Dhanoya, AS; (2012) Characterisation of plasmid DNA complexes for application in genetic immunisation. Doctoral thesis , UCL (University College London).

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Abstract

Non-viral gene delivery into mammalian cells is widely used in bio-processing for the production of recombinant proteins as well as considered for clinical trials in gene therapy and vaccination. DNA can be delivered through various non-viral methods including polymers, lipids, peptides and entrapment within nanoparticles. Non-viral gene delivery often entails nucleic acids that are bound to a polymer or polycation to form a complex referred to as polyplexes. Various factors may affect the efficiency of polyplex uptake in mammalian cells. One factor is DNA topology, which is important from a regulatory perspective whereby FDA guidelines require the majority of plasmid DNA (pDNA) (>80%) to be in its supercoiled (SC) form. Therefore the motivation of this study was to investigate the impact of DNA topology on non-viral gene delivery. In this study pDNA (6.8kb) was complexed with poly-L-lysine (PLL) (MW, 9600) to form PLL/DNA polyplexes. pDNA of three topologies; SC, open circular (OC) and linear-pDNA were complexed with PLL. Biophysical analyses which included size, surface charge, DNA binding and nuclease resistance assays revealed topology dependent results. For example SCpDNA polyplexes were smaller (<140nm); more efficiently packaged and displayed greater nuclease resistance than OC- and linear-pDNA polyplexes. DNA release from PLL was analysed although such experiments were not a time course study, rather a confirmatory assay to identify PLL-bound DNA. Polyplex uptake in Chinese hamster ovary (CHO), HeLa and dendritic cells (DCs) were studied. Uptake was monitored by fluorescent confocal microscopy, flow cytometry and reporter gene expression assays. Regardless of cell type, complexes containing SC-pDNA displayed greater reporter gene expression than OC- and linear-pDNA polyplexes. In regards to CHO cells confocal image analysis revealed SC-pDNA polyplexes associated most efficiently with host cell nuclei. SC-pDNA polyplexes were smaller and nuclease resistant than its counterparts which may facilitate uptake. Endocytic mechanisms of uptake were analysed in CHO cells. This is important as knowledge of polyplex uptake pathways could be exploited for future gene delivery studies. Polyplex nuclear import was studied in regards to importin-7 (Imp7). Imp7 is key nuclear import receptor identified in previous studies, which was a preselected candidate. Gene expression studies along with qualitative and quantitative confocal microscopy analyses indicated possible exploitation of Imp7. However live cell imaging experiments showed colocalisation between DNA and nuclei fluorescence in Imp7 KD cells which suggests other routes of nuclear import may be employed. Polyplex uptake in DCs was also studied as these are key sentinels of the immune system. SC-pDNA polyplexes displayed the most efficient uptake and gene expression profiles in DCs. Gene expression and ability to induce DC phenotypic changes was dependent on dosage and DNA topology. Therefore this study stresses the importance of DNA topology which impacts on the bio-processing of non-viral gene delivery products.

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