R16/17-Independent nNOS Anchoring Mechanism

 描述（由申请人提供）：杜氏肌营养不良症（DMD）是一种由肌营养不良蛋白缺失引起的致死性肌肉疾病。肌营养不良蛋白是将神经元型一氧化氮合酶（nNOS）锚定到肌膜的细胞骨架蛋白。在没有肌营养不良蛋白的情况下，nNOS从肌细胞膜上丢失。nNOS的肌膜定位对肌肉健康至关重要，因为它允许短寿命的血管扩张剂一氧化氮（由nNOS产生）到达周围的血管系统，以抵消运动期间的交感神经血管收缩。在肌营养不良蛋白缺陷型mdx小鼠和人类患者中，肌膜nNOS的丧失废除了收缩期间的保护性交感神经溶解，并加重了肌肉疾病。我们最近确定了肌营养不良蛋白血影蛋白样重复序列16和17（R16/17）作为nNOS结合结构域在小鼠中。R16/17由外显子42至45编码。这些外显子的框内缺失消除了患者的肌膜nNOS表达，表明R16/17也是人类的nNOS结合结构域。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的合成抗肌萎缩蛋白，并发现含有R16/17的抗肌萎缩蛋白（但不含R16/17的抗肌萎缩蛋白）改善了血流并增强了运动表现。接下来的问题是，小鼠的结果是否可以应用于大型哺乳动物。几个研究小组试图通过基因型-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破坏抗肌萎缩蛋白血影蛋白样重复序列的正常定相。由于这两个因素（nNOS定位和血影蛋白样重复定相）有助于疾病的严重程度，它已成为极具挑战性的（如果不是不可能的），以区分这两种影响。事实上，公布的结果是有争议的。我们推断，在犬DMD模型中使用正常阶段的肌营养不良蛋白（有或没有nNOS结合结构域）进行精心设计的血流动力学研究，可以对大型哺乳动物肌膜nNOS的治疗意义产生重要的见解。因此，我们将含有R16/17的抗肌萎缩蛋白引入DMD狗肌肉。出乎意料的是，它没有锚nNOS的膜，表明犬肌营养不良蛋白是独特的。这种肌营养不良蛋白的物种特异性差异以前从未观察到过。我们假设nNOS结合结构域位于犬肌营养不良蛋白的不同区域，我们进一步假设可以产生腺相关病毒（AAV）载体，以将含有nNOS结合结构域的犬肌营养不良蛋白基因递送至犬肌肉。我们的具体目标是（1）使用质粒/AAV基因转移和回复突变体纤维表位作图的组合方法定位犬抗肌萎缩蛋白nNOS结合结构域;（2）开发携带犬nNOS结合结构域的犬抗肌萎缩蛋白AAV载体。该载体将用于未来的研究，以测试肌膜nNOS是否可以有益于营养不良的大型哺乳动物（受影响的狗）。

 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).