The Rational Design, Synthesis and Evaluation of Non-Viral Gene Delivery Systems Based on Computer-Aided Drug Design

Defang Ouyang (2010). The Rational Design, Synthesis and Evaluation of Non-Viral Gene Delivery Systems Based on Computer-Aided Drug Design PhD Thesis, School of Pharmacy, The University of Queensland.

       
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Author Defang Ouyang
Thesis Title The Rational Design, Synthesis and Evaluation of Non-Viral Gene Delivery Systems Based on Computer-Aided Drug Design
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2010-08
Thesis type PhD Thesis
Supervisor Dr. Harendra S.Parekh
Prof. Sean C.Smith
Total pages 159
Total colour pages 24
Total black and white pages 135
Subjects 11 Medical and Health Sciences
Abstract/Summary Gene therapy is a promising and rapidly developing medical approach, the aim of which is to transfer therapeutic genes into target cells to replace or destroy an "abnormal" disease-causing gene. However, a significant impediment to this approach becoming a therapeutic tool in the clinic is the lack of safe and effective delivery systems, coupled with a poor universal understanding of the underlying mechanisms leading to gene delivery. Thus, the broad aim of the studies described herein is to explore the mechanisms of gene delivery from different aspects, and then design and develop novel non-viral vectors with the aim of improving our understanding of how vectors behave and how rational design can then be employed to overcome biological barriers. It is apparent from our recently published review that non-viral reducible vectors, comprising disulfide bridges lead to selective (intra- over extracellular delivery) and more efficient intracellular gene release, resulting in higher transfection efficiencies (c.f. non-reducible vectors). However, existing synthetic strategies to introduce disulfide bridges into molecules employ costly reagents and are laborious. In our research, we designed and compared three approaches toward introduction of disulfide bonds into peptides and assessed the efficiency of each via construction of model tripeptide ‘HO-Cys((S-S-(CH2)2-NH2)-Lys-Gly-H’ using Fmoc-solid phase peptide synthesis (SPPS). The first two methods describe on-resin manipulation to introduce the terminal –S-S- bond, while the third, innovative approach utilizes a newly developed disulfide bridge-ready amino acid. Development of Boc-Cys(S-S-(CH2)2-NHBoc)-OH is firstly described and its stability profile also investigated. Its application toward the facile preparation of a disulfide-based tripeptide has been successfully demonstrated. Molecular dynamics (MD) can be used for exploring the structure, dynamical process and binding energies of biomolecular complexation on the molecular level. However, existing MD simulations for polymer-gene complexation do not correlate well with biological experiments/ conditions. In this thesis, systematic investigations of the complexation process of siRNA-cationic polymer were performed by MD simulations and their effects in gene release within the cytosol were analysed. Firstly, we studied the conformational fluctuation of double-strand 21 base-pair siRNA in the aqueous environment by MD simulations to provide clues into the complexation processes that occur with other biomolecules/carriers, which added a dynamical perspective beyond the static information available from X-ray data and the effects of complexation with other biomolecules for two conformations of siRNA. Changes in the two conformations of siRNA in aqueous solution were analyzed from three perspectives these being, major groove width, inclination angle and base pair per helix. Next we studied the single cationic polymer-siRNA complexation process by MD simulations. The simulations reveal detailed molecular-level pictures of the structures and dynamics of the RNA-polycation complexes. Estimates for the binding free energy indicate that electrostatic contributions are dominant followed by van der Waals interactions. The binding free energy between 8+polymers and RNA was found to be greater than that of 4+polymers, and was in general agreement with previously published data. Reliable binding free energies were firstly introduced to provide an effective index of the ability of the polycationic carrier to bind a nucleic acid and also carry implications for the process of gene release within the cytosol. Moving forward we investigated a highly complex system whereby multiple cationic polymers-siRNA complexation was studied by MD simulations, a scenario that represents more closely, the processes occurring at a cellular level when genes and vectors associate. At lower charge ratios polymers bind quite effectively to siRNA, while at high charge ratios the complexes are saturated and there are free polymers which are unable to associate with RNA. We also observed reduced fluctuations in RNA-structures when complexed with multiple polymers in-solution compared to both free siRNA in water and the single polymer complexes. These novel simulations provide a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance progress towards rational design of non-viral gene delivery systems. Live cell imaging experiments are increasingly useful qualitative and quantitative tools for gauging delivery to cells and determining the fate of both gene and vector once delivered. In the tail-end of the thesis we explore the uptake of oligo-dendrimer complexes in NIH3T3 EpoR cells via the use of two different dyes (green and red) independently labelled to an oligonucleotide and dendrimer. Albeit unsurprisingly we demonstrated that the oligonucleotide alone is unable to translocate the cell membrane; this result being effectively reversed in the presence of preformed oligo-dendrimer complexes. A gradual increase in charge ratio further facilitated the delivery of oligo-dendrimer complexes, but we observed an inverse relationship i.e. delivery efficiency reduced on increasing oligo length. Furthermore, delivery rates for single strand oligos appeared to be better than double strand oligos, under identical conditions. Overall, our research systematically investigated the mechanisms relating to the gene delivery from chemical, physical and biological perspectives, which will effectively facilitate the rational design of gene delivery systems in the future.
Keyword gene delivery
siRNA
disulfide bond
synthesis
molecular dynamics simulation
computational chemistry
live cell imaging
Additional Notes Colour pages: 77-80, 88-90, 96, 100-104, 108-112, 119-124.

 
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Created: Sat, 26 Feb 2011, 21:08:00 EST by Defang Ouyang on behalf of Library - Information Access Service