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（GWAS）已经确定并复制了稳健的单 核苷酸多态性（SNP）与骨关节炎（OA）的发展密切相关。虽然许多 SNP已被确定为OA发展的风险，其在软骨中的作用的机制理解 体内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 将人类诱导多能干细胞（iPSC）的基因组编辑和软骨组织工程相结合 用于研究鉴定的OA SNP对生物化学和机械性质的功能性影响， 关节软骨在初步数据中，我们报道了rs 11780978的功能特征， 在患者软骨中鉴定了对基因PLEC起作用的表达数量性状位点（eQTL 关于OA我们的主要假设是，OA相关SNP rs 11780978导致 软骨细胞中的PLEC继发于该位点的表观遗传调节改变。使用CRISPR-Cas9和 dCas 9 TET介导的基因组编辑，我们将研究遗传和表观遗传调节（PLEC）的作用 功能区的敲除和低甲基化）对hiPSC的软骨形成分化的影响。 此外，我们提出，由于这种SNP导致的果胶产量减少改变了细胞对蛋白质的反应。 软骨细胞在生理或病理条件下的机械应力。这种组织工程学 方法提供了一个模型系统，可用于未来的研究，以检查具体的机制 关于OA相关SNP与软骨细胞生理学调节之间联系的假说。 这些机制的确定将有望为预防或减缓OA提供新的治疗靶点 进展

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.