Mechanistic analysis and allellic genome editing of iPSC-derived dominant LCA model

iPSC 衍生的显性 LCA 模型的机制分析和等位基因组编辑

基本信息

批准号：
10723137
负责人：
Deepak Ashok Lamba
金额：
$ 7.19万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2023-07-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10723137
关键词：
Affect Alleles Binding Binding Sites Biochemical Biological Models Blindness CRISPR therapeutics CRISPR/Cas technology CRX protein Cell Line Cell Maturation Cell physiology Child Clinic Clustered Regularly Interspaced Short Palindromic Repeats Cone DNA Binding Defect Development Disease Disease model Dominant-Negative Mutation Endonuclease I Excision Foundations Functional disorder Future Genes Genetic Transcription Goals Histologic Human In Vitro Individual Inherited Investigation Knock-out Laboratories Lead Leber&apos s amaurosis Loss of Heterozygosity Mediating Modeling Mus Mutation Newborn Infant Organoids Pathogenesis Patients Phenotype Photoreceptors Play Protein Analysis Protocols documentation RNA analysis Reporting Retina Retinal Degeneration Retinal Diseases Retinal Dystrophy School-Age Population Solid Testing Therapeutic Variant Vision Vision research Work autosome developmental disease disease phenotype disease-in-a-dish early onset effective therapy functional restoration gene replacement gene therapy genome editing human subject improved in vitro Model in vivo induced pluripotent stem cell insight loss of function mutation mouse model mutant novel strategies novel therapeutic intervention protein expression protein function protein protein interaction sight restoration stem cell derived tissues stem cells therapeutic gene tool transcription factor

项目摘要

PROJECT SUMMARY Leber congenital amaurosis (LCA) is a group of devastating early-onset retinal dystrophies affecting roughly 1/50,000 to 1/33,000 newborns. LCA-associated variants in the CRX gene result in a severe autosomal dominant form of the disease, for which no effective treatments are currently available. Importantly, both mouse and human studies suggest that haploinsufficiency is not responsible for disease manifestation in dominant CRX- associated LCA, and one copy of wildtype CRX is enough to allow for mostly normal photoreceptor maturation and function. Despite substantial progress being made in the field, there is a critical need to uncover pathophysiology and establish reliable treatment options for CRX-associated LCA. The overall goal of this proposal is to bring together two major unsolved problems in vision research: (1) the ability to accurately recapitulate dominant LCA in a scalable in vitro model system to study variant-specific disease mechanisms, and (2) the ability to efficiently and specifically eliminate dominant disease alleles, leaving healthy alleles to restore photoreceptor cell function. In Aim 1, we will develop and characterize iPSC-based disease models from two different dominant variants of CRX. We will validate disease phenotypes using retinal organoids. In Aim 2, variant-specific disease mechanisms responsible for the onset of LCA will be examined by generating a retinal organoid model system from patient-derived induced pluripotent stem cells. In Aim 3, mutant CRX alleles will be inactivated with CRISPR tools within the human retinal organoid model to study rescue of disease phenotypes. Completion of this aim will provide the field with a proof-of-concept study for the development of patient-specific CRISPR-based therapeutic strategies. Taken together, the proposed studies will contribute to our basic understanding of the pathophysiological mechanisms underlying photoreceptor dysfunction in dominant CRX-associated LCA, and will enable the development of targeted gene therapies to treat affected individuals.

项目概要莱伯先天性黑蒙（LCA）是一组破坏性的早发性视网膜营养不良，影响大约 1/50,000 至 1/33,000 名新生儿。 CRX 基因中与 LCA 相关的变异会导致严重的常染色体该疾病的主要形式，目前尚无有效的治疗方法。重要的是，两只鼠标人类研究表明单倍体不足并不是显性 CRX-疾病表现的原因相关的 LCA，以及野生型 CRX 的一份副本足以实现大多数正常的光感受器成熟和功能。尽管该领域取得了实质性进展，但仍迫切需要发现病理生理学并为 CRX 相关 LCA 建立可靠的治疗方案。本次活动的总体目标该提案旨在汇集视觉研究中两个未解决的主要问题：（1）准确地在可扩展的体外模型系统中重现主要的 LCA，以研究变异特异性疾病机制， (2) 有效、特异地消除显性疾病等位基因的能力，留下健康的等位基因恢复感光细胞功能。在目标 1 中，我们将开发和表征来自两种不同主要疾病的基于 iPSC 的疾病模型 CRX 的变体。我们将使用视网膜类器官验证疾病表型。在目标 2 中，变异特异性疾病将通过生成视网膜类器官模型系统来检查导致 LCA 发生的机制来自患者的诱导多能干细胞。在目标 3 中，突变的 CRX 等位基因将被 CRISPR 灭活人类视网膜类器官模型中用于研究疾病表型拯救的工具。这一目标的完成将为该领域提供概念验证研究，以开发基于患者特异性的基于 CRISPR 的治疗方法策略。总而言之，所提出的研究将有助于我们对病理生理学的基本理解显性 CRX 相关 LCA 中光感受器功能障碍的潜在机制，并将使开发靶向基因疗法来治疗受影响的个体。