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

被引:58
作者
Taylor, MF
Paulauskis, JD
Weller, DD
Kobzik, L
机构
[1] HARVARD UNIV, SCH PUBL HLTH, PHYSIOL PROGRAM, BOSTON, MA 02115 USA
[2] BRIGHAM & WOMENS HOSP, DEPT PATHOL, BOSTON, MA 02115 USA
[3] ANTIVIRALS INC, CORVALLIS, OR 97333 USA
关键词
D O I
10.1074/jbc.271.29.17445
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-alpha. (TNF-alpha) 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 mu M was 40.9 +/- 5.3% 50.2 +/- 4.6%, and 57.7 +/- 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 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-alpha 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.
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收藏
页码:17445 / 17452
页数:8
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