The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro

M. Stern, N. J. Caplen, J. E. Browning, U. Griesenbach, F. Sorgi, L. Huang, D. C. Gruenert, C. Marriot, Ronald Crystal, D. M. Geddes, E. W.F.W. Alton

Research output: Contribution to journalArticle

98 Citations (Scopus)

Abstract

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene β-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 μg/ml). but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 μg/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.

Original languageEnglish
Pages (from-to)91-98
Number of pages8
JournalGene Therapy
Volume5
Issue number1
Publication statusPublished - 3 Feb 1998
Externally publishedYes

Fingerprint

Expectorants
Sputum
Cystic Fibrosis
Transfection
Genes
Adenoviridae
Liposomes
DNA
Galactosidases
Acetylcysteine
Therapeutics
dornase alfa
In Vitro Techniques
Reporter Genes
Genetic Therapy
Lysine
Phosphates
Cell Line

Keywords

  • Cystic fibrosis
  • Gene therapy
  • rhDNase
  • Sputum

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Stern, M., Caplen, N. J., Browning, J. E., Griesenbach, U., Sorgi, F., Huang, L., ... Alton, E. W. F. W. (1998). The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. Gene Therapy, 5(1), 91-98.

The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. / Stern, M.; Caplen, N. J.; Browning, J. E.; Griesenbach, U.; Sorgi, F.; Huang, L.; Gruenert, D. C.; Marriot, C.; Crystal, Ronald; Geddes, D. M.; Alton, E. W.F.W.

In: Gene Therapy, Vol. 5, No. 1, 03.02.1998, p. 91-98.

Research output: Contribution to journalArticle

Stern, M, Caplen, NJ, Browning, JE, Griesenbach, U, Sorgi, F, Huang, L, Gruenert, DC, Marriot, C, Crystal, R, Geddes, DM & Alton, EWFW 1998, 'The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro', Gene Therapy, vol. 5, no. 1, pp. 91-98.
Stern M, Caplen NJ, Browning JE, Griesenbach U, Sorgi F, Huang L et al. The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. Gene Therapy. 1998 Feb 3;5(1):91-98.
Stern, M. ; Caplen, N. J. ; Browning, J. E. ; Griesenbach, U. ; Sorgi, F. ; Huang, L. ; Gruenert, D. C. ; Marriot, C. ; Crystal, Ronald ; Geddes, D. M. ; Alton, E. W.F.W. / The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. In: Gene Therapy. 1998 ; Vol. 5, No. 1. pp. 91-98.
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