Graduate Studentships

Pathology prize studentship project description:

Using gene editing in human induced pluripotent cells to investigate the role of resident macrophages in HIV-1 infection and Parkinson’s disease

  1. The impact of innate immune defences during HIV-1 infection of macrophages (with Dr MD Moore).

As one of the targets of HIV-1 infection, macrophages play a key role during the pathogenesis of the disease, particularly early after infection at the site of exposure, and later within the brain in the form of microglia.  We are interested in understanding the molecular players involved during infection of macrophages by HIV-1 and have investigated the roles of lipid rafts[1], endocytosis pathway regulators[2] and CD4 cytoplasmic domains[3] on viral entry.  Much of this work has been made possible by our development of stem cell-derived macrophages[4], enabling us to genetically modify stem cells and then assay its effects on authentic macrophages (something that is not feasible with blood-derived macrophages).  The advent of efficient and precise genetic engineering using CRISPR/Cas9 has opened up even more possibilities in this field and we are currently investigating the role of innate immune defences that are employed by macrophages to prevent HIV-1 infection (e.g. the restriction factor SAMHD1), how HIV-1 is able to circumvent them, and how they tie into the overall defence network.

2.  The role of PD-related genes in microglial physiology (with Dr S Cowley).

There is an increasing recognition that immune dysregulation contributes to the progression neurodegenerative diseases, and several neurodegenerative disease-associated genes have been identified that are expressed in brain-resident macrophages, microglia. Our lab is interested in understanding the functions of Parkinson’s Disease-related genes Lrrk2, GBA, Parkin and α-synuclein, in macrophages and microglia. We have Europe’s largest collection of PD patient-derived iPSc lines[5], including lines with mutations in each of the above genes, we have made gene KnockOut lines, isogenic corrected lines[6] and tagged-gene iPSc lines, we have developed a highly efficient methodology for making large quantities of macrophages from iPSc4 and we are developing protocols for differentiating iPSc to microglia. This unique suite of tools will enable the student to examine and manipulate the function of endogenously expressed proteins in the most authentic cell culture system available, to understand the normal physiological roles of PD-related genes in macrophages/microglia and to explore the effect of disease-causing mutations on cell function.

[1] Carter et al. HIV entry in macrophages is dependent on lipid rafts. Virology. 2009

[2] Carter et al. HIV-1 infects macrophages by exploiting an endocytic route dependent on dynamin, Rac1 and Pak1. Virology. 2011

[3] van Wilgenburg et al. The productive entry pathway of HIV-1 in macrophages is dependent on endocytosis through lipid rafts containing CD4. PLoS One. 2014

[4] van Wilgenburg, B., Browne, C., Vowles, J., and Cowley, S. (2013). Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions. PloS one 8, e71098

[5] Hartfield, E., Yamasaki-Mann, M., Ribeiro Fernandes, H., Vowles, J., James, W., Cowley, S., and Wade-Martins, R. (2014). Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons. PloS one 9, e87388.

[6] Flynn, R., Grundmann, A., Renz, P., Haenseler, W., James, W., Cowley, S., and Moore, M. (2015). CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells. Experimental hematology