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

项目总结 常染色体显性遗传性视神经萎缩(DOA)是最常见的遗传性视神经病变。 OPA1基因的突变占所有突变的60%-70%，OPA1基因编码一种类似线粒体动力蛋白的GTPase 死亡案例。虽然OPA1在全身都有表达，但仅次于功能失调的线粒体， 与DOA相关的OPA1突变的患者表现出视网膜神经节细胞(RGC)的特异性丢失。尽管 关于OPA1突变的关键问题的深入研究和DOA小鼠模型的可用性 导致DOA患者人类RGCs特异性丢失的原因仍未得到回答，目前还没有 治疗这种情况的方法。人类RGC模型将极大地促进疾病机制的研究，因为 以及药物发现方面的努力。然而，从DOA患者样本中获取RGC是不可行的，因为 DOA供体眼罕见，人视网膜中RGC稀少，分离后RGC活性差。这个 拟议的研究将通过开发和详细描述三个人来解决这一未得到满足的需求 追踪疾病从干细胞分化到RGC进展的DOA的多能干细胞(HPSC)模型 退化。我们的干细胞模型的一个重要特征是，它们利用了产生 大量高纯度的RGC显示长期存活，具有重要的生化特征， 功能、形态和转录分析。我们将该协议与CRISPR/CAS9相结合 基因组编辑以OPA1单倍体不足为模型并开发可诱导CRISPR推理(CRISPRi) 用于控制OPA1功能丧失的定时的DOA模型。我们建议用这两个互补的方式 模型与患者IPSCs来源的RGC共同研究OPA1在RGC分化和分化中的作用 退化。在未来，这些特征良好的干细胞模型可能会被用于大规模销售功能 基因组学研究以及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.