Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and
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are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43690 |
| Date | 14 January 2014 |
| Creators | Orava, Erik |
| Contributors | Gariepy, Jean |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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