Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are retinal dystrophies resulting in congenital blindness normally associated with autosomal recessive gene mutations. The onset of disease can occur as early as prenatal ocular development to birth and the characteristics include abnormal electroretinogram (ERG), reduced or absent pupillary responses, and involuntary eye movement (nystagmus). RP has later onset than LCA and is more variable in clinical findings, but results in a progressive degeneration of retinal compartments (primarily photoreceptors) leading to gradual visual impairments that can end in complete blindness. Our laboratories and others, have focused on mapping and characterizing the molecular etiologies associated with gene mutations affecting visual impairments but also the structures within the eye; however, many disease-causing mutations have limited or no animal models for studying these ocular-associated human diseases. Crumbs homolog 1 (CRB1) mutations cause 9-13% of overall LCA cases (approx. 450 patients in US) and 4-5% of autosomal recessive RP cases (~3300 patients in US) with recognizable clinical features. Importantly, CRB1-/- loss in humans is characterized by night blindness subsequently leading to loss of peripheral vision, photoreceptor degeneration, and eventual complete blindness. Investigation of the cellular retinopathology associated with CRB1-/- mutations has been hampered by the absence of appropriate cellular or animal models. One of the challenges of studying the role of CRB1 in vivo is the differential cellular expression pattern of CRB1 and CRB2 between human and mouse retinas. It?s unclear whether the paralogues, which differ in their extracellular domains, serve redundant or unique functions. To investigate this disorder, we have generated patient-specific iPSCs, which provide the foundation for studying this devastating disorder. In our preliminary work, we find that by differentiating CRB1-/- iPSCs toward a retinal fate we can generate populations of retinal progenitor cells (RPCs) and photoreceptor precursor cells that have shown an increase cellular proliferation and increase expression of key retinal cell markers. Interestingly, we have also evaluated notch and WNT signaling pathway genes and show that altered expression that correlates with the increased cell number and patients? clinical phenotype of thickened retinas. Our proposed studies will identify CRB complex orientation along the cell membrane (apical-basal localization), global gene regulations associated with CRB1 mutations, and establish a translational gene transfer model for studying phenotypes of CRB1-/--associated LCA and RP. We hypothesize that the altered notch and WNT signaling resulting from loss of CRB1 expression that disrupts the normal transition from retinal progenitors to photoreceptors, can be rescued by gene augmentation. In addition, this innovative model should provide a strong foundation for future work fully characterizing retinal development in CRB1-/- disease and also provide a platform with which to develop gene therapy for CRB-associated and other early onset blinding disorders.

Public Health Relevance

Our work aims to develop a retina model for studying the cellular and molecular abnormalities that are associated with CRB1 mutations and blinding clinical phenotypes. We will focus on identifying differences in cellular dynamics (proliferation, apoptosis, cell cycle), how mutations in the CRB complex affect gene expression in retinal cells and cell polarity (apical-basal localization), and establish an in vitro retinal developmental model with which to develop gene transfer strategies. We anticipate this system will provide a thorough description of the cellular and molecular landscape of disruptions in the Crumbs (CRB) complex in retinal cells, and increase our understanding of how biomarkers established in this system can be used as a readout for translational approaches for gene augmentation therapeutics for CRB-related and other early onset blinding diseases.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Therapeutic Approaches to Genetic Diseases Study Section (TAG)
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Shen, Grace L
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University of Pennsylvania
Schools of Medicine
United States
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