title: Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.
abstract: Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (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 thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-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.
authors.affiliation: Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Joanna Y
authors.initials: JY
authors.lastname: Chin
authors.forename: Jean Y
authors.initials: JY
authors.lastname: Kuan
authors.forename: Pallavi S
authors.initials: PS
authors.lastname: Lonkar
authors.forename: Diane S
authors.initials: DS
authors.lastname: Krause
authors.forename: Michael M
authors.initials: MM
authors.lastname: Seidman
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Peterson
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
authors.forename: Ryszard
authors.initials: R
authors.lastname: Kole
authors.forename: Peter M
authors.initials: PM
authors.lastname: Glazer