Understanding the rules of life

Category: CASE Studentships

Modulating expression of candidate genes to improve lentiviral vector production in stable cell lines

Project No.2251

Primary Supervisor

Prof Mark Smales- University of Kent

Co-Supervisor(s)

Dr Mark Chen – GSK

Dr Sabine Johnson – GSK

Summary

The use of gene therapy products for the treatment of a large range of indications and as potential vaccines in the clinic shows great promise but producing high titre viral vectors and manufacturing at scale remains a challenge and contributes to the cost of these therapies.

The Cell and Gene Therapy group at GSK and the University of Kent have recently (July 2021)

completed a GSK funded collaboration to identify cellular restriction factors on lentiviral vector (LV) production in HET293T lentiviral producing stable cell lines. As a result of the collaboration, ~20 genes were identified using RNAseq transcriptomic profiling that are either significantly increased or decreased in expression when comparing high and low LV producing clones. The proposed PhD project will use a combination of genome editing, siRNA knockdown and over-expression studies to determine the impact of manipulation of individual and multiple targets on LV production from HEK293T cells, thus furthering our understanding of the basic cell biology that underpins LV production from HEK293T cells and creating novel, industrially relevant HEK293T cell lines for enhanced LV production. The programme of research is; Phase 1: Knockdown by siRNA or over-express 10-20 candidate genes in HEK293T LV expressing cell lines. From the resulting data, select the top 5 candidate genes with the greatest impact on lentiviral production, taking consideration to avoid conflict of interest with GSK. Phase 2: Combine top 2 targets by performing double knockdown, double over-expression or knockdown + over-expression in HEK293T LV expressing cell lines, assess impact on lentiviral production. Select top combination(s). Time permitting an extension to further and more combinations will be investigated. Phase3: Assess the impact of top combination(s) form Phase 2 either alone or in combination on vector production from HEK293T cells expressing different LV targets.