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Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes

Gene Therapy volume 10, pages 1105–1115 (2003)Cite this article

Abstract

Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents. Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective. A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages. This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol. To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy. The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively. ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels. Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system. The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.

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Acknowledgements

We gratefully acknowledge Mary O'Riordan at the University of California, Berkeley, for performing RT-PCR to monitor IFN-β mRNA. Additional thanks are extended to Daniel Pothen for careful review of the manuscript, and members of the Lee lab, Daniel Portnoy, and Joel Swanson for support and helpful discussions. This work was supported by the Warner-Lambert/Parke-Davis and H Helfman Pharmacy fellowships, Vahlteich Research Fund at the University of Michigan, and by NIH Grants R29AI42084 and R01AI47173.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

    E Mathew & K-D Lee

  2. ISIS Pharmaceuticals, Carlsbad, CA, USA

    G E Hardee & C F Bennett

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Mathew, E., Hardee, G., Bennett, C. et al. Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes. Gene Ther 10, 1105–1115 (2003). https://doi.org/10.1038/sj.gt.3301966

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