These investigations are focused on the design and formulation of novel nonviral lipopolyamine vectors capable of efficiently and safely delivering DNA to the nucleus, and siRNA to the cytoplasm, in two tissue cultured (primary and cancer) cell lines. The thesis starts with a focussed literature review on the non-viral gene therapy (NVGT) vectors currently used in the formulation of DNA and siRNA. The first experimental part tests the ability of our novel lipospermines in NVGT, this includes structure-activity relationship (SAR) studies changing the: position, length, saturation or symmetry of the fatty chains of N4,N9-diacyl, N1,N12-diacyl and N4,N9-dialkyl spermines. The ability of these lipospermines in DNA condensation is investigated using ethidium bromide fluorescence-quenching, and gel electrophoresis (including gel shift and DNase protection) assays followed by nanoparticle characterization techniques (particle size and zeta potential). Transfection efficiency of pEGFP (using FACS) and cytotoxicity (using MTT) were studied in both cancer and primary cell lines and compared with Lipogen™ (N4,N9-dioleoyl spermine). Some of these novel lipospermines are shown to be as good as, but not better than N4,N9-dioleoyl spermine as efficient DNA transfecting agents. N4,N9-Dioleoyl spermine is the best transfecting agent from the all tested novel lipospermines displaying the lowest N/P ratio, highest transfection efficiency and the lowest cytotoxicity on both tested cell lines. We extended this SAR study to examine the same lipospermines in siRNA delivery. The ability of these compounds to bind siRNA was studied using the RiboGreen intercalation assay followed by similar nanoparticle characterization techniques. Transfection efficiency for delivery of Label IT® RNAi Delivery Control (using FACS) and cytotoxicity (MTT) were also studied in both cancer and primary cell lines, and compared with a market leader siRNA transfecting agent Trans-IT™. Twelve of these non-viral vectors, led by N4,N9-dieicosenoyl spermine and N4,N9-dierucoyl spermine, showed both transfection efficiency and cell viability over 75%.

In this thesis nanoparticles consisting of a calcium phosphate core encapsulated by poly(lactic-co-glycolic) acid and polyethylenimine were developed for the delivery of siRNA in vivo. The nanoparticles were efficiently endocytosed by different cell types in vitro without exhibiting cytotoxic characteristics. Without possessing endogenous immune response activating properties, the nanoparticles had a highly preferable composition for the delivery of siRNA and subsequent gene knockdown. The delivery of siRNA with nanoparticles was tested in two different murine disease models: DSS-induced colitis as model for human IBD and a TH1-induced lung inflammation as model for COPD. In IBD and COPD chemokines and cytokines are predominant players in the progression of the inflammatory response. The local interference of cytokine signalling mediated by siRNA-loaded nanoparticles might therefore be a promising new therapeutic approach. In both murine models, the aim was to deliver siRNA directed against inflammation related cytokines by nanoparticles for the local treatment of mucosal inflammation. The local administration of nanoparticles loaded with siRNA to mice suffering from intestinal or lung inflammation led to significantly decreased target gene expression on mRNA as well as protein level in biopsies from the target tissues. Furthermore, reduced cytokine levels were accompanied by diminished inflammatory pathologies and augmented clinical signs of sickness. The results of this thesis indicate that a specific and local modulation of inflammatory responses by nanoparticle-based siRNA delivery is feasible and demonstrates a major therapeutic potential.

