Dissertation
Recombinant cationic biopolymers for nucleic acid delivery
Washington State University
Doctor of Philosophy (PhD), Washington State University
12/2010
DOI:
https://doi.org/10.7273/000006087
Abstract
Delivery of nucleic acids in an efficient and safe manner remains an unmet need in the biological sciences. Whether this means using gene therapy to introduce functional genes to cells, or using RNA interference mechanisms to silence misregulated proteins, the main hindrance in these approaches to disease mitigation, is the lack of a suitable delivery platform. The first chapter is a literature review of the currently available technology for nucleic acid delivery including viral vectors as well as cationic lipids and polymers. Herein the advantages and disadvantages of both viral and non-viral delivery are discussed. In Chapter II, a biopolymer featuring a lysine-histidine (KH) condensing region fused to a targeting motif is discussed. The purpose of this study was to examine the effect of architecture on transfection efficiency of plasmid DNA through a series of in vitro biological and biochemical assays. Chapter III describes a recombinant biopolymer which features an arginine-histidine (RH) DNA condensing region, a targeting motif for HER2, and endosomal release fusogenic peptide, and a nuclear localization signal. Each domain was tested for functionality through in vitro assays. The multifunctional biopolymer demonstrated selective and efficient delivery of plasmid DNA and gene expression in SK-OV-3 cells. In Chapter IV the RH based biopolymers were characterized and evaluated in terms of their ability to deliver nucleic acids to either the cytoplasm or cell nucleus. Past delivery vectors have often emphasized extracellular barriers to entry and success meant internalization of the particle. Intracellular trafficking, however is also a key compenent in successful delivery. The objective of this study was to design a biopolymer that can be programmed via its amino acid sequence to deliver siRNA specifically to cytoplasm. By modifying the amino acid sequence, the same biopolymer can also be programmed to deliver pDNA to the cell nucleus. Intracellular trafficking was observed through fluorescent microscopy and assays were conducted to demonstrate either siRNA knockdown or delivery of a suicide gene/prodrug combination. In the research presented, we describe a non-viral biopolymer system that is able to overcome the extracellular and intracellular barriers to nucleic acid delivery.
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Details
- Title
- Recombinant cationic biopolymers for nucleic acid delivery
- Creators
- Brenda F. Canine
- Contributors
- Arash Hatefi (Chair)Margaret Black (Committee Member) - Washington State University, School of Molecular BiosciencesRaymond Reeves (Committee Member) - Washington State University, School of Molecular BiosciencesChulHee Kang (Committee Member) - Washington State University, Department of ChemistryDavid W Koh (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- College of Pharmacy and Pharmaceutical Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 162
- Identifiers
- 99901055024801842
- Language
- English
- Resource Type
- Dissertation