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Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

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URI: https://hdl.handle.net/10016/28318
ISSN: 1525-0016
DOI: https://doi.org/10.1016/j.ymthe.2019.03.007
UXXI: AR/0000023429
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clinically_ASGCT_2019.pdf (1.95 MB)
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2019-03-15
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American Society of Gene and Cell Therapy
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Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations inCOL7A1 causing recessive dystrophic epidermolysis bullosaare amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeteddeletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA(sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved veryefficient targeted deletion of COL7A1 exon 80 in recessivedystrophic epidermolysis bullosa (RDEB) patient keratinocytescarrying a highly prevalent frameshift mutation. This ex vivonon-viral approach rendered a large proportion of correctedcells producing a functional collagen VII variant. The effectivetargeting of the epidermal stem cell population enabled longterm regeneration of a properly adhesive skin upon graftingonto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sitesdid not reveal any unintended nuclease activity. Our strategycould potentially be extended to a large number of COL7A1mutation-bearing exons within the long collagenous domainof this gene, opening the way to precision medicine for RDEB.
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Crispr/Cas9, Epidermal Stem Cells, Epidermolysis Bullosa, Gene Therapy
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Bonafont, J., Mencía, Á., García, M., Torres R., Rodríguez S., Carretero M., Chacón-Solano E., Modamio-Høybjør S., Marinas L., Carlos León C., Escámez M.J., Hausser I., Río M., Murillas R. y Larcher, F. (2019). Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing. Molecular Therapy, 27 (5), pp. 1-13
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