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The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine–RGD peptide containing nuclear localisation properties

Gene Therapy volume 8, pages 1643–1653 (2001)Cite this article

Abstract

One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine–RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine–RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4°C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine–RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.

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Acknowledgements

This work was supported by the Association Française de Lutte contre la Mucoviscidose and MC was supported by a grant from this association. We are very grateful to Dr Andrew D Miller of the Department of Chemistry Imperial College, London, UK, for synthesising the [K]16RGD and [K]16RGE peptides. We thank Dr Elizabeth Lasnier for the assay of LDH.

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Authors and Affiliations

  1. INSERM U402, Faculté de Médecine Saint-Antoine, Paris, France

    M Colin, S Moritz, M Kornprobst, J Capeau & MC Brahimi-Horn

  2. Service d'Imagerie Cellulaire IFR 65, Hôpital Tenon, Paris, France

    P Fontanges

  3. UMR 955 INRA-ENVA, Paris, France

    C Delouis

  4. Department of Chemistry, Imperial College, London, UK

    M Keller & AD Miller

  5. Division of Biomedical Sciences, Section of Molecular Genetics, Imperial College, School of Medicine, London, UK

    C Coutelle

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  1. M Colin
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Colin, M., Moritz, S., Fontanges, P. et al. The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine–RGD peptide containing nuclear localisation properties. Gene Ther 8, 1643–1653 (2001). https://doi.org/10.1038/sj.gt.3301572

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  • DOI: https://doi.org/10.1038/sj.gt.3301572

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