We report the evaluation of 18-mer 2′-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5′ end to the oligospermine units (S_n-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S₂₀-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S₁₅- and S₂₀-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S₂₀-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid’s outer layer.

我们报告了18-mer 2'- O-甲基修饰的核糖寡核苷酸的评估，其中全长硫代磷酸酯骨架在5'端化学偶联到了低精胺单元上（S _n-：n = 5、15、20、25 ，和30 [精胺单位数]）作为剪接转换寡核苷酸（SSO）。这些缀合物在其结构中包含共价连接的寡阳离子部分，从而使其能够穿透细胞而无需转染载体。在细胞培养中，我们观察到荧光素标记的S _20的有效胞质和核传递-SSO通过荧光显微镜检查。在不存在和存在血清的情况下，在纳摩尔浓度下，含有超过15个精胺单元的SSO缀合物诱导出显着的无载体外显子跳跃。随着精胺单位数量的增加，结合物变得略有毒性，但活性更高。这些分子的优势在模拟活体组织的三维（3D）细胞培养（多细胞肿瘤球体[MCTS]）中得到了特别证明。与单层培养中的测定相比，载体复合的SSO在MCTS中显示出大大减少的剪接转换，而低聚精胺接枝的SSO观察到有效的外显子跳跃而没有明显的毒性（S _15-和S ₂₀-SSOs），无需载体即可转染。通过流式细胞术和共聚焦显微镜显示，荧光素标记的S ₂₀ -SSO可自由扩散并穿透MCTS的最内层细胞，而复合载体的SSO仅穿透球状体外层的细胞。