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Polymerase chain reaction (PCR) sequencing of specific viral gene segments was used to investigate the phylogenetic relationships among the orbiviruses. Sequence comparisons of the bluetongue virus (BTV) RNA3 from different regions of the world (North America, South Africa, India, Indonesian, Malaysia, Australia and the Caribbean region) showed that geographic separation had resulted in significant divergence, consistent with the evolution of distinct viral populations. There were at least 3 topotypes (Gould, 1987); the Australasian, African - American and another topotype represented by BTV 15 isolated in Australia in 1986. The topotypes of BTV had RNA3 nucleotide sequences that differed by approximately 20 per cent. Analysis of BTV-specific gene segments from animal and insect specimens showed that bluetongue viruses had entered northern Australia from South East Asia, possibly by wind-borne vectors. Nucleotide sequence comparisons were used to show the close genetic relationship between BTV 2 (Ona-A strain) from Florida and BTV 12 from Jamaica, and to investigate the reassortment of BTV genome segments in nature. The mutation rates of the BTV RNA2 and RNA3 segments were estimated to be of the order of 10(-4) nucleotide changes/site/year, similar in magnitude to that reported for other RNA viruses.

The feasibility of most gene therapy strategies depends on the efficient delivery of DNA to target cells and tissues. Current gene delivery carriers can be divided into two classes: viral and non-viral delivery systems. Although the viral carriers are highly efficient due to their invasive nature, safety concerns may restrict their application in clinical settings. Synthetic non-viral carriers attract increasing attention because they are less toxic and allow readily modification. Non-viral carrier mediated gene delivery involves several processes. They must condense DNA into small particles, allow membrane penetration and protect DNA from extracellular and intracellular degradative enzymes. In the present study, a small library of carriers containing various combinations of cell penetrating peptide TAT, SV40 large T protein nuclear localisation signal (NLS) and cationic dendrimer of 7 lysine residues (DEN) was synthesised and tested for their ability to deliver DNA to mammalian cells. We evaluated the contribution of each component as well as the combination of the components on DNA condensation, uptake and gene expression. It was found that all carriers condensed DNA and protected DNA from DNase degradation. We showed that the TAT peptide was essential, but not sufficient, for uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake. The most efficient carrier contained all three components (DEN-NLS-TAT). The carriers were able to deliver DNA in the presence of serum and were non-toxic to cells at up to 30 μM. However, for those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required a fusogenic agent, such as chloroquine, before gene expression was observed. Modifications were introduced to the initial carrier library in order to circumvent the chloroquine dependence. The addition of cell penetrating peptide penetratin, virus derived fusogenic peptide or lipoamino acid C12 enhanced either DNA uptake or endosomal release. However, none of the modified carriers were able to produce high level transgene expression in the absence of chloroquine. We also found that the carriers containing lipid components were able to deliver DNA to T-lymphocytes derived cells, which are usually resistant to transfection. However, the toxicity of the lipid-based carriers needs to be reduced before further application. We also evaluated the function of chloroquine as a gene expression enhancer. We demonstrated that chloroquine did not enhance expression solely by promoting endosomal release. This was supported by the fact that fusogenic peptide and endosomal disruptive reagents (bafilomycin A1 and monensin) did not improve gene expression. Other properties of chloroquine, such as DNA protection and transcription enhancement, may also contribute to gene expression. We characterised the uptake mechanism of DEN-NLS-TAT in HeLa cell lines. We found that the uptake of DEN-NLS-TAT/DNA complex in HeLa cell line was mainly via receptor-mediated endocytosis and caveolae endocytosis. Moreover, various intracellular processes, such as intact cytoskeleton and microtubule network, tyrosine and PI 3 kinase activity, and membrane cholesterol were also required for the uptake of the carrier/DNA complex. In conclusion, the results from the present study demonstrated that multi-component peptide-based carriers are versatile carriers for the delivery of plasmid DNA in human cells. The results have improved our understanding of the role of chloroquine as a widely used gene expression enhancer which may be useful in the future improvement of non-viral gene delivery carriers. A strategy to overcome the dependence on chloroquine for gene expression or reduce the toxicity of chloroquine will be necessary for further in vivo applications. The current carrier library may also be used to delivery other cargos such as siRNA or protein to human cells.

