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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

概括视网膜退行性 (RD) 疾病，例如色素性视网膜炎 (RP) 和莱伯先天性黑蒙（LCA），导致感光（PR）细胞功能障碍和细胞死亡，最终导致失明。生命周期评估是遗传性儿童失明的主要原因，导致出生时或出生后不久丧失视力。尽管这种情况被认为是相当罕见的，这些致盲疾病对于受影响的人来说是毁灭性的。目前的努力是正在开发基因疗法，旨在纠正 RD 中受影响的一些基因，这种方法已在动物和人类中显示出恢复 RPE65 基因表达的一些希望，但还有很多其他无法治愈的 RD 原因。另外，由于RD涉及的突变较多，有我们对公关损失如何发生的理解存在重大差距。为了解决这个问题，我们将使用人类多能具有 RD 相关等位基因的基于干细胞 (PSC) 的视网膜细胞报告系，有助于探索 PR细胞死亡。鉴于在实验室中生成人类视网膜通常需要很长的时间， LCA 退化的严重性和快速发生使其成为研究人类 RD 的有吸引力的实验模型并开发潜在的疗法。我们将研究芳烃受体相互作用蛋白样1 (AIPL1) 基因探索 LCA 患者中自然发生的突变的三个功能域视锥杆营养不良（CORD）。对不同突变的比较分析可能会带来更好的理解了解视杆细胞和视锥细胞如何死亡，并更深入地了解其他更常见的 PR 变性形式，例如年龄- 相关黄斑变性（AMD）。一个中心假设是人类 PSC 衍生的 3D 视网膜类器官 AIPL1 突变将重现人类视网膜营养不良，导致 PR 丧失。这个假设是我们最近的工作和其他研究表明人类 PSC 可以被诱导变成视网膜具有 PR 的眼杯状结构、层状形态和与实际类似的外节结构视网膜。该提案将连接两项创新技术； (1)基因组编辑生成基因匹配视网膜报告基因 PSC 衍生的具有疾病相关突变的视网膜，以及 (2) 基因校正修复遗传缺陷并促进 PR 细胞存活。鉴于 LCA 发病很早，定义这一点很重要此类治疗方案的适当时间范围。这些研究不仅会带来新的见解深入研究 RD 疾病的生物学，但也可以提供创新资源来开发治疗方法 RD的治疗。