Modelling the non-coding genome using RNA and CRISPRi/a approaches to define vascular heterogeneity in health and disease

使用 RNA 和 CRISPRi/a 方法对非编码基因组进行建模，以定义健康和疾病中的血管异质性

• 批准号: 1939057
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1939057
• 关键词: Modelling; non; coding; genome; using

Despite initially being regarded as a simple monolayer barrier, the role of the vascular endothelium in vascular homeostasis has become increasingly apparent in recent years. Its function in maintaining the structure of the vessel wall coupled with its ability to respond to physical and chemical stimuli by regulation of vascular tone, coagulation, and inflammation highlights its importance in vascular biology. Vascular endothelium dysfunction has been demonstrated to occur following tissue ischemia. In addition to the necrosis of endothelial cells in response to the initial ischaemic injury, reperfusion results in cellular abnormalities evoking apoptosis. Consequently, a potential therapeutic approach for cardiovascular disease would be to utilise endothelial-regenerating cells to improve the perfusion of ischaemic tissue. Prior attempts to initiate angiogenesis using various stem cells have been disappointing. Improved understanding of vascular endothelial cell specification is required to fully utilise the therapeutic potential of endothelial-regenerating cells.In response to the limitations in existing regenerative approaches, this project aims to characterise the influence of the non-coding genome in endothelial cell maturation and specification. The Professor Andrew Baker group have established a clinically-compliant protocol to generate mature CD31+/CD144+ endothelial cells from human embryonic stem cells, thus providing an ideal model to investigate endothelial specification. A comprehensive understanding of endothelial cell specification is particularly important when considering the highly heterogeneous nature of the vascular endothelium. Such heterogeneity exists within the various vessel types as well as in an organ specific context, thus facilitating varying physiological functions. Owing to the vascular endothelial heterogeneity, single cell RNA sequencing will be conducted in various endothelial cells types to identify coding and non-coding RNA molecules implicated in cell maturation and specification. Prospective lncRNAs will then be targeted in CRISPR-Cas9 activation and interference screening, thus demonstrating their causative effect on endothelial cell maturation. lncRNA are involved in numerous biological functions, with cell specific expression patterns being attributed to coordinating cell state and differentiation. By conducting both CRISPR-Cas9 interference and activation screens, identification of complementary data will verify the causal role of candidate lncRNAs. CRISPR-Cas9 activation will be achieved by the use of the dCas9-VPR system.The expression, regulation, and function of candidate lncRNAs and protein coding genes will then be investigated in the model system, in which human embryonic stem cells give rise to mature endothelial cells. This will involve using techniques such as RT-qPCR, RNA-FISH and possibly genome editing utilising CRISPR-Cas9 methods. Findings from in vitro experiments will then be tested in murine models of peripheral and myocardial ischaemia. Finally, ethical approval has been obtained to use RNA samples from individuals with and without vascular disease to asses the relevance of lncRNAs in human vascular pathology. Since dysfunction of the vascular endothelium is known to precede atherosclerosis, then determining the role of lncRNAs in this process may contribute valuable information to vascular pathogenesis thus informing future precision medicine approaches.

尽管最初被认为是简单的单层屏障，但血管内皮在血管稳态中的作用近年来变得越来越明显。它维持血管壁结构的功能，加上它通过调节血管张力、凝血和炎症来响应物理和化学刺激的能力，凸显了它在血管生物学中的重要性。已证明组织缺血后会发生血管内皮功能障碍。除了响应初始缺血性损伤而导致内皮细胞坏死外，再灌注还会导致细胞异常，从而引发细胞凋亡。因此，心血管疾病的潜在治疗方法是利用内皮再生细胞来改善缺血组织的灌注。先前使用各种干细胞启动血管生成的尝试均令人失望。为了充分利用内皮再生细胞的治疗潜力，需要加深对血管内皮细胞规范的了解。针对现有再生方法的局限性，该项目旨在表征非编码基因组在内皮细胞成熟和规范中的影响。 Andrew Baker教授小组建立了一种符合临床要求的方案，从人类胚胎干细胞中生成成熟的CD31+/CD144+内皮细胞，从而为研究内皮规范提供了理想的模型。考虑到血管内皮的高度异质性，对内皮细胞规格的全面理解尤为重要。这种异质性存在于各种血管类型以及器官特定环境中，从而促进不同的生理功能。由于血管内皮的异质性，将在各种内皮细胞类型中进行单细胞RNA测序，以鉴定与细胞成熟和规格相关的编码和非编码RNA分子。然后，未来的 lncRNA 将被用于 CRISPR-Cas9 激活和干扰筛选，从而证明它们对内皮细胞成熟的致病作用。 lncRNA 参与多种生物学功能，细胞特异性表达模式归因于协调细胞状态和分化。通过进行 CRISPR-Cas9 干扰和激活筛选，互补数据的识别将验证候选 lncRNA 的因果作用。 CRISPR-Cas9的激活将通过使用dCas9-VPR系统来实现。然后将在模型系统中研究候选lncRNA和蛋白质编码基因的表达、调控和功能，其中人胚胎干细胞产生成熟的内皮细胞。这将涉及使用 RT-qPCR、RNA-FISH 等技术，还可能涉及利用 CRISPR-Cas9 方法进行基因组编辑。体外实验的结果将在外周和心肌缺血的小鼠模型中进行测试。最后，已获得伦理批准，可以使用患有和未患有血管疾病的个体的 RNA 样本来评估 lncRNA 在人类血管病理学中的相关性。由于已知血管内皮功能障碍先于动脉粥样硬化发生，因此确定 lncRNA 在此过程中的作用可能为血管发病机制提供有价值的信息，从而为未来的精准医学方法提供信息。

项目成果

Unravelling atherosclerotic heterogeneity by single cell RNA sequencing.

• DOI: 10.1097/mol.0000000000000559
• 发表时间: 2018-12
• 期刊: Current Opinion in Lipidology
• 影响因子: 4.4
• 作者: I. McCracken;Richard S. Taylor;N. Henderson;J. Sluimer;A. Baker

Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.

• DOI: 10.1093/eurheartj/ehz351
• 发表时间: 2020-03-01
• 期刊: EUROPEAN HEART JOURNAL
• 影响因子: 39.3
• 作者: McCracken, Ian R.;Taylor, Richard S.;Kok, Fatma O.;de la Cuesta, Fernando;Dobie, Ross;Henderson, Beth E. P.;Mountford, Joanne C.;Caudrillier, Axelle;Henderson, Neil C.;Ponting, Chris P.;Baker, Andrew H.
• 通讯作者: Baker, Andrew H.