Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries
Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries
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DOI：
10.1186/1471-2199-8-57
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发表时间：
2007-07-02
影响因子：
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通讯作者：
Wilton, Steve D.
中科院分区：
文献类型：
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作者：
Adams, Abbie M.;Harding, Penny L.;Wilton, Steve D.
背景资料：反义寡核苷酸（AO）可以干扰前体mRNA加工过程中的外显子识别和内含子去除，并诱导成熟基因转录物中靶向外显子的切除。AO已在体外和体内用于重定向人和动物细胞中的肌营养不良蛋白前mRNA加工。靶向外显子跳跃选择外显子的肌营养不良蛋白基因转录本可以删除无义或移码突变，否则会导致杜氏肌营养不良症，最常见的儿童形式的肌肉wasting.Results：虽然许多肌营养不良蛋白外显子可以切除使用一个单一的AO，几个外显子需要两个图案被掩盖的有效或特定的外显子跳跃。当单独应用时，一些AO是无活性的，但是当AO以选择组合使用时，在转染的细胞中诱导明显的外显子切除，清楚地表明对剪接的协同效应而不是累积效应。用2种不同的化学物质，硫代磷酸酯骨架上的2 '-O-甲基修饰碱基和磷酰二胺吗啉代寡聚物，观察到AO混合物诱导有效外显子去除的必要性。类似地，当将相应序列制备为磷酰二胺吗啉代寡聚体时，也观察到外显子跳跃的其他趋势，作为2 '-O-甲基AO作用的结果，例如去除额外的侧翼外显子或重叠AO的外显子跳跃效率的变化。两个AO的组合，发现靶向外显子中的适当基序，在肌营养不良蛋白前体mRNA的加工过程中诱导非常有效的靶向外显子跳跃。这种组合效应显然是协同的，并且不受用于诱导外显子切除的AO的化学性质的影响。当将这些相同的序列作为磷酰二胺吗啉代寡聚体进行评价时，也观察到外显子跳跃效率的层次结构，该层次结构由2 '-O-甲基修饰的碱基组成的重叠AO观察到，这表明用一种化学方法建立的设计参数可以应用于另一种。
Background: Antisense oligonucleotides ( AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame- shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting.Results: Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers.Conclusion: The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other.