| Triplex-forming oligonucleotides, which bind in the major groove of duplex DNA in a site-specific manner, have been used in a variety of biological applications, including gene expression inhibition, targeted DNA cleavage or cross-linking, site-specific mutagenesis, and intermolecular and intramolecular recombination. Here we describe the design, use and validation of triplex-forming peptide nucleic acids to target two sites in the human beta-globin locus. Following a review of the biology of triplex-forming oligonucleotides, we demonstrate that we can use triplex-forming peptide nucleic acids to specifically modify the human beta-globin gene in human hematopoietic progenitor and stem cells, thereby correcting a thalassemia-causing mutation. We also describe the use of triplex-forming peptide nucleic acids to target the gamma-globin gene promoter sequence, in which we specifically induce mutations at promoter sites associated with increased fetal hemoglobin production. This approach can be used to non-pharmacologically ameliorate clinical symptoms associated with severe hemoglobinopathies. Finally, we use our recombination assay to probe the repair mechanisms by which triplex-mediated genomic modification occurs. These studies highlight the potential of triplex-forming oligonucleotides as gene-targeting agents in hematopoietic progenitor cells. |
