752 Simultaneous gene-editing and reprogramming of epidermolysis bullosa simplex fibroblasts

Abstract

Epidermolysis bullosa simplex (EBS) is an autosomal dominant skin fragility disorder caused by mutations in either Keratin (K) 5 or K14. Although current therapy for EBS is limited to wound care, advances in reprogramming somatic cells into induced pluripotent stem cells (iPSC) offer the possibility of developing new approaches for EBS treatment. The iPSC-based therapeutic approach for EBS relies on the generation of patient-specific iPSCs, which then undergo genetic editing and differentiation into skin cells suitable for transplantation. One of the main hurdles in advancing the iPSC-based therapy into the clinic is the complexity of a multi-step manufacturing process to produce genetically corrected iPSC-derived epidermal sheets. To reduce the manufacturing complexity of an iPSC-based therapy for EBS, we have combined our previously presented high-efficiency RNA-based reprogramming approach with a specific CRISPR/Cas9-mediated knock-out of the mutant allele of the K14 gene into a one-step procedure. We established a fibroblast line from an EBS patient carrying a hot-spot heterozygous C373T mutation at codon 125 in K14 and successfully generated multiple isogenic EBS iPSCs lines with a specific knock out of the mutant K14 allele (KO EBS iPSC) within 4 weeks of initiating simultaneous gene editing and reprogramming. Simultaneous reprogramming and gene correction not only reduces the complexity of the iPSC-based therapy but also improves safety of the approach by avoiding lengthy cell culture periods, drug selection, and multiple sub-cloning steps. We are currently differentiating these KO EBS iPSCs into keratinocytes to determine if these gene edited iPSC-derived keratinocytes can be used for EBS therapy by employing a mouse xenograft model.