Comprehensive characterization of keratinocytes and fibroblasts derived from genetically corrected induced pluripotent stem cells of recessive dystrophic epidermolysis bullosa patients

Summary: Abstract Body: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a hereditary, incurable disease with high morbidity and mortality characterized by severe skin blistering and scarring. RDEB results from mutations in the COL7A1 gene, causing dysfunctional type VII collagen (C7), a key component of anchoring fibrils in the basement membrane. We are developing an induced pluripotent stem cell (iPSC)-based gene therapy for RDEB, which involves combined reprogramming and CRISPR/Cas9 gene correction, iPSC differentiation, and transplantation of genetically corrected iPSC-derived skin cells. Key challenges include ensuring the safety and authenticity of iPSC-derived skin cells for clinical trials. One of the safety challenges stems from the low editing efficiency and off-target effects of CRISPR/Cas9, which risk oncogenic activation and disruption of critical genes. To address this, we adapted the whole-genome CIRCLE-Seq strategy to identify potential off-target modifications in genetically corrected RDEB iPSCs and uncovered 33 potential off-target sites in patient cells. Preliminary analysis of Oxford Nanopore sequencing data in gene edited RDEB iPSCs showed no off-target activity at the top sites with high sequence homology, supporting the safety of our gene editing approach. To confirm complete iPSC differentiation and minimize tumor risks, we performed single-cell RNA sequencing (scRNA-seq) on gene-edited iPSC-derived skin cells at different stages of the manufacturing process. The results revealed distinct clusters of keratinocytes, fibroblasts, mesenchymal stem cells, and melanocytes. These cells are being further characterized by gene and protein expression analyses. Our findings provide critical preclinical safety data for advancing to a clinical trial for RDEB. Jeffrey Inen¹, Maryna Pavlova¹, Jocelyn Castillo Flores¹, Patrick McGrath², Chann Han¹, Dennis Roop¹, Anna L. Brucker¹, Ganna Bilousova¹, Igor Kogut¹ 1. Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States. 2. Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics