Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells
Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells
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DOI：
10.1038/sj.gt.3302313
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发表时间：
2004-09-01
期刊：
影响因子：
5.1
通讯作者：
van Deutekom, JCT
中科院分区：
文献类型：
--
作者：
Aartsma-Rus, A;Kaman, WE;van Deutekom, JCT
作为杜氏肌营养不良症 (DMD) 的小分子药物，反义寡核苷酸 (AON) 已被证明可以通过诱导特定的外显子跳跃来恢复 DMD 转录本被破坏的阅读框。这使得合成具有很大程度上功能性的贝克型肌营养不良症 (BMD) 样肌营养不良蛋白，并有可能将严重的 DMD 转化为较温和的 BMD 表型。到目前为止，我们已经使用了 20-O-甲基硫代磷酸酯 (2OMePS) AON。在这里，我们评估了含有吗啉代磷酸二酰胺、锁核酸 (LNA) 或肽核酸 (PNA) 主链的不同 AON 类似物的跳跃效率。与 PNA 和吗啉相比，LNA 尚未作为剪接调节剂进行测试。与针对外显子 46 的最有效的 2OMePS AON 相比，LNA 在人类对照（85% 对 20%）和外显子 45 缺失 DMD 患者（98% 对 75%）的肌管中诱导了更高的跳跃水平。吗啉诱导的跳跃水平仅为 5 - 6%，而 PNA 似乎无效。对包含多达三个错配的 LNA 和 2OMePS AON 的进一步比较分析表明，LNA 在诱导更高的跳跃效率的同时，显示出较低的序列特异性。这种限制增加了人类基因组其他部位产生不利影响的风险。等待寡化学的进一步改进，因此我们认为 2OMePS AON 目前是最有利的化合物，至少对于目标 DMD 外显子 46 跳跃来说是这样。
As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 20-O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholino-induced skipping levels were only 5 - 6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficiencies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping.