Patient-derived cornea organoids as drug repurposing models for aniridia-associated keratopathy

Background and aim: Aniridia-associated keratopathy (AAK) is a serious ocular surface disease resulting from PAX6 haploinsufficiency due to mutations in the PAX6 gene. This study aims to investigate the efficacy of drug repurposing using a corneal organoid model developed from patient-derived iPSCs and to elucidate the pathophysiology of AAK. Methods: A 90-day stepwise differentiation protocol was used to generate corneal organoids from iPSC cell lines developed from aniridia patients and a healthy control. The produced corneal organoids were characterized using histology, immunofluorescence, qPCR, western blot, and transcriptomics. Two known agents, Duloxetine and Ataluren, were tested for corneal organoids for the restoring PAX6 protein expression. Results: Histological analyses showed that the corneal organoids had a similar architecture to the native corneal tissue. Corneal epithelial, stromal, and endothelial cell biomarker staining showed positive expressions. AAK corneal organoids exhibited features that indicate the AAK disease phenotype, such as thickening of the epithelial cell layers and decrease in expressions of PAX6, ΔNP63, and keratocan genes. An increase in PAX6 protein was observed in organoids produced from AAK1 after duloxetine treatment and in organoids produced from AAK2 following ataluren treatment. AAK3 did not respond to either agent, indicating that there was no mutation-specific drug activity. Transcriptomic analyses showed clear corneal differentiation and absence of retina or lens profile in organoids. Conclusion: This study presents patient-specific organoid models for AAK using iPSCs and offers insight into mutation effects and PAX6 restoration following drug repurposing. The findings form the basis of personalized treatments for congenital aniridia.