CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.

Beau R Webber,Ron T McElmurry,Chong Jai Kim,Jakub Tolar,Amber N McElroy,Kirk Twaroski,Anthony P DeFeo,Megan J Riddle,Dharmeshkumar D Patel,Cara-lin Lonetree,Lily Xia,Christopher J Lees,Cindy Eide,Mark J Osborn,Bruce R Blazar

doi:10.1038/npjregenmed.2016.14

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.

Highlights

Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic disorder resulting from mutations in the type VII collagen gene (COL7A1) on chromosome 3
As further verification of successful reprogramming, the OCT4 and NANOG gene promoters were observed to be hypomethylated, and in vivo teratomas derived from gene-corrected induced pluripotent stem cells (iPSCs) contained representative tissues from all three germ layers. (Supplementary Figures S6b and c). These results demonstrate that clustered regularly interspaced palindromic repeats (CRISPR)/Cas[9] genome modification and Sendai virus reprogramming allow for precision repair and iPSC generation
RDEB is associated with a loss of the functional integrity of the dermal-epidermal junction, which results in painful erosions and blistering

Summary

Introduction

Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic disorder resulting from mutations in the type VII collagen gene (COL7A1) on chromosome 3. To demonstrate that iPSCs derived from CRISPR/ Cas[9] gene-corrected RDEB fibroblasts are capable of differentiating into therapeutically relevant cell populations in vitro, we utilised protocols to produce keratinocytes under fully defined, feeder-free conditions.[18,19,20] Two-dimensional culture of iPSCs in the presence of retinoic acid (RA) and bone morphogenic protein[4] (BMP-4) resulted in the formation of cells with characteristic epidermal morphology (Figure 2).

Results

Conclusion