title: In vitro efficacy of morpholino-modified antisense oligomers directed against tumor necrosis factor-alpha 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-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 microM 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-alpha production by LPS-stimulated macrophages (RAW 264.7 cells). Incubation of cells with 25 microM 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.
authors.affiliation: Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
authors.forename: M F
authors.initials: MF
authors.lastname: Taylor
authors.forename: J D
authors.initials: JD
authors.lastname: Paulauskis
authors.forename: D D
authors.initials: DD
authors.lastname: Weller
authors.forename: L
authors.initials: L
authors.lastname: Kobzik
mesh_terms: D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003062:Codon; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D009025:Morpholines; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010710:Phosphates; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012156:Reticulocytes / Q000378:metabolism; D013329:Structure-Activity Relationship; D013873:Thionucleotides; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003062:Codon; D009025:Morpholines; D016376:Oligonucleotides, Antisense; D010710:Phosphates; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases