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- Title
Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells.
- Authors
Chin, Joanna Y; Reza, Faisal; Glazer, Peter M
- Abstract
Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes.
- Subjects
PEPTIDE nucleic acids; GAMMA globin; HEMATOPOIETIC stem cells; PROGENITOR cells; RECOMBINANT DNA; GENE therapy
- Publication
Molecular Therapy, 2013, Vol 21, Issue 3, p580
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1038/mt.2012.262