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

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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.