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

项目摘要 Leber先天性黑蒙（LCA）是一组破坏性的早发性视网膜营养不良， 大约是新生儿的1/50,000到1/33,000。LCA相关的CRX基因变异导致严重的常染色体 这种疾病的主要形式，目前没有有效的治疗方法。重要的是，两个鼠标 人类研究表明，单倍不足不是显性CRX疾病表现的原因， 相关的LCA，并且一个野生型CRX拷贝足以允许大多数正常的感光细胞成熟 和功能尽管在这一领域取得了重大进展，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。这个项目的总体目标是 建议将视觉研究中两个主要未解决的问题结合在一起：（1）准确识别 在可扩展的体外模型系统中重现显性LCA以研究变体特异性疾病机制， 和（2）有效和特异性消除显性疾病等位基因，留下健康等位基因的能力， 恢复感光细胞功能。 在目标1中，我们将从两种不同的主导疾病模型开发和表征基于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，变体特异性疾病 负责LCA的发病机制将通过产生视网膜类器官模型系统进行检查 从患者衍生的诱导多能干细胞中。在目标3中，突变CRX等位基因将用CRISPR失活， 人类视网膜类器官模型中的工具，以研究疾病表型的拯救。实现这一目标将 为开发基于患者特异性CRISPR的治疗药物提供概念验证研究 战略布局 综上所述，这些研究将有助于我们对病理生理学的基本理解。 在显性CRX相关的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。

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.