Manufacturing lentiviral vectors for gene therapies: Optimisation of cellular factories

Lentiviral vectors based on HIV-1 have been used as efficient gene transfer tools for the past two decades and have demonstrated a therapeutic benefit to patients in a number of gene therapy clinical trials. However, production of lentiviral vectors remains a severe limitation to treatment of larger patient populations. The design of lentiviral vectors was based on understanding developed from researching the life cycle of HIV-1. This has demonstrated an interaction of the virus with cellular physiology of the host cell via the structural protein Gag. It is also proposed that HIV-1 may utilise conserved organelles involved in autophagy as sites of packaging. Autophagy is a homeostatic mechanism that relies upon controlled maturation, acidification and turnover of double membrane structures to relieve the cell of stress. HEK293 cells are currently the gold standard cellular factories used to produce lentiviral vectors. Whilst these cells are commonly used there is little known about their cell biology especially at the virus-host cell autophagy interface. Autophagy appears to play an important role during production of therapeutic proteins such as monoclonal antibodies. In this project the student will extend these studies by exploring the interaction between HIV Gag and autophagy in HEK293 cells during lentiviral vector production. This interaction may create a detrimental cycle that impacts both the cell and viral vector production but insight into this must be obtained. The overall aim of this project is to obtain fundamental understanding of HEK293 cell biology and Gag trafficking throughout the cell during lentiviral vector production. This knowledge will identify potential limitations in lentiviral vector production and define targets for HEK293 cell engineering or optimisation to generate improved lentiviral production processes for clinical application.