Cardiovascular and Pulmonary Branch, Division of Intramural Research, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Gene Ther. 2013 Mar;20(3):274-82. doi: 10.1038/gt.2012.38. Epub 2012 May 3.
Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.
杜氏肌营养不良症 (DMD) 心肌病患者目前尚无治疗选择。我们评估了经心内膜导管传递表达与特定顺式作用剪接信号互补的小核 U7 RNA（U7smOPT）的重组腺相关病毒 (rAAV)。消除特定外显子会恢复开放阅读框，从而导致截短的肌营养不良蛋白的翻译。为了在临床上相关的 DMD 模型中测试这种方法，金毛寻回猎犬肌营养不良症 (GRMD) 狗通过冠状动脉内或经心内膜途径接受血清型 6 rAAV-U7smOPT。经心内膜注射使用注射尖端导管和 X 射线融合磁共振成像 (XFM) 路线图进行荧光引导。治疗 3 个月后，对组织进行 DNA、RNA、肌营养不良蛋白和组织学分析。虽然冠状动脉内递送未导致有效转导，但经心内膜注射和 XFM 引导使每个动物可进行 30±10 次非重叠注射。所有测试样本均检测到载体 DNA，范围为每个细胞<1 到>3000 个载体基因组拷贝。RNA 分析、western blot 分析和免疫组织化学分析表明，在治疗的心肌中广泛表达了跳过的 RNA 和肌营养不良蛋白。在 3 个月的随访中，左心室功能保持不变。这些结果表明，XFM 实现了 rAAV-U7smOPT 的有效经心内膜传递。这种方法在受影响的狗中恢复了肌营养不良蛋白的开放阅读框，具有潜在的临床应用价值。
