Anti-inflammatory gene therapy directed at the airway epithelium.
Cystic fibrosis (CF) is characterised by chronic airway inflammation. Pro-inflammatory mediators in the lung are regulated by the transcription factor nuclear factor kappa B (NFkappaB). We have assessed the effect of adenovirus and liposome-mediated overexpression of the NFkappaB inhibitor IkappaBalpha, as well as liposome-mediated transfection with oligonucleotides resembling NFkappaB consensus binding sites (decoys) in a cystic fibrosis airway epithelial cell line (CFTE). Electrophoretic mobility shift assays (EMSA) were used to assess NFkappaB activity and secretion of the pro-inflammatory cytokine interleukin-8 (IL-8) was measured by ELISA. At a MOI of 30, Ad-IkappaBalpha significantly decreased IL-8 secretion to 60% and 43% of control unstimulated and TNF-alpha stimulated cells, respectively. At this MOI, approximately 70% of cells are transduced. EMSA showed an approximately 50% decrease in NFkappaB activation. Liposome-mediated transfection of IkappaBalpha did not reduce IL-8 secretion, probably due to low transfection efficiency (approximately 5% of cells). Liposome-mediated transfection of CFTE cells with rhodamine-labeled decoy oligonucleotides indicated a transfection efficiency close to 100%. TNF-alpha stimulated IL-8 secretion was reduced by approximately 40% using this approach. EMSA confirmed a significant decrease of NFkappaB activation. Decoy oligonucleotides may be a promising approach for reduction of NFkappaB-mediated pulmonary inflammation. Gene Therapy (2000) 7, 306-313.
Griesenbach
U
U
Department of Gene Therapy, National Heart and Lung Institute, London, UK.
Scheid
P
P
Hillery
E
E
de Martin
R
R
Huang
L
L
Geddes
D M
DM
Alton
E W
EW
D000256:Adenoviridae / Q000235:genetics; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism / Q000628:therapy*; D004586:Electrophoresis; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008081:Liposomes / Q000235:genetics / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics*; D009841:Oligonucleotides / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000235:genetics
D016209:Interleukin-8; D008081:Liposomes; D016328:NF-kappa B; D009841:Oligonucleotides