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In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors

Gene Therapy volume 17, pages 227–237 (2010)Cite this article

Abstract

In cystic fibrosis (CF), respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress toward gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF, we examined the feasibility of using recombinant SV40-derived vectors (rSV40s), which may circumvent some of these obstacles. To accommodate the large cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We, therefore, tested whether ‘gutless’ rSV40s could be packaged and were able to express a functional human CFTR cDNA. The results from our in vitro analysis determined that rSV40–CFTR was able to successfully result in the expression of CFTR protein, which localized to the plasma membrane and restored channel function to CFTR-deficient cells. Similarly, in vivo experiments delivering rSV40–CFTR to the lungs of Cftr−/− mice resulted in a reduction of the pathology associated with intra-tracheal P. aeruginosa challenge. rSV40–CFTR-treated mice had less weight loss when compared with control-treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the broncho-alveolar lavage. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40–CFTR to CF gene therapy is warranted.

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References

  1. Schwiebert EM, Benos DJ, Fuller CM . Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein. Am J Med 1998; 104: 576–590.

    Article  CAS  PubMed  Google Scholar 

  2. Southern KW . delta F508 in cystic fibrosis: willing but not able. Arch Dis Child 1997; 76: 278–282.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Flotte TR . Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Therapy 2004; 11: 805–810.

    Article  CAS  PubMed  Google Scholar 

  4. Ferrari S, Geddes DM, Alton EW . Barriers to and new approaches for gene therapy and gene delivery in cystic fibrosis. Adv Drug Deliv Rev 2002; 54: 1373–1393.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Driskell RA, Engelhardt JF . Current status of gene therapy for inherited lung diseases. Annu Rev Physiol 2003; 65: 585–612.

    Article  CAS  PubMed  Google Scholar 

  6. Griesenbach U, Ferrari S, Geddes DM, Alton EW . Gene therapy progress and prospects: cystic fibrosis. Gene Therapy 2002; 9: 1344–1350.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Flotte TR . Recombinant adeno-associated virus vectors for cystic fibrosis gene therapy. Curr Opin Mol Ther 2001; 3: 497–502.

    CAS  PubMed  Google Scholar 

  8. Mueller C, Flotte TR . Gene therapy for cystic fibrosis. Clin Rev Allergy Immunol 2008; 35: 164–178.

    Article  CAS  PubMed  Google Scholar 

  9. Harvey BG, Leopold PL, Hackett NR, Grasso TM, Williams PM, Tucker AL et al. Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus. J Clin Invest 1999; 104: 1245–1255.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Virella-Lowell I, Poirier A, Chesnut KA, Brantly M, Flotte TR . Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients. Gene Therapy 2000; 7: 1783–1789.

    Article  CAS  PubMed  Google Scholar 

  11. Zeitlin PL . Cystic fibrosis gene therapy trials and tribulations. Mol Ther 2000; 1: 5–6.

    Article  CAS  PubMed  Google Scholar 

  12. Strayer D, Branco F, Zern MA, Yam P, Calarota SA, Nichols CN et al. Durability of transgene expression and vector integration: recombinant SV40-derived gene therapy vectors. Mol Ther 2002; 6: 227–237.

    Article  CAS  PubMed  Google Scholar 

  13. Strayer DS, Zern MA, Chowdhury JR . What can SV40-derived vectors do for gene therapy? Curr Opin Mol Ther 2002; 4: 313–323.

    CAS  PubMed  Google Scholar 

  14. Norkin LC . Simian virus 40 infection via MHC class I molecules and caveolae. Immunol Rev 1999; 168: 13–22.

    Article  CAS  PubMed  Google Scholar 

  15. Pelkmans L, Puntener D, Helenius A . Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 2002; 296: 535–539.

    Article  CAS  PubMed  Google Scholar 

  16. Kondo R, Feitelson MA, Strayer DS . Use of SV40 to immunize against hepatitis B surface antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent. Gene Therapy 1998; 5: 575–582.

    Article  CAS  PubMed  Google Scholar 

  17. Strayer DS, Lamothe M, Wei D, Milano J, Kondo R . Generation of recombinant SV40 vectors for gene transfer. Methods Mol Biol 2001; 165: 103–117.

    CAS  PubMed  Google Scholar 

  18. Schoumacher RA, Ram J, Iannuzzi MC, Bradbury NA, Wallace RW, Hon CT et al. A cystic fibrosis pancreatic adenocarcinoma cell line. Proc Natl Acad Sci USA 1990; 87: 4012–4016.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, Balamuth N et al. Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med 2002; 8: 485–492.

