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Title: Quantitative imaging of plasmid DNA in human airway epithelial cells following non-viral gene transfer
Author: Singh, Charanjit
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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The UK CF Gene Therapy Consortium is interested in non-viral gene therapy for cystic fibrosis (CF). To provide insight into the intracellular bottlenecks plasmid DNA (pDNA) encounters on its way to the nuclei of airway epithelial cells, I sought to quantitatively describe the intracellular fate of non-virally transferred pDNA using a clinically relevant human airway epithelial cell air-liquid-interface (ALI) model. Plasmid DNA was tagged with nanogold particles or fluorescent quantum dots (QDots) for use in transmission electron microscopy (TEM) or confocal microscopy studies, respectively. Conjugation of pDNA with either nanogold or QDots did not affect the biological activity. The number of cells showing cytoplasmicaly-localised nanogold-conjugated pDNA increased with increasing transfection time. Interestingly, there was an equal distribution of pDNA in cytoplasm and nuclei of cells transfected for 60 minutes. In the parallel confocal study, QDot-pDNA was visible in nuclei (~12 m below the apical surface) after transfection for only 15 minutes, consistent with our earlier observations. Approximately 8% (n= 349 nuclei from 9 independent ALIs) of nuclei contained QDot-pDNA in ALIs transfected for 15 minutes. I next collected z-stacks of QDot-pDNA-transfected ALIs every 10 minutes up to a maximum of 120 minutes. As expected, some QDot-pDNA remained on the apical surface throughout the transfection period. Importantly, by 60 minutes, there was a time-dependent net movement of the QDot-fluorescence downwards through the epithelium with the number of nuclei containing QDot-pDNA increasing as early as 30 minutes reaching maximum levels of 8.2±4.8% (n=256 nuclei from 7 ALIs). In conclusion, the results from the TEM and confocal studies showed good agreement for the numbers of nuclei containing pDNA at early transfection times (15, 30 and 60 minutes). These data, in a clinically relevant model, should help focus efforts on increasing gene transfer efficiency by finding the strategies to overcome cytoplasmic and nuclear barriers.
Supervisor: Munkonge, Felix ; Alton, Eric Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral