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

项目总结 Leber先天性黑色素沉着症是一组破坏性的早发性视网膜营养不良，影响 大约1/50,000到1/33,000名新生儿。CRX基因的LCA相关变异导致严重的常染色体 这种疾病的主要形式，目前还没有有效的治疗方法。重要的是，这两个鼠标 人类研究表明，单倍体不足与显性CRX的疾病表现无关。 相关的LCA和一个野生型CRX的拷贝足以允许大部分正常的光感受器成熟 和功能。尽管在这一领域取得了实质性进展，但仍有迫切需要发现 并为CRX相关的LCA建立可靠的治疗方案。这个项目的总体目标是 建议将视觉研究中两个尚未解决的主要问题集中在一起：(1)准确地 在可扩展的体外模型系统中概括显性LCA，以研究变种特有的疾病机制， 以及(2)有效和特异地消除显性疾病等位基因的能力，将健康的等位基因留给 恢复感光细胞功能。 在目标1中，我们将从两个不同的显性基因开发和表征基于IPSC的疾病模型 CRX的变种。我们将使用视网膜器官来验证疾病表型。在目标2中，变种特有疾病 将通过建立视网膜器官模型系统来研究LCA的发病机制 来自患者诱导的多能干细胞。在目标3中，突变的CRX等位基因将被CRISPR灭活 人类视网膜器官模型中研究疾病表型抢救的工具。完成这一目标将 为开发以患者为基础的CRISPR治疗方法提供概念验证研究 战略。 综上所述，建议的研究将有助于我们对病理生理的基本了解。 显性CRX相关LCA中光感受器功能障碍的潜在机制，并将使 开发有针对性的基因疗法来治疗受影响的个体。

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.