A Novel Model System to Identify Cellular and Molecular Defects Underlying Rare Genetic Disorders

Abstract

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) is a disorder caused by autosomal-dominant mutations in the TP63 gene. AEC is characterised by the presence of severe and painful skin erosions that can take years to heal. Current treatment options for these devastating lesions are limited, highlighting the need for new therapeutic strategies. We previously generated keratinocytes from patient-derived induced pluripotent stem cells (iPSC-K) and identified defects in several cell adhesion complexes, including desmosomes, hemidesmosomes and focal adhesions. In the present study, we developed a complementary in vitro model using NTERT keratinocytes transduced with lentiviral constructs expressing AEC-related TP63 mutations (N-AEC). This model allows for the large-scale production of disease-relevant material, overcoming the limitations of iPSC-derived keratinocytes, which have the characteristics of primary keratinocytes, including limited cell doublings and lifespan. We demonstrate that N-AEC keratinocytes exhibit key defects observed in AEC iPSC-K and AEC patient skin, including downregulation of cell adhesion proteins. In addition, 3D epidermal equivalents generated from these cells replicate pathological features seen in AEC patient skin, such as intra-epidermal cysts, reduced desmosomal protein expression and altered expression of differentiation markers. Our N-AEC model provides a valuable tool for investigating the mechanisms underlying skin fragility in AEC and other genetic skin disorders and advances the potential for novel therapeutic development.