Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration
Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration
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DOI：
10.1073/pnas.0504505102
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发表时间：
2006-01-10
影响因子：
11.1
通讯作者：
Rando, TA
中科院分区：
文献类型：
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作者：
Bertoni, C;Jarrahian, S;Rando, TA
质粒介导的基因治疗可以恢复Duchenne肌营养不良模型mdx小鼠骨骼肌中肌营养不良蛋白的表达。然而，足够的长期表达和分布的肌营养不良蛋白仍然是将这项技术转化为可行的治疗杜氏肌营养不良的障碍。为了改善质粒介导的肌肉疾病基因治疗，我们使用噬菌体整合酶（phi C31）研究了靶向质粒整合的效果，该酶可以介导适当修饰的质粒整合到哺乳动物基因组中。利用荧光素erase表达质粒，利用生物发光成像技术无创地监测了质粒基因在活体小鼠体内的表达。共注射整合酶质粒可使荧光素酶的持续表达水平提高5- 10倍。同样，质粒介导的肌营养不良蛋白在mdx肌肉中的表达通过整合增强。利用营养不良蛋白和荧光素酶质粒的组合，我们分析了营养不良蛋白在营养不良肌肉中的表达的功能益处。肌营养不良蛋白的表达减缓了与肌肉变性相关的荧光素酶表达的丧失，并且通过靶向整合肌营养不良蛋白质粒增强了这种保护作用。在整合酶存在的情况下，肌营养不良蛋白的表达分布在更长的单个纤维上，这与增强的抗退行性变化的保护有关。这些数据表明，肌营养不良蛋白的表达水平和分布对获得治疗效果都很重要，并且可以通过靶向质粒整合来提高疗效。
Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (phi C31) that can mediate the integration of suitably modified plasmids into the mammalian genome. Using a lucif erase expression plasmid, we monitored plasmid gene expression noninvasively in living mice by bioluminescence imaging. Coinjection of an integrase plasmid resulted in 5- to 10-fold higher levels of sustained luciferase expression. Likewise, plasmid-mediated dystrophin expression in mdx muscle was enhanced by integration. Using a combination of dystrophin and luciferase plasmids, we analyzed the functional benefit of dystrophin expression in the dystrophic muscle. The expression of dystrophin slowed the loss of luciferase expression associated with muscle degeneration, and that protection was enhanced by targeted integration of the dystrophin plasmid. In the presence of integrase, dystrophin expression was distributed along a much greater length of individual fibers, and this was associated with increased protection against degenerative changes. These data demonstrate the importance of both the level and distribution of dystrophin expression to achieve therapeutic efficacy, and that the efficacy can be enhanced by targeted plasmid integration.