Quantitative imaging of the dystrophin-glycoprotein complex

肌营养不良蛋白-糖蛋白复合物的定量成像

• 批准号: 10011760
• 负责人: Philip Doble
• 金额: $ 15.74万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-06 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011760
• 关键词: Affect; Antibodies; Becker Muscular Dystrophy; Benchmarking; Biological; Biopsy; Birth; Clinical; Clinical Research; Complex; DNA; Data; Development; Drug Approval; Duchenne muscular dystrophy; Dystroglycan; Dystrophin; Engineering; FDA approved; Fiber; Fibrosis; Foundations; Genes; Genetic Diseases; Glycoproteins; Human; Image; Immunofluorescence Immunologic; Immunohistochemistry; Impairment; Individual; Intercalating Agents; Intervention; Label; Lectin; Mass Spectrum Analysis; Measures; Methods; Mus; Muscle; Muscle Fibers; Mutation; Myosin ATPase; Outcome Measure; Patients; Proteins; Public Health; Quality Control; Rare Earth Metals; Reagent; Resources; SSPN gene; Sampling; Solid; Speed; Structure; Testing; Therapeutic Intervention; Tissues; Transgenic Mice; Transgenic Organisms; Validation; Western Blotting; clinical translation; clinically relevant; clinically translatable; drug efficacy; improved; male; mdx mouse; medication safety; member; mouse model; multiplexed imaging; muscle degeneration; mutant; novel; novel strategies; protein complex; quantitative imaging; reconstitution

PROJECT SUMMARY Many exciting therapeutic interventions for Duchenne muscular dystrophy are emerging, but the challenges of quantitating functional dystrophin are complicating drug approvals and delaying the pace of clinical translation. To overcome this obstacle, we are developing a multiplexed imaging-mass spectrometry (I-MS) method to simultaneously quantitate and localize dystrophin and other members of the DGC in single muscle sections. Our preliminary data showing successful quantitation and localization of dystrophin and myosin in mouse muscle sections supports the feasibility of our proposed approach. This approach improves on the quantitation of Western blots and simultaneously provides the localization of dystrophin and other DGC proteins that was previously done by separate immunohistochemistry. Specific Aim 1: Development and validation of rare earth element labeled antibodies for simplex imaging-mass spectrometry of dystrophin and other DGC components. 32 primary antibodies, DNA intercalators or lectins will be labeled with specific REEs. Each REE-labeled antibody will be validated individually by comparing Western blot, immunofluorescence and I-MS using wild-type, heterozygous mdx, homozygous mdx mouse and normal human muscle samples. This aim will: 1) develop a robust pipeline for antibody labeling, quality control and validation for use in I-MS, 2) benchmark this new approach to conventional approaches, and 3) provide the solid foundation required for multiplex I-MS in Aim 2. Specific Aim 2: Multiplex imaging-mass spectrometry of dystrophin and other DGC components. We will assemble a panel of the validated antibodies from Aim 1 and use them to simultaneously measure dystrophin and other DGC components in single muscle tissue sections. We have selected tissues of varying dystrophin and DGC component levels including: 1) transgenic mouse models engineered for increasing levels of human sarcospan, 2) patient carriers of a mutant dystrophin gene and 3) patients with Becker's muscular dystrophy. This aim will: 1) optimize and validate the multiplexing of reagents from Aim 1, 2) test the sensitivity of this approach on clinically relevant samples and 3) demonstrate clinical translatability. Our novel application of imaging-mass spectrometry will facilitate the absolute quantitation and sarcolemmal localization of functional dystrophin and up to 32 DGC members in very small muscle samples. Clinical research in DMD will benefit from the ability to accurately quantitate and image functional dystrophin and the DGC, which will make the best use of valuable patient resources, provide improved information on drug efficacy and safety, and speed the pace of clinical translation.

项目总结 针对Duchenne肌营养不良症的许多令人兴奋的治疗干预措施正在涌现，但 功能性肌营养不良蛋白的量化使药物审批复杂化，并延缓了临床翻译的步伐。 为了克服这一障碍，我们正在开发一种多路成像-质谱仪(I-MS)方法来 同时在单个肌肉切片中定量和定位dystrophin和DGC的其他成员。 我们的初步数据显示，在小鼠体内成功地定量和定位了肌营养不良蛋白和肌球蛋白 肌肉切片支持我们提出的方法的可行性。这种方法改进了量化方法。 并同时提供了dystrophin和其他DGC蛋白的定位，这是 以前由单独的免疫组织化学完成。 特异性目标1：单纯性肺炎稀土标记抗体的研制与验证 Dstrophin和其他DGC组分的成像-质谱学。32种一抗，DNA 嵌入剂或凝集素将被标记上特定的稀土。每个REE标记的抗体都将得到验证 通过比较野生型、杂合型MDX的蛋白质印迹、免疫荧光和I-MS， 纯合MDX小鼠和正常人类肌肉样本。这一目标将：1)开发一条强大的管道，以 用于I-MS的抗体标记、质量控制和验证，2)基准这一新方法 传统方法，以及3)为目标2中的多路I-MS提供所需的坚实基础。 特定目标2：Dstrophin和其他DGC组分的多重成像-质谱学。我们 将组装一组来自Aim 1的有效抗体，并用它们同时测量 肌营养不良蛋白和其他DGC组分在单个肌肉组织切片中。我们选择了各种不同的组织 Dstrophin和DGC成分水平包括：1)为提高水平而设计的转基因小鼠模型 2)突变的Dstrophin基因携带者和3)Becker肌肉患者 营养不良。这一目标将：1)优化和验证来自目标1的试剂的多重；2)测试灵敏度 这种方法在临床相关样本上的应用，以及3)证明了临床可译性。 我们的新的成像-质谱学的应用将促进绝对定量和肌膜 在非常小的肌肉样本中定位功能性肌营养不良蛋白和多达32个DGC成员。临床 DMD的研究将受益于准确定量和成像功能性Dstrophin和 DGC将充分利用宝贵的患者资源，提供更好的药物信息 有效性和安全性，加快了临床翻译的步伐。