Correction of the Δf508 mutation in the cystic fibrosis transmembrane conductance regulator gene by zinc-finger nuclease homology-directed repair

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Abstract

The use of zinc-finger nucleases (ZFNs) to permanently and precisely modify the human genome offers a potential alternative to cDNA-based gene therapy. The ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is observed in *70% of patients with cystic fibrosis (CF) and is a candidate for ZFN-mediated repair. Here, we report the modular design and synthesis of a pair of ZFNs that can create a double-stranded break (DSB) 203 bp upstream of the ΔF508 lesion, resulting in a nonhomologous end-joining (NHEJ) frequency of 7.8%. In spite of this relatively long distance between the DSB and the DF508 mutation, homology-directed repair (HDR) could be detected when using a DNA donor containing part of the wildtype (WT) CFTR. The ZFN target half-sites in CFTR are separated by a 4-bp spacer, but efficient cleavage of synthetic targets with either a 4-or 6-bp spacer was observed in vitro. These ZFNs may be suitable for a genomeediting strategy using a partial cDNA sequence-containing exons 10-24 of CFTR to restore CFTR function to cells containing not only the ΔF508 mutation but also potentially any mutation in or downstream of exon 10. © Mary Ann Liebert, Inc.

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Lee, C. M., Flynn, R., Hollywood, J. A., Scallan, M. F., & Harrison, P. T. (2012). Correction of the Δf508 mutation in the cystic fibrosis transmembrane conductance regulator gene by zinc-finger nuclease homology-directed repair. BioResearch Open Access, 1(3), 99–103. https://doi.org/10.1089/biores.2012.0218

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