A cellular disease model toward gene therapy of -dependent lamellar ichthyosis.

Lamellar ichthyosis (LI) is a chronic disease, mostly caused by mutations in the gene, marked by impaired skin barrier formation. No definitive therapies are available, and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, such as combined cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the gene in human primary clonogenic keratinocytes. Gene-edited cells showed complete absence of transglutaminase 1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI . Using a self-inactivating γ-retroviral (SINγ-RV) vector expressing transgenic under the control of its own promoter, we tested an gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected -null keratinocytes displayed proper TG1 expression, enzymatic activity, and cornified envelope formation and, hence, restored proper epidermal architecture. Single-cell multiomics analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial for epidermal regeneration. This study serves as a proof of concept for assessing the potential of this therapeutic approach in treating -dependent LI.