R16/17-Independent nNOS Anchoring Mechanism

 描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种由肌营养不良蛋白缺失引起的致命性肌肉疾病。肌营养不良蛋白是一种细胞骨架蛋白，将神经元型一氧化氮合酶(NNOS)锚定在肌膜上。如果没有dystrophin，nNOS就会从肌肉细胞膜上消失。肌膜上nNOS的定位对肌肉健康至关重要，因为它允许短暂的血管扩张剂一氧化氮(由nNOS产生)到达周围的血管系统，以对抗运动中的交感血管收缩。在缺乏dystrophin的mdx小鼠和人类患者中，肌膜nNOS的丢失取消了收缩过程中保护性的交感神经溶解，并加剧了肌肉疾病。最近，我们在小鼠中发现了抗肌营养不良蛋白类似的重复序列16和17(R16/17)作为nNOS结合域。R16/17由外显子42至45编码。这些外显子的框内缺失取消了患者肌膜nNOS的表达，表明R16/17也是人类的nNOS结合域。为了研究肌膜nNOS的治疗效果，我们在mdx小鼠中表达了含有或不含有R16/17的合成抗肌营养不良蛋白，发现含有R16/17的抗肌营养不良蛋白(但不含R16/17的)改善了血流，提高了运动能力。眼下的下一个问题是，老鼠的研究结果是否可以推广到大型哺乳动物身上。几个小组试图通过基因-表型分析来梳理出nNOS与人类患者疾病严重程度的相关性。不幸的是，破坏nNOS结合的缺失突变通常会扰乱dystrophin血影蛋白样重复序列的正常阶段。由于这两个因素(nNOS定位和像幽灵蛋白一样的重复阶段)都有助于疾病的严重性，因此将这两种影响分开已经变得极其具有挑战性(如果不是不可能的话)。事实上，公布的结果是有争议的。我们认为，在犬DMD模型中使用正常阶段性肌营养不良蛋白(有或没有nNOS结合域)进行设计良好的血流动力学研究，可以对大型哺乳动物肌膜nNOS的治疗意义产生关键的洞察。因此，我们将含有R16/17的抗肌营养不良蛋白引入到DMD犬肌肉中。出乎意料的是，它没有将nNOS固定在细胞膜上，这表明犬肌营养不良蛋白是独一无二的。在营养不良蛋白中，这种物种特异性差异以前从未观察到过。我们假设nNOS结合区位于犬dystrophin中的不同区域，并进一步假设腺相关病毒(AAV)载体可以将含有nNOS结合区的犬dystrophin基因运送到狗肌肉中。我们的具体目标是：(1)利用载体/AAV基因转移和可逆性纤维表位定位相结合的方法来定位犬dystrophin nNOS结合区；(2)构建携带犬nNOS结合区的犬dystrophin AAV载体。该载体将用于未来的研究，以测试肌膜nNOS是否能使营养不良的大型哺乳动物(受影响的狗)受益。

 DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the loss of dystrophin. Dystrophin is a cytoskeleton protein that anchors neuronal nitric oxide synthase (nNOS) to the sarcolemma. Without dystrophin, nNOS is lost from the muscle cell membrane. Sarcolemmal localization of nNOS is crucial for muscle health because it allows short-lived vasodilator nitric oxide (generated by nNOS) to reach the surrounding vasculature to counteract sympathetic vasoconstriction during exercise. In dystrophin-deficient mdx mice and human patients, the loss of sarcolemmal nNOS abolishes protective sympatholysis during contraction and aggravates muscle disease. We recently identified dystrophin spectrin-like repeats 16 and 17 (R16/17) as the nNOS-binding domain in mice. R16/17 is encoded by exons 42 to 45. In-frame deletion of these exons abolishes sarcolemmal nNOS expression in patients, suggesting R16/17 is also the nNOS-binding domain in human. To study the therapeutic effect of sarcolemmal nNOS, we expressed synthetic dystrophins with or without R16/17 in mdx mice and found that R16/17-containing dystrophins (but not the ones without R16/17) improved blood flow and enhanced exercise performance. The immediate next question is whether the mouse results can be translated to large mammals. Several groups have tried to tease out the relevance of nNOS to disease severity in human patients by genotype-phenotype analysis. Unfortunately, deletion mutations that destroy nNOS binding often disrupt the normal phasing of dystrophin spectrin-like repeats. Since both factors (nNOS localization and spectrin-like repeat phasing) contribute to disease severity, it has become extremely challenging (if not impossible) to separate the two effects. Indeed, the published results are controversial. We reasoned that a well-designed hemodynamic study in the canine DMD model using normally phased dystrophins (with or without the nNOS-binding domain) can yield critical insight on the therapeutic significance of sarcolemmal nNOS in large mammals. Hence, we introduced R16/17-containing dystrophin to DMD dog muscle. Unexpectedly, it did not anchor nNOS to the membrane, suggesting canine dystrophin is unique. Such species-specific difference in dystrophin has never been observed before. We hypothesize that the nNOS-binding domain is located in a different region in dog dystrophin and we further hypothesize that an adeno-associated virus (AAV) vector can be generated to deliver an nNOS-binding domain-containing canine dystrophin gene to dog muscle. Our specific aims are (1) to localize the canine dystrophin nNOS-binding domain using a combinatory approach of plasmid/AAV gene transfer and the epitope mapping of revertant fibers; (2) to develop a dog dystrophin AAV vector that carries the canine nNOS-binding domain. This vector will be used in future studies to test whether sarcolemmal nNOS can benefit a dystrophic large mammal (affected dogs).