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

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.

Gene Therapy for Epidermolysis Bullosa

“…These results stimulated exploration of nonviral gene transfer methods including transposon, integrase (Ortiz-Urda et al, 2003b, 2003c, and adeno-associated virus . Clustered regularly interspaced short palindromic repeats (i.e., CRISPR)/Cas9-and transcription activator-like effector nuclease (TALEN)-based gene correction methodologies have also become active areas of exploration in EB therapy (Osborn et al, 2013(Osborn et al, , 2018Webber et al, 2016). Despite these new areas of study, ex vivo retroviral therapy has remained of high interest because of the efficiency of gene transfer to primary cells.…”

Gene Editing–Mediated Disruption of Epidermolytic Ichthyosis–Associated KRT10 Alleles Restores Filament Stability in Keratinocytes

“…These aspects and the frequently dominant nature of EI engender the development of gene editingebased therapies. Gene editing has been used to permanently correct dominant Luan et al, 2018) and recessive (Hainzl et al, 2017;Webber et al, 2016;Wu et al, 2017) epidermolysis bullosaerelated mutations via knockout and homologous recombination.…”

Anex vivoRNAtrans‐splicing strategy to correct human generalized severe epidermolysis bullosa simplex

“…Such genome editing approaches might involve CRISPR (clustered regularly interspaced short palindromic repeats) or TALEN (transcription activator‐like effector nuclease) technologies. These would either alter sequences at the genetic level, thereby activating premature termination codons to knock down a specific mutated allele, or induce homology‐directed repair if an appropriate template is provided …”