872 One-step correction through CRISPR/Cas9-mediated HDR and reprograming in induced pluripotent stem cells from a patient with a highly recurrent mutation in COL7A1

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the mucosa after minor trauma. RDEB caused by wide variety mutations in COL7A1 encoding type VII collagen, major component anchoring fibrils which form key attachment structures dermal-epidermal adhesion. In our study, we investigated therapeutic potential combined CRISPR/Cas-9-based correction reprogramming primary RDEB-Fibroblasts (Fs) harboring highly recurrent c.425A>G (p.Lys142Arg) mutation exon 3 COL7A1. To achieve correction, designed gRNA specifically targeting activity was estimated to be 70% RDEB-Fs. Combined iPSCs were carried out one step. iPSC performed transfecting an integration-free mRNA microRNA cocktail factors (OCT4, SOX2, cMYC, KLF4, NANOG, LIN28 miR-367/302s) under feeder-free conditions. First, Cas9 mRNA, 100nt donor template ssODN delivered single lipofectamin-based transfection into Then RNA transfections every other day 14 days. At 18, clones selected characterized their pluripotency differentiation capacity three germ layers. We able differentiate them cell types interest:i) Fs expressing specific CD surface markers, CD10, CD73, CD90, COL1 COL3 ii) Keratinocytes basal keratins KRT5 KRT14. Finally, gene-correction RDEB-iPSCs scored at genomic DNA level Taqman-ddPCR up 70%. Demonstration functional collagen through generation analysis 3D-skin equivalents ongoing perspective developing transplantable models suitable clinical application.