Targeted p63 isoform modulation corrects dominant mutations in AEC syndrome without disrupting epidermal homeostasis [human RNA-seq]

The transcription factor p63 is a master regulator of stratified epithelial development. Its disruption leads to severe congenital defects affecting the skin, limbs, and craniofacial structures. Among p63-related disorders, Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome is caused by dominant mutations predominantly in the SAM/TID domains of the TP63 gene, affecting the p63a isoform. These mutations promote protein aggregation and transcriptional dysregulation, resulting in debilitating skin erosions. To explore a therapeutic strategy based on isoform switching, we developed a conditional mouse model in which exon 13 was deleted, replacing p63a with the shorter p63ß isoform, which is expressed in the skin, albeit at lower levels . Despite the essential role of p63a in limb and palate formation, we found that p63ß is sufficient to sustain epidermal development, postnatal skin homeostasis, and wound healing. At the molecular level, the switch from p63a to p63ß preserved chromatin binding and global transcriptional programs in keratinocytes. We next applied CRISPR/Cas9-mediated exon 13 deletion in human primary keratinocytes. This isoform conversion maintained proliferation and gene expression. Importantly, in AEC patient-derived keratinocytes, p63ß expression rescued protein aggregation, restored mechanical integrity, and normalized key epidermal gene expression. Together, these findings demonstrate that p63ß can functionally compensate for p63a in the skin and establish isoform switch as a promising therapeutic strategy for AEC syndrome. Overall design: NHEK and mutant (I537T) human keratinocytes (HK) were nucleofected with Cas9 with or without sgRNA1 and sgRNA2 for the generation of p63alpha KO. After 72 hours from nucleofection, RNA was isolated using Direct-Zol micro prep kit (Zymo Research, R2061)