Triplex-forming peptide nucleic acids induce heritable elevations in gamma-globin expression in hematopoietic progenitor cells

Mol Ther. 2013 Mar;21(3):580-7. doi: 10.1038/mt.2012.262. Epub 2013 Jan 22.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adult
  • Animals
  • Antigens, CD34 / metabolism
  • Cell Line
  • Chromosomes, Artificial, Yeast / metabolism
  • DNA / chemistry
  • DNA / genetics*
  • Fetal Hemoglobin / genetics
  • Fetal Hemoglobin / metabolism
  • Gene Expression Regulation
  • Genetic Therapy
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism*
  • Hemoglobinopathies / therapy
  • Humans
  • K562 Cells
  • Mice
  • Mice, Transgenic
  • Mutation
  • Peptide Nucleic Acids / chemistry
  • Peptide Nucleic Acids / genetics*
  • Promoter Regions, Genetic
  • Transfection
  • gamma-Globins / genetics*
  • gamma-Globins / metabolism

Substances

  • Antigens, CD34
  • Peptide Nucleic Acids
  • gamma-Globins
  • triplex DNA
  • DNA
  • Fetal Hemoglobin