Bennett C F, Chiang M Y, Chan H, Shoemaker J E, Mirabelli C K
Department of Molecular and Cellular Biology, ISIS Pharmaceuticals, Carlsbad, California 92008.
Mol Pharmacol. 1992 Jun;41(6):1023-33.
We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.
我们研究了一种阳离子脂质制剂在人脐静脉内皮细胞中增强反义寡核苷酸活性的作用。发现一种含有阳离子脂质N-[1-(2,3-二油酰氧基)丙基]-N,N,N-三甲基氯化铵（DOTMA）的脂质体制剂可使与人类细胞间黏附分子-1的AUG翻译起始密码子杂交的反义寡核苷酸（ISIS 1570）的效力提高至少1000倍。在存在8 microM DOTMA的情况下，当寡核苷酸浓度为0.01至5 microM时，与细胞结合的35S-ISIS 1570比不存在DOTMA时多6至15倍。35S-ISIS 1570与细胞的结合以及反义活性均随DOTMA浓度的增加以及与阳离子脂质孵育时间的延长而增加。使用荧光素标记的ISIS 1570来评估在存在和不存在DOTMA的情况下寡核苷酸在细胞内的分布。在不存在DOTMA的情况下，寡核苷酸定位于细胞胞质中的离散结构，产生点状荧光模式。在存在DOTMA的情况下，细胞荧光明显增加，寡核苷酸定位于细胞核内以及胞质中的离散结构。在存在DOTMA的情况下，寡核苷酸在细胞核中的积累是时间和温度依赖性的。用莫能菌素预孵育细胞可抑制核积累，但用氯喹、NH4Cl、诺考达唑、秋水仙酰胺或布雷菲德菌素A预孵育则不能。这些数据表明，阳离子脂质通过增加与细胞结合的寡核苷酸量并改变寡核苷酸的细胞内分布来增加反义活性。
