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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：
10532032
负责人：
Farshid Guilak
金额：
$ 20.75万
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/10532032
关键词：
Affect Alleles Anabolism Automobile Driving Biochemical Biological Models CRISPR/Cas technology Cartilage Cartilage Matrix Cartilage injury Catabolism Cells Chondrocytes Chromosome 8 Complex CpG Cluster Cytoskeletal Proteins DNA Methylation Data Data Reporting Degenerative polyarthritis Development Epigenetic Process Equilibrium Event Future GTP-Binding Protein alpha Subunits, Gs 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 Risk Role Secondary to Signal Transduction Signaling Protein Single Nucleotide Polymorphism Specimen System Testing Tissue Engineering Tissues United Kingdom articular cartilage biobank cartilage development cohort disability epigenetic regulation epigenome epigenome editing epigenomics 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

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.

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