Nucleic Acids Research Advance Access originally published online on April 13, 2009
Nucleic Acids Research 2009 37(11):3635-3644; doi:10.1093/nar/gkp217
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2009, Vol. 37, No. 11 3635-3644
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Targeted correction of a thalassemia-associated β-globin mutation induced by pseudo-complementary peptide nucleic acids
1Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, 2Lineberger Comprehensive Cancer Center and Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA and 3Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, Blegdamsvej 3, Copenhagen, DK-2200, Denmark
*To whom correspondence should be addressed. Tel: +1 203 737 2788; Fax: +1 203 785 6309; Email: peter.glazer{at}yale.edu
Received March 6, 2008. Revised February 17, 2009. Accepted March 19, 2009.
β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.