CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa
Authors
Beau R Webber, Mark J Osborn, Amber N McElroy, Kirk Twaroski, Cara-lin Lonetree, Anthony P DeFeo, Lily Xia, Cindy Eide, Christopher J Lees, Ron T McElmurry, Megan J Riddle, Chong Jai Kim, Dharmeshkumar D Patel, Bruce R Blazar and Jakub Tolar
Institution
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota
Country
United States
Year
2016
Journal
Nature – Regenerative Medicine
Abstract
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene 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-causing COL7A1 mutation 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
differentiation in vitro in 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.