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Title: An evaluation of viral nanoparticles for siRNA delivery
Author: Galaway, Francis Ashley
ISNI:       0000 0004 2740 2974
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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At the beginning of the 2oth century Paul Ehrlich proposed the concept of 'magic bullets' to fight disease in the human body. At the start of the 21st century there are now a range of highly selective biologics. Amongst these are the emerging RNAi therapeutics that offer gene specific treatment. There is a need for delivery systems that will localise these RNAi therapeutics at the disease site and facilitate their cellular internalisation. To fully capitalise on the potential of RNAi therapeutics the delivery systems will need to be platform technologies that can be applied to a broad range of diseases. This project explored the potential of the bacteriophage MS2 as a platform delivery technology for RNAi therapeutics. The 19 nucleotide translational repressor sequence of the bacteriophage MS2 genome was synthesised as part of a si RNA. The double stranded 21 nucleotide siRNA was specific for a 21 nucleotide sequence in the mRNA of the onco-protein Bcl2. The chimeric siRNA triggered the assembly of recombinant MS2 coat protein into virus-like particles in vitro. The siRNA was stable and protected from nuclease-mediated degradation inside the capsids:The human transferrin protein was then covalently linked to the virus-like particles to form synthetic virions. The effect of these synthetic virions on human epithelial cancer cells was investigated in vitro using flow cytometry. The synthetic virions were targeted for receptor- mediated endocytosis and the siRNA accumulated in the cancer cells where it reduced Bcl2 expression. All the cells that internalised a detectable quantity of the siRNA in the initial 24 hours were apoptotic within 48 hours. Increasing the synthetic virion dose improved the number of cancer cells with the lethal intracellular quantity of siRNA, but also caused off-target effects. In order to test synthetic virions incorporating other siRNA sequences, a high- throughput screening system was developed. It used automated confocal microscopy technology and image recognition software to observe and map individual cells from adherent monolayers. Different targeter and siRNA combinations can now be tested as part of the MS2 platform technology in a high-throughput manner.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available