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.

概括 视网膜变性（RD）疾病，例如色素性视网膜炎（RP）和Leber先天性瘤 （LCA），引起光感受器（PR）细胞的功能障碍和细胞死亡，最终导致失明。 LCA 是继承儿童失明的主要原因，导致出生后或出生后不久失去视力。尽管 这被认为是很少见的，这些盲目疾病对受影响的人来说是毁灭性的。当前的努力是 被建立开发旨在纠正RD中某些基因的基因疗法和这种方法 在动物和人类中表现出一些恢复RPE65基因表达的希望，但是有很多 RD的其他原因无法治愈。此外，由于RD涉及的许多突变， 我们对公关损失的理解有很大的差距。为了解决这个问题，我们将使用人类多能 带有RD相关等位基因的基于干细胞（PSC）的视网膜细胞再生蛋白线，以帮助探索机制 PR细胞死亡。鉴于在实验室中产生人类视网膜所需的通常很长的时间 LCA中的严重程度和变性的快速发作使其成为研究人RD的有吸引力的实验模型 并开发潜在的疗法。我们将研究芳基烃受体相互作用的蛋白质样（AIPL1） 基因探索三个功能域，这些功能结构域具有LCA和LCA患者的自然突变 锥杆营养不良（绳索）。对不同突变的比较分析可能会导致更好的理解 关于杆和锥体如何死亡以及对其他更常见的PR变性形式的洞察力，例如年龄 相关的黄斑变性（AMD）。一个中心假设是人PSC衍生的3D视网膜器官 随着AIPL1突变，将概括人类视网膜营养不良，导致PR损失。这个假设是 在我们最近的工作和其他工作的支持下，可以将人类PSC哄骗成为视网膜 带有PRS的眼部样结构，层状形态和类似于实际的外部段结构 视网膜。该提议将桥接两种创新技术。 （1）基因组编辑以产生遗传 匹配的视网膜报道器PSC衍生出具有疾病相关突变的视网膜，（2）基因纠正 修复遗传缺陷并促进PR细胞存活。鉴于LCA的早期发作很重要 适合此类治疗选择的时间窗口。这些研究不仅会带来新的见解 进入RD疾病的生物学，但也可以提供创新的资源来开发疗法 Rd的处理。