Selective Disruption of Mutant TP63 Alleles Restores Corneal Epithelial proliferation in EEC Syndrome

Abstract

Ectrodactyly-Ectodermal Dysplasia-Cleft Lip/Palate (EEC) syndrome is a rare disorder caused by dominant-negative mutations in the TP63 gene, frequently leading to limbal stem cell deficiency (LSCD) and progressive corneal degeneration. Current therapeutic strategies are limited, primarily due to impaired epithelial renewal and poor proliferative capacity of patient-derived cells. We have recently shown that decreasing the expression of the mutated allele by means of siRNA-mediated silencing can restore epithelial cell proliferation. However, the clinical utility of this approach is hindered by the presence of different TP63 mutations causing EEC syndrome, and the need for continuous siRNA administration to achieve sustained gene silencing. To address these challenges, we employed a CRISPR/Cas9-based genome editing strategy to disrupt mutant TP63 alleles in human induced pluripotent stem cells (hiPSCs) derived from EEC patients carrying R279H and R304Q mutations. Targeted editing of exon 6 induced frameshift mutations that activated nonsense-mediated mRNA decay, leading to a significant reduction in mutant transcript levels. Edited hiPSC-derived corneal epithelial cells exhibited improved cell proliferation compared to unedited isogenic controls. These findings demonstrate the feasibility and therapeutic potential of allele-specific genome editing to correct TP63-associated epithelial defects in EEC syndrome paving the way toward future regenerative therapies for TP63-related corneal diseases.