Disease modeling and CRISPR/Cas9-mediated rescue of dominant Leber congenital amaurosis retinal phenotypes

项目总结/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， 1/50，000至1/33，000新生儿。LCA相关的CRX基因变异导致严重的常染色体显性遗传 这种疾病的形式，目前没有有效的治疗方法。因此，尽管大量 随着该领域取得的进展，迫切需要揭示病理生理学并建立可靠的 CRX相关LCA的治疗选择。该提案的总体目标是将两大 视觉研究中尚未解决的问题：（1）在可扩展的体外实验中准确重现主导LCA的能力 模型系统来研究变异特异性疾病机制，以及（2）有效和特异性地 消除显性疾病等位基因，留下健康等位基因来恢复感光细胞功能。在目标1中， 变异特异性疾病的发病机制负责LCA将检查通过产生视网膜 来自患者来源的诱导多能干细胞的类器官模型系统。这些研究将建立一个模型 系统的LCA的机制表征，并将提供洞察替代治疗策略， 这种疾病。在Aim 2中，突变CRX等位基因将在人类视网膜类器官内用CRISPR工具失活。 研究拯救疾病表型的模型。重要的是，小鼠和人类的研究都表明， 在显性CRX相关的LCA中，单倍不足不负责疾病表现， 野生型CRX足以允许大多数正常的感光细胞成熟和功能。完成本 目的是为该领域提供一个概念验证研究，以开发基于患者特异性CRISPR的 治疗策略总的来说，拟议的研究将有助于我们对 显性CRX相关LCA中感光细胞功能障碍的病理生理机制，以及 将使靶向基因疗法的发展，以治疗受影响的个人。

PROJECT SUMMARY/ABSTRACT 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. Therefore, 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, 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. These studies will establish a model system for mechanistic characterization of LCA and will provide insight into alternative treatment strategies for this disease. In Aim 2, mutant CRX alleles will be inactivated with CRISPR tools within the human retinal organoid model to study rescue of disease phenotypes. 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. 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.