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

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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.