A 3D osteoarthritis model targeting patient populations with high risk genetic polymorphisms

摘要 骨关节炎(OA)是一种退行性关节疾病，在美国估计有3000万成年人受到影响。 并造成每年超过1300亿美元的经济负担。尽管办公自动化的负担是巨大的， 目前的非手术治疗只是姑息性的，目前还没有治疗骨性关节炎的药物(DMOADs) 存在着解决问题的办法。在识别DMOAD方面缺乏成功通常被归因于变量 骨关节炎发生的原因和可用于筛选潜在DMOADs的人软骨的缺乏。至 增加识别DMOAD的可能性，我们建议基于以下因素研究分段的OA人群 明确了骨性关节炎发生的遗传易感性。为了生产几乎无限来源的人类软骨 在DMOAD筛查中，我们将使用诱导多能干细胞(IPSCs)作为软骨的细胞来源 组织工程学。该项目的目标是创建一种平台筛选技术来识别 骨性关节炎相关遗传危险因素的治疗要求。我们的方法是创建一个三维的 骨性关节炎的体外模型，该模型利用已被修改以包含明确的遗传变异的IPSC系。在……里面 目的1我们将使用基因组编辑技术来产生与OA相关的单核苷酸多态 (SNPs)存在于HiPSCs基因组中。由这些编辑的细胞形成的工程化软骨将被表征 用生化和微机械方法检测炎性细胞因子诱导的骨性关节炎样变 软骨的变化。由此产生的体外OA模型将通过测量基质降解来验证， 机械性能的丧失和炎症介质的产生。在目标2中，我们基于IPSC的模型 OA将转移到96孔板格式，以促进OA药物筛选平台的开发。 一套已知的抑制炎症降解的模型疗法将用于验证 该模型用于定义高通量的办公自动化进程读数。最后，两个新的生物活性文库 在我们的体外模型中，化合物将被筛选出它们减缓与OA相关的降解的能力。这 该提案将有助于阐明基因变异导致骨关节炎风险增加的机制，并将 通过提供识别的平台技术，促进定制的骨关节炎疗法的发展 基于已定义的危险因素的治疗效果。

Abstract Osteoarthritis (OA) is a degenerative joint disease that affects an estimated 30 million adults in the United States and results in an economic burden of over $130 billion per year. Although the burden of OA is immense, current non-surgical treatments are only palliative, and no disease-modifying OA drugs (DMOADs) presently exist to address the problem. This lack of success in identifying DMOADs is frequently attributed to the variable causes of OA initiation and the dearth of human cartilage available for screening potential DMOADs. To increase the likelihood of identifying DMOADs, we propose to study a segmented OA population based on defined genetic predisposition to OA development. To produce a nearly unlimited source of human cartilage for use in DMOAD screening, we will use induced pluripotent stem cells (iPSCs) as a cell source for cartilage tissue engineering. The goal of this project is to create a platform screening technology to identify the therapeutic requirements of OA-associated genetic risk factors. Our approach is to create a three-dimensional in vitro model of OA which utilizes iPSC lines that have been modified to contain defined genetic variations. In Aim 1 we will employ genome editing technology to generate OA-associated single-nucleotide polymorphisms (SNPs) in the genome of hiPSCs. Engineered cartilage formed from these edited cells will be characterized using biochemical and micromechanical assays and then treated with inflammatory cytokines to induce OA-like changes in the cartilage. The resulting in vitro OA model will be validated by measuring matrix degradation, loss of mechanical properties, and production of inflammatory mediators. In Aim 2, our iPSC-based model of OA will be transferred to a 96-well plate format to facilitate the development of an OA drug screening platform. A set of model therapeutics known to inhibit inflammatory degradation will be used to validate the sensitivity of the model and to define high-throughput readouts of OA progression. Finally, two libraries of novel bioactive compounds will be screened for their ability to slow OA-associated degradation in our in vitro model. This proposal will help elucidate the mechanism by which genetic variants result in increased risk for OA and will catalyze the development of tailored OA therapeutics by providing a platform technology for identifying therapeutic effectiveness based on defined risk factors.