Huntington's disease (HD) can be regarded as a model neurodegenerative disorder to screen potential genetic based therapeutics and their carriers. It is an autosomal dominant disorder caused by a mutation in a single gene that leads to progressive neurodegeneration caused in part by protein misfolding. The mutation codes for an expanded polyglutamine tract within the Huntingtin gene (HTT) which leads to neuronal loss through a pathological cascade of events. Current treatment strategies include symptom management but no disease-modifying therapies exist. Research has shown that nucleic acid based therapeutics aimed at decreasing HTT expression can prevent, or reverse, the phenotype. Translating such therapies to the clinic is hindered by the blood brain barrier (BBB) and the lack of an easily administrable, non-toxic, immunologically inert delivery vehicle capable of bypassing the BBB. This study examines a range of nucleic acid based therapeutics for their potency and toxicity, and evaluates extracellular vesicles (EVs) as a delivery vehicle through investigation of the biodistribution of brain targeted EVs and an analysis of EV loading. A small interfering RNA (siRNA) targeting a region upstream of the repeat induced potent non-toxic silencing of HTT. In examining EVs as a carrier for therapeutics for neurological diseases including HD, it was found that targeting can increase brain accumulation of EVs but that the physiological characteristics of EVs which make them susceptible to clearance by the reticuloendothelial system (RES) must be further evaluated to bioengineer modified EVs to avoid fast clearance. Lastly, loading by electroporation was found to induce siRNA aggregation which can lead to overestimation of loading by increasing siRNA content in an ultracentrifugation pellet, but cholesterol-conjugated siRNA mixed with EVs was capable of generating silencing in vitro. This thesis examined EV based treatment for HD through the selection of a nucleic acid cargo to silence HTT, and examination of EVs as a delivery vehicle via biodistribution and loading studies. If the loading can be optimised and fast clearance avoided, there is promise in the use of EVs as a carrier of siRNA for HD.

In dieser Arbeit wurde mit Hilfe von small-molecule Inhibitoren die Rolle von clathrin- und dynaminabhängigen Endozytosemechanismen bei der Aufnahme von anti-Amphiphysin-Autoantikörpern am Zellkulturmodell primärer hippocampaler Neurone untersucht. Hierbei konnte eine Beeinflussung der Autoaantikörperaufnahme durch die Intervention gezeigt werden. Außerdem erfolgte der Versuch der Etablierung eines siRNA knockdowns unter Zuhilfenahme unterschiedlicher Traansfektionsreaaagenzien. / This thesis investigated the role of clathrin and dynamin dependent endocytosis in internalisation of anti-amphiphysin-autoantibodies in primary mouse hippocampal neurons by using small molecule inhibitors. An influence in the uptake due to small molecule treatment can be shown. Furthermore an attempt of siRNA knockdown establishment was performed using different transfection reagents.

SUMOylation is a type of post-translational protein modification similar to ubiquitination and it involves the covalent attachment of a small ubiquitin-like modifier (SUMO) protein to the lysine residue of a target substrate. While there is strong evidence for the role of protein ubiquitination in the formation of fear-based memories, few studies have been conducted examining the role that SUMOylation plays in this same process. The amygdala is the main site of storage for emotional memories and there is strong evidence that protein ubiquitination is critical for fear memory formation in this region. However, it has not previously been studied whether protein SUMOylation in the amygdala is also involved in fear memory formation. Additionally, although there is evidence to support sex differences in ubiquitin signaling during fear memory formation in the amygdala, whether males and females differ in their need for protein SUMOylation during fear memory formation has not been investigated. We have found significant sex differences in protein SUMOylation in the amygdala both at baseline (rest) and during fear memory formation. Western blot analysis revealed higher resting levels of SUMOylated proteins in females when compared to males, though both sexes showed global increases following fear conditioning. A SUMOylation-specific proteomic analysis discovered that only females had increased protein targeting with SUMO following fear conditioning, with four proteins being identified that gained SUMOylation modifications, the main target being a heat shock protein. One heat shock protein in males was identified as having lower SUMOylation levels following fear conditioning. This suggests sex differences in the interaction and targeting of proteins by SUMOylation following fear conditioning. We also inhibited the function of the only E2 conjugase for SUMOylation, Ube2i, via siRNA in the amygdala and found impaired fear memory in males but enhanced fear memory in females, though the latter only occurred under high siRNA concentrations. Interestingly, western blot analysis revealed that knockdown of Ube2i caused an increase in protein SUMOylation levels in females but a decrease in males, indicating that compensation is likely occurring in females. This suggests that in females, protein SUMOylation may be critical for basal cellular functioning, which precludes us from directly determining its role in fear memory formation. