Adeno-Associated Viral Vectors for CF Gene Therapy

  • Terence R. Flotte
  • Isabel Virella-Lowell
  • Kye A. Chesnut
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 70)


AAV is a nonpathogenic human parvovirus that has a natural mechanism for long-term persistence in human cells. Wild-type AAV is unique in that it undergoes stable integration of its DNA into a specific region of human chromosome 19, the AAVS1 site (1, 2, 3, 4), a process that our group has also recently described in vivo in rhesus macques (5). This site specificity appears to be mediated by specific interactions between the nonstructural protein, Rep, and the sequence-specific binding elements within the AAVS1 site (6). AAV vectors have been developed by deleting the two AAV genes, rep and cap from the viral genome, and substituting transgenes such as CFTR (7,8). Since the rep gene is deleted, these vectors generally do not integrate in a site-specific manner, yet they do persist long-term in mammalian cells through a complicated process that involves both episomal persistence and random-site integration (9, 10, 11).


Cystic Fibrosis Fetal Bovine Serum Medium Vector Genome Biological Titer Infectious Center Assay 
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  1. 1.
    Gregory, R. J., Cheng, S. H., Rich, D. P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K. W., Welsh, M. J., and Smith, A. E. (1990) Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347, 382–386.CrossRefPubMedGoogle Scholar
  2. 2.
    Goldman, M., Anderson, G., Stolzenberg, E. D., Kari, U. P., Zasloff, M., and Wilson, J. M. (1997) Human β-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88, 553–560.CrossRefPubMedGoogle Scholar
  3. 3.
    Smith, J. J., Travis, S. M., Greenberg, E. P., and Welsh, M. J. (1996) Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85, 229–236.CrossRefPubMedGoogle Scholar
  4. 4.
    Ellison, R. T. and Giehl, T. J. (1991) Killing of Gram-negative bacteria by lactoferrin and lysozyme. J. Clin. Invest. 88, 1080–1091.CrossRefPubMedGoogle Scholar
  5. 5.
    Raphael, G. D., Jeney, E. V., Baraniuk, J. N., Kim, I., Meredith, S. D., and Kaliner, M. A. (1989) Pathophysiology of rhinitis. Lactoferrin and lysozyme in nasal secretions. J. Clin. Invest. 84, 1528–1535.CrossRefPubMedGoogle Scholar
  6. 6.
    McCray, P. B. and Bentley, L. (1997) Human airway epithelia express a β-defensin. Am J Respir. Cell Mol. Biol. 16, 343–349.PubMedGoogle Scholar
  7. 7.
    Singh, P. K., Jia, H. P., Wiles, K., Hesselberth, J., Liu, L., Conway, B. D., et al. (1998) Production of β-defensins by human airway epithelia. Proc. Natl. Acad. Sci. USA 95, 14,961–14,966.CrossRefPubMedGoogle Scholar
  8. 8.
    Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S. S., Daher, K., Bainton, D. F., and Lehrer, R. I. (1985) Defensins. Natural peptide antibiotics of human neutro-phils. J. Clin. Invest. 76, 1427–1435.CrossRefPubMedGoogle Scholar
  9. 9.
    Schnapp, D. and Harris, A. (1998) Antibacterial peptides in bronchoalveolar lav-age fluid. Am. J. Respir. Cell Mol. Biol. 19, 352–356.PubMedGoogle Scholar
  10. 10.
    Hiemstra, P. S., Maassen, R. J., Stolk, J., Heinzel-Wieland, R., Steffens, G. J., and Dijkman, J. H. (1996) Antibacterial activity of antileukoprotease. Infect. Immun. 64,4520–4524PubMedGoogle Scholar
  11. 11.
