| Autor: |
Jacków J; Department of Dermatology, Columbia University, New York, NY 10032., Guo Z; Department of Dermatology, Columbia University, New York, NY 10032., Hansen C; Department of Dermatology, Columbia University, New York, NY 10032., Abaci HE; Department of Dermatology, Columbia University, New York, NY 10032., Doucet YS; Department of Dermatology, Columbia University, New York, NY 10032., Shin JU; Department of Dermatology, Columbia University, New York, NY 10032., Hayashi R; Department of Dermatology, Columbia University, New York, NY 10032., DeLorenzo D; Department of Dermatology, Columbia University, New York, NY 10032., Kabata Y; Division of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan., Shinkuma S; Division of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan., Salas-Alanis JC; Dystrophic Epidermolysis Bullosa Research Association MEXICO, Guadalupe 67150, Mexico., Christiano AM; Department of Dermatology, Columbia University, New York, NY 10032.; Department of Genetics and Development, Columbia University, New York, NY 10032. |
| Abstrakt: |
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. |
