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

OPA1在视网膜神经节细胞分化和显性视神经萎缩发病机制中的作用

• 批准号: 10705002
• 负责人: Donald J. Zack
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705002
• 关键词: ATOH7 gene; Action Potentials; Autosomal Dominant Optic Atrophy; Biochemical; Biological Models; CRISPR/Cas technology; Cell Death; Cell Death Induction; Cell Differentiation process; Cell Survival; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Comparative Study; Competence; Cytoprotection; Defect; Development; Diagnosis; Disease; Disease Progression; Dynamin; Energy Metabolism; Exhibits; Eye; Eye diseases; Family; Future; Ganglion Cell Layer; Gene Dosage; Genes; Genetic Transcription; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Human; Induction of Apoptosis; Inherited; Lead; Methods; Mitochondria; Modeling; Morphology; Mus; Mutation; Neurons; Neuroprotective Agents; OPA1 gene; Optic Disk; Oxidative Phosphorylation; Oxidative Stress; Oxidative Stress Induction; Pathogenesis; Patients; Pilot Projects; Population; Predisposition; Process; Proteins; Protocols documentation; Reactive Oxygen Species; Retina; Retinal Ganglion Cells; Role; SOX11 gene; Sales; Sampling; Secondary to; Site; Specific qualifier value; Study models; System; Techniques; Testing; Thinness; Undifferentiated; cell injury; clinically relevant; disease mechanisms study; drug discovery; experimental study; functional genomics; genome editing; high throughput screening; human embryonic stem cell; human pluripotent stem cell; human stem cells; in vivo; induced pluripotent stem cell; inhibitor; loss of function; mitochondrial dysfunction; mouse model; mutant; optic nerve disorder; programs; protective pathway; regenerative agent; resilience; retinal ganglion cell degeneration; retinal nerve fiber layer; single-cell RNA sequencing; stem cell differentiation; stem cell model; stem cells; tert-Butylhydroperoxide; transcription factor; transcriptomics

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.

项目概要 常染色体显性视神经萎缩（DOA）是最常诊断的遗传性视神经病。 OPA1 基因（编码 GTP 酶等线粒体动力蛋白）的突变占所有突变的 60-70% DOA 案例。尽管 OPA1 在全身表达，继发于线粒体功能障碍， 患有 DOA 相关 OPA1 突变的患者会特别表现出视网膜神经节细胞 (RGC) 的丧失。尽管 深入研究和 DOA 小鼠模型的可用性，关于 OPA1 突变如何发生的关键问题 导致 DOA 患者人类 RGC 特异性缺失的原因仍然没有答案，目前还没有 针对这种情况的治疗。人类 RGC 模型将极大地促进疾病机制的研究，例如 以及药物发现工作。然而，从 DOA 患者样本中获取 RGC 是不可行的，因为 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 活力较差。这 拟议的研究将通过开发和详细描述三种人类特征来解决这一未满足的需求 DOA 的多能干细胞 (hPSC) 模型可追踪从干细胞分化到 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，具有长期存活的特性，对于生化、 功能、形态学和转录组分析。我们将此协议与 CRISPR/Cas9 结合起来 基因组编辑以模拟 OPA1 单倍体不足并开发了诱导型 CRISPR 推理 (CRISPRi) DOA模型控制OPA1功能丧失的时间。我们建议使用这两个互补的 模型与源自患者 iPSC 的 RGC 一起研究 OPA1 在 RGC 分化和 退化。未来，这些特征良好的干细胞模型可用于大规模销售功能性 基因组学研究以及神经保护和再生药物的高通量筛选。