Manufacturing Lentiviral Vectors for Gene Therapies: Optimisation of Cellular Factories

Abstract

The use of Lentiviral Vectors (LVV) as tools for gene delivery has gained momentum in recent years due to successes in clinical trials for rare diseases. Production of high-titre LVVs is a limiting factor in their wider application in diseases with large patient populations, such as cancer. LVVs are commonly produced by the transient transfection of HEK293T cells, however this process is costly and inefficient. An alternative stable, scalable manufacturing system for LVV production is necessary. Before the development of a stable cell line can be achieved, it is vital to gain an understanding of how individual vector components interact with HEK293T cells at a molecular level. HIV-1 GagPol is the major viral structural protein constituting approximately 70% of a bona fide virion. It is not known how synthetic GagPol, utilised during LVV production at GSK, interacts with HEK293T cells. Given that HIV-1 utilises several host cell systems for its own propagation, it is hypothesised that synthetic GagPol may have similar effects, potentially stressing the capacity of a HEK293T cell to carry out typical homeostatic functions. This could be detrimental to HEK293T cells in terms of causing cellular stress due to the burden of ectopic protein production, and may also impact negatively on GagPol production. This thesis therefore aimed to develop an understanding of the interactions between synthetic GagPol and HEK293T cellular physiology in order to define approaches to identify and intervene in molecular bottlenecks associated with LVV production. To do this, plasmid constructs encoding GagPol and fluorescently-tagged GagPol were generated and used in HEK293T transient transfections, where a maximum GagPol production titre of over 1250 ng p24/mL was achieved. HEK293T cellular physiology was examined during high-level GagPol production. Activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathway, in addition to the induction of apoptosis and autophagy were observed. The effects of chemical inhibitors of cellular stresses were examined, with chemical inhibitors of apoptosis leading to a modest increase in GagPol production, in addition to increased cell viability. These investigations with chemical inhibitors of cellular stress illuminate the potential for genetic targets of manipulation within HEK293T cells that could enable increased GagPol production which would, in turn, be beneficial for LVV production.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alan Dickson (Supervisor) & Neil Dixon (Supervisor)