Exploring the role of hypoxia signaling and its inhibition as a therapy for Facioscapulohumeral muscular dystrophy

探索缺氧信号传导及其抑制作为面肩肱型肌营养不良症治疗的作用

基本信息

批准号：
10599956
负责人：
Justin Cohen
金额：
$ 7.38万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10599956
关键词：
ACTA1 gene Affect Apoptosis Attenuated Autophagocytosis Biological Assay Biological Markers Biopsy CRISPR screen CRISPR-mediated transcriptional activation CRISPR/Cas technology Cell Death Cell Death Induction Cell Death Inhibition Cell Line Chromosome 4 Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Complex D4Z4 Disease Embryonic Development Energy Metabolism Epigenetic Process FDA approved FRAP1 gene Facioscapulohumeral Muscular Dystrophy Functional disorder Gene Activation Generations Genes Genetic Genetic Diseases Genetic Engineering Genetic Screening Genetic Transcription Genus Hippocampus Goals HIF1A gene Hand Strength Haplotypes Head Histology Human Hypoxia Hypoxia Pathway Immune Individual Knock-out Knowledge Lifting Link Measurement Measures Mediating Metabolic Metabolic dysfunction Microscopy Modeling Molecular Mus Muscle Muscle Cells Muscle Contraction Muscle function Myopathy Neuromuscular Diseases Oxidative Phosphorylation Oxidative Stress Oxygen Consumption PIK3CG gene Pathogenicity Pathologic Pathology Pathway interactions Patients Phenotype Physiological Prevalence Proteins Quality of life Reactive Oxygen Species Reporting Reproducibility Research Role SDZ RAD Screening Result Signal Pathway Signal Transduction Skeletal Muscle Techniques Testing Therapeutic Therapeutic Agents Tissues Titrations Toxic effect Transcript Transgenes Transplantation Wait Time Xenograft procedure arm derepression design epigenetic silencing experience extracellular gene therapy genome-wide hypoxia inducible factor 1 in vivo inducible Cre knock-down loss of function mTOR Inhibitor mTOR inhibition metabolic profile mouse model muscle physiology muscular dystrophy mouse model mutation screening new therapeutic target novel overexpression permissiveness pharmacologic pre-clinical assessment prevent promoter response skeletal muscle wasting small molecule small molecule inhibitor therapeutic evaluation therapeutic target therapy development transcription factor transcriptome sequencing treadmill

项目摘要

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder for which there is currently no treatment or cure. Affected individuals have difficulty accomplishing everyday tasks such as lifting one’s arms above their head, impacting quality of life. Much research has been done trying to elucidate the mechanism of disease pathology but there has been little congruence apart from apoptosis via DUX4, the toxic protein associated with FSHD. We took advantage of this phenotype to design a genome-wide complimentary CRISPR-Cas9 loss of function and activation screens for genetic modifiers of DUX4 toxicity. These screens identified hypoxia signaling as a novel pathway relating to FSHD pathology with therapeutic potential. Small molecule inhibitors that target modifiers of this pathway, including the FDA-approved compound everolimus, reduced DUX4-mediated cell death and FSHD biomarkers, demonstrating the viability of targeting hypoxia signaling as a potential therapy. In this project we intend to further explore the mechanism of how hypoxia signaling relates to DUX4-mediated pathology (Specific aims 1). The PI3K/Akt/mTOR signaling axis that interacts with hypoxia signaling will be modulated through knock-down and/or pharmacological inhibition for their effect on DUX4-mediated apoptosis. The consequence of autophagy modulation through the mTOR inhibitor everolimus will be explored. The relationship between DUX4-induced hypoxia and metabolic dysfunction will be assessed through RNAseq, Seahorse assays of oxygen consumption and extracellular acidification rates, modulation of oxidative phosphorylation and reactive oxygen species generation. We will additionally test the therapeutic potential of everolimus in a DUX4-inducible mouse model of FSHD (Specific aims 2). We will measure effects on muscle function through treadmill endurance, grip strength and ex vivo contractile force. Using microscopy, we will examine effects on muscle histology and markers of hypoxic signaling while exploring if this pathology is affected by the metabolic profile of the individual muscles. Lastly, effect on FSHD biomarkers will be assessed both in the mouse model and validated using patient biopsy myocytes. If successful, this project will elucidate a new mechanism of FSHD pathology for therapeutic targeting and identify everolimus as a therapeutically promising compound whose FDA status allows for a shorter timeframe before patient availability.

抽象的 facioscapulohumeral肌肉营养不良（FSHD）是一种神经肌肉疾病，目前没有该疾病治疗或治愈。受影响的人很难完成每天的任务，例如举起武器在他们的头顶上，影响着生活质量。已经做了很多研究，以阐明疾病病理学，但除了通过dux4（有毒蛋白）凋亡外，几乎没有一致性与FSHD相关。我们利用这种表型设计了全基因组的免费 CRISPR-CAS9功能和激活筛选的遗传修饰剂的遗传修饰剂的损失。这些屏幕鉴定出缺氧信号传导是与FSHD病理有关的新途径，具有治疗潜力。小的靶向该途径的修饰剂的分子抑制剂，包括FDA批准的化合物依维莫司（Everolimus）降低了DUX4介导的细胞死亡和FSHD生物标志物，证明了靶向缺氧的生存能力信号传导是一种潜在的疗法。在这个项目中，我们打算进一步探讨缺氧的机制与DUX4介导的病理有关的信号传导（特定目的1）。 PI3K/AKT/MTOR信号传导轴与缺氧信号相互作用将通过敲击和/或药理抑制来调节它们对DUX4介导的细胞凋亡的影响。通过MTOR自噬调制的结果将探索抑制剂依维莫司。 DUX4诱导的缺氧与代谢之间的关系功能障碍将通过RNASEQ，氧气消耗和细胞外的海马测定法进行评估酸化速率，氧化物磷酸化的调节和活性氧的产生。我们将另外，在DUX4诱导小鼠FSHD中测试依维莫司的治疗潜力（特定目标2）。我们将通过跑步机耐力，抓地力和离体来衡量对肌肉功能的影响收缩力。使用显微镜，我们将检查对肌肉组织学和低氧标志的影响在探索该病理学是否受单个肌肉的代谢特征影响时发出信号。最后，对FSHD生物标志物的影响将在小鼠模型中进行评估，并使用患者活检进行验证心肌细胞。如果成功，该项目将阐明FSHD病理的新机制靶向并识别依依他木斯是一种具有治疗有希望的化合物，其FDA状态允许患者可用性之前的时间范围较短。