Pluripotent Stem Cell Derived 3D Retinas for Studies of Early Onset Retinal Degeneration

多能干细胞衍生的 3D 视网膜用于研究早发性视网膜变性

• 批准号: 10372095
• 负责人: KARL J WAHLIN
• 金额: $ 38.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10372095
• 关键词: 3-Dimensional; ARA9 protein; Address; Affect; Age related macular degeneration; Alleles; Animals; Apoptosis; Binding; Binding Proteins; Biological Models; Biology; Birth; Blindness; CRISPR/Cas technology; Cell Cycle Proteins; Cell Death; Cell Survival; Cells; Childhood; Clinical; Complex; Cone; Cyclic GMP; DNA Repair; Data; Defect; Disease; Electrophysiology (science); Experimental Models; Eye diseases; Fluorescence; Freezing; Functional disorder; Gene Expression; Genes; Goals; Human; Hydrocarbons; Inherited; Knock-out; Knowledge; Label; Laboratories; Lead; Leber' s amaurosis; Measures; Modeling; Molecular; Monitor; Morphology; Mus; Mutation; Organoids; Pathogenesis; Pathway interactions; Patients; Phenotype; Photoreceptors; Pluripotent Stem Cells; Population; Primates; Property; Proteins; RIPK1 gene; RPE65 protein; Recombinant adeno-associated virus (rAAV); Recovery of Function; Reporter; Resources; Retina; Retinal Degeneration; Retinal Diseases; Retinal Dystrophy; Retinitis Pigmentosa; Rod; Role; Severities; Structure; Study models; System; Tacrolimus Binding Proteins; Testing; Time; Tissues; Variant; Vertebrate Photoreceptors; Viral; Western Blotting; Work; base; cell type; comparative; cone-rod dystrophy; disease phenotype; disease-in-a-dish; early onset; gene correction; gene therapy; genome editing; human disease; human pluripotent stem cell; human stem cells; in vivo; inherited retinal degeneration; innovation; innovative technologies; insight; knockout gene; live cell imaging; mRNA Expression; mutant; photoreceptor degeneration; polyproline; prevent; repaired; response; selective expression; stem cell derived tissues; stem cells; therapy development; transcriptome sequencing

SUMMARY Retinal degenerative (RD) diseases, such as Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), cause dysfunction and cell death of photoreceptor (PR) cells, ultimately leading to blindness. LCA is the leading cause of inherited childhood blindness resulting in a loss of vision at or soon after birth. Though this is considered to be quite rare, these blinding diseases are devastating for those affected. Current efforts are being made to develop gene-therapies aimed at correcting some of the genes affected in RD and this approach has shown some promise in animals and humans for restoring RPE65 gene expression, but there are many other causes of RD for which there is no cure. In addition, due to the many mutations involved in RD, there are significant gaps in our understanding of how PR loss occurs. To address this, we will use human pluripotent stem cell (PSC) based retinal cell-reporter lines with RD-associated alleles to help explore the mechanisms of PR cell death. Given the typically long period of time required to generate human retinas in the laboratory, the severity and rapid onset of degeneration in LCA makes it an attractive experimental model to study human RD and to develop potential therapies. We will study the aryl hydrocarbon receptor interacting protein-like1 (AIPL1) gene to explore three functional domains that harbor naturally occurring mutations in patients with LCA and cone-rod dystrophy (CORD). A comparative analysis of different mutations might lead to a better understanding of how rods and cones die and greater insight into other more common forms of PR degeneration, such as age- related macular degeneration (AMD). A central hypothesis is that human PSC derived 3D retina organoids with AIPL1 mutations will recapitulate human retinal dystrophy resulting in PR loss. This hypothesis is supported by our recent work, and others, showing that human PSCs can be coaxed into becoming retinal eyecup-like structures with PRs, a laminar morphology and outer segment structures that are similar to an actual retina. This proposal will bridge two innovative technologies; (1) genome-editing to generate genetically matched retinal reporter PSC derived retinas with disease-associated mutations and (2) gene-correction to repair genetic defects and promote PR cell survival. Given the very early onset of LCA it is important to define the appropriate windows of time for such treatment options. Not only will these studies lead to new insights into the biology of RD disease, but could also provide an innovative resource to develop therapies for the treatment of RD.

摘要 视网膜退行性疾病(RD)，如视网膜色素变性(RP)和Leber先天性黑发 (LCA)，导致光感受器(PR)细胞功能障碍和细胞死亡，最终导致失明。生命周期评价 是导致儿童遗传性失明的主要原因，导致出生时或出生后不久失明。尽管 这被认为是相当罕见的，这些致盲疾病对受影响的人来说是毁灭性的。目前的努力是 被要求开发基因疗法，目的是纠正一些受RD影响的基因，以及这种方法 在动物和人类身上显示了一些恢复RPE65基因表达的希望，但还有很多 其他无法治愈的RD原因。此外，由于RD涉及的突变很多，因此有 我们对公关损失是如何发生的理解上存在很大差距。为了解决这个问题，我们将利用人类的多能性 基于干细胞(PSC)的携带RD相关等位基因的视网膜细胞报告系有助于探讨视网膜脱离的机制 公关细胞死亡。考虑到在实验室生成人类视网膜通常需要很长的时间， LCA的严重程度和快速退变使其成为研究人类RD的一个有吸引力的实验模型 并开发潜在的治疗方法。我们将研究芳烃受体相互作用蛋白样蛋白1(AIPL1) 基因探索LCA和LCA患者存在自然发生突变的三个功能域 视锥-杆状营养不良(脐带)。对不同突变的比较分析可能会让我们更好地理解 了解视杆和视锥是如何死亡的，并更深入地了解其他更常见的PR退化形式，如年龄- 相关性黄斑变性(AMD)。一个中心假设是人类PSC起源于3D视网膜器质 AIPL1基因突变会重现人类视网膜营养不良，导致PR丢失。这一假设是 由我们最近的工作和其他研究支持，表明人类的PSCs可以被诱使变成视网膜 具有PR的眼球状结构，层状形态和类似于实际的外节结构 视网膜。这项提议将连接两项创新技术：(1)基因组编辑以产生基因 匹配的视网膜报告PSC衍生的视网膜与疾病相关的突变和(2)基因矫正 修复基因缺陷，促进PR细胞存活。鉴于LCA的发病非常早，重要的是要定义 这种治疗选择的适当时间窗口。这些研究不仅将带来新的见解 对RD疾病的生物学研究，但也可以为开发治疗方法提供创新资源 RD的治疗。