Nuclease-stimulated homologous recombination at the human β-globin gene
Version of Record online: 25 FEB 2014
© 2013 The Authors. Journal of Gene Medicine published by John Wiley & Sons, Ltd.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
The Journal of Gene Medicine
Volume 16, Issue 1-2, pages 1–10, January-February 2014
How to Cite
Vannocci, T., Kurata, H., de la Fuente, J., Roberts, I. A. and Porter, A. C. G. (2014), Nuclease-stimulated homologous recombination at the human β-globin gene. J. Gene Med., 16: 1–10. doi: 10.1002/jgm.2751
- Issue online: 25 FEB 2014
- Version of Record online: 25 FEB 2014
- Accepted manuscript online: 19 DEC 2013 10:02AM EST
- Manuscript Accepted: 11 DEC 2013
- Manuscript Revised: 9 DEC 2013
- Manuscript Received: 6 SEP 2013
- gene targeting;
- gene therapy;
- sickle cell anaemia;
- zinc finger nuclease
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.
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.
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.
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. © 2013 The Authors. Journal of Gene Medicine published by John Wiley & Sons, Ltd.