title: Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.
abstract: Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.
authors.affiliation: Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.|Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Chamorro
authors.affiliation: Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.|Department of Biomedical Engineering, Carlos III University (UC3M), Madrid, Spain.
authors.forename: Angeles
authors.initials: A
authors.lastname: Mencía
authors.affiliation: Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.|Present address: Institute of Cellular Medicine, University of Newcastle, Newcastle Upon Tyne, UK.
authors.forename: David
authors.initials: D
authors.lastname: Almarza
authors.affiliation: Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.|Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.|Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER) U714, Madrid, Spain.
authors.forename: Blanca
authors.initials: B
authors.lastname: Duarte
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Köln, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Köln, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Sallach
authors.affiliation: Institute of Pathology, Universitätsklinikum Heidelberg, Heidelberg, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Hausser
authors.affiliation: Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.|Department of Biomedical Engineering, Carlos III University (UC3M), Madrid, Spain.|Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER) U714, Madrid, Spain.
authors.forename: Marcela
authors.initials: M
authors.lastname: Del Río
authors.affiliation: Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.|Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.|Department of Biomedical Engineering, Carlos III University (UC3M), Madrid, Spain.|Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER) U714, Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Larcher
authors.affiliation: Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.|Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.|Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER) U714, Madrid, Spain.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Murillas