Additive Gene and Stem Cell Therapy for Mutation-Independent Treatment of p63 Disorders

Ectrodactyly–ectodermal dysplasia-clefting (EEC) syndrome is a rare genetic disorder characterized by dominant-negative mutations in the TP63 gene, leading to ectodermal and limb malformations and premature senescence of epithelial structures, particularly the corneal epithelium. This causes progressive epithelial degeneration, often resulting in limbal stem cell deficiency (LSCD) and blindness. Currently, no curative treatments exist, underscoring the need for long-lasting therapeutic strategies aimed at preservation and functional restoration of epithelial stem cells. This study provides a proof of concept for a combined gene and stem cell therapy approach to correct TP63-related disorders through the transplantation of autologous, genetically modified keratinocyte stem cells. We engineered a self-inactivating (SIN) lentiviral vector expressing ΔNp63α-EGFP under the control of the epithelial-specific K14 regulatory elements to achieve physiological, lineage-restricted expression of wild-type ΔNp63α in oral mucosa epithelial stem cells (OMESCs). Transduced mutant cells were evaluated alongside wild-type controls through lifespan assays, colony-forming efficiency (CFE) assays, confocal microscopy, qPCR, and organotypic 3D cultures. Transgene expression was selectively activated in p63-positive epithelial progenitor cells, confirming the tissue specificity of the K14 enhancer. This led to an extended proliferative lifespan and improved clonogenic potential while maintaining cell stratification capability. Integration site analysis revealed an integration profile consistent with that expected for SIN lentiviral vectors. These results support the feasibility of a mutation-independent gene addition strategy to restore ΔNp63α function in epithelial stem cells through tight transcriptional regulation, thereby minimizing off-target effects. Overall, this approach offers a promising therapeutic platform for the long-term correction of TP63-related disorders, particularly for ocular surface reconstruction in patients with EEC syndrome.