Molecular Biotechnology

, Volume 3, Issue 3, pp 237–248

Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy

  • Jacqueline Guy
  • Dubravka Drabek
  • Michael Antoniou


The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement.

The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal “break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape.

Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.

Index entries

Cell transfection receptor mediated endocytosis gene therapy 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Miller, A. D. (1992) Human gene therapy comes of age.Nature 357, 455–460.PubMedCrossRefGoogle Scholar
  2. 2.
    Graham, F. L and van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5.Virology 52, 456–467.PubMedCrossRefGoogle Scholar
  3. 3.
    Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA.Mol. Cell. Biol. 7, 2745–2752.PubMedGoogle Scholar
  4. 4.
    McCutchan, J. H. and Pagano, J. S. (1968) Enhancement of the infectivity of Simian Virus 40 Deoxyribonucleic acid with Diethylamino-ethyl-Dextran.J. Natl. Can. Inst. 41, 351–356.Google Scholar
  5. 5.
    Lake, R. A. and Owen, M. J. (1991) Transfection of the chloramphenicol-acetyltransferase gene into eukaryotic cells using diethyl-aminoethyl (DEAE)dextran, inGene Transfer and Transfection Protocols (Murray, E. J., ed.), Humana, Clifton, NJ, pp. 23–33.CrossRefGoogle Scholar
  6. 6.
    Kawai, S. and Nishizawa, M. (1984) A new procedure for DNA transfection with polycation and dimethyl sulphoxide.Mol. Cell. Biol. 4, 1172–1174.PubMedGoogle Scholar
  7. 7.
    Morgan, T. L. Maher, V. M., and McCormick, J. J. (1980) Optimal parameters for the polybrene-induced DNA transfection of diploid human fibroblasts.In Vitro Cell. Dev. Biol. 22, 317–319.CrossRefGoogle Scholar
  8. 8.
    Chaney, W. G., Howard, D. R., Pollard, J. W., Sallustio, S., and Stanley, P. (1986) High-frequency transfection of CHO cells using polybrene.Somatic Cell Mol. Genet. 12, 237–244.CrossRefGoogle Scholar
  9. 9.
    Aubin, R. J., Weinfeld, M., and Peterson, M. C. (1988) Factors influencing the efficiency and reproducibility of polybrene-assisted gene transfer.Somatic Cell. Mol. Genet. 14, 155–167.CrossRefGoogle Scholar
  10. 10.
    Wienhues, U., Hosokawa, K., Höveler, A., Siegmann, B., and Doerfler, W. (1987) A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells.DNA 6, 81–89.PubMedGoogle Scholar
  11. 11.
    Fraley, R., Subramani, S., Berg, P., and Papahadjopoulos, D. (1980) Introduction of liposome-encapsulated SV40 DNA into cells.J. Biol. Chem. 255, 10,431–10,435.Google Scholar
  12. 12.
    Schaefer-Ridder, M., Wang, Y., and Hofschneider, P. H. (1982) Liposomes as gene carriers: efficient transformation of mouse L-cells by thymidine kinase gene.Science 215, 166–168.PubMedCrossRefGoogle Scholar
  13. 13.
    Itani, T., Ariga, H., Yamaguchi, N., Tadakuma, T., and Yasuda, T. (1987) A simple and efficient liposome method for transfection of DNA into mammalian cells grown in suspension.Gene 26, 267–276.Google Scholar
  14. 14.
    Kaneda, Y., Iwai, K., and Ucheda, T. (1989) Increased expression of DNA cointroduced with nuclear protein in adult rat liver.Science 243, 375–378.PubMedCrossRefGoogle Scholar
  15. 15.
    Lapidot, M. and Loyter, A. (1990) Fusion-mediated microinjection of liposome-enclosed DNA into cultured cells with the aid of influenza virus glycoproteins.Exp. Cell. Res. 189, 241–246.PubMedCrossRefGoogle Scholar
