Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Abstract

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene-editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.

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

“…This could be achieved in a similar manner to the highly successful gene replacement therapy that was recently used to treat almost the entire epidermis of a boy with junctional epidermolysis bullosa (Hirsch et al, 2017). Although demonstrated in a limited fashion in this study, epidermal generation following single-cell clonal expansion of gene-edited KCs is feasible (Mencía et al, 2018). It is necessary to monitor off-target activity, a key limitation of gene-editing therapies, where off-target activity may prove detrimental (Cho et al, 2014;Park et al, 2014).…”

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

“…However, the associated phenotypes of many mutations may be treated through the implementation of several approaches. The simplest and most efficient genome editing strategies employ EJ-based DSB repair for gene disruption [31][32][33][34] and gene reframing [35][36][37]. As EJ-based strategies do not require a DNA donor template, potentially detrimental integrations and DNA-associated toxicity are avoided.…”

“…Genome editing-mediated reading frame restoration of pathogenic alleles is an effective therapeutic strategy for Duchenne muscular dystrophy [63][64][65] and recessive DEB (RDEB) [35][36][37]. Exon reframing can be achieved in an identical manner to gene disruption approaches, as approximately one third of all nuclease-induced indels lead to codon restoration of frameshift mutations.…”

Epidermolysis bullosa: Advances in research and treatment

“…For genes such as COL7A1 with a size of >30 kb and disease‐causing mutations spread almost along the entire length of the gene, this is challenging. Among the first targets chosen from our laboratory and others to develop efficient nucleases were therefore hot‐spot mutations such as the c.6527insC in exon 80 of COL7A1 , which is highly prevalent in the Spanish dystrophic EB patient population …”

“…First attempts of gene editing mutations associated with EB used meganucleases . These were soon replaced by approaches employing the more specific transcription activator‐like effector nucleases (TALEN) . Due to the straightforward use and rapid advancement of the technology, CRISPR is today dominating the gene editing field.…”