IN-VITRO EFFICACY OF MORPHOLINO-MODIFIED ANTISENSE OLIGOMERS DIRECTEDAGAINST TUMOR-NECROSIS-FACTOR-ALPHA MESSENGER-RNA

Chemical modification of antisense oligonucleotides to increase nuclea se resistance may improve their efficacy within enzyme-rich cellular t argets (e.g. macrophages). We evaluated a panel of morpholino antisens e oligomers (M-AS) for their ability to inhibit macrophage tumor necro sis factor-alpha. (TNF-alpha) release and compared them to phosphodies ter (O-AS) and phosphorothioate (S-AS) types of oligonucleotides, M-AS inhibited translation in vitro (rabbit reticulocyte lysate) of target mRNA at concentrations as low as 200 nM (e.g. percent inhibition by M -AS 2 at 0.2, 1.0, and 2.0 mu M was 40.9 +/- 5.3% 50.2 +/- 4.6%, and 5 7.7 +/- 3.6%, respectively, n = 4,p less than or equal to 0.002 versus control). Similarly, M-AS 2 effectively, albeit partially, inhibited TNF-alpha production by LPS-stimulated macrophages (RAW 264.7 cells). Incubation of cells with 25 mu M M-AS 2 resulted in 32.6 +/- 2.6% (n = 3, p = 0.002 versus control) decrease in TNF-alpha release. In contra st, S-AS inhibited translation of the target mRNA in the rabbit reticu locyte lysate assay, but not in the cell-based assay. In fact, S-AS no nspecifically augmented TNF-alpha release, O-AS were without effect in either system, Uptake studies with fluorescent M-AS revealed that inh ibitory effects were seen despite relatively low cellular uptake (intr acellular concentration 30.5 +/- 6.7 nM; efficiency of uptake 0.1%). I n contrast, flow cytometric and confocal analysis revealed that S-AS w ere avidly taken up by RAW 264.7 cells, confirming that their lack of efficacy was not due to lack of uptake, With improved methods of deliv ery, M-AS may represent an important therapeutic modality.