Establishment of high-titre, stable LV producer cells with high-level genome RNA

One of the most efficient gene transfer methodologies to deliver therapeutic DNA into patients is the use of engineered viruses, such as lentiviral vectors or LVs. To produce safe, non-replicating lentiviral vectors, you need to express multiple components from separate DNA in the producer cells, including gag-pol for viral core structure and enzymes and envelopes. The other essential component is the vector genome itself that encodes therapeutic genes to be delivered to patients' cells. Innovation in cell line development has led to the establishment of producer cell lines where all of the viral vector components, including the vector genome, are stably integrated into the host cell. The development of these cell lines are key activities at Oxford Biomedica and at UCL. Stable producer cell lines are desirable as they represent the more scalable and robust manufacturing process.

While all components need to be expressed at high levels in the LV producer cell, this project focuses on the level of vector genome RNA in the producer cell. A key hypothesis here is that high-titre LV production requires high level of vector genome RNA in LV producers. However, it has been challenging to stably express high enough level of RNA genome, increasing proportion of fully-packaged, functional LVs against 'empty' LV particles in the producer culture supernatant.

Both UCL and OXB have been exploring methods to boost genome RNA level; specifically this project will explore the use of many LVGenome copies as a way to achieve high levels of vector genome. This will support high-titre LV production and help create a reproducible platform for establishing genetically defined (safer), high-titre, stable LV producers. Alternative strategies towards high vector genome expression will be explored. The student will investigate the biological/molecular mechanism(s) behind these strategies.

Besides these cell engineering, the student will also investigate vector analytics and scale-down manufacture to demonstrate the quality and scalability of cells engineered in this project. In addition to common LV analyses (functional and physical titres, contaminant detection/quantification etc), single particle analyses with upcoming analytic technologies will be explored.