Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

Abstract

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.

DOI: 10.1073/pnas.0711793105

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@article{Chin2008CorrectionOA, title={Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.}, author={Joanna Y. Chin and Jean Y. Kuan and Pallavi S Lonkar and Diane S Krause and Michael M. Seidman and Kenneth R. Peterson and Peter E. Nielsen and R. Kole and Peter M. Glazer}, journal={Proceedings of the National Academy of Sciences of the United States of America}, year={2008}, volume={105 36}, pages={13514-9} }