CRISPR-based genome and epigenome perturbation in stem cells to understand and reverse osteoarthritis risk

骨关节炎（OA）是一种复杂的多因素疾病，其特征在于关节内关节软骨的疼痛性破坏。这种疾病影响老年人，但有很强的遗传成分（30-50%的遗传率）。迄今为止，通过全基因组关联研究（GWAS），超过150个SNP与该疾病显著相关。骨质疏松症（OP）是一种无症状疾病，其特征是骨密度损失，导致患者骨折发生率增加。OP还具有很强的遗传成分（遗传率估计在50-80%之间），并且已经鉴定了超过500个与疾病风险增加显著相关的SNP。历史上，由于所采用的实验方法（在基因分型微阵列上使用标签SNP）和所报道的基因座的遗传复杂性（即，高连锁不平衡区）。然而，遗传和表观遗传的相互作用，可以通过共定位的数量性状基因座（QTL）与报告的变体，和功能的后续研究的目的基因和发病机制的鉴定。Rice小组最近发表的一项研究发现，虽然OA是一种影响老年人的疾病，但OA发展的功能风险可能在骨骼发生过程中预先编程。这项小规模的靶向研究提供了证据，证明一些位点可能在生命早期发挥其功能影响，而另一些位点可能在生命后期导致疾病。本项目试图在这些发现的基础上，通过对OA患者髋关节软骨和骨样本中的mQTL进行表观基因组研究，（无OA，但推测为骨质疏松症（OP），胎儿髋骨和软骨。我们试图确定集群的位点，可能会共享时空的影响。样品（n=312）通过Illumina全球筛选阵列（GSA）进行基因分型，并且使用人类甲基化EPIC阵列确定其甲基化状态。我们将分析通过这种测序产生的数据，以确定mQTL，并进行信号的共定位分析，旨在优先考虑参与OA发展的基因和途径。通过询问胎儿软骨和骨的表观基因组，与OA患者的老年软骨和骨进行比较，我们将获得对OA和OP的发育起源的独特见解。然后，我们将继续进行功能研究，以验证我们的发现，并揭示所识别的基因如何导致疾病的分子机制。这将形成使用原代人关节软骨细胞和成骨细胞进行体内分析的基础（hAC和hOB）、永生化脂肪源性干细胞（ad-MSC）、永生化软骨细胞和成骨细胞（TC28a2，SaOS 2）和CRISPR-Cas9工具箱使用精确编辑来询问基因组和表观基因组。在圣路易斯华盛顿大学的Farshid Guilak教授的一个月的实习中，我们将把我们的发现从ad-MSCs转化为人类诱导多能干细胞，这可以产生患者匹配的OA模型，这项工作的首要目标是确定疾病背后的功能性遗传机制，其影响的组织特异性，以及它们发挥作用的生命过程中的时间点。这项工作将为疾病修饰OA药物（DMOAD）的开发铺平道路，目前缺乏药理学治疗。这也可以允许早期治疗干预，这对OA和OP等进展性疾病至关重要。

Osteoarthritis (OA) is a complex multi-factorial disorder, characterised by the painful breakdown of articular cartilage within the joints. The disease affects older individuals yet has a strong genetic component (30-50% heritability). To date, over 150 SNPs have been significantly associated with the disease via genome wide association studies (GWAS). Osteoporosis (OP) is a silent disease, characterised by a loss of bone density, leading to an increased incidence of fracture in patients. OP also has a strong genetic component (heritability estimates between 50-80%) and over 500 SNPs have been identified which significantly associate with increased disease risk.Historically, GWAS have been difficult to interpret due to both the experimental methodology employed (the use of tag SNPs on genotyping microarrays) and the genetic complexity at the reported loci (i.e., areas of high linkage disequilibrium). However, genetic and epigenetic interactions can facilitate the identification of the target gene and mechanisms of pathogenesis through co-localization of quantitative trait loci (QTLs) with the reported variants, and functional follow up studies. A study recently published by the Rice group identified that, whilst OA is a disease affecting older people, the functional risk of OA development may be pre-programmed during skeletogenesis. This small-scale targeted study provided evidence that some loci may exert their functional impact early in life, with others contributing to disease later in the life course.This project seeks to build on these findings by carrying out an epigenome-wide study of mQTLs in both cartilage and bone samples from the hips of OA patients, of those with neck of femur fracture (no OA, but presumably osteoporosis (OP) and in foetal hip bone and cartilage. We seek to identify clusters of loci which may share spatiotemporal impacts. The samples (n=312) have undergone genotyping via Illumina Global Screening Array (GSA), and their methylation status determined using an HumanMethylation EPIC array. We will analyse the data generated by this sequencing to identify mQTLs and undergo co-localisation analysis of the signals, aiming to prioritise genes and pathways that are involved in the development of OA. By interrogating the epigenome of the foetal cartilage and bone in comparison to the aged cartilage and bone from OA patients, we will gain a unique insight into the developmental origins of OA and OP.We will then move on to functional studies to validate our findings and uncover the molecular mechanism behind how the identified genes contribute to disease. This will form the basis of in vivo analysis using primary human articular chondrocytes and osteoblasts (hACs and hOBs), immortalised adipose-derived stem cells (ad-MSCs), immortalised chondrocytes and osteoblasts (TC28a2, SaOS2) and a CRISPR-Cas9 toolbox to interrogate the genome and epigenome using precision editing.During a planned 3-month placement with Prof. Farshid Guilak at the University of Washington in St. Louis we will translate our findings from ad-MSCs into human induced pluripotent stem cells, which can generate patient-matched OA models, to validate our findings.The overarching aim of this work is to identify the functional genetic mechanisms underlying disease, the tissue-specificity of their impact, and the timepoint in the life course at which they exert their effects. This work will pave the way for the development of disease modifying OA drugs (DMOADs), where currently there is a lack of pharmacological cures. This may also allow for earlier treatment intervention, vital in progressive diseases such as OA and OP.