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Antigene effect in K562 cells of a PEG-conjugated triplex-forming oligonucleotide targeted to the bcr/abl oncogene
Valentina Rapozzi1, Susanna Cogoi, Paola Spessotto
1Department of Biomedical Sciences and Technologies, School of Medicine, Piazzale Kolbe 4, 33100 Udine, Italy.
Abstract:
Triplex-forming oligonucleotides are able to modulate gene expression by site-specific binding to genomic DNA. Their use as therapeutic agents is limited by inefficient cellular uptake, scarce nuclear internalization, and oligonucleotide self-aggregation. In this study, we demonstrate that a 13-mer AG motif oligonucleotide covalently linked to a high-molecular mass (9000 Da) polyethylene glycol (PEG ODN(13)) exhibits uptake and biological properties that are superior to those of the nonconjugated isosequence analogue (free ODN(13)). Band-shift and footprinting experiments showed that PEG ODN(13) forms a stable triple helix (apparent K(d) between 10(-6) and 10(-7) M in 50 mM Tris-acetate, 10 mM MgCl(2), pH 7.4, 37 degrees C) with a natural polypurine-polypyrimidine target located in the 5' flanking region of the human bcr/abl oncogene. Confocal laser microscopy performed on unfixed live cells stained with hexidium iodide as well as on glass-fixed cells stained with propidium iodide showed that fluorescein-labeled PEG ODN(13) is far more efficiently taken up and internalized in the nucleus by K562 and HeLa cells than the nonconjugated free ODN(13). It was found that PEG ODN(13) specifically downregulated the transcription of bcr/abl mRNA at 65 +/- 5% with respect to control and inhibited cell growth by 32 +/- 3% in a 3 day liquid culture assay. Moreover, PEG ODN(13) was more resistant against S1 and fetal bovine serum nucleases than free ODN(13), and less inclined to self-associate into multistrand structures in solution. Taken together, these results provide useful elements for designing artificial transcription repressors with enhanced potency in vivo.
Insights
Polyethylene glycol (PEG) conjugation enhances triplex-forming oligonucleotides (TFOs) for improved cellular uptake and nuclear delivery. This PEG-conjugated TFO effectively downregulates bcr/abl oncogene transcription and inhibits cancer cell growth.
Area of Science:
- Molecular Biology
- Oligonucleotide Therapeutics
- Gene Regulation
Background:
- Triplex-forming oligonucleotides (TFOs) can modulate gene expression via DNA binding.
- Therapeutic applications of TFOs are hindered by poor cellular uptake, nuclear entry, and self-aggregation.
Purpose of the Study:
- To evaluate the efficacy of a polyethylene glycol (PEG)-conjugated TFO (PEG ODN(13)) compared to its unconjugated counterpart.
- To assess the impact of PEGylation on TFO cellular internalization, DNA binding, stability, and biological activity.
Main Methods:
- Synthesis and characterization of PEG ODN(13) and free ODN(13).
- Band-shift and footprinting assays for DNA binding affinity.
- Confocal laser microscopy for cellular uptake and nuclear localization.
- Quantitative analysis of bcr/abl mRNA levels and cell growth inhibition assays.
- Nuclease resistance and self-aggregation studies.
Main Results:
- PEG ODN(13) demonstrated significantly enhanced cellular uptake and nuclear internalization in K562 and HeLa cells compared to free ODN(13).
- PEG ODN(13) formed a stable triplex with the bcr/abl oncogene promoter region.
- PEG ODN(13) specifically downregulated bcr/abl mRNA transcription by 65% and inhibited cell growth by 32%, while exhibiting increased nuclease resistance and reduced self-aggregation.
Conclusions:
- Covalent linkage of high-molecular mass PEG to TFOs significantly improves their pharmacokinetic and pharmacodynamic properties.
- PEGylated TFOs represent a promising strategy for developing potent artificial transcription repressors with enhanced in vivo efficacy.
- The findings provide a foundation for designing improved oligonucleotide-based therapeutics targeting specific gene sequences.
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