Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is caused by heterozygous missense point mutations in the p63 gene, an important transcription factor during embryogenesis and for stem cell differentiation in stratified epithelia. Most of the cases are sporadic, related to de novo mutations arising during early-stage development. Familial cases show an autosomic dominant inheritance. The major cause of visual morbidity is limbal stem cell failure, which develops in the second to third decade of life. Patients often show ocular surface alterations, such as recurrent blepharitis and conjunctivitis, superficial microlesions of the cornea, and spontaneous corneal perforation and ulceration, leading to progressive corneal clouding and eventually visual loss. No definitive cures are currently available, and treatments to alleviate symptoms are only palliative. In this review, we will discuss the proposed therapeutic strategies that have been tested or are under development for the management of the ocular defects in patients affected by EEC syndrome: (i) gene therapy-based approaches by means of Allele-Specific (AS) siRNAs to correct the p63 mutations; (ii) cell therapy-based approaches to replenish the pool of limbal stem cells; and (iii) drug therapy to correct/bypass the genetic defect. However, as the number of patients with EEC syndrome is too limited, further studies are still necessary to prove the effectiveness (and safety) of these innovative therapeutic approaches to counteract the premature differentiation of limbal stem cells.

Background/Aims: The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) and Ankyloblepharon–ectodermal defect–cleft lip/palate (AEC) syndromes are rare autosomal dominant diseases caused by heterozygous mutations in the p63 gene. Patients are characterized by abnormalities of the skin, teeth, and hair and have limb defects, orofacial clefting and ectodermal dysplasia. In addition, they often show ocular surface alterations, leading to progressive corneal clouding and eventually blindness. Here, we present 8 cases describing patients affected by EEC (n = 6, with 5 sporadic and 1 familial cases) and AEC (n = 2, both sporadic cases) syndromes. We attempt to provide a description of the ocular disease progression over the years. Methods: Clinical examinations and monitoring of ocular parameters for the assessment of limbal stem cell deficiency were constantly performed on patients between 2009 and 2023. Quantitative data and comparison with existing cases described in the literature are reported. Results: The therapies supplied to patients were essential for the management of the symptoms, but unfortunately did not halt the progression of the pathology. Conclusions: A constant monitoring of the patients would help avoid the sudden worsening of symptoms. If the progression of the disease slows down, it would allow for the development of newer therapeutic strategies aimed at correcting the genetic defect.

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.

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.

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.