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Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential

Gene Therapy volume 10, pages 72–83 (2003)Cite this article

Abstract

Listeriolysin O (LLO), a sulfhydryl-activated pore-forming protein from Listeria monocytogenes, was tested and utilized for promoting plasmid DNA (pDNA) delivery into the cytosol of cells in culture. To render pDNA-complexing capability to LLO, the unique cysteine 484 of LLO was conjugated to polycationic peptide protamine (PN) at a 1:1 molar ratio through a reversible, endosome-labile disulfide bond. The sulfhydryl-oxidized LLO construct, LLO-s-s-PN, completely lacked its pore-forming activity, yet regained its original activity upon reduction. The enhanced cytosolic delivery using this construct therefore relies on the requisite reduction of the disulfide bond in LLO-s-s-PN by endogenous cellular reducing capacity. Condensed PN/pDNA complexes incorporating LLO-s-s-PN were tested for their enhanced gene delivery capability monitoring reporter gene expression in HEK293, RAW264.7, P388D1 cell lines and bone-marrow-derived macrophages in the presence of serum. Dramatic enhancement was observed for all tested complexes with varying weight ratios. The effect was most prominent at 0.64–0.80 (w/w) of PN/pDNA upon replacing 1–4% of PN with LLO-s-s-PN, resulting in approximately three orders of magnitude higher luciferase expression compared to PN/pDNA without apparent toxicity. These results demonstrate that incorporation of endosomolytic LLO into pDNA delivery systems in a controlled fashion is a promising approach of enhancing delivery into the cytosol of target cells in gene delivery strategies.

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References

  1. Wagner E et al. Transferrin–polycation conjugates as carriers for DNA uptake into cells. Proc Natl Acad Sci USA 1990; 87: 3410–3414.

    Article  CAS  Google Scholar 

  2. Wu GY, Wu CH . Receptor-mediated gene delivery and expression in vivo. J Biol Chem 1988; 263: 14 621–14 624.

    Google Scholar 

  3. Boussif O et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995; 92: 7297–7301.

    Article  CAS  Google Scholar 

  4. Lee RJ, Huang L . Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumor cell-specific gene transfer. J Biol Chem 1996; 271: 8481–8487.

    Article  CAS  Google Scholar 

  5. Gao X, Huang L . Potentiation of cationic liposome-mediated gene delivery by polycations. Biochemistry (Mosc) 1996; 35: 1027–1036.

    Article  CAS  Google Scholar 

  6. Zauner W, Wagner E, Ogris M . Polylysine-based transfection systems utilizing receptor-mediated delivery. Adv Drug Deliv Rev 1998; 30: 97–113.

    Article  CAS  Google Scholar 

  7. Wagner E . Application of membrane-active peptides for nonviral gene delivery. Adv Drug Deliv Rev 1999; 38: 279–289.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Wagner E et al. Coupling of adenovirus to transferring–polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc Natl Acad Sci USA 1992; 89: 6099–6103.

    Article  CAS  Google Scholar 

  10. Wyman TB et al. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997; 36: 3008–3017.

    Article  CAS  Google Scholar 

  11. Legendre JY, Szoka Jr. FC . Cyclic amphipathic peptide–DNA complexes mediate high-efficiency transfection of adherent mammalian cells. Proc Natl Acad Sci USA 1993; 90: 893–897.

    Article  CAS  Google Scholar 

  12. Nishikawa M et al. Hepatocyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system. Gene Ther 2000; 7: 548–555.

    Article  CAS  Google Scholar 

  13. Finlay BB, Cossart P . Exploitation of mammalian host cell functions by bacterial pathogens. Science 1997; 276: 718–725.

