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Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli

Gene Therapy volume 15, pages 434–442 (2008)Cite this article

Abstract

Bacteria-mediated gene transfer (‘bactofection’) has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o−) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 × 108 E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

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References

  1. Davies JC, Alton EW . Airway gene therapy. Adv Genet 2005; 54: 291–314.

    Article  CAS  Google Scholar 

  2. Griesenbach U, Geddes DM, Alton EW . Gene therapy progress and prospects: cystic fibrosis. Gene Therapy 2006; 13: 1061–1067.

    Article  CAS  Google Scholar 

  3. Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J et al. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial. Lancet 1999; 353: 947–954.

    Article  CAS  Google Scholar 

  4. Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE, Welsh MJ . Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993; 75: 207–216.

    Article  CAS  Google Scholar 

  5. Sizemore DR, Branstrom AA, Sadoff JC . Attenuated bacteria as a DNA delivery vehicle for DNA-mediated immunization. Vaccine 1997; 15: 804–807.

    Article  CAS  Google Scholar 

  6. Vecino WH, Quanquin NM, Martinez-Sobrido L, Fernandez-Sesma A, Garcia-Sastre A, Jacobs Jr WR et al. Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge. Virology 2004; 325: 192–199.

    Article  CAS  Google Scholar 

  7. Bauer H, Darji A, Chakraborty T, Weiss S . Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids. Gene Therapy 2005; 12: 364–372.

    Article  CAS  Google Scholar 

  8. Darji A, Zur LS, Garbe AI, Chakraborty T, Weiss S . Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. FEMS Immunol Med Microbiol 2000; 27: 341–349.

    Article  CAS  Google Scholar 

  9. Dietrich G, Bubert A, Gentschev I, Sokolovic Z, Simm A, Catic A et al. Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat Biotechnol 1998; 16: 181–185.

    Article  CAS  Google Scholar 

  10. Krusch S, Domann E, Frings M, Zelmer A, Diener M, Chakraborty T et al. Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells. J Gene Med 2002; 4: 655–667.

    Article  CAS  Google Scholar 

  11. Pasetti MF, Anderson RJ, Noriega FR, Levine MM, Sztein MB . Attenuated deltaguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin Immunol 1999; 92: 76–89.

    Article  CAS  Google Scholar 

  12. Castagliuolo I, Beggiao E, Brun P, Barzon L, Goussard S, Manganelli R et al. Engineered E. coli delivers therapeutic genes to the colonic mucosa. Gene Therapy 2005; 12: 1070–1078.

    Article  CAS  Google Scholar 

  13. Grillot-Courvalin C, Goussard S, Huetz F, Ojcius DM, Courvalin P . Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 16: 862–866.

    Article  CAS  Google Scholar 

  14. Fajac I, Grosse S, Collombet JM, Thevenot G, Goussard S, Danel C et al. Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells. J Control Release 2004; 97: 371–381.

    Article  CAS  Google Scholar 

  15. Shiau AL, Chu CY, Su WC, Wu CL . Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses. Vaccine 2001; 19: 3277–3284.

    Article  CAS  Google Scholar 

  16. Goussard S, Grillot-Courvalin C, Courvalin P . Eukaryotic promoters can direct protein synthesis in Gram-negative bacteria. J Mol Microbiol Biotechnol 2003; 6: 211–218.

    Article  CAS  Google Scholar 

  17. Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, Grillot-Courvalin C et al. Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 2004; 11: 1224–1233.

    Article  CAS  Google Scholar 

  18. Hense M, Domann E, Krusch S, Wachholz P, Dittmar KE, Rohde M et al. Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell Microbiol 2001; 3: 599–609.

    Article  CAS  Google Scholar 

  19. Pilgrim S, Stritzker J, Schoen C, Kolb-Maurer A, Geginat G, Loessner MJ et al. Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery. Gene Therapy 200; 10: 2036–2045.

    Article  Google Scholar 

  20. Laner A, Goussard S, Ramalho AS, Schwarz T, Amaral MD, Courvalin P et al. Bacterial transfer of large functional genomic DNA into human cells. Gene Therapy 2005; 12: 1559–1572.

    Article  CAS  Google Scholar 

  21. Zelmer A, Krusch S, Koschinski A, Rohde M, Repp H, Chakraborty T et al. Functional transfer of eukaryotic expression plasmids to mammalian cells by Listeria monocytogenes: a mechanistic approach. J Gene Med 2005; 7: 1097–1112.

    Article  Google Scholar 

  22. Blundell RA, Harrison DJ . Integrin characterization in pulmonary bronchioles. Exp Mol Pathol 2005; 79: 74–78.

    Article  CAS  Google Scholar 

  23. Griesenbach U, Boyton RJ, Somerton L, Garcia SE, Ferrari S, Owaki T et al. Effect of tolerance induction to immunodominant T-cell epitopes of Sendai virus on gene expression following repeat administration to lung. Gene Therapy 2006; 13: 449–456.

    Article  CAS  Google Scholar 

  24. Griesenbach U, Kitson C, Escudero GS, Farley R, Singh C, Somerton L et al. Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo. Respir Res 2006; 7: 26.

    Article  Google Scholar 

  25. Courvalin P, Goussard S, Grillot-Courvalin C . Gene transfer from bacteria to mammalian cells. C R Acad Sci III 1995; 318: 1207–1212.

    CAS  PubMed  Google Scholar 

  26. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066–1073.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Lucinda Somerton (Department of Gene Therapy, Faculty of Medicine, Imperial College, London, UK) and Ed Inett (Royal Brompton Hospital, London, UK) for their help with this project. The study was funded by the Cystic Fibrosis Trust and the CF Trust Dr Benjamin Angel Fellowship (UG).

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Authors and Affiliations

  1. Department of Gene Therapy, Faculty of Medicine, National Heart and Lung Institute, Imperial College, London, UK

    M D B Larsen, U Griesenbach, D M Geddes & E W F W Alton

  2. UK Cystic Fibrosis Gene Therapy Consortium, London, UK

    M D B Larsen, U Griesenbach & E W F W Alton

  3. Unité des Agents Antibactériens, Institut Pasteur, Paris, France

    S Goussard, P Courvalin & C Grillot-Courvalin

  4. Department of Laboratory Medicine, California Pacific Medical Center Research Institute, University of California, San Francisco, CA, USA

    D C Gruenert

  5. Department of Medicine, University of Vermont, Burlington, VT, USA

    D C Gruenert

  6. Genzyme Corporation, Framingham, MA, USA

    R K Scheule & S H Cheng

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  1. M D B Larsen
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  2. U Griesenbach
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  7. S H Cheng
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  8. P Courvalin
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  9. C Grillot-Courvalin
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  10. E W F W Alton
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Correspondence to U Griesenbach.

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Larsen, M., Griesenbach, U., Goussard, S. et al. Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli. Gene Ther 15, 434–442 (2008). https://doi.org/10.1038/sj.gt.3303090

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  • DOI: https://doi.org/10.1038/sj.gt.3303090

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