In vitro efficacy of morpholino-modified antisense oligomers directed against tumor necrosis factor-α mRNA

Abstract

Chemical modification of antisense oligonucleotides to increase nuclease resistance may improve their efficacy within enzyme-rich cellular targets (e.g. macrophages). We evaluated a panel of morpholino antisense oligomers (M-AS) for their ability to inhibit macrophage tumor necrosis factor-α (TNF- α) release and compared them to phosphodiester (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 μM was 40.9 ± 5.3%, 50.2 ± 4.6%, and 57.7 ± 3.6%, respectively, n = 4, p ≤ 0.002 versus control). Similarly, M-AS 2 effectively, albeit partially, inhibited TNF-α production by LPS-stimulated macrophages (RAW 264.7 cells). Incubation of cells with 25 μM M-AS 2 resulted in 32.6 ± 2.6% (n = 3, p = 0.002 versus control) decrease in TNF-α release. In contrast, S-AS inhibited translation of the target mRNA in the rabbit reticulocyte lysate assay, but not in the cell- based assay. In fact, S-AS nonspecifically augmented TNF-α release. O-AS were without effect in either system. Uptake studies with fluorescent M-AS revealed that inhibitory effects were seen despite relatively low cellular uptake (intracellular concentration 30.5 ± 6.7 nM; efficiency of uptake 0.1%). In contrast, flow cytometric and confocal analysis revealed that S-AS were 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 delivery, M- AS may represent an important therapeutic modality.