Genome and epigenome editing of induced pluripotent stem cells for investigating osteoarthritis risk alleles

摘要 在大型队列中进行的全基因组关联研究发现并复制了健壮的单基因 核苷酸多态(SNPs)与骨关节炎(OA)的发展密切相关。虽然很多人 单核苷酸多态已被认为是骨性关节炎发生的风险，这是对其在软骨中作用的一种机械性理解 动态平衡和机械生物学仍然难以捉摸。我们建议使用一种新的体外系统 将人诱导多能干细胞基因组编辑与软骨组织工程相结合 为研究已鉴定的OA SNPs对生物化学和力学性能的功能影响 关节软骨。在初步数据中，我们报告了rs11780978的功能特征，其中我们 在患者软骨中发现了一个作用于Plec基因的表达数量性状基因座 和OA在一起。我们的主要假设是，与OA相关的SNP rs11780978导致 在软骨细胞中的PLEC继发于该基因的表观遗传调控的改变。使用CRISPR-Cas9和 DCas9TET介导的基因组编辑，我们将检查遗传和表观遗传调节(PLEC)的作用 功能区域的敲除和低甲基化)对HiPSCs向软骨分化的影响。 此外，我们提出，由于这种SNP导致的plectin产量的减少改变了 在生理或病理条件下对软骨细胞施加机械应力。这种组织工程学 方法提供了一个模型系统，可以在未来的研究中使用来检查特定的机制 关于骨性关节炎相关SNPs与软骨细胞生理调节之间联系的假说。 对这些机制的识别将有望导致预防或延缓骨性关节炎的新的治疗目标 进步。

Abstract Genome wide association studies (GWAS) in large cohorts have identified and replicated robust single nucleotide polymorphisms (SNPs) with a strong association to osteoarthritis (OA) development. While many SNPs have been identified as risks for OA development, a mechanistic understanding of their role in cartilage homeostasis and mechanobiology has remained elusive. We propose to use a novel in vitro system combining genome editing of human induced pluripotent stem cells (iPSCs) and cartilage tissue engineering for studying the functional effect of identified OA SNPs on the biochemical and mechanical properties of articular cartilage. In preliminary data, we reported a functional characterization of rs11780978 in which we identified an expression quantitative trait locus (eQTL) operating on the gene PLEC in the cartilage of patients with OA. Our primary hypothesis is that OA-associated SNP rs11780978 results in reduced expression of PLEC in chondrocytes secondary to altered epigenetic regulation at this locus. Using CRISPR-Cas9 and dCas9TET-mediated genome editing, we will examine the role of genetic and epigenetic modulation (PLEC knockout and hypomethylation of the functional region) on the chondrogenic differentiation of hiPSCs. Furthermore, we propose that reduced production of plectin due to this SNP alters the response of chondrocytes to mechanical stress under physiologic or pathologic conditions. This tissue-engineering approach provides a model system that can be used in future studies to examine specific mechanistic hypothesis on the link between OA-associated SNPs and the regulation of chondrocyte physiology. Identification of these mechanisms will hopefully lead to new therapeutic targets for preventing or slowing OA progression.