COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

Abstract

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

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

“…TALEN_6.21 demonstrated high on-target activity in immortalized KCs from a patient with EI (56.8%), compared with a recent geneediting approach targeting KRT5 in KCs derived from a patient with epidermolysis bullosa simplex (25%) (Aushev et al, 2017). These high efficiencies were achieved without the use of selection methods commonly employed in gene editing of KCs (Hainzl et al, 2017;Kocher et al, 2017).…”

“…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.…”

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 …”

“…Due to the straightforward use and rapid advancement of the technology, CRISPR is today dominating the gene editing field. Whereas most CRISPR approaches harnessed the DNA double‐strand break inducing Streptococcus pyogenes Cas9 (SpCas9) nuclease variants, we have recently developed double nicking approaches based on single‐strand DNA breaks (nicks) at the targeting site with the intention to reduce off‐target site effects . Compared to the extremely high numbers of off‐target site modifications inherent to replacement gene therapies resulting from multiple random integrations in the treated cells, off‐target mutations are rare or non‐existent upon designer nuclease treatment …”

“…We demonstrated delivery of functional RTMs into murine epidermis and upper dermis with a gene gun . Specific RTMs for several genes involved in EB have been developed at the EB House Austria ( COL7A1 , Col17A1 , KRT14 and PLEC ) and holds US and European patents on improved RTMs and their uses (US8735366, EP2320952).…”