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation. / Master of Science / SUMOylation is a modification of protein which plays a key role in various biological processes and is similar to the protein modification process called ubiquitination, which has been implicated in the formation of fear-based memories for traumatic events. Despite this and the established role of SUMOylation in genomic stability, cell proliferation, and migration, less is known about its role in the process of memory formation. Importantly, ubiquitination and SUMOylation of proteins often work in tandem to regulate cell signaling and recent evidence suggests that SUMOylation may also be involved in fear memory formation. However, the role of protein SUMOylation in regulating fear memory formation in the amygdala, the primary site of storage for emotional memories, has never been directly examined. Additionally, there is also a significant gap in the literature regarding whether sex differences exist for the requirement of protein SUMOylation in fear memory formation. We have found that there are significant differences between the sexes regarding protein SUMOylation during fear memory formation in the amygdala. Western blot analysis showed that females have higher resting (baseline) levels of SUMOylated proteins in the amygdala compared to males, though both sexes showed global increases in protein SUMOylation following fear conditioning. In addition, a proteomic analysis revealed that four proteins in females gained a SUMOylation modification following fear conditioning. In contrast, one protein was identified in males which lost a SUMOylation modification, together suggesting unique targeting of proteins by SUMOylation across sexes during fear memory formation. Further, when the function of an essential enzyme for protein SUMOylation was inhibited in vivo, fear memory in males was impaired but enhanced in females. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation.

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Autophagy, referring to macroautophagy, is an evolutionarily conserved degradation pathway. Through autophagy cells can degrade damaged organelles, lipid vesicles and misfolded protein aggregates with implications in various pathological conditions, including neurodegenerative diseases, cancers, and infectious diseases. Autophagy is one of the major intracellular membrane-trafficking processes and its morphology includes the initiation, maturation, transportation and degradation of autophagosomes, where the double-membrane autophagosomes package the cargo for degradation. Therefore, understanding how autophagosomes form and are regulated is important in this field. Here we conducted a genome-wide siRNA screen using a high-throughput imaging system to identify undiscovered regulators in autophagosome formation. In this study, HEK293 cells stably expressing GFP-DFCP1 (GFP-tagged zinc finger FYVE-type containing 1) were used and amino acid starvation was used to induce autophagy. After the first round of primary screening, a small-scale screen was conducted with the same conditions including 384 candidates and additionally with these candidates HEK293 cells stably expressing GFPLC3 (GFP-tagged microtubule associated protein 1 light chain 3) were used to monitor the late steps of the autophagy process. From these rounds of screening, 39 candidates were selected and validated by investigating early autophagosome markers for their effects on autophagosome formation. Finally, five of the best candidates were confirmed based on their depletion effects on autophagy. Among these five candidates, DCAKD (dephospho-CoA kinase domain containing), WDR91 (WD repeat domain 91) and WDR65 were further investigated. Preliminary data using both RNAi and CRISPR Cas9 showed that DCAKD affected the accumulation of some early autophagy markers on initiation membranes in autophagosome formation. Interestingly, protein levels of canonical autophagy markers remained unchanged in DCAKD-deficient cells. Another candidate WDR91 showed the ability to mediate endosomal and lysosomal PtdIns3P and mildly affect autophagy initiation. WDR65 also inhibited early autophagy events. However, the detailed mechanism for these proteins are yet to be determined. In summary, our work provided more understanding on the egulation of autophagosome formation, as well as a list of potentially novel regulators.

