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

骨关节炎（OA）是一种疼痛和致残的关节疾病，具有巨大的全球医疗保健和社会经济负担。OA的治疗主要局限于缓解疼痛。由于缺乏适当的临床前试验系统，目前还没有批准的改善疾病的OA药物（dmoad）。我们开发了一种永生化的人克隆间充质干细胞（MSC）系，能够分化成成骨细胞和软骨形成软骨细胞，这是骨性关节炎的主要关节组织。从这个MSC细胞系中，我们设计了成骨和软骨分化状态的荧光素酶报告因子（分别是碱性磷酸酶和II型胶原启动子驱动的报告因子）和炎症反应（NF-KB启动子驱动的报告因子）的荧光素酶报告因子。在这里，我们将确定开发基于msc的报告系统作为识别dmoad的筛选平台的可行性。目的验证现有的MSC报告细胞系设计一个新的双重（健康/疾病）报告细胞系在中低筛选中测试报告细胞系功能测试已确定的化合物开发用于扩大规模和商业化的筛选平台实验方法我们将对现有的报告细胞系进行全面验证，以表征体内可译性（通过RNAseq）和在大规模筛选中使用的适用性（通过对化合物的稳健性测试）训练集)。我们将设计一个双重报告器，用于同时读出健康（aggrecan启动子报告）和疾病（ADAMTS5启动子报告）。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，鉴定出一系列成骨和软骨形成的调节因子。这将定义更广泛的化学物质，这将使我们能够在中通量分析中展示概念验证，并为MSC分化的调节提供机制见解。通过这些筛选确定的化合物将被用于深入分析成骨和软骨分化分析的影响。后续工作将侧重于扩大规模、产量和商业化，葛兰素史克将成为首批潜在终端用户。该项目填补了在努力解决一项紧迫但尚未得到满足的全球医疗保健需求方面的一个关键空白。OA的发病率目前估计为2.5亿人，预计将上升为全球残疾的主要原因。研究工具是独一无二的。这项工作有可能确定关节组织健康的新机制，并为OA药物发现提供非常有价值的资源。

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.