The ability to safely delivery efficacious amounts of nucleic acids to cells and tissues remains an important goal for the gene therapy field. Viruses are very efficient at delivering DNA, but safety concerns limit their clinical use. Nonviral vectors are not as efficient at DNA delivery, but have a better safety profile. Limiting the efficaciousness of nonviral vectors are the numerous extra and intracellular barriers that must be overcome for successful DNA delivery in vivo. While single polymers can successfully transfect immortalized cell lines in vitro, multicomponent gene delivery systems are required for delivery in vivo. Key in the development of multicomponent systems is their syntheses. Optimization of a nonviral gene delivery system requires the development of methodologies that incorporate the different components in a controlled fashion, generating homogeneous gene delivery vectors. Such syntheses ensure every polymer has the different components required for successful delivery. The amount of each component and location within the gene delivery system can also be varied systemically, allowing optimization of the vector.
The overall scope of this thesis is to develop a chemical method to iteratively couple gene delivery peptides through reducible disulfide bonds. The synthesis of such polypeptides allows the triggered disassembly of a polypeptide polyplexed with DNA upon cellular uptake. To synthesize homogeneous gene delivery polypeptides, a novel iterative reducible ligation strategy was developed, based upon the use of a thiazolidine masked cysteine. Initial studies demonstrated that a thiazolidine could be unmasked to a cysteine in the presence of a disulfide bond without side reaction, though the reported thiazolidine hydrolysis conditions of aqueous methoxyamine were insufficiently robust for high yielding ligations. Discovery of a novel silver trifluoromethanesulfonate hydrolysis led to an efficient process for generating reducible polypeptides, as evidenced in the synthesis of a 4 component polypeptide.
Due to the success of the thiazolidine mediated iterative ligation strategy, cysteines were replaced by penicillamines to produce more stable disulfide bonds. The mild thiazolidine hydrolysis and subsequent peptide conjugation reactions led to attempt the iterative ligation strategy on a solid support, eliminating purification steps that lowered the yields in the solution phase methodology. Initial progress at generating gene delivery peptides that could be incorporated into the synthetic strategy included the generation of a tri-orthogonal cysteine protecting scheme that allowed a third cysteine to be derivatized with a targeting ligand or stealthing polymer. Due to the use of terminal cysteines in the iterative ligation strategy, a PEG stealthing polymer could be placed in the center of a polyacridine gene delivery peptide with only a small decrease in the ability to condense and protect DNA during systemic circulation. A convergent synthesis was also developed that was able to synthesize large polypeptides in fewer linear steps. The synthetic methodology of thiazolidine mediated iterative reducible ligation developed in this thesis is important in the gene therapy field as it allows the construction of polypeptides that can be systemically optimized, potentially resulting in highly efficacious nonviral gene delivery.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-3450 |
| Date | 01 December 2012 |
| Creators | Ericson, Mark David |
| 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 2012 Mark Ericson |
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