Non-viral gene delivery is plagued by low transfection levels compared to viral delivery. The nuclear envelope presents a significant obstacle for non-viral vectors. A peptide-based nuclear localizing sequence has been incorporated into non-viral vectors to traverse the nuclear envelope. Here, we selected a photo-chemical method for covalently labeling the peptide onto plasmid DNA. The hypothesis of this work was to incorporate a nuclear localizing sequence into a non-viral delivery vector, demonstrate increased nuclear uptake and show a subsequent increase in transgene expression both in vitro and in vivo.
We focused on pursuing in vitro and in vivo methods by which to test non-viral vectors for increases in gene expression based on the nuclear localizing sequence. Hydrodynamic dosing and intramuscular dosing (followed by electroporation) are two efficient delivery routes for dosing DNA in vivo. Through preliminary experiments, we became confident that whole animal bioluminescent imaging was a reliable and quantitative method by which to detect luciferase expression by either delivery route. Moving forward, both hydrodynamic and intramuscular dosing would be used to test formulations for nuclear localizing ability in vivo.
Nuclear localizing peptides containing a photo-activatable functionality were synthesized and characterized. We quantitatively explored the photo-labeling capabilities on plasmid DNA via a radioactive peptide. In vitro, tissue culture-based experiments were carried out to show increased nuclear uptake by confocal microscopy as well as increased transgene expression. Throughout the literature, achieving an increase in expression by incorporating a nuclear localizing sequence into a non-viral vector has been elusive. The complexity of achieving this goal is increased when considering an in vivo system for improving gene transfer efficiency. Several strategies have been explored to demonstrate an increase in reporter gene expression from this type of non-viral vector, and the methods developed herein can be applied to other nuclear localizing vectors.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-1397 |
| Date | 01 January 2008 |
| Creators | Rettig, Garrett Richard |
| Contributors | Rice, Kevin G. |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright 2008 Garrett Richard Rettig |
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