Unveiling the molecular mechanisms in TPM3-related myopathy and therapies

项目摘要 常染色体显性遗传性TPM3相关肌病主要由TPM3基因错义突变引起 这会导致肌肉萎缩和虚弱，并严重影响患者的生活质量。到目前为止，还没有 TPM3相关性肌病的治疗。 K99指导阶段：目前，缺乏有代表性的动物模型系统限制了 TPM3相关肌病的新病理生理学发现以及潜在的治疗方法。要克服这一点 GAP，我们最近产生了基于CRISPR的TPM3小鼠，它们携带最常见的突变之一 在病人身上见过。我们的目标是(1)研究人源化小鼠模型的肌肉病理学。 TPM3相关性肌病，以及(2)解剖慢、快肌核转录变化 人口。此外，我们还鉴定了一种缺乏tpm3的斑马鱼，表现出类似的特征。 肌病的病理生理学特征与互补性疾病的发展前景 这种方法在哺乳动物模型系统中是不可想象的。我们的目标也是(3)产生 基于CRISPR的tpm3斑马鱼携带在患者中看到的相同的最普遍的突变。 R00独立期：斑马鱼成为加速发现潜力的有力典范 治疗学。我们的目标是(4)建立一种针对tpm~3斑马鱼的药物筛选方法。等位基因特异性RNA 干预也已成为治疗显性遗传病(如与TPM3相关的疾病)的有效策略 肌病。根据我们的初步数据，TPM3单倍体不足不是可能的潜在机制 肌病。为了补充我们在斑马鱼上的研究，我们的目标是筛选抑制基因表达的siRNA。 在体外实验中不降低野生型等位基因的突变型等位基因的表达，并测试其在 在我们的TPM3小鼠体内。 我的长期目标是成为一名独立的研究员，拥有一个结合了基因和 研究神经肌肉疾病和开发治疗方法的分子工具。在K99期间 在指导阶段，我将在概念、技术和职业发展方面获得更多技能，这将 使我能够在我自己的研究小组中成功地过渡到一个独立的职位。 我的短期目标是(1)在研究肌肉病理学的实验程序中获得更多的技能 在老鼠和斑马鱼方面，(2)获得大规模数据分析方面的专业知识，(3)提高我在 设计神经肌肉疾病的治疗策略，(4)获得独立的终身教职轨道 助理教授职位，以及(5)在本提案提出后5年内成功获得R01资金。

Project Summary Autosomal dominant TPM3-related myopathy is primarily caused by missense mutations in the TPM3 gene that lead to muscle atrophy and weakness, and substantially affect patient’s quality of life. So far, there is no treatment for TPM3-related myopathy. K99 mentored phase: Currently, the absence of representative animal model systems limits the emergence of new pathophysiological findings in TPM3-related myopathy as well as potential therapeutics. To overcome this gap, we have recently generated CRISPR-based Tpm3 mice that carry one of the most prevalent mutations seen in patients. Our objectives are (1) to study the muscle pathology in the humanized mouse model of TPM3-related myopathy, and (2) to dissect the transcriptional changes in slow and fast myonuclear populations. Furthermore, we have characterized a tpm3-deficient zebrafish that show similar pathophysiological features of myopathy and holds promise for the development of complementary approaches that would be inconceivable with mammalian model systems. We also aim (3) to generate CRISPR-based tpm3 zebrafish that carry the same most prevalent mutation seen in patients. R00 independent phase: Zebrafish have become a powerful model to accelerate the discovery of potential therapeutics. Our objective is (4) to develop a drug screening assay in tpm3 zebrafish. Allele-specific RNA interference has also emerged as a powerful strategy for dominant inherited diseases such as TPM3-related myopathy. Based on our preliminary data, TPM3 haploinsufficiency is not the likely underlying mechanism of the myopathy. To complement our study in zebrafish, we aim (5) to screen for siRNAs that suppress the mutant allele expression without reducing the wild-type allele in in vitro experiments and to test their efficacy in vivo in our Tpm3 mouse. My long-term goal is to become an independent investigator with a lab that combines the genetic and molecular tools to study the neuromuscular diseases and develop therapeutic approaches. During the K99 mentored phase, I will gain additional skills in conceptual, technical and career development aspects which will enable me to make a successful transition to an independent position with my own research group. My short-term goals are (1) to acquire further skills in experimental procedures to study the muscle pathology in mouse and zebrafish, (2) to gain expertise in large-scale data analysis, (3) to improve my knowledge in the design of therapeutic strategies for neuromuscular disorders, (4) to obtain an independent tenure-track assistant professor position, and (5) to successfully obtain R01 funding within 5 years of this proposal.