Correction of the ?F508 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 ?F508 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 ?F508 lesion, resulting in a nonhomologous end-joining (NHEJ) frequency of 7.8%. In spite of this relatively long distance between the DSB and the ?F508 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 ?F508 mutation but also potentially any mutation in or downstream of exon 10.

SUBMITTER: Lee CM 

PROVIDER: S-EPMC3559198 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature