Targeted exon skipping in transgenic hDMD mice:: A model for direct preclinical screening of human-specific antisense oligonucleotides
Targeted exon skipping in transgenic hDMD mice:: A model for direct preclinical screening of human-specific antisense oligonucleotides
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DOI：
10.1016/j.ymthe.2004.05.031
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发表时间：
2004-08-01
影响因子：
12.4
通讯作者：
van Deutekom, JCT
中科院分区：
文献类型：
--
作者：
Bremmer-Bout, M;Aartsma-Rus, A;van Deutekom, JCT
反义寡核苷酸（AONs）的框架恢复外显子跳跃的治疗潜力最近在一系列杜氏肌营养不良症（DMD）患者的培养肌肉细胞中得到了证明。为了促进临床应用，需要在动物模型中进行体内研究，以开发安全有效的aon给药方法。然而，由于外显子跳跃是一种序列特异性治疗，因此希望直接针对人类DMD基因。因此，我们在携带全尺寸人类基因（hDMD）的转基因小鼠中建立了人类序列特异性外显子跳变。我们首先比较了不同给药试剂对野生型小鼠肌内注射AON的效率和毒性。在3.6 nmol AON剂量和使用聚乙烯亚胺时，注射后第二周跳跃水平累积到3%，持续4周。我们观察到这种长期效应与肌内AON持续存在的相关性。在肌纤维再生中，可以获得更高的效率（高达9%）。最后，在hDMD小鼠中使用优化的方案，我们能够在不影响内源性小鼠基因的情况下诱导人类DMD外显子的特异性跳跃。这些数据突出了该疗法的高序列特异性，并将hDMD小鼠作为优化人类特异性外显子跳跃的独特模型。
The therapeutic potential of frame-restoring exon skipping by antisense oligonucleotides (AONs) has recently been demonstrated in cultured muscle cells from a series of Duchenne muscular dystrophy (DMD) patients. To facilitate clinical application, in vivo studies in animal models are required to develop safe and efficient AON-delivery methods. However, since exon skipping is a sequence-specific therapy, it is desirable to target the human DMD gene directly. We therefore set up human sequence-specific exon skipping in transgenic mice carrying the full-size human gene (hDMD). We initially compared the efficiency and toxicity of intramuscular AON injections using different delivery reagents in wild-type mice. At a dose of 3.6 nmol AON and using polyethylenimine, the skipping levels accumulated up to 3% in the second week postinjection and lasted for 4 weeks. We observed a correlation of this long-term effect with the intramuscular persistence of the AON. In regenerating myofibers higher efficiencies (up to 9%) could be obtained. Finally, using the optimized protocols in hDMD mice, we were able to induce the specific skipping of human DMD exons without affecting the endogenous mouse gene. These data highlight the high sequence specificity of this therapy and present the hDMD mouse as a unique model to optimize human-specific exon skipping in vivo.