RT Journal Article
T1 Gene editing for the efficient correction of a recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa keratinocytes
A1 Chamorro, Cristina
A1 Mencía Rodríguez, Ángeles
A1 Almarza, David
A1 Duarte, Blanca
A1 Büning, Hildegard
A1 Sallach, Jessica
A1 Hausser, Ingrid
A1 Río Nechaevsky, Marcela del
A1 Larcher Laguzzi, Fernando
A1 Murillas, Rodolfo
AB 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.
PB Elsevier
SN 2162-2531
YR 2016
FD 2016-01
LK https://hdl.handle.net/10016/38921
UL https://hdl.handle.net/10016/38921
LA eng
NO This work was supported in part by grants SAF2014-54885-R from MINECO to MDR; S2010/BMD-2420 and S2010/ BMD-2359 from Dirección General de Investigación de la Comunidad de Madrid to MDR and FL respectively; PI14/00931 from Instituto de Salud Carlos III to FL and by funding from Center for Molecular Medicine Cologne (CMMC) of the University of Cologne to H.B. There are no conflicts of interest.
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RD 27 jul. 2024