The ability to deliver DNA to target cells creating therapeutic effects remains an important goal in the field of gene therapy. A majority of clinical trials to overcome this issue have utilized viral vectors due to their efficiency at DNA delivery and ability to create high levels of gene expression. However, their inherent toxicity and a several clinical trials leading to patients contracting new diseases from the treatment have greatly hindered the progress of viral gene therapy. Non-viral gene delivery agents have a much better safety profile, but are also much less efficient at delivering DNA, leading to low gene expression. The reason for this low expression is the numerous barriers that must be overcome to achieve gene expression: circulation, tissue specific accumulation, internalization, release of DNA cargo, and nuclear localization. While peptides are currently being improved upon, enhancing binding and the ability to protect DNA, they are still deficient when it comes to tissue specificity. Numerous targeting methods, including the use of lectins, antibodies, aptamers, and peptides, have been designed to deliver molecules to a specific research. Research to incorporate targeting ligands onto non-viral gene delivery vectors is abundant in the literature; however, successful site specific gene delivery has not been achieved. The somatostatin receptor 2 (SSTR2) ligand, octreotide, is a well-researched eight amino acid peptide that has extensive SAR data available. Also, the receptors have been well characterized and octreotide is used clinically in the radioscintigraphy imaging of brain tumors. While well researched, there are unexplored opportunities to utilize octreotide to enhance non-viral gene delivery vectors. The overall scope of this thesis is to develop and synthesize non-viral gene delivery peptides conjugated to octreotide creating receptor mediated targeting of DNA polyplexes to create tissue specific accumulation. Initial experiments indicated that attachment of octreotide to the polycationic peptide WK18 does not inhibit affinity for the SSTR2 receptor. Therefore, peptides were designed and synthesized to attach octreotide onto polyacridine peptide (Acr-Lys)6. Polyplex characteristics were unchanged by the incorporation of octreotide, and exhibited very low genotoxic effects compared to the in vitro gene delivery agent PEI. Competitive binding assays suggested a stoichiometric, ligand, and temperature dependent accumulation of polyplex on SSTR2 expressing cells, but gene expression could not be achieved. The success of (Acr-Lys)6octreotide, led to the synthesis of a di-maleimide-PEG attached to each end by (Acr-Lys4)3Acr-Lys-Cys or Cys-Gly5octreotide in attempts to create distance, and better ligand availability for the receptor, by expressing octreotide away from the polyplex. Testing of this peptide in PEGylated polyplex ad-mixtures verified that separating the DNA binding peptide from octreotide did lead to better inhibition of binding to DAOY cells in a competitive binding study. However, transfection assays with this compound showed background levels of gene expression. Although gene expression was not achieved, the synthetic strategy to create a molecule incorporating a DNA binding peptide, ligand, and PEG to create better ligand presentation to its receptor when incorporated into PEGylated polyplexes is an important step in the design of gene delivery vectors.

The 21st century has been called the age of genomic medicine, yet gene therapy for medicinal use remains a theory. One reason that there are no safe and effective treatments for human disease is the lack of a vehicle capable of delivering genetic material to a specific target. In nature we observe gene pathology by viral vectors, which deliver their own genetic material to specific host cells efficient at spreading the viral blueprint throughout the organism. The aim of my research into gene therapy has been to develop a synthetic vector with the delivery capability of viral vectors found in nature. This includes the ability to protect genetic cargo from modification and degradation in vivo, target to a desired cell type within a specific tissue, facilitating absorption into the cell, and delivery to the nucleus, where expression of genetic material occurs. The goal of this thesis project was to synthesize a novel vector which would selectively target the dendritic cell SIGN receptor, mirroring the method of pathogens such as HIV, which target this receptor and subsequently the immune system, resulting in chronic infection. The vector we designed contains two major components, the high mannose N-glycan Man9GlcNAc2Asn, and a peptide composed of nine amino acids: four lysine spacing residues, four lysines derivatized with acridine on the epsilon amine of their side chains, and a cysteine for conjugation to the glycan. This compound, the Man9-AcrLys Glycopeptide, was engineered to intercalate into plasmid DNA via the acridine functional groups and to bind the DC-SIGN receptor through the glycan's mannose residues. The vehicle was tested in vitro in CHO cells bearing a recombinant DC-SIGN receptor in the context of luciferase reporter gene delivery. We found that under equal treatment conditions, DC-SIGN (+) CHO cells expressed more luciferase and were 100-fold more luminescent than control DC-SIGN (-) CHO cells. My delivery method was further analyzed in a cell-sorting FACS experiment. I covalently labeled pGL3 reporter plasmid with a fluorophore, and transfected the CHO cells under typical transfection conditions. The experimental results confirmed preferential DC-SIGN mediated gene delivery.

