A cellular disease model towards gene therapy of TGM1-dependent Lamellar Ichthyosis

Lamellar Ichthyosis (LI) is a chronic disease, mostly caused by mutations in TGM1 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, as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene edited cells showed complete absence of Transglutaminase-1 (TG1) expression and recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a SIN?-RV vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity and cornified envelopes formation, hence restored proper epidermal architecture. Single cell multiomic analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial to epidermal regeneration. This study serves as a proof-of-concept for assessing the potential of this therapeutic approach in treating TGM1 dependent LI. Overall design: Primary human keratinocytes from healthy donor were transduced with a self-inactivating retroviral vector carryng the TGM1 coding sequence under the control of its own promoter (endogenous TGM1 promoter). Multiomic analysys were performed on these cells for the simultaneous evaluation of provirus integration and transcriptome profiling within a single cell. For the analisys of keratinocytes population we used a previously defined reference (DOI: 10.1038/s41467-021-22779-9). TGM1-proviral integrations were identified by mapping scATAC-seq data of TGM1-transduced cells to a custom human reference genome that includes the sequence of the SIN?-RV-TGM1 provirus.