title: Nuclease-stimulated homologous recombination at the human β-globin gene.
abstract: BACKGROUND
  Mutations in the β-globin gene (HBB) cause haemoglobinopathies where current treatments have serious limitations. Gene correction by homologous recombination (HR) is an attractive approach to gene therapy for such diseases and is stimulated by gene-specific endonucleases, including zinc finger nucleases (ZFNs). Customised nucleases targeting HBB have previously been shown to promote HR-mediated HBB modification in 0.3–60% of drug-selected cells, although frequencies among unselected cells, more relevant to the goal of correcting HBB in primary stem cells, have not been reported.
  
  
  METHODS
  ZFNs targeting HBB were tested for HBB binding (two-hybrid assay) or HBB cleavage followed by inaccurate end joining (surveyor assay)in bacteria or human cancer cell lines, respectively. ZFN-stimulated HR was measured in cell lines by a modified fluorescence-based reporter assay or by targeted insertion of a drug-resistance marker into endogenous HBB confirmed by Southern analyses.
  
  
  RESULTS
  Although the ZFNs that we assembled in-house showed limited potential, a commercially commissioned nuclease (ZFN4) enhanced HR mediated HBB modification in up to 95% of drug-selected cells. Among unselected cells, however, this frequency was less than 0.2%. Furthermore, ZFN4 cleaved HBB at an efficiency of 1–2% (surveyor assay) and enhanced the HR reporter assay 20-fold less efficiently than a control endonuclease.
  
  
  CONCLUSIONS
  With ZFN4, we achieved higher efficiencies of HR-mediated HBB modification than previously reported for drug-selected cells. Our measurements of ZFN4-induced HR in unselected cells, however, suggest that improved nucleases must be developed if therapeutic HBB correction is to be achievable in primary stem cells.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Vannocci
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurata
authors.forename: Josu
authors.initials: J
authors.lastname: de la Fuente
authors.forename: Irene A
authors.initials: IA
authors.lastname: Roberts
authors.forename: Andrew C G
authors.initials: AC
authors.lastname: Porter
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy; D045744:Cell Line, Tumor; D059766:DNA End-Joining Repair; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D015316:Genetic Therapy; D059765:Homologous Recombination; D006801:Humans; D009154:Mutation; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics / Q000628:therapy
chemical_list: D014157:Transcription Factors; D055544:beta-Globins; D004706:Endodeoxyribonucleases; C000593171:ZFN4 protein, human