title: Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).
abstract: Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco, San Francisco, CA, USA.
authors.forename: R Geoffrey
authors.initials: RG
authors.lastname: Sargent
authors.forename: Shingo
authors.initials: S
authors.lastname: Suzuki
authors.forename: Dieter C
authors.initials: DC
authors.lastname: Gruenert
mesh_terms: D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004720:Endonucleases / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D059765:Homologous Recombination; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004720:Endonucleases