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  • Nonviral Transfer Technology
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Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells

Gene Therapy volume 7, pages 139–152 (2000)Cite this article

Abstract

The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some correlated with an increase in the rate of transfer of the pDNA to lysosomes, a decrease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes–lysosomes was beneficial for expression. After 30 min both the peptide and pDNA localized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellular mechanisms by which vectors transfer to and traffic in cells should help design improved vectors.

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References

  1. Coutelle C . Gene therapy for cystic fibrosis. Strategies, problems and perspectives. In: Strauss M, Barranger JA (eds) Concepts in Gene Therapy Walter de Gruyter & Co: Berlin, New York 1997 313–343

    Google Scholar 

  2. Lee ER et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung Hum Gene Ther 1996 7: 1701–1717

    Article  CAS  PubMed  Google Scholar 

  3. Lasic DD, Templeton NS . Liposomes in gene therapy Adv Drug Del Rev 1996 20: 221–266

    Article  CAS  Google Scholar 

  4. Hart SL et al. Gene delivery and expression mediated by an integrin-binding peptide Gene Therapy 1995 2: 552–554

    CAS  PubMed  Google Scholar 

  5. Zhang F et al. A transfecting peptide derived from adenovirus fiber protein Gene Therapy 1999 6: 171–181

    Article  CAS  PubMed  Google Scholar 

  6. Colin M et al. Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells Gene Therapy 1998 5: 1488–1498

    Article  CAS  PubMed  Google Scholar 

  7. Harbottle RP et al. An Arg–Gly–Asp/oligolysine peptide: a prototype construct for integrin mediated gene delivery Hum Gene Ther 1998 9: 1037–1047

    Article  CAS  PubMed  Google Scholar 

  8. Hart SL et al. Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector Hum Gene Ther 1998 9: 575–585

    Article  CAS  PubMed  Google Scholar 

  9. Matsui H, Johnson LG, Randell SH, Boucher RC . Loss of binding and entry of liposome–DNA complexes decreases transfection efficiency in differentiated airway epithelial cells J Biol Chem 1997 272: 1117–1126

    Article  CAS  PubMed  Google Scholar 

  10. Zabner J et al. Cellular and molecular barriers to gene transfer by a cationic lipid J Biol Chem 1995 270: 18997–19007

    Article  CAS  PubMed  Google Scholar 

  11. Labat-Moleur F et al. An electron microscopy study into the mechanism of gene transfer with polyamines Gene Therapy 1996 3: 1010–1017

    CAS  PubMed  Google Scholar 

  12. Zhou X, Huang L . DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action Biochim Biophys Acta 1994 1189: 195–203

    Article  CAS  PubMed  Google Scholar 

  13. Griffiths G, Gruenberg J . The arguments for pre-existing early and late endosomes Trends Cell Biol 1991 1: 5–9

    Article  CAS  PubMed  Google Scholar 

  14. Riezman H, Woodman PG, van Meer G, Marsh M . Molecular mechanisms of endocytosis Cell 1997 91: 731–738

    Article  CAS  PubMed  Google Scholar 

  15. Misteli T, Spector DL . The cellular organization of gene expression Curr Opin Cell Biol 1998 10: 323–331

    Article  CAS  PubMed  Google Scholar 

  16. Wickham TJ, Mathias P, Cheresh, DA, Nemerow GR . Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment Cell 1993 73: 309–319

    Article  CAS  PubMed  Google Scholar 

  17. Larkin JM, Donzell WC, Anderson RGW . Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices J Cell Biol 1986 103: 2619–2627

    Article  CAS  PubMed  Google Scholar 

  18. Heuser JE, Anderson RGW . Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation J Cell Biol 1989 108: 389–400

    Article  CAS  PubMed  Google Scholar 

  19. Li G et al. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5 Proc Natl Acad Sci USA 1995 92: 10207–10211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kurashima K et al. Endosomal recycling of the Na+/K+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway J Biol Chem 1998 273: 20828–20836

    Article  CAS  PubMed  Google Scholar 

  21. Salisbury JL, Keller GA . Structural investigations on the role of microfilaments in ligand translocation Meth Enzymol 1983 98: 368–375

    Article  CAS  Google Scholar 

  22. Brown EJ . Phagocytosis BioEssays 1995 17: 109–117

    Article  CAS  PubMed  Google Scholar 

  23. Leopold PL et al. Fluorescent virons: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells Hum Gene Ther 1998 9: 367–378

    Article  CAS  PubMed  Google Scholar 

  24. Nicolau C, Sene C . Liposome-mediated DNA transfer in eukaryotic cells. Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage Biochim Biophys Acta 1982 721: 185–190

    Article  CAS  PubMed  Google Scholar 

  25. Watanabe Y et al. Highly efficient transfection into primary cultured mouse hepatocytes by use of cation-liposomes: an application for immunization J Biochem 1994 116: 1220–1226

    Article  CAS  PubMed  Google Scholar 

  26. Memmo LM, McKeown-Longo P . The αvβ5 integrin functions as an endocytic receptor for vitronectin J Cell Sci 1998 111: 425–433

