842 Combining the reprogramming of RDEB fibroblasts with mono- and bi-allelic correction of the mutant COL7A1 gene into a one-step procedure

Abstract

Coupling the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) with targeted gene correction using CRISPR/Cas9 offers the possibility of developing a new stem cell-based approach for the treatment of inherited skin blistering diseases such as Recessive Dystrophic Epidermolysis Bullosa (RDEB). The conventional iPSC-based therapy for RDEB involves multiple sequential steps such as reprogramming, gene correction and iPSC differentiation. Each of these steps complicates the manufacturing process and is associated with lengthy cell culture periods that increase the risk of mutation accumulation and karyotypic instability in the final epidermal cell product. To reduce the number of steps associated with the generation of genetically corrected RDEB iPSCs, we combined our previously reported high-efficiency RNA-based reprogramming and a specific CRISPR/Cas9-mediated correction of the mutant COL7A1 gene (homozygous COL7A1c.7485+5G>A) into a one-step procedure. Employing this novel approach for simultaneous reprogramming and gene editing, we successfully generated multiple RDEB iPSC clones with mono- and bi-allelic correction of COL7A1c.7485+5G>A with an efficiency reaching 3-5%. We are currently differentiating these genetically corrected RDEB iPSCs into keratinocytes and fibroblasts to determine the restoration of collagen 7 function in these iPSC-derived cells.