies and high throughput screening for neuroprotective and regenerative agents.