Mathew E, Hardee G E, Bennett C F, Lee K-D
Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA.
Gene Ther. 2003 Jul;10(13):1105-15. doi: 10.1038/sj.gt.3301966.
Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents. Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective. A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages. This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol. To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy. The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively. ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels. Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system. The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.
反义寡脱氧核苷酸（ODNs）作为序列特异性治疗剂具有巨大潜力。要使ODNs具有治疗效果，足够浓度的完整ODN必须绕过膜屏障并进入细胞质和细胞核。设计了一种细胞质递送策略以提高骨髓来源巨噬细胞中ODN的递送效率。这种基于脂质体的制剂利用单核细胞增生李斯特菌的溶细胞性溶血素李斯特菌溶素O（LLO）来介导ODN从内吞小室逃逸到细胞质中。为了监测ODN的细胞质递送，使用落射荧光显微镜观察荧光标记的ODN的亚细胞运输。分别使用流式细胞术和Northern印迹分析来测量递送后靶蛋白和mRNA的表达。封装在LLO脂质体中的针对小鼠细胞间粘附分子-1（ICAM-1）的ODN被释放到细胞质中并运输到细胞核，在蛋白质和mRNA水平上有效且特异性地抑制激活诱导的ICAM-1表达。没有LLO的递送导致ODN隔离在囊泡区室中，导致对ICAM-1表达的抑制很小，这支持了使用该系统进行有效细胞质递送对LLO的需求。数据清楚地表明，LLO介导的ODN从细胞内囊泡的逃逸是在培养的巨噬细胞中实现完全治疗性反义活性的有效方法。
