Revectorisation of HIV 1 and lentiviral vector production in alternative host cell lines

The research question is 'Can I identify an envelope glycoprotein that can be used to generate vectors to efficiently target CD4+ T cells in vivo?'. The envelope will need to be stable and highly selective for CD4+ T cells compared to other cell types during transduction. HIV-1 envelopes, which have a natural tropism to CD4, will be characterised. Other viral envelopes and binding machinery such as DARPins will be assayed to obtain a pseudotyped vector that can achieve the best titre in cell lines and PBMCs. Ultimately, the newly developed vector will enable genetic modification of CD4+ T cells to express chimeric antigen receptors, which can then target certain cancers.

Firstly, I will identify envelope candidates for targeting T cells from data available at King's and literature, with the goal of targeting CD4+ T cells and specifically, a subpopulation of Tmemory stem cells due to their strong ability to differentiate into CAR-T cells upon gene delivery via lentiviral vectors. M-tropic HIV-1 envelopes have a natural tropism to this T cell subpopulation, as well as macrophages. However, some T or dual-tropic envelopes may have greater stability or infectivity in CD4+ T cells, therefore I will not limit to only one type of tropic envelope. Envelope-pseudotyped vectors will be titrated on cell lines to identify an envelope that binds cells expressing CD4 and CXCR4/CCR5. Cell lines such as U87-MG ectopically expressing CD4 and CCR5 or CXCR4 will initially be used before moving onto T cell lines.

PBMCs from multiple donors will be isolated and the vectors will be used to transduce these cells. After infection, cells will be stained with a cocktail of antibodies conjugated to different fluorophores. These antibodies will recognise cell-specific receptors to identify cell type. The cells will then be assessed using flow cytometry. This will allow quantification of transduction within different cell types and subpopulations.