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：
10319983
负责人：
Deepak Ashok Lamba
金额：
$ 39.16万
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 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10319983
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 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 research Work base 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.

项目摘要 Leber先天性症（LCA）是一群毁灭性的早期发作的视网膜营养不良，影响大约1/50,000至1/33,000新生儿。 CRX基因中与LCA相关的变体导致严重的常染色体该疾病的主要形式，目前尚无有效治疗。重要的是，两种鼠标人类的研究表明，单倍不足概不负责主要CRX的疾病表现。相关的LCA和一份Wildtype CRX的副本足以使大多数正常的光感受器成熟和功能。尽管该领域取得了重大进展，但仍有至关重要的需要发现病理生理学并为CRX相关LCA建立可靠的治疗选择。总体目标建议是在视力研究中汇总两个主要的未解决问题：（1）准确的能力在可伸缩的体外模型系统中概括了显性LCA，以研究变异特异性疾病机制，（2）有效，特异性地消除显性疾病等位基因的能力，使健康等位基因到恢复感光细胞功能。在AIM 1中，我们将开发并表征来自两个不同主要优势的基于IPSC的疾病模型 CRX的变体。我们将使用视网膜器官验证疾病表型。在AIM 2中，变异特异性疾病通过产生视网膜器官模型系统，将检查负责LCA发作的机制来自患者衍生的诱导多能干细胞。在AIM 3中，突变的CRX等位基因将被CRISPR灭活人类视网膜器官模型中的工具研究了疾病表型的拯救。这个目标的完成为该领域提供概念验证研究，以开发基于患者的CRISPR治疗策略。综上所述，拟议的研究将有助于我们对病理生理的基本理解主要与CRX相关的LCA中光感受器功能障碍的机制，并将启用开发靶向基因疗法以治疗受影响的个体。