The pharmacologic treatment of painful neuropathy continues to pose problems and challenges in clinical practice. This is largely due to a limited understanding of the underlying etiologies of such neuropathic pain and insufficient knowledge of the optimal effective doses that would cause only minimal systemic side effects. The use of molecular methods, such as gene deletion from knockout mice and the development of cellular mini-pumps for the delivery of biologic antinociceptive molecules have led to a better understanding of the underlying mechanisms involved in the induction of intractable neuropathic pain. It is now known that the initiation of an excitatory cascade after injury or disease leads to the induction of various second messenger systems, and the loss or down-regulation of the endogenous inhibitory spinal system and central sensitization, both of which cause such pain. Currently, there are novel approaches that use genetic therapy in the management of neuropathic pain. Two such approaches which have been determined to be safe are proposed to be investigated in this study using animal models of pain. The first approach involves cell-mediated delivery of antinociceptive molecules to the cerebrospinal fluid using cultivated spinal progenitor cells transplanted into the subarachnoid space. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the hind paw of rats. 1x106 spinal progenitor cells (SPCs) were implanted intrathecally on the third day after the CCI surgery. The behavioral response to thermal hyperalgesia was observed and recorded during the 14 days post surgery. Various techniques were utilized to trace the progenitor cells, confirm the differentiation, and identify the neurotransmitters involved. Glutamic acid decarboxylase (GAD) immunoreactivity was revealed for 65% of the cultivated SPCs in our study. We also determined that transplanted cells could survive more than four weeks post intrathecal implantation. Significant reductions were demonstrated for responses to thermal stimuli for the CCI rats that had received intrathecal SPC transplantation. A novel intrathecal delivery with SPCs reduced CCI-induced neuropathic pain. The second approach involves the use of a newly developed intrathecal electroporation probe in the delivery of antinociceptive peptides to reduce expression of endogenous nociceptive molecules in the spinal cord. To investigate the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection, pE-GFP C1 vector was used to achieve the goal. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of, followed by 5 electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and western blotting. At day 1, 3 or 7 following electroporation a clear green fluorescence protein (GFP) expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and western blotting. The expression of GFP protein was peaked between 3-7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery. Furthermore, the optimal pulse characteristics (voltage, pulse duration, number of shocks) were investigated for in vivo electroporation for gene transfer into the spinal cord. The expression of pre-opiomelanocortin (POMC) gene from electroporated plasmid DNA was then evaluated in this study using RT-PCR and western blot. We conclude that the optimal conditions for electroporation are a pulse voltage of 200 V, 75-ms duration, 925-ms interval, for five iterations. Also, electroporation treatment for neuropathic pain was attempted for CCI rats using plasmid DNA that expresses the POMC gene. Intrathecal administrations of the POMC plasmid elevated spinal beta-endorphin levels, as manifested in significantly elevated pain threshold for the CCI limbs. We also tested whether intrathecal electric stimulation would reduce the tolerance of chronic morphine usage and the severity of precipitated morphine withdrawal symptoms. Rats received intrathecal electrode catheter implantation and a continuous intrathecal infusion of morphine (2 nmol/hr) or saline for seven days. Intrathecal electric stimulations (0, 20V, 200V) were performed once daily during the same period. Daily tail flick and intrathecal morphine challenge tests were performed to assess the effect of intrathecal electric stimulation on antinociception and tolerance of morphine. Naloxone withdrawal (2mg/kg) was performed to assess morphine dependence, and changes in spinal neurotransmitters were monitored by microdialysis. The antinoceptive effect of intrathecal morphine was increased by 200V electric stimulation. The magnitude of tolerance was decreased in the rats receiving 2 nmol/hr infusion with daily intrathecal electric stimulation. The severity of naloxone-induced withdrawal symptom was lower in the rats receiving 200V stimulation. Intrathecal stimulation thus enhances analgesia and attenuates naloxone-induced withdrawal symptoms in rats receiving chronic intrathecal morphine infusion. Increases in spinal glycine release may be the underlying mechanisms. The promise is that, both approaches attenuate or reverse persistent nociceptive states; they could be exploited for use in the development of gene therapy for the management of pain.

abstract: Gene delivery is a broadly applicable tool that has applications in gene therapy, production of therapeutic proteins, and as a study tool to understand biological pathways. However, for successful gene delivery, the gene and its carrier must bypass or traverse a number of formidable obstacles before successfully entering the cell’s nucleus where the host cell’s machinery can be utilized to express a protein encoded by the gene of interest. The vast majority of work in the gene delivery field focuses on overcoming these barriers by creative synthesis of nanoparticle delivery vehicles or conjugation of targeting moieties to the nucleic acid or delivery vehicle, but little work focuses on modifying the target cell’s behavior to make it more amenable to transfection. In this work, a number of kinase enzymes have been identified by inhibition to be targets for enhancing polymer-mediated transgene expression (chapter 2), including the lead target which appears to affect intracellular trafficking of delivered nucleic acid cargo. The subsequent sections (chapters 3 and 4) of this work focus on targeting epigenetic modifying enzymes to enhance polymer-mediated transgene expression, and a number of candidate enzymes have been identified. Some mechanistic evaluation of these targets have been carried out and discussion of ongoing experiments and future directions to better understand the mechanistic descriptions behind the phenomena are discussed. The overall goal is to enhance non-viral (polymer-mediated) transgene expression by modulating cellular behavior for general gene delivery applications. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2016

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