Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides
Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides
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DOI：
10.1089/104303402317322276
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发表时间：
2002-04-10
影响因子：
4.2
通讯作者：
Rando, TA
中科院分区：
文献类型：
--
作者：
Bertoni, C;Rando, TA
在MDX小鼠和Duchenne肌营养不良症患者中，dystrophin基因点突变会导致dystrophin缺乏和肌营养不良症。作为基因治疗由点突变引起的肌营养不良症的一种方法，我们研究了RNA-DNA嵌合寡核苷酸(嵌合体)诱导MDX小鼠肌营养不良蛋白基因修复的能力。我们先前已经证明，肌肉注射后，靶向嵌合体可以修复分化的肌纤维中外显子23的点突变。为了使肌营养不良患者长期受益，任何基因治疗技术都必须不仅针对分化的肌纤维，还必须针对参与正在进行的肌肉修复的未分化的肌肉前体细胞。目前的研究重点是测试嵌合体是否可以修复MDX肌肉前体细胞中的dystrophin突变。最初的研究是通过将靶向嵌合体导入体外培养的MDX成肌细胞来完成的。在这些细胞中，基因修复在DNA、RNA和蛋白质水平上被证明，而用对照嵌合体处理的细胞不会导致基因校正。在用靶向嵌合体处理的MDX细胞分化后，免疫印迹分析显示全长抗肌营养不良蛋白表达。通过对独立培养细胞的定量分析，dystrophin的表达量在野生型细胞中的2%到15%之间，这为体外基因转化的效果提供了一个衡量标准。为了将评估扩展到体内的肌肉前体细胞，我们将靶向和对照嵌合体注射到mdx小鼠的肌肉中。当肌肉前体细胞随后被注入靶向嵌合体的肌肉时，我们发现这些细胞中也发生了基因修复。综上所述，这些结果进一步表明，嵌合体介导的基因修复可能是一种有效的基因治疗方法，用于由点突变引起的肌营养不良症。
Point mutations in the dystrophin gene cause dystrophin deficiency and muscular dystrophy in the mdx mouse and a subset of patients with Duchenne muscular dystrophy. As an approach to gene therapy for muscular dystrophies due to point mutations, we have studied the ability of RNA-DNA chimeric oligonucleotides (chimeraplasts) to induce repair of the dystrophin gene in mdx mice. We have previously demonstrated that targeting chimeraplasts can repair the exon 23 point mutation in differentiated myofibers in vivo after intramuscular injection. For long-term benefit to patients with muscular dystrophy, any gene therapy technology must target not only differentiated myofibers but also undifferentiated muscle precursor cells that are involved in ongoing muscle repair. The focus of the current studies was to test whether chimeraplasts could repair the dystrophin mutation in mdx muscle precursor cells. Initial studies were done by transfecting a targeting chimeraplast into mdx myoblasts in vitro. Gene repair was demonstrated at the DNA, RNA, and protein levels in these cells, whereas treatment of the cells with a control chimeraplast resulted in no gene correction. After differentiation of mdx cells that had been treated with a targeting chimeraplast, immunoblot analysis demonstrated full-length dystrophin expression. By quantitative analysis of independent cultures, the amount of dystrophin expressed ranged from 2 to 15% of that expressed in wild-type cells, providing a measure of the efficacy of gene conversion in vitro. To extend the assessment to muscle precursor cells in vivo, we injected targeting and control chimeraplasts into muscles of mdx mice. When muscle precursor cells were subsequently derived from muscles injected with a targeting chimeraplast, we found that gene repair had occurred in these cells as well. These results, taken together, further demonstrate that chimeraplast-mediated gene repair may be effective as an approach to gene therapy for muscular dystrophies due to point mutations.