Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3,LAMB3,or\ud LAMC2,which together encode laminin-332, a hetero-trimeric protein consisting ofa3,b3, andg2chain. In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life. We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protoco

Functional defects in type VII collagen, caused by premature termination codons on both alleles of the COL7A1 gene, are responsible for the severe autosomal recessive types of the skin blistering disease, recessive dystrophic epidermolysis bullosa (RDEB). The full-length COL7A1 complementary DNA (cDNA) is about 9 kb, a size that is hardly accommodated by therapeutically used retroviral vectors. Although there have been successful attempts to produce functional type VII collagen protein in model systems of RDEB, the risk of genetic rearrangements of the large repetitive cDNA sequence may hamper the clinical application of full-length COL7A1 cDNA in the human system. Therefore, we used trans-splicing to reduce the size of the COL7A1 transcript. Retroviral transduction of RDEB keratinocytes with a 3' pre-trans-splicing molecule resulted in correction of full-length type VII collagen expression. Unlike parental RDEB keratinocytes, transduced cells displayed normal morphology and reduced invasive capacity. Moreover, transduced cells showed normal localization of type VII collagen at the basement membrane zone in skin equivalents, where it assembled into anchoring fibril-like structures. Thus, using trans-splicing we achieved correction of an RDEB phenotype in vitro, which marks an important step toward its application in gene therapy in vivo.

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.