Efficacy of adenoviral TNF alpha antisense is enhanced by a macrophage specific promoter.
Macrophage-derived TNF alpha is a critical mediator of inflammation and destruction in diseases such as rheumatoid arthritis and Crohn's disease. These studies were undertaken to develop an effective adenovirus-based strategy to specifically suppress TNF alpha in primary human macrophages. A variety of promoters and LTRs were evaluated for effective expression in the macrophage cell line RAW 264.7. The CMV promoter and the Visna LTR were the most strongly expressed and were therefore used to drive the expression of TNF alpha antisense fragments. In transient transfection assays, the antisense fragment terminating at the 3' end of the first exon (216 bp) was superior to the others (70 and 750 bp), when expressed under the control of either the CMV promoter or the Visna LTR. Adenoviral vectors expressing the 216 bp TNF alpha antisense fragment, controlled by the CMV promoter or the Visna LTR, were both effective at suppressing LPS-induced TNF alpha secretion by primary human macrophages. However, the Visna LTR was more effective not only at suppressing LPS-induced TNF alpha secretion, but also IL-6, which is highly sensitive to TNF alpha secretion. These results demonstrate that effective, specific, suppression of TNF alpha in macrophages is possible, employing a directed antisense approach and a promoter system that is highly efficient in human macrophages.
Sidiropoulos
P
P
Department of Medicine, Division of Rheumatology Northwestern University Medical School and the VA Chicago Health Care System, Lakeside Division, Chicago, IL, USA.
Liu
H
H
Mungre
S
S
Anderson
L
L
Thimmapaya
B
B
Pope
R M
RM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016375:Antisense Elements (Genetics) / Q000235:genetics*; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
D016375:Antisense Elements (Genetics); D015850:Interleukin-6; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha