Taylor M F, Weller D D, Kobzik L
Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Antisense Nucleic Acid Drug Dev. 1998 Jun;8(3):199-205. doi: 10.1089/oli.1.1998.8.199.
Antisense oligomers can inhibit expression of a single gene in a sequence-specific manner. As a result, these sequences are being developed both as powerful experimental tools in the laboratory and as a novel class of therapeutic agents. In this study, we evaluated a panel of morpholino antisense (M-AS) oligomers for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha) production by primary murine alveolar macrophages (AMs) and compared them with the more commonly used phosphorothioate oligonucleotides (S-AS). We found that 25 microM of morpholino oligomers whose sequence spanned the AUG (M-AS 2, M-AS 2me, and M-AS 5) start codon of TNF-alpha significantly inhibited TNF production on stimulation by both lipopolysaccharides (LPS) (36.6 +/- 3.2%, 27.3 +/- 3.0%, and 37.7 +/- 2.0% inhibition, respectively), whereas S-AS targeted toward the same region were ineffective. M-AS 2 and M-AS 2me also significantly inhibited TNF production in AMs stimulated by adherence to a solid substrate (28.7 +/- 2.2% and 29.4 +/- 8.3% inhibition, respectively). Increasing the concentration of M-AS 2 and M-AS 2me to 50 microM improved their efficacy in both LPS-stimulated (42.7 +/- 1.5% and 45.9 +/- 2.1% inhibition, respectively) and adherence-stimulated (52.6 +/- 0.7% and 41.7 +/- 2.9% inhibition, respectively) AMs. In contrast, we showed that neither an antisense sequence targeted to a region upstream of the AUG site (M-AS 4) nor the nonsense control sequences M-NS 1 and M-NS 2 significantly inhibited TNF-alpha production by AMs on exposure to either stimulus. The data indicate that morpholino oligomers inhibit TNF-alpha production by murine AMs in a sequence-dependent and dose-dependent manner.
反义寡聚物能够以序列特异性方式抑制单个基因的表达。因此，这些序列正被开发成为实验室中强大的实验工具以及一类新型治疗药物。在本研究中，我们评估了一组吗啉代反义（M-AS）寡聚物抑制原代小鼠肺泡巨噬细胞（AM）产生肿瘤坏死因子-α（TNF-α）的能力，并将它们与更常用的硫代磷酸酯寡核苷酸（S-AS）进行比较。我们发现，25微摩尔序列跨越TNF-α起始密码子AUG（M-AS 2、M-AS 2me和M-AS 5）的吗啉代寡聚物在脂多糖（LPS）刺激下显著抑制TNF产生（分别抑制36.6±3.2%、27.3±3.0%和37.7±2.0%），而靶向相同区域的S-AS则无效。M-AS 2和M-AS 2me在通过粘附到固体基质刺激的AM中也显著抑制TNF产生（分别抑制28.7±2.2%和29.4±8.3%）。将M-AS 2和M-AS 2me的浓度增加到50微摩尔可提高它们在LPS刺激（分别抑制42.7±1.5%和45.9±2.1%）和粘附刺激（分别抑制52.6±0.7%和41.7±2.9%）的AM中的效力。相比之下，我们表明靶向AUG位点上游区域的反义序列（M-AS 4）以及无义对照序列M-NS 1和M-NS 2在暴露于任何一种刺激时均未显著抑制AM产生TNF-α。数据表明吗啉代寡聚物以序列依赖性和剂量依赖性方式抑制小鼠AM产生TNF-α。
