Redwood-Sawyerr, Chileab; (2025) Applying synthetic biology to improve biologics production by mammalian cell transient transfection. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Transient transfection (TT) is the introduction of DNA into cells for temporary transgene expression. TT has seen improved titres to 2 g/L, expanding its possible use-cases in biologics production. Stem-loop linear (SLL) DNA, routinely synthesized in vitro, has demonstrated superior transgene expression in TT compared to its parental plasmids. Here, a novel protelomerase, TelA, has been used to generate SLL in vivo. Diagnostic digests indicated the generation of SLL DNA-specific bands. polyethylenimine (PEI)-mediated vector condensation was assessed via dynamic light scattering (DLS), revealing a 33.3% reduction in plasmid DNA diameter and up to a 60.1% reduction for SLL DNA. However, due to the lack of optimization in the condensation conditions, a direct comparison between plasmid and SLL DNA was not possible. Preliminary studies correlating particle size and polyplex uptake suggested distinct uptake dynamics for SLL DNA of varying lengths. Since TT efficiency is influenced by cell passage number, understanding this correlation is advantageous. Here, -galactosidase expression, an age-related biomarker for primary cells, indicated increased expression for high passage number for HeLa and HEK293T cells. Additionally, a positive correlation of cell fluorescence and passage number was observed when these cells were incubated with tetracycline. HeLa cells are increasingly being explored as industrial TT expression hosts for biologics production. In this study, initial efforts toward developing transfection-sensitive HeLa cells were undertaken to facilitate the design of transfection-sensitive synthetic genetic networks (SGNs). The Trensor-T stable population exhibited sensitivity to PEI-mediated transfection. Transfection-enhancing chemicals (TECs) provide insights into the molecular pathways underlying transfection sensitivity. Trensor-T responded to trichostatin A which was identified as a TEC, improving GFP reporter expression by 2.5-fold. Taken together, this thesis presents novel approaches for generating SLL DNA in vivo to improve TT, characterizing TT expression hosts, and initial steps to design develop transfection-sensitive SGNs.

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