    Article  CAS  Google Scholar 

  14. Dietrich G 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 

  15. Sizemore DR, Branstrom AA, Sadoff JC . Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 1995; 270: 299–302.

    Article  CAS  Google Scholar 

  16. Darji A et al. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 1997; 91: 765–775.

    Article  CAS  Google Scholar 

  17. Smith AE . Viral vectors in gene therapy. Annu Rev Microbiol 1995; 49: 807–838.

    Article  CAS  Google Scholar 

  18. Jacobs T, Darji A, Weiss S, Chakraborty T . In: Alouf JE, Freer JH (eds), The Comprehensive Sourcebook of Bacterial Protein Toxins. Academic Press: London, 1999, pp 511–521.

    Google Scholar 

  19. Portnoy DA, Jones S . The cell biology of Listeria monocytogenes infection (escape from a vacuole). Ann N Y Acad Sci 1994; 730: 15–25.

    Article  CAS  Google Scholar 

  20. Gottschalk S, Tweten RK, Smith LC, Woo SL . Efficient gene delivery and expression in mammalian cells using DNA coupled with perfringolysin O. Gene Ther 1995; 2: 498–503.

    CAS  PubMed  Google Scholar 

  21. Alouf JE, Geoffroy C . In: Alouf JE, Freer JH (eds), Sourcebook of Bacterial Protein Toxins. Academic Press: London, 1991, pp 147–186.

    Google Scholar 

  22. Cowell JL, Grushoff-Kosyk PS, Bernheimer AW . Purification of cereolysin and the electrophoretic separation of the active (reduced) and inactive (oxidized) forms of the purified toxin. Infect Immun 1976; 14: 144–154.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Iwamoto M, Ohno-Iwashita Y, Ando S . Role of the essential thiol group in the thiol-activated cytolysin from Clostridium perfringens. Eur J Biochem 1987; 167: 425–430.

    Article  CAS  Google Scholar 

  24. Lee KD, Oh YK, Portnoy DA, Swanson JA . Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes. J Biol Chem 1996; 271: 7249–7252.

    Article  CAS  Google Scholar 

  25. Mandal M, Lee KD, . Listeriolysin O-liposome-mediated cytosotic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency, activity and tumor protectionListeria monocytogenes. Biochim Biophys Acta 2002; 1563: 7–17.

    Article  CAS  Google Scholar 

  26. Mandal M, Lee KD . Listeriolysin O-liposome-mediated cytosotic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency, activity and tumor protection. Biochim Biophys Acta 2002; 1563: 7–17.

    Article  CAS  Google Scholar 

  27. Ando T, Yamasaki M, Suzuki K . Protamines. Isolation, characterization, structure and function. Mol Biol Biochem Biophys 1973; 12: 1–114.

    Article  CAS  Google Scholar 

  28. Sorgi FL, Bhattacharya S, Huang L . Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther 1997; 4: 961–968.

    Article  CAS  Google Scholar 

  29. Yang JP, Huang L . Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther 1997; 4: 950–960.

    Article  CAS  Google Scholar 

  30. Perales JC et al. Biochemical and functional characterization of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor. J Biol Chem 1997; 272: 7398–7407.

    Article  CAS  Google Scholar 

  31. Sekiya K, Danbara H, Futaesaku Y . [Mechanism of pore formation on erythrocyte membrane by streptolysin-O]. Kansenshogaku Zasshi 1993; 67: 736–740.

    Article  CAS  Google Scholar 

  32. Beauregard KE, Lee KD, Collier RJ, Swanson JA . pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J Exp Med 1997; 186: 1159–1163.

    Article  CAS  Google Scholar 

  33. Portnoy DA, Tweten RK, Kehoe M, Bielecki J . Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells. Infect Immun 1992; 60: 2710–2717.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Geoffroy C, Gaillard JL, Alouf JE, Berche P . Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect Immun 1987; 55: 1641–1646.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Feener EP, Shen WC, Ryser HJ . Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J Biol Chem 1990; 265: 18 780–18 785.

    Google Scholar 

  36. Shen WC, Ryser HJ, LaManna L . Disulfide spacer between methotrexate and poly(D-lysine). A probe for exploring the reductive process in endocytosis. J Biol Chem 1985; 260: 10 905–10 908.

