Corrias M V, Guarnaccia F, Ponzoni M
Laboratory of Oncology, G. Gaslini Children's Hospital, Genoa, Italy.
J Neurooncol. 1997 Jan;31(1-2):171-80. doi: 10.1023/a:1005726623591.
To evaluate the real effectiveness of various chemical modifications in enhancing the ability of antisense molecules to inhibit gene expression, the toxicity, stability, uptake, and intracellular localization of an identical sequence, synthetized either with a phosphodiester or a phosphorothioate backbone, with or without a cholesteryl moiety linked to the 3'-end, were compared in three different human neuroblastoma cell lines. The toxicity, assessed by inhibition of cell viability, greatly depend on the presence of the lipid moiety and to a less extent on the cell line used. At high doses all the antisenses caused a necrotic lysis of plasma membranes. Typical features of apoptotic cell death were never observed. The presence of the lipid moiety enhanced the uptake of antisense molecules while the phosphorothioate backbone, as expected, conferred higher stability. At late times, therefore, the combination of lipid conjugation and phosphorothioate backbone seems to be the most effective in obtaining a consistent antisense accumulation inside the cells. The presence of the cholesteryl moiety also caused a stronger association of the antisense to membraneous compartments, so that a quite different biodistribution occurred among the four antisenses tested. However, the actual amount of antisense molecules found inside NB cells was low in all the conditions tested. Only following cellular permeabilization a significant uptake was obtained, making the use of delivery system mandatory to achieve an efficient inhibition of highly expressed genes.
为了评估各种化学修饰在增强反义分子抑制基因表达能力方面的实际效果，在三种不同的人神经母细胞瘤细胞系中，比较了具有磷酸二酯或硫代磷酸酯骨架、带有或不带有连接到3'-末端的胆固醇部分的相同序列的毒性、稳定性、摄取和细胞内定位。通过抑制细胞活力评估的毒性在很大程度上取决于脂质部分的存在，在较小程度上取决于所使用的细胞系。在高剂量下，所有反义分子都会导致质膜坏死溶解。从未观察到凋亡细胞死亡的典型特征。脂质部分的存在增强了反义分子的摄取，而硫代磷酸酯骨架如预期的那样赋予了更高的稳定性。因此，在后期，脂质缀合和硫代磷酸酯骨架的组合似乎在细胞内获得一致的反义积累方面最有效。胆固醇部分的存在还导致反义分子与膜性区室的更强结合，因此在所测试的四种反义分子之间发生了相当不同的生物分布。然而，在所有测试条件下，在NB细胞内发现的反义分子的实际量都很低。只有在细胞通透后才能获得显著的摄取，这使得使用递送系统成为实现对高表达基因有效抑制的必要条件。