This PhD project explored the role of tricarboxylic acid (TCA) cycle enzymes in regulating Chinese hamster ovary (CHO) cell metabolism with respect to growth and recombinant protein expression. It was hypothesised that regulation of central metabolism can influence CHO cell productivity in terms of biomass and protein production. Thus the aim of these studies was to identify the key metabolic reactions of the TCA cycle associated with growth and protein expression in CHO cells. The gene expression of all TCA cycle genes was independently knocked-down using RNAi technology. The small interfering RNA (siRNA) mediated silencing of 11 TCA cycle genes significantly reduced cellular growth along with a decline in adenylate energy charges and an increase in catabolic reduction charges. The gene profiling of glucose and amino acid metabolism (not targeted by siRNA) suggested siRNA mediated knock-down of targeted TCA cycle genes led to cellular stress along with an enhanced rate of glycolysis leading to channelling of glucose for the generation of pyruvate. For the purpose of estimating intracellular metabolites, quenching and extraction method using ammonium bicarbonate and methanol was optimised to use with UCB CHO-K1 cell line and static transient siRNA transfections. A gas chromatography-mass spectrometry (GC-MS) analysis post-silencing of the aconitase gene, which catalyses the conversion of citrate to isocitrate in the TCA cycle, yielded higher MS peak intensities of at least four metabolites (gluconic acid, lysine, threonine and leucine) 72 h post-transfection in comparison to the controls. Transient knock-down of gene expression of seven TCA cycle genes in a recombinant stable cell line (expressing a rabbit monoclonal antibody) reduced cellular growth and altered the energy charges leading to a decline in antibody expression. Although silencing of the pyruvate dehydrogenase E1 gene, which is the component of the pyruvate dehydrogenase complex connecting glycolysis to the TCA cycle, did not affect cell viability, a reduction in antibody expression was recorded. Seven TCA cycle genes which demonstrated the most significant effect on cellular growth and energy charges were transiently over-expressed along with a monoclonal antibody in CHO-K1 cells with addition of their corresponding preceding intermediates. No differences in protein expression and cell specific productivity were observed compared to the control transfections. These results could be due to limitations of the effects of transient transfections for enhancing the metabolic activity of CHO cells. The aconitase gene demonstrated the most significant effect on CHO cell growth and proliferation in this study, therefore this gene was proposed as a novel selection marker for a metabolic selection system for the generation of recombinant therapeutics. This PhD project also established metabolite analysis tools and siRNA protocols for future metabolomic studies for investigating the intracellular CHO metabolism. The findings validated the hypothesis that TCA cycle plays an important role in CHO cell growth and recombinant protein production. The key metabolic genes affecting cellular growth and altering energy metabolism can be further explored for generation of an energy efficient CHO host-cell line (by over-expression of key TCA cycle genes) for enhanced recombinant protein production.

Influenza A is a single-stranded, multi-segmented, negative sense RNA virus of the family Orthomyxoviridae and is the causative agent of seasonal Influenza. Influenza viruses cause significant impacts on a global scale regarding public health and economics. Annual influenza virus infections in the United States account for over 200,000 hospitalizations, up to 49,000 deaths, and an $87.1 billion economic burden. Influenza A virus has caused several pandemics since the turn of the 20th century. The effects of Influenza on public health and economics, compounded with low efficacy of the annual vaccine and emerging antiviral resistance, brings to light the need for an effort to stem these impacts, prevent pandemics, and protect public health by developing novel treatments. This project proposes an alternative approach to combatting Influenza by targeting host factors hijacked during infection that, if inhibited, significantly impair viral RNA expression, but result in low host toxicity. The host factors we examined include RNA export factors (XpoT and Xpo5) and RNA helicases (UAP56 and URH49). We selected paralogs URH49 (DDX39A) and UAP56 (DDX39B) because previous studies suggest differing roles during infection, but we theorize that their high degree of sequence similarity, similar function, and association with many of the same cellular factors may allow them to substitute for one another if one is inhibited. CRISPR was considered as the primary method to evaluate the effect of