Department of Surgery, Comprehensive Cancer Center, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Curr Pharm Biotechnol. 2009 Sep;10(6):579-88. doi: 10.2174/138920109789069279. Epub 2009 Sep 1.
Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. Unlike phosphorothioates (PS ODN) and other charged oligonucleotides, little is known about the uptake characteristics of neutral oligomers. The purpose of this study was to understand the kinetics of PMO transport in cells and correlate with antisense activity. In contrast to primary cells and some transformed cell lines which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. Differential PMO uptake was also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T and B cells. In addition, PMO localization was observed to be heterogeneous within a population of uptake permissive cells. Unassisted PMO delivery targeting specific genes was correlated with functional antisense efficacy in experiments showing correction of pre-mRNA missplicing and inhibition of target enzyme activity in cells in culture. PMO internalization in uptake-permissive cells was identified to be specific, saturable, and energy-dependent, suggesting a receptor mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.
磷酰二胺吗啉寡聚物（PMO）具有中性化学性质，被广泛用作细胞培养模型中选择性抑制基因表达的工具，目前正在进行人体临床试验。与磷酸硫代酯（PS ODN）和其他带电寡核苷酸不同，对于中性寡聚物的摄取特性知之甚少。本研究的目的是了解 PMO 在细胞中的转运动力学，并与反义活性相关联。与摄取允许的原代细胞和一些转化细胞系相反，已建立的癌细胞系摄取非常差，偶尔出现弥漫性细胞内模式。在免疫细胞中也观察到 PMO 的摄取差异，树突状细胞和单核细胞的摄取率明显高于 T 和 B 细胞。此外，在摄取允许的细胞群体中，PMO 的定位也表现出不均匀性。在显示纠正前体 mRNA 剪接错误和抑制培养细胞中靶酶活性的实验中，针对特定基因的非辅助 PMO 递送与功能性反义功效相关。在摄取允许的细胞中，PMO 的内化被确定为特异性、饱和性和能量依赖性的，这表明存在受体介导的摄取机制。了解 PMO 转运应该有助于设计更有效的合成反义寡聚物作为治疗剂。
