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Pharmacy

Permanent URI for this collectionhttps://uwspace.uwaterloo.ca/handle/10012/9947

This is the collection for the University of Waterloo's School of Pharmacy.

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Browsing Pharmacy by Author "Aucoin, Marc"

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    The construction and characterization of a synthetic SARS VLP comprised of betacoronavirus consensus sequence-encoded viral components
    (University of Waterloo, 2022-12-22) Ng, Andrew; Slavcev, Roderick; Aucoin, Marc
    In 2019, a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China leading to the COVID-19 pandemic. Although there was an outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) in 2003 and 2012, respectively, a vaccine against the two SARS viruses were not given high priority due to low interest. The lack of investment into a SARS vaccine in the past has placed researchers in the position to race against time to produce a COVID-19 vaccine in a span of several months. The three coronavirus outbreaks and the emergence of several strains of COVID-19 suggests that future outbreaks are inevitable. Although the COVID-19 pandemic is still a concern, the time to start preparing for the next pandemic-causing coronavirus is now. A “universal vaccine”, which may provide cross-protective abilities, may be a highly effective approach in the protection against current and future coronaviruses, removing the need to generate new vaccines to address each emerging strain or variant. Universal vaccines function by relying on viral components that are highly conserved among various strains. A consensus sequence, which is a single sequence that represents several related sequences, may function to confer universal protection. Virus-like particles (VLPs) have been shown to be a highly safe and promising vaccine platform that can mimic the native virus and activate an immune response comparable to natural infections. This project will explore whether a consensus sequence based on the similarities of SARS-CoV, MERS-CoV and SARS-CoV-2, including its emerging variants, can produce a viable VLP that assembles to serve as a potential cross-protective vaccine candidate.
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    Construction and Characterization of a Targeting M13-Based Phagemid Carrying an Anti-Angiogenic DNA-Encoded Virus-Like Particle
    (University of Waterloo, 2024-09-18) Li, Jiayang; Slavcev, Roderick; Aucoin, Marc
    Over the past several years, molecular targeted therapy has emerged as a promising strategy for cancer treatment. Unlike broad-spectrum cytotoxic drugs used in conventional chemotherapy, targeted therapy aims to address specific molecular alterations unique to cancer cells. To develop effective targeted therapies, numerous delivery platforms have been investigated to optimize safety, specificity, and efficiency. The work presented here investigates the construction and characterization of a miniphagemid-mediated cancer therapy delivering anti-angiogenetic DNA-encoded virus-like particles (VLPs). VLPs have shown a robust ability to stimulate potent immune responses and overcome the immunosuppressive state of the tumour microenvironment (TME). Additionally, the filamentous bacteriophage (phage) M13 has been explored as a safe and efficient vehicle for delivering therapeutic genes and drugs. Phage-based vectors (phagemids) can be engineered to transfer exogenous genetic material to mammalian cells safely, as they possess no natural tropism. The present study aims to combine the advantages of both VLPs and phagemids to construct a hybrid biological platform for the specific delivery of DNA encoding VLP-displaying anti-tumour peptides, specifically VGB4, to tumour cells via M13 – a filamentous phage capable of phagemid production as well as phage display. The VGB4 peptide has demonstrated potent ability to inhibit angiogenesis in the tumour vasculature by blocking the downstream signalling pathways of vascular endothelial growth factor receptor (VEGFR). The human papillomavirus (HPV) type 16 L1 capsid gene with an inserted VGB4 peptide sequence was cloned into a miniaturized phagemid (miniphagemid) engineered by our lab. This genetically engineered miniphagemid was produced in Escherichia coli using a novel non-packaging M13 helper plasmid. The helper plasmid not only complements phagemid packaging but also enables the display of a cell-specific targeting ligand, the epidermal growth factor (EGF), which promotes receptor-mediated endocytosis for specific phage uptake by tumour cells overexpressing epidermal growth factor receptors (EGFRs). This thesis project investigated the formation of VGB4-displaying HPV VLPs within HEK 293T and HeLa cells. Our results demonstrated that the EGF-displaying miniphagemid improves gene delivery to cells compared to non-displaying miniphagemids. Furthermore, the VGB4-displaying HPV VLPs do not form in cells treated with miniphagemids, but these VLPs are successfully formed in cells treated with the precursor phagemids encoding the same gene cassette. Overall, this study highlights the necessity for further investigation and optimization to enhance miniphagemid-mediated gene transfer by overcoming cellular barriers, paving the way for its application as a novel targeted gene therapy for cancer.
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    Design of a DNA-Encoded Human Papilloma Virus-Like Particle Displaying a Vascular Endothelial Growth Factor Antagonistic Peptide for Characterization in Mammalian Cells
    (University of Waterloo, 2022-12-21) Pushparajah, Deborah; Slavcev, Roderick; Aucoin, Marc
    Cancer immunotherapy has evolved as an effective platform for the treatment of a variety of cancer types by enhancing and/or modulating the functionality of the immune system to target cancer cells and consequently mitigate tumour growth. Within the last decade, self-assembled viral-derived protein complexes, known as virus-like particles (VLPs), have been extensively studied for their application toward cancer immunotherapy. These structural viral-mimicking particles possess the capability to engender potent immune responses, and in doing so, combat the immunosuppressive tumour microenvironment. VLP-based vaccines have been commercialized, however, VLPs encoded as a genetic sequence, such as a DNA-VLP strategy, for delivery and subsequent in vivo formation, has not been licensed to date. This would enable transcription and translation of the introduced genetic sequence into viral structural proteins, assembly of the expressed proteins to form VLPs, and successive immune response stimulation, characterizing this type of treatment modality as both an immunotherapeutic and gene therapeutic. Delivery of VLPs encoded as a genetic sequence, opposed to conventional VLP delivery, enables viral structural protein expression and assembly into VLPs directly within cancer and immune cells, promoting enhanced cell-mediated immune responses, which can contribute to a greater extent towards tumour eradication. Here, we are seeking to apply this concept toward the design of a DNA-VLP gene cassette to produce human papillomavirus (HPV) 16 VLPs as a gene therapy-based cancer immunotherapeutic. The DNA-VLP gene cassettes were designed to encode the major capsid protein of HPV16, known as L1, along with an inserted peptide. This peptide, known as VGB, is characterized as an anti-angiogenic molecule that has previously demonstrated active reduction of cancer cell proliferation and tumour growth. Transfection of the designed DNA-VLP gene cassettes was conducted within mammalian cells, which successfully encoded the HPV16 L1 protein, in addition to possible in vitro assembly of VGB-displaying HPV16 L1 VLPs. This was validated via western blot analysis, enzyme-linked immunosorbent assay experimentation, and visualization using transmission electron microscopy. Potential display of the VGB peptide within surface exposed regions of the VLPs was observed by increased binding towards VGB’s targeted receptor, VEGFR. The prospective for cell lysis contributed by the accumulation of VLPs within mammalian cells was not validated, as decreased cell growth and viability subsequent to transfection were not observed. Overall, the characterization of VGB-displaying HPV16 L1 VLPs encoded within the designed DNA-VLP gene cassettes, promotes further investigation to employ this as a potential gene therapy-based cancer immunotherapeutic for future clinical applications.