    CAS  PubMed  Google Scholar 

  27. Timar J et al. Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the αIIbβ3-integrin in melanoma cells J Cell Sci 1995 108: 2175–2186

    CAS  PubMed  Google Scholar 

  28. Pol A, Ortega D, Enrich C . Identification and distribution of proteins in isolated endosomal fractions of rat liver: involvement in endocytosis, recycling and transcytosis Biochem J 1997 323: 435–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Friend DS, Papahadjopoulos D, Debs RJ . Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes Biochim Biophys Acta 1996 1278: 41–50

    Article  PubMed  Google Scholar 

  30. Isberg RR, Tran Van Hhieu G . The mechanism of phagocytic uptake promoted by invasin–integrin interaction Trends Cell Biol 1995 5: 120–124

    Article  CAS  PubMed  Google Scholar 

  31. Maxfield FR . Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts J Cell Biol 1982 95: 676–681

    Article  CAS  PubMed  Google Scholar 

  32. Wagner E et al. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin–polylysine–DNA complexes: toward a synthetic virus-like gene-transfer vehicle Proc Natl Acad Sci USA 1992 89: 7934–7938

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Midoux P et al. Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells Nucleic Acids Res 1993 21: 871–878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Erbacher P, Roche AC, Monsigny M, Midoux P . Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes Exp Cell Res 1996 225: 186–194

    Article  CAS  PubMed  Google Scholar 

  35. Fominaya J, Uherek C, Wels W . A chimeric fusion protein containing transforming growth factor-α mediates gene transfer via binding to the EGF receptor Gene Therapy 1998 5: 521–530

    Article  CAS  PubMed  Google Scholar 

  36. Legendre J-Y, Szoka FC Jr . Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes Pharm Res 1992 9: 1235–1242

    Article  CAS  PubMed  Google Scholar 

  37. Farhood H, Serbina N, Huang L . The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer Biochim Biophys Acta 1995 1235: 289–295

    Article  PubMed  Google Scholar 

  38. Meunier-Durmont C et al. Mechanism of adenovirus improvement of cationic liposome-mediated gene transfer Biochem Biophys Acta 1997 1330: 8–16

    Article  Google Scholar 

  39. Fasbender A et al. Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo J Biol Chem 1997 272: 6479–6489

    Article  CAS  PubMed  Google Scholar 

  40. Wattiaux R et al. Cationic lipids delay the transfer of plasmid DNA to lysosomes Biochem Biophys Res Commun 1996 227: 448–454

    Article  CAS  PubMed  Google Scholar 

  41. Gruenert DC et al. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40 Proc Natl Acad Sci USA 1998 85: 5951–5955

    Article  Google Scholar 

  42. Wheeler VC, Coutelle C . Nondegradative in vitro labelling of plasmid DNA Anal Biochem 1994 225: 374–376

    Article  Google Scholar 

  43. Ghosh RN, Gelman, DL, Maxfield FR . Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy J Cell Sci 1994 107: 2177–2189

    CAS  PubMed  Google Scholar 

  44. Bradford M . A rapid and sensitive method for the quantification of microgram quantities of protein utilizing protein-dye binding Anal Biochem 1976 72: 248–254

    Article  CAS  PubMed  Google Scholar 

  45. Whitman M et al. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation Nature 1985 315: 239–242

    Article  CAS  PubMed  Google Scholar 

  46. Galloway CJ et al. Acidification of macrophage and fibroblast endocytic vesicles in vitro Proc Natl Acad Sci USA 1983 80: 3334–3338

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Green SA, Zimmer K-P, Griffiths G, Mellman I . Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins J Cell Biol 1987 105: 1227–1240

    Article  CAS  PubMed  Google Scholar 

  48. Cesarone CF, Bolognesi C, Santi L . Improved microfluorometric DNA determination in biological material using 33258 Hoechst Anal Biochem 1979 100: 188–197

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the ‘Institut National de la Santé et de la Recherche Médicale’ (INSERM) and from the ‘Association Française de Lutte contre la Mucoviscidose’. We thank Dr E Coudrier of CNRS UMR 144 at the ‘Institut Curie’ for advice on subcellular fractionation. Our thanks to the members of the INSERM units 482 and 489 at the St-Antoine and Tenon Hospitals, respectively for access to their electron microscope. We also thank Dr M Caron, Dr E Lasnier, Ms M Auclair and Ms L Grivot of INSERM U402. CC is funded by the Muller Bequest/CF-Trust, the MRC and the March of Dimes Birth Defects Foundation. RPH and AK are supported by the March of Dimes Birth Defects Foundation (19-FY97-0632).

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Authors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale U 402, Faculté de Médecine Saint-Antoine, Paris, France

    M Colin, M Kornprobst, J Capeau & M C Brahimi-Horn

  2. Institut National de la Santé et de la Recherche Médicale CJF 96.07, Faculté de Médecine Saint-Antoine, Paris, France

    M Maurice & G Trugnan

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

    R P Harbottle, A Knight & C Coutelle

  4. Department of Chemistry Imperial College, London, UK

    R G Cooper & A D Miller

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Colin, M., Maurice, M., Trugnan, G. et al. Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells. Gene Ther 7, 139–152 (2000). https://doi.org/10.1038/sj.gt.3301056

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