The goal of non-viral gene delivery is to treat illnesses stemming from gene deficiencies or overexpression without the use of viruses, which can cause severe immunogenic response. Many barriers face the delivery of DNA both in vivo and in vitro and must be overcome by the development of a complex multi-component carrier designed to address each challenge. While it is intuitive to develop a carrier in vitro, the requirements for in vivo gene delivery differ greatly, and often a non-viral carrier optimized in vitro will fail in the bloodstream in vivo due to high surface charge, which encourages blood protein binding, or dissociation of the polyplex leaving the DNA vulnerable to nucleases. It is evident that development of a non-viral gene delivery vector for use in vivo requires an easily amended platform to develop the carrier and a reproducible, calibrated assay to determine the expression of polyplexed DNA in vivo. Polyacridine peptides conjugated to polyethylene glycol (PEG) are a unique and characterizable set of carrier molecules that can be modified by peptide synthesis and various PEGylation strategies. Through the use of bioluminescence imaging and hydrodynamic stimulation (HS), a physical method that provides high levels of expression with small doses of DNA, it is possible to determine the state of polyplexed DNA in the bloodstream after various periods of circulation. The goal of this thesis was to overcome the first barrier of a systemically administered gene delivery system by developing a carrier molecule that reversibly binds to DNA and stabilizes it against metabolism in the bloodstream while avoiding undesirable biodistribution properties. The PEGylated polyacridine peptides presented herein were modified in response to each polyplex's in vivo performance based on pharmacokinetics, biodistribution, and gene expression by HS in mice after intravenous dosing. Modifications to the DNA-binding motif of the peptide were addressed initially along with various formulation strategies. Because PEG is installed to stealth polyplex surface properties, the effect of PEG attributes was also examined through optimization of PEG conjugation, size, and position. The results demonstrate the development of long circulating polyplexes that completely stabilize 1 µg of DNA in the bloodstream for five hours. This result provides a necessary prerequisite to allowing targeted accumulation of a polyplex at the site of action, which is the next step toward a fully-effective, systemically-administered non-viral gene delivery system.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-4908 |
| Date | 01 July 2013 |
| Creators | Kizzire, Koby Lynn |
| 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 2013 Koby Kizzire |
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