knockout of these factors on viral RNA expression and host cell toxicity. CRISPR is an RNA-guided mechanism for gene editing and can be used to make null mutations in targeted host genes. However, CRISPR proved to be a significant challenge and, while we could not conclusively confirm whether the CRISPR plasmids were effective at targeting our genes of interest, our initial results were not promising and we did not pursue this approach further. As an alternative, host RNA export factors were evaluated using siRNA to knockdown the factor prior to influenza infection. RNA was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The potential of inhibiting UAP56 or URH49 as a novel therapeutic target was determined using a visual assessment of cell death. We found that siRNA-mediated knockdown of XpoT and Xpo5 did not have any impact on viral RNA synthesis early during infection. siRNA against UAP56 and DDX39 (targets both UAP56 and URH49) resulted in significant impairment in viral RNA synthesis, confirming previously established work suggesting that UAP56 and URH49 have important roles during infection. Importantly, these helicases play an interferon (IFN) independent role to enhance viral replication, as indicated by analysis in IFN deficient VERO cells. A viability assay relying on trypan blue exclusion did not yield trustworthy results, so a visual assessment of cell death was done. The visual assessment confirms previously-established observations that Nxf1 siRNA treatments result in a high degree of cell death, indicating the toxic nature of Nxf1 inhibition. Cells treated with UAP56 or DDX39 siRNAs demonstrated little to no additional toxicity compared to the non-target control, suggesting they can be inhibited to serve as antiviral targets.

Applications of nanotechnology in medicine, also known as nanomedicine, is a rapidly growing field as it holds great potential in the development of novel therapeutics toward treatment of various diseases. Shell crosslinked knedel-like nanoparticles (SCKs) that are self assembled from amphiphilic block copolymers into polymeric micelles followed by crosslinking selectively throughout the shell domain have been investigated as theranostic agents for the delivery of nucleic acids and incorporation of imaging probes. The main focus of this dissertation is to design and develop unique multifunctional bio-synthetic hybrid nanoparticles that can carry agents for radiolabeling, moieties for inducing stealth properties to minimize protein adsorption in vivo, ligands for site-specific targeting, therapeutic payloads, and are optimized for efficient delivery of cargoes intracellularly and to the target sites toward constructing novel nanoscopic objects for therapy and diagnosis. Alteration of polymeric building blocks of the nanoparticles provides opportunities for precise control over the sizes, shapes, compositions, structures and properties of the nanoparticles. To ensure ideal performance of nanoparticles as theranostic agents, it is critical to ensure high intracellular bioavailability of the therapeutic payload conjugated to nanoparticles. Special efforts were made by employing well-defined multi-step polymerization and polymer modification reactions that involved conjugation of peptide nucleic acids (PNAs) to chain terminus of poly(ethylene glycol) (PEG) chain grafts such that they were presented at the outermost surface of SCKs. Additionally, chemical modification reactions were performed on the polymer backbone to integrate positive charges onto the shell of the nanoparticles to afford cationic SCKs (cSCKs) for facilitating cellular entry and electrostatic interactions with negatively charged nucleic acids. Covalent conjugation of F3, a tumor homing peptide, post-assembly of the nanoparticles enhanced cellular uptake and knockdown of nucleolin (a shuttling protein overexpressed at the sites of angiogenesis) and thus inhibiting tumor cell growth. Furthermore, these polymer precursors of the cSCKs were modified with partial to full incorporation of histamines to facilitate their endosomal escape for efficient delivery into the cytosol. The cSCKs were further templated onto high aspect ratio anionic cylinders to form hierarchically-assembled nanostructures that bring together individual components with unique functions, such as one carrying a therapeutic payload and the other with sites for radiolabeling. These higher order nanoobjects enhance circulation in vivo, have capabilities to package nucleic acids electrostatically and contain sites for radiolabeling, providing an overall advantage over the individual components, which could each facilitate only one or the other of the combined functions. Hierarchically-assembled nanostructures were investigated for their cellular uptake, transfection behavior and radiolabeling efficiency, as the next generation of theranostic agents.