  16. 16.
    Nussbaum, O., Rott, R., and Loyter, A. (1992) Fusion of influenza virus particles with liposomes: requirement for cholesterol and virus receptors to allow fusion with and lysis of nautral but not of negatively charged liposomes.J. Gen. Virol. 73, 2831–2837.PubMedGoogle Scholar
  17. 17.
    Feigner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenze, M., Northrop, J. P, Ringold, G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA transfection procedure.Proc. Natl. Acad. Sci. USA 84, 7413–7417.CrossRefGoogle Scholar
  18. 18.
    Kinosita, K. and Tsong, T. Y. (1977) Voltage-induced pore formation and hemolysis of human erythrocytes.Biochim. Biophys. Acta. 471, 227–242.PubMedCrossRefGoogle Scholar
  19. 19.
    Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A., and Kucherlapati, R. S. (1985) Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination.Nature 317, 230–234.PubMedCrossRefGoogle Scholar
  20. 20.
    Chu Hayakawa, H., Berg, P. (1987) Electroporation for the efficient transfection of mammalian cells with DNA.Nucleic Acids Res. 15, 1311–1326.CrossRefGoogle Scholar
  21. 21.
    Wright, S. and Huang, L. (1989) Antibody-directed liposomes as drug-delivery vehicles.Adv. Drug Deliv. Rev. 3, 343–389.CrossRefGoogle Scholar
  22. 22.
    Yoshimura, K, Rosenfeld, M. A., Nakamura, H., Scherer, E. M., Pavirani, A., Lecocq, J-P., and Crystal, R. G. (1992) Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer.Nucleic Acids Res. 20, 3233–3240.PubMedCrossRefGoogle Scholar
  23. 23.
    Hyde, S. C., Gill, D. R., Higgins, C. F., Trezise, A. E., MacVanish, L. J., Cuthbert, A. W., Ratcliff, R., Evans, M. J., and Colledge, W. H. (1993) Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy.Nature 362, 250–255.PubMedCrossRefGoogle Scholar
  24. 24.
    Mulligan, R. C. (1993) The basic science of gene therapy.Science 260, 926–932.PubMedCrossRefGoogle Scholar
  25. 25.
    Muzyczka, N. (1992) Use of adeno-associated virus as a general transduction vector for mammalian cells, inCurrent Topics Microbiology Immunology, vol. 158 (Muzyczka, N., ed.), Springer-Verlag, Berlin, pp. 97–129.Google Scholar
  26. 26.
    Spiess, M. (1990) The asialoglycoprotein receptor: a model for endocytic transport receptors.Biochemistry 29, 10,009–10,018.CrossRefGoogle Scholar
  27. 27.
    Huebers, H. and Finch, C. (1987) The physiology of transferrin and transferrin receptors.Physiol. Rev. 67, 520–582.PubMedGoogle Scholar
  28. 28.
    Cotten, M., Wagner, E., and Birnstiel, M. L. (1993) Receptor mediated transport of DNA into eukaryotic cells, inMethods in Enymology, vol. 217 (Wu, R., ed.), Academic, San Diego, CA, pp. 618–644.Google Scholar
  29. 29.
    Wagner, E., Zenke, M., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells.Proc. Natl. Acad. Sci. USA 87, 3410–3414.PubMedCrossRefGoogle Scholar
  30. 30.
    Zenke, M., Steinlein, P., Wagner, E., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells.Proc. Natl. Acad. Sci. USA 87, 3655–3659.PubMedCrossRefGoogle Scholar
  31. 31.
    Cotten, M., Längle-Roualt, F., Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that effect the survival of transfected DNA or modulate transferrin receptor levels.Proc. Natl. Acad. Sci. USA 87, 4033–4037.PubMedCrossRefGoogle Scholar
  32. 32.
    Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M. L. (1991) Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells.Proc. Natl. Acad. Sci. USA 88, 4255–4259.PubMedCrossRefGoogle Scholar
  33. 33.
    Wu, G. Y. and Wu, C. H. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system.J. Biol. Chem. 262, 4429–4432.PubMedGoogle Scholar
  34. 34.
    Cristiano, R. J., Smith, L. C., and Woo, S. L. C. (1993) Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.Proc. Natl. Acad. Sci. USA 90, 2122–2126.PubMedCrossRefGoogle Scholar
  35. 35.
    Cristiano, R. J., Smith, L. C, Kay, M. A., Brinkley, B. R., and Woo, S. L. C. (1993) Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilising a conjugated adenovirus-DNA complex.Proc. Natl. Acad. Sci. USA 90, 11,548–11,552.Google Scholar
  36. 36.
    Rosenkranz, A. A., Yachmenev, S. V., Jans, D. A., Serabryakova, N. V., Murav’ev, V. I., Peters, R., and Sobolev, A. S. (1992) Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct.Exp. Cell. Res. 199, 323–329.PubMedCrossRefGoogle Scholar
  37. 37.
    Midoux, P., Mendes, C., Legrand, A., Raimond, J., Mayer, R., Monsigny, M., and Roche, A. C. (1993) Specific gene transfer mediated by lactosylated polyl-lysine into hepatoma cells.Nucleic Acids Res. 21, 871–878.PubMedCrossRefGoogle Scholar
  38. 38.
    Trubetskoy, V. S., Torchilin, V. P., Kennel, S. J., and Huang, L. (1992) Use of N-terminal modified Poly(llysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.Bioconjugate Chem. 3, 323–327.CrossRefGoogle Scholar
  39. 39.
    Wagner, E., Cotten, M., Mechtler, K., Kirlappos, H., and Birnstiel, M. L. (1991) DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.Bioconjugate Chem. 2, 226–231.CrossRefGoogle Scholar
  40. 40.
    Gilliland, D. G., Steplewski, Z., Collier, R. J., Mitchell, K. F., Chang, T. H. C., and Koprowski, H. (1980) Antibody-directed cytotoxic agents: use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells.Proc. Natl. Acad. Sci. USA 77, 4539–4543.PubMedCrossRefGoogle Scholar
  41. 41.
    Cotten, M., Wagner, E., Zatloukal, K., Phillips, S., Curiel, D. T., and Birnstiel, M. L. (1992) High-efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosomedisruption activity of defective or chemically inactivated adenovirus particles.Proc. Natl. Acad. Sci. USA 89, 6094–6098.PubMedCrossRefGoogle Scholar
  42. 42.
    Curiel, D. T., Agarwal, S., Wagner, E., and Cotten, M. (1991) Adenovirus enhancement of transferrinpolylysine-mediated gene delivery.Proc. Natl. Acad. Sci. USA 88, 8850–8854.PubMedCrossRefGoogle Scholar
  43. 43.
    Liang, T. J., Makdisi, W. J., Sun, S., Hasegawa, K., Zhang, Y., Wands, J. R., Wu, C. H., and Wu, G. Y. (1993) Targeted transfection and expression of hepatitus B viral DNA in human hepatoma cells.J. Clin. Invest. 91, 1241–1246.PubMedCrossRefGoogle Scholar
  44. 44.
    Seth, P., Fitzgerald, D. J. P., Willingham, M. C., and Pastan, I. (1984) Role of a low-pH environment in adenovirus enhancement of the toxicity of a Pseudomonas exotoxin-epidermal growth factor conjugate.J. Virol. 51, 650–655.PubMedGoogle Scholar
  45. 45.
    Seth, P., Fitzgerald, D., Ginsberg, H., Willingham, M., and Pastan, I. (1984) Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor.Mol. Cell. Biol. 4, 1528–1533.PubMedGoogle Scholar
  46. 46.
    Cotten, M., Wagner, E., Zatloukal, K., and Birnstiel, M. L. (1993) Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.J. Virol. 67, 3777–3785.PubMedGoogle Scholar