    Google Scholar 

  37. Papini E, Rappuoli R, Murgia M, Montecucco C . Cell penetration of diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol. J Biol Chem 1993; 268: 1567–1574.

    CAS  PubMed  Google Scholar 

  38. Ryser HJ, Mandel R, Ghani F . Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells. J Biol Chem 1991; 266: 18 439–18 442.

    Google Scholar 

  39. Mandel R et al. Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase. Proc Natl Acad Sci USA 1993; 90: 4112–4116.

    Article  CAS  Google Scholar 

  40. Maric M et al. Defective antigen processing in GILT-free mice. Science 2001; 294: 1361–1365.

    Article  CAS  Google Scholar 

  41. Phan UT, Arunachalam B, Cresswell P . Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action. J Biol Chem 2000; 275: 25 907–25 914.

    Article  Google Scholar 

  42. Luster AD, Weinshank RL, Feinman R, Ravetch JV . Molecular and biochemical characterization of a novel gamma-interferon-inducible protein. J Biol Chem 1988; 263: 12 036–12 043.

    Google Scholar 

  43. Gainey D, Short S, McCoy KL . Intracellular location of cysteine transport activity correlates with productive processing of antigen disulfide. J Cell Physiol 1996; 168: 248–254.

    Article  CAS  Google Scholar 

  44. Merkel BJ, Mandel R, Ryser HJ, McCoy KL . Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds. J Immunol 1995; 154: 128–136.

    CAS  PubMed  Google Scholar 

  45. Zychlinsky A, Prevost MC, Sansonetti PJ . Shigella flexneri induces apoptosis in infected macrophages. Nature 1992; 358: 167–169.

    Article  CAS  Google Scholar 

  46. Tsang AW, Oestergaard K, Myers JT, Swanson JA . Altered membrane trafficking in activated bone marrow-derived macrophages. J Leukoc Biol 2000; 68: 487–494.

    CAS  PubMed  Google Scholar 

  47. Gedde MM, Higgins DE, Tilney LG, Portnoy DA . Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes. Infect Immun 2000; 68: 999–1003.

    Article  CAS  Google Scholar 

  48. Glomski IJ et al. The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells. J Cell Biol 2002; 156: 1029–1038.

    Article  CAS  Google Scholar 

  49. Connor J, Schroit AJ . Transbilayer movement of phosphatidylserine in erythrocytes: inhibition of transport and preferential labeling of a 31000-dalton protein by sulfhydryl reactive reagents. Biochemistry 1988; 27: 848–851.

    Article  CAS  Google Scholar 

  50. Gusse M et al. Purification and characterization of nuclear basic proteins of human sperm. Biochim Biophys Acta 1986; 884: 124–134.

    Article  CAS  Google Scholar 

  51. Portnoy DA, Jacks PS, Hinrichs DJ . Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med 1988; 167: 1459–1471.

    Article  CAS  Google Scholar 

  52. Racoosin EL, Swanson JA . Macropinosome maturation and fusion with tubular lysosomes in macrophages. J Cell Biol 1993; 121: 1011–1020.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Dr Daniel Portnoy (UC Berkeley) for the LLO-his plasmid and Dr Gary Nabel (National Institutes of Health) for the GFP-luciferase plasmid. We thank Dr Joel Swanson (University of Michigan) and members of the Lee and Amidon Labs for helpful discussions. G Saito was supported by a fund from Sankyo Co. Ltd (Tokyo, Japan). This work was supported by NIH grants R29AI42084 and R01AI47173, and Rackham Fund and Vahlteich Research Fund at the University of Michigan.

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

    G Saito, G L Amidon & K-D Lee

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Saito, G., Amidon, G. & Lee, KD. Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential. Gene Ther 10, 72–83 (2003). https://doi.org/10.1038/sj.gt.3301859

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