Antisense oligodeoxynucleotides targeting internal exon sequences efficiently regulate TNF-alpha expression.
ABSTRACT Exon sequences upstream of splice sites play a critical role in mRNA processing, which is dependent on spliceosome interactions with these sites. Using antisense oligodeoxynucleotides (ODN), we targeted these and other sequences of the proinflammatory tumor necrosis factor-alpha (TNF-alpha) gene because it is multiply spliced and has been difficult to regulate with ODN in the past. ODN targeting exon sequences upstream of the donor splice sites of internal exons 2 (ORF4) and 3 (ORF6) significantly reduced TNF-alpha levels in stimulated U937 cells by 62%+/-7% and 51%+/-9%, respectively, in a dose-dependent manner but did not affect interleukin-6 (IL-6) levels. In contrast, ODN targeting the exon sequences downstream of the acceptor splice sites of exons 1, 2, and 3 failed to reduce TNF-alpha levels significantly under the same conditions. End-phosphorothioated ORF4 (ORF4-PE) significantly reduced TNF-alpha mRNA levels by greater than 80% (p < 0.001) and protein levels by 60% (p < 0.001) in U937 cells. ORF4-PE reduced newly synthesized TNF-alpha protein levels by >80% in lipopolysaccharide (LPS)-stimulated human macrophages, by greater than 60% in phorbol myristate acetate/phyto-hemagglutinin (PMA/PHA)-stimulated human peripheral blood mononuclear cells (PBMC), and by approximately 50% in LPS-stimulated murine monocytes. These results suggest that exon sequences flanking donor splice sites are highly susceptible target domains for antisense inhibition of TNF-alpha gene expression.
Mayne
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Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.
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McKenna
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Power
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D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D005091:Exons; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D012326:RNA Splicing; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics; D020298:U937 Cells / Q000166:cytology / Q000187:drug effects
D020319:Oligodeoxyribonucleotides, Antisense; D014409:Tumor Necrosis Factor-alpha