title: Correction of the ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Zinc-Finger Nuclease Homology-Directed Repair.
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 ΔF508 mutation, homology-directed repair (HDR) could be detected when using a DNA donor containing part of the wild-type (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 genome-editing 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.
authors.affiliation: Department of Physiology, University College Cork , Cork, Ireland . ; Department of Microbiology, University College Cork , Cork, Ireland .
authors.forename: Ciaran M
authors.initials: CM
authors.lastname: Lee
authors.forename: Rowan
authors.initials: R
authors.lastname: Flynn
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Hollywood
authors.forename: Martina F
authors.initials: MF
authors.lastname: Scallan
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Harrison
keywords: AAV vectors; DNA repair; gene therapy; viral vectors; zinc-finger nuclease