    Brogden, K. A., Ackermann, M., and Huttner, K. M. (1998) Detection of anionic antimicrobial peptides in ovine bronchoalveolar lavage fluid and respiratory epithelium. Infect. Immun. 66, 5948–5954.PubMedGoogle Scholar
  12. 12.
    Brogden, K. A., Ackermann, M. R., McCray, P. B., Jr., and Huttner, K. M. (1999) Differences in the concentrations of small, anionic, antimicrobial peptides in bronchoalveolar lavage fluid and in respiratory epithelia of patients with and without cystic fibrosis. Infect. Immun. 67, 4256–4259.PubMedGoogle Scholar
  13. 13.
    Samaranayake, Y. H., Samaranayake, L. P., Wu, P. C., and So, M. (1997) The antifungal effect of lactoferrin and lysozyme on Candida krusei and Candida albicans. APMIS 105, 875–883.CrossRefPubMedGoogle Scholar
  14. 14.
    Kalfa, V. C. and Brogden, K. A. (1999) Anionic antimicrobial peptide-lysozyme interactions in innate pulmonary immunity. Int. J. Antimicrob. Agents 13, 47–51.CrossRefPubMedGoogle Scholar
  15. 15.
    Harder, J., Bartels, J., Christophers, E., and Schroeder, J.-M. (1997) A peptide antibiotic from human skin. Nature 387, 861,862.CrossRefGoogle Scholar
  16. 16.
    Zhao, C. Q., Wang, I., and Lehrer, R. I. (1996) Widespread expression of β-defensin HBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396, 319–322.CrossRefPubMedGoogle Scholar
  17. 17.
    Knowles, M. R., Robinson, J. M., Wood, R. E., Pue, C. A., Mentz, W. M., Wager, G. C., Gatzy, J. T., and Boucher, R. C. (1997) Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest. 100, 2588–2595.CrossRefPubMedGoogle Scholar
  18. 18.
    Matsui, H., Grubb, B. R., Tarran, R., Randell, S. H., Gatzy, J. T., Davis, C. W., and Boucher, R. C. (1998) Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95, 1005–1015.CrossRefPubMedGoogle Scholar
  19. 19.
    Cole, A. M., Wu, M., Kim, Y.-H., and Ganz, T. (2000) Microanalysis of antimicrobial properties of human fluids. J. Microbiol. Meth. 41, 135–143.CrossRefGoogle Scholar
  20. 20.
    Cole, A. M., Dewan, P., and Ganz, T. (1999) Innate antimicrobial activity of nasal secretions. Infect. Immun. 67, 3267–3275.PubMedGoogle Scholar
  21. 21.
    Osserman, E. F. and Lawlor, D. P. (1966) Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J. Exp. Med. 124, 921–952.CrossRefPubMedGoogle Scholar
  22. 22.
    Tang, Y. Q., Yuan, J., Miller, C. J., and Selsted, M. E. (1999) Isolation, characterization, cDNA cloning, and antimicrobial properties of two distinct subfamilies of a-defensins from rhesus macaque leukocytes. Infect. Immun. 67, 6139–6144.PubMedGoogle Scholar
  23. 23.
    Valore, E. V. and Ganz, T. (1997) Laboratory production of antimicrobial pep-tides in native conformation, in Antimicrobial Peptide Protocols, vol. 78, (Shafer, W. M., ed.), Humana Press, Totowa, pp. 115–131.CrossRefGoogle Scholar
  24. 24.
    Soong, L. B., Ganz, T., Ellison, A., and Caughey, G. H. (1997) Purification and characterization of defensins from cystic fibrosis sputum. Inflamm. Res, 46,98–102.CrossRefPubMedGoogle Scholar
  25. 25.
    Valore, E. V., Park, C. H., Quayle, A. J., Wiles, K. R., McCray, P. B., and Ganz, T. (1998) Human β-defensin-1: an antimicrobial peptide of urogenital tissues. J. Clin.Invest. 101, 1633–1642.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Terence R. Flotte
    • 1
  • Isabel Virella-Lowell
    • 1
  • Kye A. Chesnut
    • 1
  1. 1.Powell Gene Therapy CenterUniversity of FloridaGainesville

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