708. Gene Targeting with Triplex-Forming Oligonucletides in the Pyrimidine Motif
Authors: Jennifer M. Kalish, Melissa P. Knauert, Denise C. Hegan, Peter M. Glazer
Journal: Molecular Therapy (2006)

Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence- specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Initial studies demonstrated the ability of TFOs in both motifs to deliver mutagenic agents in a DNA site-specific manner. It was found that purine motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the mutated gene via the effect of the triple helix itself. Gene modification with TFOs includes induced recombination between a DNA target and a donor DNA molecule, a process that allows a TFO to exert an effect at a distance from the third-strand binding site. In this work, we report the ability of a N3->P5 phosphoramidate-modified pyrimidine motif TFO to induce repair and recombination in the absence of any DNA-reactive conjugate. We establish the ability of this modified TFO when co- transfected with short, single-stranded donor DNAs to induce gene correction at a single-copy chromosomal locus in mammalian cells at frequencies up to 0.1%. We demonstrate sequence, target site, and dose-dependent effects of the TFO. Additionally, we show that the induction of recombination is independent of the binding motif and the orientation of the TFO (parallel versus anti-parallel), but is dependent on the donor fragment being homologous to the transcribed strand of the duplex. This work expands the number of targets available for triplex-mediated gene targeting and shows effective targeting at a chromosomal locus. Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence- specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Initial studies demonstrated the ability of TFOs in both motifs to deliver mutagenic agents in a DNA site-specific manner. It was found that purine motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the mutated gene via the effect of the triple helix itself. Gene modification with TFOs includes induced recombination between a DNA target and a donor DNA molecule, a process that allows a TFO to exert an effect at a distance from the third-strand binding site. In this work, we report the ability of a N3->P5 phosphoramidate-modified pyrimidine motif TFO to induce repair and recombination in the absence of any DNA-reactive conjugate. We establish the ability of this modified TFO when co- transfected with short, single-stranded donor DNAs to induce gene correction at a single-copy chromosomal locus in mammalian cells at frequencies up to 0.1%. We demonstrate sequence, target site, and dose-dependent effects of the TFO. Additionally, we show that the induction of recombination is independent of the binding motif and the orientation of the TFO (parallel versus anti-parallel), but is dependent on the donor fragment being homologous to the transcribed strand of the duplex. This work expands the number of targets available for triplex-mediated gene targeting and shows effective targeting at a chromosomal locus.