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Title: Engineering liposome-siRNA vectors for anti-angiogenic tumour therapeutics by gene silencing
Author: Podesta, Jennifer Elizabeth
ISNI:       0000 0004 2690 8666
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2010
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RNA interference (RNAi) is an endogenous cellular pathway that can be activated by small interfering RNA (siRNA) and can lead to knockdown of a target gene through sequence- directed mRNA degradation. Naked, unmodified siRNA is poorly taken up by cells and can be degraded by serum nucleases. Therefore, there is a requirement for a robust delivery system which protects siRNA from the biological milieu; facilitates its uptake into the cell; and releases the siRNA intact into the cytoplasm where it can enter the RNAi pathway. In this work, cationic liposomes were explored for use as delivery systems for siRNA. Conventional DOTAP:Cholesterol (2:1) and sterically-stabilised DOTAP:Cholesterol:DSPE- PEG2000 (2:1:0.5) liposomes were complexed with siRNA at charge ratios from 1:1-8:1 (N/P). The effects of charge ratio were studied with respect to complex size and biological activity in vitro. The presence of PEG on the liposome surface decreased siRNA interaction and abolished biological activity. These observations led to the development of a novel encapsulation method that was able to overcome the physical barrier imposed by PEG and reinstate the delivery of functional siRNA. siRNA offers the possibility for targeting gene products that are disease-causing or are up-regulated in disease. Two different therapeutic strategies were employed: inducing cytotoxicity (to specifically kill cancer cells); and knocking down VEGFR2 (to block angiogenesis). Polo-like kinase 1 (PLK1) is essential to mitotic progression and siRNA- mediated silencing causes dose- and time-dependent apoptosis. VEGFR2 is expressed by vascular endothelial cells in solid tumours and is a validated target for anti-angiogenic therapy. Liposome-mediated siRNA delivery was validated in human cervical (HeLa) and lung (Calu6) carcinomas cells, and murine endothelial cells (SVEC 4-10 and B10D10). Moreover, by combining these two siRNAs a novel cocktail was developed which offered dual therapeutic functionality. The biodistribution of liposomes and L/siRNA complexes was assessed using radio- and fluorescently-labeled liposomes. Live imaging of fluorescent complexes showed they remained intact and that liposomes were able to alter the fate of siRNA in vivo. Finally, the in vivo biological activity of siRNA was investigated in human lung cancer xenografts using two routes of administration. Intratumoural administration of L/siRNA complexes failed to induce a therapeutic outcome, however comparison with carbon nanotubes demonstrated that siRNA could elicit a biological effect. Furthermore, intravenous administration of siRNA produced a sequence-specific, dose-dependent decrease in survival. Taken together, this study demonstrates that cationic liposome delivery of biologically active siRNA in vitro is dependent on complex formation with respect to charge ratio, inclusion of PEG and methodology. Furthermore, cationic liposomes are able to maintain stable complexes with siRNA in vivo and provide a shield from nuclease degradation potentiating the delivery of a therapeutically relevant, biologically active siRNA cocktail.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available