R16/17-Independent nNOS Anchoring Mechanism

R16/17 独立的 nNOS 锚定机制

基本信息

批准号：
9035085
负责人：
Dongsheng Duan
金额：
$ 19.45万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9035085
关键词：
Address Affect Animal Model Attenuated Binding Biological Assay Biology Blood flow Budgets Canis familiaris Cell membrane Childhood Clinical Data Complementary DNA Cytoskeleton Data Deletion Mutation Dependovirus Duchenne muscular dystrophy Dystrophin Engineering Epitope Mapping Exercise Exons Fiber Figs - dietary Funding Future Gene Delivery Gene Transfer Genes Genetic screening method Goals Hand Health Human In Situ Ischemia Length Life Limb structure Mammals Maps Mediating Membrane Modeling Mus Muscle Muscle Cells Muscular Dystrophies Myopathy Nitric Oxide Nitric Oxide Synthase Type I Patients Performance Phase Phenotype Plasmids Proteins Protocols documentation Publishing Recruitment Activity Review Literature Sarcolemma Series Severity of illness Spectrin Symptoms Testing Therapeutic Therapeutic Effect Therapeutic Studies Time Transfection Translating Vasodilator Agents adeno-associated viral vector design gene therapy genotyped patients hemodynamics improved in vivo insight mdx mouse mini-dystrophin novel public health relevance restoration translational study vasoconstriction vector

项目摘要

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).

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命性肌肉疾病，肌营养不良蛋白是将神经元一氧化氮合酶 (nNOS) 锚定在肌膜上的细胞骨架蛋白，如果没有肌营养不良蛋白，nNOS 就会缺失。 nNOS 的肌膜定位对于肌肉健康至关重要，因为它可以产生短暂的血管舒张作用。在肌营养不良蛋白缺乏的 mdx 小鼠和人类患者中，肌膜 nNOS 的丧失会消除收缩过程中的保护性交感神经作用，并加剧肌肉疾病。就像小鼠中 nNOS 结合域的重复序列 16 和 17 (R16/17) 一样。 R16/17 由外显子 42 至 45 编码。这些外显子的框内删除消除了患者肌膜 nNOS 的表达，表明 R16/17 也是人类 nNOS 结合域。为了研究肌膜 nNOS 的治疗效果，我们表达了 R16/17。在 mdx 小鼠中合成含有或不含 R16/17 的肌营养不良蛋白，并发现含有 R16/17 的肌营养不良蛋白（但不含有 R16/17 的肌营养不良蛋白） R16/17）改善了血液流动并增强了运动表现，接下来的问题是小鼠的结果是否可以转化为大型哺乳动物。一些研究小组试图通过基因型-表型分析来弄清楚 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肌营养不良蛋白血影蛋白样重复的正常定相，因为这两个因素（nNOS 定位和血影蛋白样重复定相）都会导致疾病严重程度，因此它已变得极其严重。事实上，我们认为，使用正相肌营养不良蛋白（有或没有 nNOS 结合域）在犬 DMD 模型中进行精心设计的血流动力学研究可能会产生不同的结果。因此，我们将含有 R16/17 的肌营养不良蛋白引入到 DMD 狗的肌肉中，但出乎意料的是，它并没有将 nNOS 锚定在其上。膜，表明犬肌营养不良蛋白是独特的。我们以前从未观察到肌营养不良蛋白的这种物种特异性差异，我们发现，nNOS 结合域位于狗肌营养不良蛋白的不同区域，并且我们进一步发现，腺相关病毒（可以生成 AAV）载体，将含有 nNOS 结合域的犬肌营养不良蛋白基因递送至狗的肌肉。我们的具体目标是 (1) 使用组合来定位犬肌营养不良蛋白 nNOS 结合域。质粒/AAV基因转移方法和回复纤维表位作图；（2）开发携带犬nNOS结合域的狗肌营养不良蛋白AAV载体，该载体将用于测试肌膜nNOS是否有益于肌营养不良蛋白AAV载体。营养不良的大型哺乳动物（受影响的狗）。