title: CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.
abstract: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to theCOL7A1gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causingCOL7A1mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiationin vitroin a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Beau R
authors.initials: BR
authors.lastname: Webber
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.|Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.|Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.|Asan-Minnesota Institute for Innovating Transplantation, Seoul, Republic of Korea.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Osborn
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Amber N
authors.initials: AN
authors.lastname: McElroy
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Kirk
authors.initials: K
authors.lastname: Twaroski
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Cara-Lin
authors.initials: CL
authors.lastname: Lonetree
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: DeFeo
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Lily
authors.initials: L
authors.lastname: Xia
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Cindy
authors.initials: C
authors.lastname: Eide
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Lees
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Ron T
authors.initials: RT
authors.lastname: McElmurry
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Riddle
authors.affiliation: Asan-Minnesota Institute for Innovating Transplantation, Seoul, Republic of Korea.
authors.forename: Chong Jai
authors.initials: CJ
authors.lastname: Kim
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Dharmeshkumar D
authors.initials: DD
authors.lastname: Patel
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.|Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Blazar
authors.affiliation: Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.|Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.|Asan-Minnesota Institute for Innovating Transplantation, Seoul, Republic of Korea.
authors.forename: Jakub
authors.initials: J
authors.lastname: Tolar