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

项目摘要/摘要 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，主要影响 1/50,000至1/33,000名新生儿。CRX基因LCA相关变异导致严重常染色体显性遗传 这种疾病的一种形式，目前没有有效的治疗方法。因此，尽管有大量的 在这一领域取得了进展，迫切需要发现病理生理学并建立可靠的 CRX相关LCA的治疗选择。这项提案的总体目标是将两大 视觉研究中未解决的问题：(1)在可伸缩的体外实验中准确概括优势LCA的能力 研究变种特有疾病机制的模型系统，以及(2)高效和特异地 消除显性疾病等位基因，留下健康的等位基因来恢复感光细胞的功能。在目标1中， 导致LCA发病的变异特异性疾病机制将通过产生视网膜来检查 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 LCA的机械特征系统，并将提供对替代治疗策略的洞察力 这种病。在目标2中，突变的CRX等位基因将用CRISPR工具在人类视网膜器质中灭活 研究疾病表型抢救的模型。重要的是，老鼠和人类的研究都表明 单倍体功能不全不是显性CRX相关LCA疾病表现的原因，并且只有一个拷贝 野生型CRX足以使大部分正常的光感受器成熟和功能。完成这项工作 AIM将为该领域开发基于患者的特定CRISPR提供概念验证研究 治疗策略。综上所述，拟议的研究将有助于我们对 显性CRX相关LCA中光感受器功能障碍的病理生理机制 将能够开发有针对性的基因疗法来治疗受影响的个人。

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.