    Article  CAS  PubMed  Google Scholar 

  20. West MR, Molloy CR . A microplate assay measuring chloride ion channel activity. Anal Biochem 1996; 241: 51–58.

    Article  CAS  PubMed  Google Scholar 

  21. Heeckeren A, Walenga R, Konstan MW, Bonfield T, Davis PB, Ferkol T . Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J Clin Invest 1997; 100: 2810–2815.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Sirninger J, Muller C, Braag S, Tang Q, Yue H, Detrisac C et al. Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector. Hum Gene Ther 2004; 15: 832–841.

    CAS  PubMed  Google Scholar 

  23. Dajani R, Zhang Y, Taft PJ, Travis SM, Starner TD, Olsen A et al. Lysozyme secretion by submucosal glands protects the airway from bacterial infection. Am J Respir Cell Mol Biol 2005; 32: 548–552.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Akinbi HT, Epaud R, Bhatt H, Weaver TE . Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice. J Immunol 2000; 165: 5760–5766.

    Article  CAS  PubMed  Google Scholar 

  25. Wiley SR, Kraus RJ, Zuo F, Murray EE, Loritz K, Mertz JE . SV40 early-to-late switch involves titration of cellular transcriptional repressors. Genes Dev 1993; 7: 2206–2219.

    Article  CAS  PubMed  Google Scholar 

  26. Pasyk EA, Foskett JK . Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J Biol Chem 1995; 270: 12347–12350.

    Article  CAS  PubMed  Google Scholar 

  27. Johnson LG, Olsen JC, Sarkadi B, Moore KL, Swanstrom R, Boucher RC . Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet 1992; 2: 21–25.

    Article  CAS  PubMed  Google Scholar 

  28. Dorin JR, Farley R, Webb S, Smith SN, Farini E, Delaney SJ et al. A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction. Gene Therapy 1996; 3: 797–801.

    CAS  PubMed  Google Scholar 

  29. Flotte TR . Recombinant adeno-associated virus gene therapy for cystic fibrosis and alpha(1)-antitrypsin deficiency. Chest 2002; 121 (3 Suppl): 98S–102S.

    Article  CAS  PubMed  Google Scholar 

  30. Mueller C, Flotte TR . Clinical gene therapy using recombinant adeno-associated virus vectors. Gene Therapy 2008; 15: 858–863.

    Article  CAS  PubMed  Google Scholar 

  31. Sauter BV, Parashar B, Chowdhury NR, Kadakol A, Ilan Y, Singh H et al. A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats. Gastroenterology 2000; 119: 1348–1357.

    Article  CAS  PubMed  Google Scholar 

  32. Middleton PG, Alton EW . Gene therapy for cystic fibrosis: which postman, which box? [see comments]. Thorax 1998; 53: 197–199.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Ferkol T, Perales JC, Eckman E, Kaetzel CS, Hanson RW, Davis PB . Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor. J Clin Invest 1995; 95: 493–502.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Strayer DS, Milano J . SV40 mediates stable gene transfer in vivo. Gene Therapy 1996; 3: 581–587.

    CAS  PubMed  Google Scholar 

  35. Ruiz FE, Clancy JP, Perricone MA, Bebok Z, Hong JS, Cheng SH et al. A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis. Hum Gene Ther 2001; 12: 751–761.

    Article  CAS  PubMed  Google Scholar 

  36. Zahm JM, Debordeaux C, Maurer C, Hubert D, Dusser D, Bonnet N et al. Improved activity of an actin-resistant DNase I variant on the cystic fibrosis airway secretions. Am J Respir Crit Care Med 2001; 163: 1153–1157.

    Article  CAS  PubMed  Google Scholar 

  37. Louboutin JP, Liu B, Chekmasova AA, Reyes BA, van Bockstaele EJ, Strayer DS . Delivering genes to the organ-localized immune system: long-term results of direct intramarrow transduction. J Gene Med 2007; 9: 843–851.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants AI48244, RR13156, AI41399, and R01HL69877 from the National Institutes of Health as well as by a fellowship from the Parker B Francis Foundation and the Diabetes and Endocrinology Research Center of the University of Massachusetts Medical School (supported by Grant P30 DK32520). We are grateful to Miss Maria Lamothe and Mr Charles Ko for technical assistance. Dr Janet S Butel, Baylor College of Medicine, generously provided us with the original SV40 genomic constructs from which these vectors were derived. We are grateful to Dr John Engelhardt for the human CFTR cDNA used in these studies.

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Authors and Affiliations

  1. Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA

    C Mueller, S Braag & T R Flotte

  2. Department of Pathology, Jefferson Medical College, Philadelphia, PA, USA

    M S Strayer, F Branco, J-P Louboutin & D S Strayer

  3. Department of Veterinary Pathology, Louisiana State University, Baton Rouge Louisiana, LA, USA

    J Sirninger

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  1. C Mueller
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Correspondence to C Mueller.

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Mueller, C., Strayer, M., Sirninger, J. et al. In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors. Gene Ther 17, 227–237 (2010). https://doi.org/10.1038/gt.2009.137

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