Abstract：
Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by the absence of functional dystrophin. There are multiple ongoing clinical trials for DMD that are testing gene therapy treatments consisting of adeno-associated viral (AAV) vectors carrying miniaturized versions of dystrophin optimized for function, termed micro-dystrophins (μDys). Utrophin, the fetal homolog of dystrophin, has repeatedly been reported to be upregulated in human DMD muscle as a compensatory mechanism, but whether µDys displaces full-length utrophin is unknown. In this study, dystrophin/utrophin-deficient mice with transgenic overexpression of full-length utrophin in skeletal muscles were systemically administered low doses of either AAV6-CK8e-Hinge3-µDys (μDysH3) or AAV6-CK8e-μDys5 (μDys5). We used immunofluorescence to qualitatively assess the localization of μDys with transgenic utrophin and neuronal nitric oxide synthase (nNOS) in quadriceps muscles. μDys protein resulting from both gene therapies co-localized at myofiber membranes with transgenic utrophin. We also confirmed the sarcolemmal co-localization of nNOS with μDys5, but not with transgenic utrophin expression or μDysH3. Transgenic utrophin expression and μDys proteins produced from both therapies stabilize the dystrophin-glycoprotein complex as observed by sarcolemmal localization of β-dystroglycan. This study suggests that µDys gene therapy will likely not inhibit any endogenous compensation by utrophin in DMD muscle.
摘要：
杜氏肌营养不良(DMD)是一种由功能性肌营养不良蛋白缺失引起的破坏性肌肉疾病。目前正在进行的多项DMD临床试验正在测试基因疗法，这些疗法由腺相关病毒（AAV）载体组成，这些载体携带针对功能进行优化的肌营养不良蛋白的小型化版本。称为微肌营养不良蛋白(μDys)。Utrophin,肌营养不良蛋白的胎儿同源物，反复报道，作为一种代偿机制，在人类DMD肌肉中被上调，但μDys是否置换全长的乌托芬是未知的。在这项研究中,在骨骼肌中具有全长乌托素转基因过表达的肌营养不良蛋白/乌托素缺陷小鼠全身施用低剂量的AAV6-CK8e-Hinge3-µDys（μDysH3）或AAV6-CK8e-μDys5（μDys5）。我们使用免疫荧光定性评估了μDys在股四头肌中的转基因乌托素和神经元一氧化氮合酶（nNOS）的定位。由两种基因疗法产生的μDys蛋白与转基因乌托邦蛋白共定位在肌纤维膜上。我们还证实了nNOS与μDys5的肌膜共定位，但与转基因乌托邦蛋白表达或μDysH3无关。从两种疗法产生的转基因乌托邦蛋白表达和μDys蛋白稳定了肌营养不良蛋白-糖蛋白复合物，如通过β-肌营养不良聚糖的肌膜定位所观察到的。这项研究表明，μDys基因治疗可能不会抑制DMD肌肉中utrophin的任何内源性补偿。
