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

背景骨性关节炎是一种疼痛和致残的关节疾病，在全球范围内造成巨大的医疗和社会经济负担。骨性关节炎的治疗主要局限于止痛。由于缺乏适当的临床前测试系统，目前还没有获得批准的治疗骨性关节炎的药物(DMOADs)。我们开发了一种永生化的人类克隆性间充质干细胞(MSC)系，能够分化为成骨细胞和软骨形成软骨细胞，这是OA影响的主要关节组织。从该间充质干细胞系中，我们设计了成骨和成软骨分化状态(碱性磷酸酶和II型胶原启动子驱动的报告)和炎症反应(核因子-KB启动子驱动的报告)的荧光和分泌型荧光素酶报告。在这里，我们将确定开发基于MSC的报告系统作为鉴定DMOAD的筛查平台的可行性。目标验证现有的MSC报道线设计一种新的双重(健康/疾病)报道线在低、中全程筛选中测试记者对确定的化合物进行功能测试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我们现有的报道线进行全面验证，以确定可在体内翻译的特性(通过RNAseq)和可用于大规模筛选的适用性(通过对化合物训练集的稳健性测试)。我们将设计一个同时读出健康(aggrecan启动子记者)和疾病(ADAMTS5启动子记者)的双重记者。使用这种双重报告进行筛选，有可能确定决定组织形成和降解的平衡的途径，这驱动了OA的发病机制。我们将结合CRISPR/Cas-9基因编辑和/或RNA沉默，使用一组小的、注释良好的化学修饰剂来识别成骨和软骨生成的调节因子。这将定义更广泛的化学物质，使我们能够在中通量分析中展示概念验证，并提供对MSC分化调控的机械性见解。通过这些筛选确定的化合物将被用于深入分析成骨和成软骨分化试验中的影响。随后的工作将集中在扩大规模、产能和商业化方面，GSK是第一批潜在的最终用户。时间该项目解决了在努力解决紧急但尚未得到满足的全球医疗保健需求方面的一个关键差距。骨性关节炎的发病率目前估计为2.5亿人，预计将上升为世界范围内残疾的主要原因。这项拟议的工作有可能确定负责关节组织健康的新机制，并为骨性关节炎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.