Genome Editing of Stem Cells for Analysis of Osteoarthritis Causal Variants

描述(由申请人提供)： 摘要骨性关节炎(OA)在美国估计有2700万人受到影响，其特征是关节疼痛和软骨丢失。发生骨性关节炎的危险因素之一是在基因组的某些位置有特殊的遗传变异，例如生长和分化因子5(GDF5)基因调控区的特定单核苷酸多态性。因果变异增加骨性关节炎风险的机制尚不清楚，部分原因是骨性关节炎患者之间存在大量的遗传差异，生活方式的差异也影响骨性关节炎的发展。除了所讨论的因果变异外，完全匹配的软骨组织的产生将允许深入了解特定的遗传变异如何影响软骨对骨关节炎刺激的反应，如炎症细胞因子。此外，它还将为体外药物筛选提供一个重要工具，这些药物旨在确定为具有特定基因特征的患者优化的候选药物。我们建议利用诱导多能干细胞的基因组编辑和软骨组织工程技术开发一种新的体外系统，用于研究已确定的骨性关节炎原因变体对关节软骨生化和力学特性的功能影响。我们的假设是，在随后的软骨分化之前，用户定义的IPSCs中精确的基因变化将改变软骨的产生，并使组织更容易受到促炎细胞因子白介素1α(IL-1α)的影响，后者参与了OA的发病机制。基因组编辑将使用针对特定基因座的工程核酸酶进行，以通过同源重组刺激基因靶向。我们将对工程软骨的生化成分和力学性能进行分析。此外，我们还将使用RNA-Seq来检测遗传变化如何影响全球基因表达。我们的第一个目标将是研究GDF5的T/C单核苷酸多态rs143383，作为这项普遍适用的技术的初始例子。我们的第二个目标将是制造代表基因组区域中五种不同变体的软骨，通过全基因组关联研究，该区域与OA风险增加有关。这一目标的一个目标将是确定哪种基因变化是导致骨性关节炎风险增加的原因。总而言之，这项工作将建立一个严格的体外系统，以测试特定的遗传变异对骨性关节炎发育的影响。这一知识有可能为发现与具有特定遗传风险因素的患者相匹配的骨性关节炎疗法提供一种平台技术。

DESCRIPTION (provided by applicant): Summary Osteoarthritis (OA) affects an estimated 27 million people in the United States and is characterized by joint pain and cartilage loss. One of the risk factors for developing OA is having particular genetic variations at certain locations in the genome, such as a specific single nucleotide polymorphism in the regulatory region of the growth and differentiation factor 5 (GDF5) gene. The mechanism by which a causal variant increases the risk for OA is unclear, partly because there is substantial genetic variation among OA patients and lifestyle differences also affect the development of OA. The production of cartilage tissue that is perfectly matched except for the causal variant in question would allow for insight into how a specific genetic variant affects the response of cartilage to OA stimuli such as inflammatory cytokines. Additionally, it would provide an important tool for in vitro drug screens that seek to identify candidate drugs optimized for patients with particular genetic profiles. We propose to develop a novel in vitro system for studying the functional effect of identified OA causal variants on the biochemical and mechanical properties of articular cartilage using genome editing of induced pluripotent stem cells (iPSCs) and cartilage tissue engineering. Our hypothesis is that user-defined precise genetic changes in iPSCs before subsequent chondrogenic differentiation will alter the cartilage production and render the tissue more susceptible to the pro-inflammatory cytokine interleukin-1 alpha (IL-1α), which has been implicated in the pathogenesis of OA. Genome editing will be performed with engineered nucleases targeted to specific loci to stimulate gene targeting by homologous recombination. We will analyze the engineered cartilage for both biochemical composition and mechanical properties. Furthermore, we will also use RNA-Seq to detect how the genetic changes affect global gene expression. Our first aim will be to pursue the T/C single nucleotide polymorphism rs143383 of GDF5 as an initial example of this generally applicable technique. Our second aim will be to make cartilage that represents five different variants in a region of the genome that has been associated with an increased risk for OA by a genome-wide association study. One goal of this aim will be to determine which of the genetic changes is responsible for the increased OA risk. Together, this work will establish a rigorous in vitro system for testing the effect of particular genetic variants on OA development. This knowledge has the potential to provide a platform technology for discovering OA therapeutics that are matched to patients with particular genetic risk factors.