  47. 47.
    Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D. T., and Birnstiel, M. L. (1992) Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptormediated gene delivery and expression of transfected genes.Proc. Natl. Acad. Sci. USA 89, 6099–6103.PubMedCrossRefGoogle Scholar
  48. 48.
    Gao, L., Wagner, E., Cotten, M., Agarwal, S., Harris, C., Rømer, M. U., Miller, M., Hu, P-C., and Curiel, D. (1993) Direct in vivo gene transfer to airway epithelium employing adenovirus-polylysine DNA complexes.Hum. Gene Ther. 4, 17–24.PubMedGoogle Scholar
  49. 49.
    Michael, S. I., Huang, C-H., Rømer, M. U., Wagner, E., Hu, P-C., and Curiel, D. T. (1993) Binding-incompetent adenovirus facilitates molecular conjugatemediated gene transfer by the receptor-mediated endocytosis pathway.J. Biol. Chem. 268, 6866–6869.PubMedGoogle Scholar
  50. 50.
    Wiley, D. C. and Skehel, J. J. (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus.Annu. Rev. Biochem. 56, 365–394.PubMedCrossRefGoogle Scholar
  51. 51.
    Wharton, S. A., Martin, S. R., Ruigrok, R. W. H., Skehel, J. J., and Wiley, D. C. (1988) Membrane fusion by peptide analogues of influenza virus haemagglutinin.J. Gen. Virol. 69, 1847–1857.PubMedGoogle Scholar
  52. 52.
    Wagner, E., Plank, C., Zatloukal, K., Cotten, M., and Birnstiel, M. L. (1992) Influenza viras haemagglutinin 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 89, 7934–7938.PubMedCrossRefGoogle Scholar
  53. 53.
    Wu, G. Y. and Wu, C. H. (1988) Receptor-mediated gene delivery and expression in vivo.J. Biol. Chem. 263, 14,621–14,624.Google Scholar
  54. 54.
    Chowdhury, N. R., Wu, C. H., Wu, G. Y., Yerneni, P. C, Bommineni, V. R., and Chowdhury, J. R. (1993) Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo.J. Biol. Chem. 268, 11,265–11,271.Google Scholar
  55. 55.
    Wu, G. Y., Wilson, J. M., Shalaby, F., Grossman, M., Shafritz, D. A., and Wu, C. H. (1991) Receptormediated gene delivery in vivo.J. Biol. Chem. 266, 14,338–14,342.Google Scholar
  56. 56.
    Plank, C., Zatloukal, K., Cotten, M., Mechtler, K., and Wagner, E. (1992) Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetraantennary galactose ligand.Bioconjugate Chem. 3, 533–539.CrossRefGoogle Scholar
  57. 57.
    Grosveld, F., Blom van Assendelft, G., Greaves, D., and Kollias, G. (1987) Position-independent high level expression of the human β-globin gene in transgenic mice.Cell 51, 975–985.PubMedCrossRefGoogle Scholar
  58. 58.
    Lang, G., Wotton, D., Owen, M. J., Sewell, W. A., Brown, M. H., Mason, D. Y., Crumpton, M. J., and Kioussis, D. (1988) The structure of the human CD2 gene and its expression in transgenic mice.EMBO J. 7, 1675–1682.PubMedGoogle Scholar
  59. 59.
    Bonifer, C., Vidal, M., Grosveld, F., and Sippel, A. E. (1990) Tissue-specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice.EMBO J. 9, 2843–2848.PubMedGoogle Scholar
  60. 60.
    Carson, S. and Wiles, M. V. (1993) Far upstream regions of class II MHC Ea are necessary for position-independent, copy-dependent expression of Ea transgene.Nucleic Acids Res. 21, 2065–2072.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1995

Authors and Affiliations

  • Jacqueline Guy
    • 1
  • Dubravka Drabek
    • 1
  • Michael Antoniou
    • 1
  1. 1.Laboratory of Gene Structure and ExpressionNational Institute for Medical ResearchLondonUK

Personalised recommendations