Publication: Gene editing for the efficient correction of a recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa keratinocytes
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2016-01
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Elsevier
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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.
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AAV vectors, Epidermolysis bullosa, Gene editing, Indels, Homologous recombination, Talen
Bibliographic citation
Chamorro, C., Mencía, Á., Almarza, D., Duarte, B., Büning, H., Sallach, J., Hausser, I., Del Río, M., Larcher, F., & Murillas, R. (2016). Gene editing for the efficient correction of a recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa keratinocytes. Molecular Therapy - Nucleic Acids, 5, e307.