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

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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. Further, recent advances in the use of mRNAs as treatments and preventative vaccines also requires further development of their design and manufacturing to reduce cost and improve the potency of such therapies.At the University of Kent we have used HET293 cells to produce viral vectors, particularly adenovirus associated virus (AAV), for the delivery of gene therapies and through our studies and the literature identified cellular targets that are increased or decreased in expression when comparing conditions leading to lower or amounts of correctly assembled and packaged AAVs. We have also developed approaches to design mRNAs that when produce by in vitro transcription (IVTmRNAs) give higher protein expression for use as therapies or vaccine candidates. 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 AAV production from HEK293 cells, thus furthering our understanding of the basic cell biology that underpins AAV production from HEK293 cells and creating novel, industrially relevant HEK293 cell lines for enhanced AAV production. We will also work with the researchers at Southampton to determine if enzymes capable of mRNA editing impact the function of IVTmRNAs. The programme of research is; Phase 1: Knockdown by siRNA or over-express 10-20 candidate genes in HEK293 cell lines and determine the impact on AAV production and genome packaging. From the resulting data, select the top 5 candidate genes with the greatest impact on AAV production. Phase 2: Combine top 2 targets by performing double knockdown, double over-expression or knockdown + over-expression in HEK293 expressing cell lines, assess impact on AAV production. Select top combination(s). Phase 3: Assess impact of IVTmRNA sequence on RNA editing and protein expression from these IVTmRNAs in HEK293 cells. Phase4: Assess the impact of top combination(s) form Phase 2 either alone or in combination on AAV vector production from HEK293 cells.