title: Triplex-forming peptide nucleic acids induce heritable elevations in gamma-globin expression in hematopoietic progenitor cells.
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.
authors: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
authors: Joanna Y
authors: JY
authors: Chin
authors: Faisal
authors: F
authors: Reza
authors: Peter M
authors: PM
authors: Glazer
mesh_terms: D000328:Adult; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D005319:Fetal Hemoglobin / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006453:Hemoglobinopathies / Q000628:therapy; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection; D055545:gamma-Globins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D020135:Peptide Nucleic Acids; D055545:gamma-Globins; C051802:triplex DNA; D004247:DNA; D005319:Fetal Hemoglobin