Bestrophinopathies are a class of inherited blinding diseases that cause debilitating central vision loss. The study of these diseases plays a central role in efforts to understand and treat a variety of related retinal diseases, including age related macular degeneration. Bestrophinopathies are diagnosed clinically as five distinct conditions that are linked to over 200 separate mutations in the BEST1 gene. Efforts to understand the specific genotype-to-mechanism relationships?that explain the diverse clinical presentation of these conditions?have been hampered by a deficiency of accurate disease models. Recently described patient- specific induced pluripotent stem cell (iPSC) disease models have incrementally improved the representativeness these model systems. Unfortunately, patient-derived iPSC disease models remain poorly suited to large-scale genotype-specific studies necessary to evaluate personalized therapies for the hundreds of reported bestrophinopathy mutations. Existing models are hampered by limited accessibility of patient- samples and from confounding effects of variable genetic backgrounds in patient-derived iPSCs. This work aims to address the challenges of accessibility and genetic variability by using gene-editing to create a selection of iPSC lines, each with a unique disease-associated mutation, from a single commercially available wild-type (WT) iPSC line.
In Aim 1, we will evaluate the disease phenotype of two gene edited iPSC derived disease models in comparison to bestrophinopathy patient-derived iPSC models.
In Aim 2, we will apply our genotype-specific disease models to evaluate the extent that a proof-of-principle gene therapy strategy can rescue quantifiable disease phenotypes. These studies will inform the study and treatment of bestrophinopathy as well as other genetic diseases of the retina.

Public Health Relevance

Bestrophinopathies are a collection of inherited retinal and macular dystrophies that cause debilitating irreversible vision loss. These diseases are linked to over 200 specific mutations in the BEST1 gene and they play an important role in efforts to study an array of blinding diseases, such as age related macular degeneration. Interrogation of genotype-specific disease mechanisms in bestrophinopathy is limited by the availability of representative disease models. The proposed work aims to address these challenges by applying advances in human gene-editing to improve the accuracy and accessibility of pluripotent stem cell derived ?disease-in-a-dish? models of bestrophinopathy. These models will be applied to study disease mechanisms and to evaluate proof-of-principle gene therapies.

National Institute of Health (NIH)
National Eye Institute (NEI)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1)
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Agarwal, Neeraj
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University of Wisconsin Madison
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United States
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Steyer, Benjamin; Cory, Evan; Saha, Krishanu (2018) Developing precision medicine using scarless genome editing of human pluripotent stem cells. Drug Discov Today Technol 28:3-12
Steyer, Benjamin; Bu, Qian; Cory, Evan et al. (2018) Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection. Stem Cell Reports 10:642-654