CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells

Abstract

Significance This study demonstrates a therapeutic option using induced pluripotent stem cells (iPSCs), gene editing, and tissue engineering techniques for the development of a long-lasting treatment that will result in the permanent closure of nonhealing wounds in dystrophic epidermolysis bullosa (DEB), especially for recessive DEB (RDEB). Here, we demonstrated that the combination of iPSCs, gene editing, and tissue engineering allows us to perform efficient gene correction by homology-directed repair with the CRISPR-Cas9 gRNA ribonucleoprotein complex system, differentiate the gene-corrected RDEB patient iPSCs into skin cells, and generate human skin equivalents (HSEs) that show skin integrity and type VII collagen restoration after grafting them onto nude mice. The results from this study serve as a foundation to translate this treatment into the clinic. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal–epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient’s quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.