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

诱导多能干细胞的基因组和表观基因组编辑用于研究骨关节炎风险等位基因

基本信息

批准号：
10707979
负责人：
Farshid Guilak
金额：
$ 17.11万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-22 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10707979
关键词：
Acceleration Affect Alleles Anabolism Automobile Driving Biochemical Biological Models CRISPR/Cas technology Cartilage Cartilage Matrix Cartilage injury Catabolism Chondrocytes Chromosome 8 Complex Cytoskeletal Proteins DNA Methylation Data Degenerative polyarthritis Development Epigenetic Process Equilibrium Event Future Gene Frequency Genes Genetic Genetic Polymorphism Genetic Risk Genetic Variation Genome Genotype Goals Hip Osteoarthritis Histology Homeostasis Human Human Engineering Hypertrophy Immunohistochemistry In Vitro Introns Knock-out Lead Link Mechanical Stress Mechanics Mediating Methylation Minor Molecular Pathologic Pathway interactions Patients Phenotype Physiological Physiology Play Process Production Quantitative Trait Loci Regulation Regulatory Pathway Reporting Risk Role Secondary to Signal Pathway Signal Transduction Single Nucleotide Polymorphism Specimen System Testing Tissue Engineering Tissues United Kingdom articular cartilage biobank cartilage development cell type cohort comparison control disability epigenetic regulation epigenome epigenome editing gene regulatory network genetic variant genome editing genome wide association study in vitro Model induced pluripotent stem cell interest mechanical load mechanical properties mechanotransduction new therapeutic target novel plectin prevent response risk variant single-cell RNA sequencing stem cells transcriptome transcriptome sequencing variant of interest

项目摘要

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.

抽象的大型队列中的全基因组关联研究 (GWAS) 已确定并复制了稳健的单项研究核苷酸多态性（SNP）与骨关节炎（OA）的发展密切相关。虽然很多 SNP 已被确定为 OA 发展的风险，这是对其在软骨中作用的机械理解稳态和机械生物学仍然难以捉摸。我们建议使用一种新颖的体外系统将人类诱导多能干细胞 (iPSC) 的基因组编辑与软骨组织工程相结合用于研究已鉴定的 OA SNP 对生物化学和机械特性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，其中我们确定了对患者软骨中的 PLEC 基因起作用的表达数量性状基因座 (eQTL) 与办公自动化。我们的主要假设是 OA 相关 SNP rs11780978 导致软骨细胞中的 PLEC 继发于该位点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的作用（PLEC 功能区的敲除和低甲基化）对 hiPSC 软骨分化的影响。此外，我们建议，由于该 SNP 导致的凝集素产量减少会改变软骨细胞在生理或病理条件下承受机械应力。这种组织工程该方法提供了一个模型系统，可用于未来的研究以检查特定的机制 OA 相关 SNP 与软骨细胞生理调节之间联系的假设。这些机制的识别有望带来预防或减缓 OA 的新治疗靶点进展。