In vitro modeling of aniridia‐related PAX6 haploinsufficiency by the use of CRISPR/Cas9 on limbal epithelial cells

Abstract

PurposeHaploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by iris hypoplasia and reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia‐related keratopathy (ARK), caused by a combination of factors including limbal stem‐cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub‐epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation.MethodsWe used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation into one allele of the PAX6 gene in TERT‐immortalized limbal cells, which remain identical to primary limbal cells able to differentiate into corneal cells upon calcium raise at confluency.ResultsA resulting mutated clone, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency, was further characterized. Sequencing analysis showed that no off‐target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of recombinant PAX6 protein was able to activate endogenous PAX6 gene and, as a consequence, rescue partially the phenotype.ConclusionsOur in vitro model will be powerful to identify drugs that could rescue the corneal defect.