Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients
Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients
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DOI：
10.1093/hmg/ddg100
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发表时间：
2003-04-15
影响因子：
3.5
通讯作者：
van Deutekom, JCT
中科院分区：
文献类型：
--
作者：
Aartsma-Rus, A;Janson, AAM;van Deutekom, JCT
Duchenne肌营养不良症(DMD)患者的肌营养不良蛋白缺乏导致严重进行性肌肉变性是由DMD基因的移码突变引起的。我们正在开发一种阅读框架校正疗法，旨在反义诱导跳过前mRNA中的靶向外显子。尽管引入了(更大的)缺失，但仍产生了一个框内转录本，允许合成略微较短但功能较强的肌营养不良蛋白，就像在最轻微的Becker肌营养不良症(BMD)中发现的那样。我们最近已经证明了在对照肌肉细胞中反义诱导跳过DMD转录本中的许多外显子的有效性和高效性。原则上，这将在莱顿DMD突变数据库中报告的超过75%的患者中恢复阅读框架。在这项研究中，我们实际上在6名携带不同缺失和无义突变的DMD患者的培养肌肉细胞中展示了这一策略的广泛治疗适用性。在每种情况下，目标外显子的特定跳跃都被诱导，在超过75%的细胞中恢复了抗肌营养不良蛋白的合成。该蛋白在转染后16h即可检测到，然后在2天内在细胞膜上增加到显著水平，并保持至少一周。最后，从Dystrophin-糖蛋白复合体中恢复了四个相关蛋白的膜表达，进一步表明了它的正确功能。这些结果证明了临床适用的基于小分子的治疗方法的重要进展。
The dystrophin deficiency leading to the severely progressing muscle degeneration in Duchenne muscular dystrophy (DMD) patients is caused by frame-shifting mutations in the DMD gene. We are developing a reading frame correction therapy aimed at the antisense-induced skipping of targeted exons from the pre-mRNA. Despite introducing a (larger) deletion, an in-frame transcript is generated that allows the synthesis of a slightly shorter, but largely functional dystrophin as found in the mostly milder Becker muscular dystrophy (BMD). We have recently demonstrated both the efficacy and,high efficiency of the antisense-induced skipping of numerous exons from the DMD transcript in, control muscle cells. In principle, this would restore the reading frame in over 75% of the patients reported in the Leiden DMD mutation database. In this study, we in fact demonstrate the broad therapeutic applicability of,this strategy in cultured muscle cells from six DMD patients carrying different deletions and a nonsense mutation. In each case, the specific skipping of the targeted exon was induced, restoring dystrophin synthesis in over 75% of cells. The protein was detectable as soon as 16 h post-transfection, then increased to significant levels at the membrane within 2 days, and was maintained for at least a week. Finally, its proper function was further suggested by the restored membranal expression of four associated proteins from the dystrophin-glycoprotein complex. These results document important progress towards a clinically applicable, small-molecule based therapy.