ΔNp63α is a master regulator of epithelial development, driving the expansion of progenitor cells in stratified epithelia. Mutations in ΔNp63α are associated with squamous cell carcinomas (SCCs), basal cell carcinomas (BCCs), and ectodermal dysplasia syndromes, including ectrodactyly-ectodermal dysplasia-clefting (EEC) and ankyloblepharon-ectodermal dysplasia-clefting (AEC). Although ΔNp63α functions as a nuclear transcription factor, the mechanisms governing its nuclear import remain poorly understood. Here, by combining imaging, biochemical, structural, and functional approaches, we demonstrate that ΔNp63α nuclear import is mediated by the importin (IMP) α/β1 heterodimer. We also show that ΔNp63α has evolved a robust and mutation-tolerant mechanism to preserve nuclear localization, which is required for DNA binding and transcriptional activity. Despite the identification of a canonical bipartite nuclear localization signal (NLS), comprising two stretches of basic amino acids located between the DNA binding and oligomerization domains, each stretch is independently sufficient to engage both IMPα binding sites upon homodimerization. Consequently, unlike most known bipartite NLSs, only simultaneous substitution of both stretches abolished IMPα binding, impaired nuclear accumulation, and thereby prevented DNA binding and transcriptional activity. Given that several p63 isoforms lacking the N-terminal basic stretch have been described, and multiple variants within the NLS region are present in the Genome Aggregation Database, p63 appears to have evolved a resilient nuclear import mechanism that preserves nuclear localization despite mutations affecting its NLS.

Background: The melanocortin-1 receptor (MC1R) gene is a major determinant of skin and hair color. Although several MC1R variants increase the risk of skin tumors regardless of pigmentation, their role as genetic modifiers in ocular diseases remains poorly understood. Methods: Here, we analyzed a cohort of 48 Caucasian individuals using a custom next-generation sequencing (NGS) panel targeted for oculocutaneous disorders. Results: MC1R variants were identified in two probands selected from this cohort, exhibiting distinctly divergent pathological phenotypes. The first patient, an adult male with a pheomelanin-related trait and a history of early-onset skin cancer without ocular abnormalities, carried compound heterozygous MC1R variants: the novel frameshift c.537dupC (p.Ile180Hisfs*59) and the known c.880G>C (p.Asp294His). Conversely, the second patient, a young boy with severe vision-threatening features—including congenital nystagmus, foveal hypoplasia, and an atypical visual-pathway misrouting pattern—carried a candidate digenic genotype combining the same MC1R c.880G>C variant with the albinism-related c.1209G>T (p.Arg403Ser) variant in the tyrosinase (TYR) gene. Conclusions: While MC1R variants have previously been proposed as phenotypic modifiers in albinism, primarily in combination with the oculocutaneous type II melanosomal transmembrane protein (OCA2) encoding gene, our findings support a potential role for MC1R as a candidate modifier interacting with TYR in neuroretinal development. Although functional validation is required to establish causality, incorporating MC1R screening in patients with unresolved foveal hypoplasia may help clarify complex oligogenic mechanisms and optimize phenotypic classification.

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.

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 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 revelaed 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.