Williams-Fegredo, Thomas Edward; (2024) Developing strategies to enhance transfection efficiency and mitigate auto-transduction in transient lentiviral vector bioprocessing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy to treat a wide range of diseases. Demand for efficient, large-scale, lentiviral vector manufacture is growing as more therapies reach late-stage clinical trials and are commercialised. Most viral vectors are currently manufactured via transient gene expression-based bioprocesses, but the multifactorial and highly sensitive transient transfection unit operation, which is a critical step in this process, can be complex to scale-up. There is a need for robust and scalable transfection processes to enhance process productivity but several challenges, related to transfection reagent stability, transfection complex preparation times and transfection volumes, complicate the large-scale manufacture of viral vectors. As part of this research, it was demonstrated that the growth kinetics of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, could be controlled by regulating the pH, ionic strength and liposome concentration of transfection formulations. This enabled cationic lipid-based transfection complexes which exhibited a superior stability to be generated; these complexes could be generated at high concentrations, achieving up to a 6-fold reduction in the overall transfection volume, compared to complexes prepared using standard preparation methodologies. Transfection-based manufacturing processes were found to be additionally complicated through the accumulation of inhibitory extracellular glycosaminoglycans in the conditioned medium of suspension cell cultures, particularly when culturing cells at higher densities. The selective enzymatic degradation of chondroitin sulphate-based glycosaminoglycans via treatment with chondroitinase ABC significantly enhanced transfection performance and lentiviral vector production. In addition to enhancing transfection efficiencies, this project also sought to tackle an existing process inefficiency. The unintended auto-transduction of viral vector-producing cells by newly synthesised lentiviral vector particles during manufacturing processes was found to account for the loss of over 60% of the total functional vector particles produced, an unaddressed and under-recognised inefficiency likely widespread throughout the industry. The auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G glycoprotein was effectively inhibited by reducing the extracellular pH. Introducing a post-transfection pH shift to pH 6.7-6.8 during vector production effectively impaired the ability of the vector to interact with its target receptor. This resulted in a 6.7-fold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and improved lentiviral vector production kinetics. The strategies developed in this work provide robust, scalable, cost-effective and industrially relevant approaches to address existing inefficiencies in lentiviral vector bioprocessing by improving transfection performance and mitigating vector loss to auto-transduction.

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