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Abstract

Aniridia is a rare, congenital, blinding disorder, caused by mutations in the paired box 6 (PAX6) gene. People with aniridia are born with poor vision, which deteriorates towards blindness in early adulthood due to glaucoma and aniridia associated keratopathy. Glaucoma can be managed by conventional treatments, however interventions for keratopathy fail to provide lasting vision. Aniridia is caused by haploinsufficiency, where the underlying cause of the phenotype is insufficient production of PAX6. Therefore, PAX6 augmentation is a possible approach to treating aniridia. Gene therapy, defined as the manipulation of gene expression, or repair of abnormal genes, has recently demonstrated clinical success, providing therapeutics for genetic disorders such as lipoprotein lipase, Leber's congenital amaurosis, spinal muscular atrophy, and hemophilia B. All of these gene therapies use recombinant adeno associated viruses (rAAV) as a vector to transfer genes to patient cells, augmenting protein production. However, PAX6 is a potent morphogen, and using gene transfer technologies to express PAX6 ectopically risks detrimental effects in off target cells. Therefore, towards developing a PAX6 gene therapy for aniridia, I tested PAX6 minimal promoters (MiniPromoters) to restrict gene expression from rAAV to cells that endogenously express PAX6. Using regulatory regions from the PAX6 gene, seven MiniPromoters were tested, and four were found to restrict expression to the four cell types that express PAX6 in the adult retina. Most gene therapies begin in a mouse model before they are tested clinically. Therefore, before I began testing PAX6 gene transfer, I studied the Small eye (Sey) mouse model of aniridia to define therapeutic targets for PAX6 gene transfer, and to quantify phenotypic features of clinical interest. These studies identified the cornea as a target with a clinically relevant phenotype, epithelial thinning, which I could target for gene transfer. Finally, I tested PAX6 gene transfer to the Sey mouse cornea by injecting rAAV encoding a PAX6 open reading frame (ORF) directly into the cornea. These tests revealed that PAX6 gene transfer to the cornea transiently reverses corneal epithelial thinning in Sey mice, and lays the foundation for the development of a gene therapy for aniridia.