SPECIFIC SUPPRESSION OF HUMAN TUMOR-NECROSIS-FACTOR-ALPHA SYNTHESIS BY ANTISENSE OLIGODEOXYNUCLEOTIDES

Recent clinical studies using neutralizing antibodies point to a key r ole for tumor necrosis factor-alpha (TNF-alpha) in chronic inflammator y diseases, Antisense technique is a recent approach aiming at inhibit ion of single proteins, Previously, we described nonspecific induction of TNF by phosphorothioate oligonucleotides. In this study, we establ ished an in vitro model that allows specific inhibition of TNF synthes is, bypassing TNF induction, Freshly isolated human monocytes were inc ubated with oligonucleotides and the cationic lipid lipofectin in diff erent ratios, TNF synthesis was stimulated with lipopolysaccharide and quantified by a specific radioimmunoassay (RIA). Among all sequences tested, one of the antisense oligonucleotides complementary to the tra nslation initiation region of TNF mRNA (5'-CAT GCT TTC AGT GCT CAT-3') revealed highest efficacy, At 2 mu M, the antisense oligonucleotide i nhibited TNF synthesis by up to 79%, A concentration as low as 250 nM of the antisense oligonucleotide was effective. Scrambled controls and controls with different, defined degrees of mismatches confirmed a se quence-specific action. Examination with confocal fluorescence microsc opy showed a marked difference comparing lipofectin-mediated vs, spont aneous uptake, This study defines criteria that form the prerequisite necessary for design and application of antisense oligonucleotides aga inst TNF in vivo.