Development of an osteoarthritis drug screening platform using engineered stem cell lines to identify novel mechanisms of disease biology

使用工程干细胞系开发骨关节炎药物筛选平台，以确定疾病生物学的新机制

• 批准号: 2753493
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2753493
• 关键词: Development; osteoarthritis; drug; screening; platform

BackgroundOsteoarthritis (OA) is a painful and disabling disease of articulating joints with substantial global healthcare and socioeconomic burden. Management of OA is primarily limited to pain relief. There are no approved disease-modifying OA drugs (DMOADs), hampered by the lack of appropriate pre-clinical test systems. We have developed an immortalised human clonal mesenchymal stem cell (MSC) line capable of differentiating into bone-forming osteoblasts and cartilage-forming chondrocytes, the primary joint tissues affected in OA. From this MSC line we engineered fluorescent and secreted luciferase reporters of osteogenic and chondrogenic differentiation status (alkaline phosphatase and type II collagen promoter-driven reporters respectively) and inflammatory responses (NF-KB promoter-driven reporter). Here, we will determine the feasibility of developing the MSC-based reporter system as a screening platform to identify DMOADs. ObjectivesValidate existing MSC reporter linesEngineer a new dual (health/disease) reporter lineTest reporters in low- and medium-throughout screensFunctionally test compounds identifiedDevelop screening platform for scale-up and commercialisationExperimental ApproachWe will undertake a comprehensive validation of our existing reporter lines to characterise both in vivo translatability (via RNAseq) and applicability to use in scaled screening (via robustness testing against chemical compound training sets). We will engineer a dual reporter for simultaneous readouts of health (aggrecan promoter reporter) and disease (ADAMTS5 promoter reporter). Screening using this dual reporter, has the potential to identify pathways that determine the balance of tissue formation versus degradation, which drives OA pathogenesis. We will identify a selection of regulators of osteogenesis and chondrogenesis using a small, well-annotated set of chemical modifiers in conjunction with CRISPR/Cas-9 gene editing and/or RNA silencing. This will define a wider set of chemicals, which will enable us to demonstrate proof-of-concept in medium-throughput assays and provide mechanistic insight into the regulation of MSC differentiation. Compounds identified by these screens will be taken forward for in-depth analysis of effects in osteogenic and chondrogenic differentiation assays. Later work will focus on scale-up, throughput and commercialisation with GSK as the first potential end-users. TimelinessThe project addresses a key gap in efforts to tackle an urgent yet unmet global healthcare need. The incidence of OA, currently estimated at 250 million affected individuals, is predicted to rise to become a leading cause of disability worldwide.NoveltyThe research tools are unique. The proposed work has the potential to identify novel mechanisms responsible for joint tissue health and provide a highly valuable resource of OA drug discovery.

骨关节炎（Osteoarthritis，OA）是一种疼痛性关节疾病，给全球带来巨大的医疗和社会经济负担。OA的管理主要限于疼痛缓解。由于缺乏适当的临床前测试系统，没有批准的疾病修饰OA药物（DMOAD）。我们已经开发了一种永生化的人克隆间充质干细胞（MSC）系，能够分化成骨形成成骨细胞和软骨形成软骨细胞，OA中受影响的主要关节组织。从这个MSC系，我们工程化成骨和软骨分化状态的荧光和分泌的荧光素酶报告基因（分别为碱性磷酸酶和II型胶原启动子驱动的报告基因）和炎症反应（NF-κ B启动子驱动的报告基因）。在这里，我们将确定开发基于MSC的报告系统作为筛选平台来识别DMOAD的可行性。目标扩展现有的MSC报告器线路设计一个新的双（健康/疾病）报告基因线在低通量和中通量筛选中测试报告基因识别的功能测试化合物开发用于规模扩大和商业化的筛选平台实验方法我们将对现有报告基因线进行全面验证，以验证体内可翻译性和（通过RNAseq）和在规模化筛选中使用的适用性（通过针对化合物训练集的稳健性测试）。我们将设计一种双报告基因，用于同时读出健康（聚集蛋白聚糖启动子报告基因）和疾病（ADAMTS 5启动子报告基因）。使用这种双重报告基因进行筛选，有可能确定决定组织形成与降解平衡的途径，从而驱动OA发病机制。我们将使用一组小的、注释良好的化学修饰剂结合CRISPR/Cas-9基因编辑和/或RNA沉默来确定骨生成和软骨生成的调节剂。这将定义一组更广泛的化学物质，这将使我们能够在中等通量测定中证明概念验证，并提供对MSC分化调控的机制见解。通过这些筛选鉴定的化合物将被用于深入分析成骨和软骨分化试验中的作用。后期工作将集中在规模扩大，吞吐量和商业化与GSK作为第一个潜在的最终用户。及时性该项目解决了在努力解决紧迫但未满足的全球医疗保健需求方面的一个关键差距。OA的发病率，目前估计有2.5亿受影响的人，预计将上升成为全球残疾的主要原因。拟议的工作有可能确定负责关节组织健康的新机制，并提供一个非常有价值的OA药物发现资源。