EFFECT OF TNF-ALPHA ANTISENSE OLIGOMERS ON CYTOKINE PRODUCTION BY PRIMARY MURINE ALVEOLAR MACROPHAGES

Antisense oligomers can inhibit expression of a single gene in a seque nce-specific manner, As a result, these sequences are being developed both as powerful experimental tools in the laboratory and as a novel c lass of therapeutic agents. In this study, we evaluated a panel of mor pholino antisense (M-AS) oligomers for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha) production by primary murine alveola r macrophages (AMs) and compared them with the more commonly used phos phorothioate oligonucleotides (S-AS). We found that 25 mu M of morphol ino oligomers whose sequence spanned the AUG (M-AS 2, M-AS 2me, and M- AS 5) start codon of TNF-alpha significantly inhibited TNF production on stimulation by both lipopolysaccharides (LPS) (36.6 +/- 3.2%, 27.3 +/- 3.0%, and 37.7 +/- 2.0% inhibition, respectively), whereas S-AS ta rgeted toward the same region were ineffective. M-AS 2 and M-AS 2me al so significantly inhibited TNF production in AMs stimulated by adheren ce to a solid substrate (28.7 +/- 2.2% and 29.4 +/- 8.3% inhibition, r espectively). Increasing the concentration of M-AS 2 and M-AS 2me to 5 0 mu M improved their efficacy in both UPS-stimulated (42.7 +/- 1.5% a nd 45.9 +/- 2.1% inhibition, respectively) and adherence-stimulated (5 2.6 +/- 0.7% and 41.7 +/- 2.9% inhibition, respectively) AMs, In contr ast, we showed that neither an antisense sequence targeted to a region upstream of the AUG site (M-AS 4) nor the nonsense control sequences M-NS 1 and M-NS 2 significantly inhibited TNF-alpha production by AMs on exposure to either stimulus. The data indicate that morpholino olig omers inhibit TNF-alpha production by murine AMs in a sequence-depende nt and dose-dependent manner.