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  • Viral Transfer Technology
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Long-term silencing of retroviral vectors is resistant to reversal by trichostatin A and 5-azacytidine

Gene Therapy volume 7, pages 653–663 (2000)Cite this article

Abstract

One problem limiting the development of long-term gene replacement therapy is gene silencing. A variety of experiments have implicated DNA methylation and histone deacetylation in gene silencing and shown that the agents 5-azacytidine (5-Aza) and trichostatin A (TSA) are able to reverse these effects. To begin to investigate clinically relevant strategies to reverse silencing with these drugs, we transduced the MEL and FDCP-1 hematopoietic cell lines with Moloney murine leukemia virus (MMLV) and Harvey murine sarcoma virus (HMSV)-based retroviral vectors carrying the β-galactosidase/neomycin resistance fusion gene (β-geo). Fifty-one clones were isolated under G418 selection over 2 weeks and then allowed to grow without selection as β-gal activity was monitored over time. More than 80% of these clones showed significant silencing over a period of 70–80 days. The clones were then exposed to a wide range of 5-Aza and TSA concentrations, both alone and in combination, in an effort to reverse silencing. Despite demonstration that the agents were able to decrease DNA methylation and increase histone acetylation, significant reversal of long-term silencing was not seen under any experimental condition. These results suggest that long-term retroviral silencing involves mechanisms in addition to DNA methylation and histone acetylation and that new pharmacologic strategies are needed to overcome the silencing process.

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Acknowledgements

We particularly wish to thank Dr Steven Fiering for insightful discussions and assistance with the flow cytometric β-galactosidase assay. We appreciate the laboratory assistance of Judy Macnab, Michael Nemeth and Michael Layon. We also thank Dr Philip Soriano for providing the β-geo gene construct, Dr Brian Sorrentino for providing the HMSV vector backbone and Dr Ruth Craig for assistance with FDCP-1 cell cultures. This research was supported by NIH F32 HL10083 (JMM) and the Marie Wilkinson Fund. CHL also receives support under NIH RO1 HL52243.

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  1. Department of Medicine, Dartmouth Medical School, Hanover, NH, USA

    J M McInerney & C H Lowrey

  2. Department of Pharmacology/Toxicology, Dartmouth Medical School, Hanover, NH, USA

    J R Nawrocki & C H Lowrey

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McInerney, J., Nawrocki, J. & Lowrey, C. Long-term silencing of retroviral vectors is resistant to reversal by trichostatin A and 5-azacytidine. Gene Ther 7, 653–663 (2000). https://doi.org/10.1038/sj.gt.3301155

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