Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy.
Gene Ther. 2010 Mar;17(3):432-8. doi: 10.1038/gt.2009.145. Epub 2009 Nov 12.
Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as alpha-, beta-, gamma- and delta-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.
目前，针对杜氏肌营养不良症（DMD）的潜在可行治疗方法已近在咫尺。实际上，临床试验目前正在进行中。仍有两个关键方面需要解决：最大限度地提高治疗效果和确定适当和合理的疗效指标。然而，这些试验的终点仍然是对接受治疗的男孩进行疼痛的肌肉活检，以显示和量化治疗后蛋白质的恢复情况。在这项研究中，我们表明 PMMA/N-异丙基丙烯酰胺+（NIPAM）纳米颗粒（ZM2）能够非常有效地结合和传递反义寡核苷酸（AONs）。ZM2-AON 复合物的系统注射恢复了 mdx 小鼠骨骼肌和心肌中的肌营养不良蛋白合成，使多达 40%的肌纤维中能够定位蛋白质。通过 RealTime 测定，mdx 外显子 23 跳跃水平高达 20%，并且通过逆转录-PCR 和 Western blot 证实了肌营养不良蛋白的恢复。此外，我们还证实，ZM2-AON 处理的 mdx 小鼠的背侧皮肤竖毛肌平滑肌中也发生了肌营养不良蛋白的恢复，与未处理的动物相比。这一发现表明，竖毛肌平滑肌是一种有吸引力的生物标志物候选物和新的低侵入性治疗终点。此外，该标志物也适合随后监测 DMD 患者的治疗效果。此外，我们还证明了其他几种细胞膜蛋白，如 alpha、beta、gamma 和 delta-肌聚糖，在人类皮肤竖毛肌平滑肌中的表达，从而表明这种肌肉作为其他肌肉营养不良症的生物标志物的潜力，这些肌肉营养不良症目前或即将成为临床试验的对象。
