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

Background

The vascular endothelium is essential to the maintenance of vessel wall structure, tone and function. It is, however, intimately involved in abnormalities of blood vessel function, including the adverse remodeling associated with the response to vascular damage and in tissue ischemia (peripheral and myocardial). Regenerative approaches to cardiovascular disease include use of endothelial cells to generate improved vasculature in tissue ischemia. However, to data, clinical data using a variety of cell types to induce vascular regeneration, such as mesenchymal stem cells and CD34+ cells has, in general, been poor. We are interested in the use of human embryonic stem cells to generate endothelium. Indeed, we have established a clinically-compliant protocol to establish large quantities of “mature” CD31+/CD144+ endothelial cells. This highly reproducible system can also be used to understand the basis of endothelial cell maturation and specification. Critically, in vivo, the endothelial lineage is a very heterogeneous cell type with not only venous, arterial and lymphatic specification, but also heterogeneity in organ-specific and context-specific settings. Thus understanding of the pathways and systems that impact maturation and specification and associated heterogeneity is of substantial current interest in the field and impacts on potential use of regenerative approaches in precision medicine. We propose that a radical approach, using the very latest techniques and technologies, will be required to identify and quantify the protein-coding and noncoding molecular cues that control endothelial cell specification. To understand and unravel this molecular complexity requires an interdisciplinary approach using novel datasets, computational approaches and assessment of lncRNA function in cellular and whole organism experiments.

Aims

We will address the following 4 aims via the proposed experimental and analytical approaches

Aim 1: Generation of scRNAseq datasets to inform computational approaches. We will perform single cell RNA sequencing to identify molecular signatures of specification to arterial, venous and lymphatic endothelium. We will use dimensionality reduction and pseudotime approaches to predict networks of protein-coding and noncoding RNA molecules that determine cell type commitment and specification.

Aim 2: CRISPRi/a screens to identify causal lncRNA in endothelial cell specification. Using CRISPR interference/activation with guide RNAs (as pooled lentiviral plasmid libraries) which target the promoters of lncRNAs we will identify lncRNAs that are required for the specification of arterial, venous or lymphatic cells. To ensure the RNA-dependence of these loci we will require the cellular phenotypes from activation and interference screens to be complementary.

Aim 3: Generate proof-of-concept experiments in hES cells and in vivo. We will use our model system to ascertain the expression, regulation and function of candidate lncRNAs, protein-coding genes, using qRT-PCR, RNA-FISH and other techniques, potentially using CRISPR-Cas9 genome engineering approaches. We will additionally explore in vivo experimentation in mouse ischemic models, including peripheral and myocardial models.

Aim 4: Assess relevance to human vascular pathology and precision medicine using material from patients with cardiovascular disease. We possess ethical approval for RNA samples from patients with and without diseases of the vasculature, and will use these for assessment of lncRNA expression. This will be undertaken in collaboration with Prof David Newby (Royal Infirmary of Edinburgh).

Training Outcomes

The student will develop an interdisciplinary skill sets, as evidenced by the complementing research of both supervisors involving in-depth computational genomics and transcriptomics, human physiology and pathology.

This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

All applications should be made via the University of Edinburgh, irrespective of project location:

http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919

Please note you must apply to one of the projects and you are encouraged to contact the primary supervisor prior to making your application. Additional information on the application process if available from the link above.

For more information about Precision Medicine visit:

http://www.ed.ac.uk/usher/precision-medicine