Precision Medicine DTP - Developmental consequences and potential therapeutic exploitation of genetic PI 3-kinase activation

This exciting project at the interface of cell biology and bioinformatics offers the opportunity for training in state-of-the art human disease modelling using human induced pluripotent stem cells (iPSCs), in design and execution of scRNAseq experiments, and in analysis of data generated from start to finish of the bioinformatic pipeline. It will draw on a supervisory team including authorities in human rare disease genetics, endothelial and haematopoietic stem cell development, and computational analysis of complex transcriptomic datasets. Deep knowledge of the PI3K signalling pathway, blood cell and endothelial development will be acquired, with exposure to use of iPSC-derived endothelial cells in models of vascular injury.
The focus will be on highly deforming human growth disorders caused by mosaic activating mutations in the PIK3CA gene, encoding the catalytic subunit of phosphoinositide 3-kinase alpha (PI3Kalpha)(1). This project builds on the observation that PIK3CA-driven overgrowth shows a highly skewed distribution, favouring some mesoderm and neurectoderm-derived tissues (2). Vascular overgrowth is particularly common, but although endothelial cells (ECs) are exquisitely regulated by PI3Kalpha, PIK3CA mutations have been identified in capillary, venous and lymphatic but not arterial ECs. Whether mutations in PIK3CA are in all lineages, but only pathogenic in some, or whether negative selection occurs is unknown (2). A particularly interesting developmental bifurcation occurs in haemogenic endothelium, which is a source of haematopoietic cells, where strong PIK3CA mutations are almost never found.
iPSCs allow study of the effect of PI3KCA mutations on survival, differentiation, migration and other behaviours of lineages including EC and haematopoietic cells. Highly characterised wild type iPSCs have been edited using CRISPR/CAS9 to mimic disease-related PIK3CA genotypes, generating multiple independent PIK3CA mutated lines. These will be studied using state-of-the art single cell sequencing and bioinformatic analysis to interrogate how activating PIK3CA mutations perturb normal differentiation. Published protocols driving iPSC differentiation to ECs (3) or HSCs (4) will be used. EC protocols yield a heterogeneous population including multiple EC lineages, offering the perfect opportunity to address the effect of PIK3CA mutations on critical developmental decisions(3). Numerous independent PIK3CA mutant and control iPSCs will first be studied, with single cell RNA sequencing (scRNAseq) used to assess differences in the cell profile at each differentiation stage, using analytic approaches including velocity and pseudotime clustering to understand dynamics of key developmental transitions. Reversibility by inhibition of PI3Kalpha will be assessed, and the effect of different protocols favouring venous or other EC subtypes will be evaluated. Candidate downstream mediators will be sought before skewed bifurcations. PIKCA genotype will be also called in scRNAseq datasets and used to investigated differentiation of different admixtures of wildtype and mutant cells, mimicking the human disease state. These experiments will unravel the complex cellular and molecular mechanisms of a debilitating human disease and will give translational insight into PIK3CA-related vascular overgrowth. This will have implications for personalized medicine for affected patients, and will also give new insights into the role of PI3K in vascular and blood development, cancer and normal tissue growth and regeneration.

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. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow.

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 must contact the primary supervisor prior to making your application. Additional information on the application process is available from the link above.

For more information about Precision Medicine visit:
http://www.ed.ac.uk/usher/precision-medicine