期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 105, 期 36, 页码 13514-13519出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0711793105
关键词
beta-thalassemia; gene correction; triplex-forming oligonucleotides; gene targeting
资金
- Yale Center of Excellence in Molecular Hematology
- National Institutes of Health [DK072442, R01CA64186, R01HL082655, 5T32GM07205]
Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing betathalassemia. Triplex-forming DNA oligonucleoticles (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 thalassernia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent proteinbeta-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.
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