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Title: Development of a viral and a non-viral based gene transfer systems using the yeast Saccharomyces cerevisiae
Author: Bowden, Jonathan Kirk
ISNI:       0000 0004 6348 6837
Awarding Body: Brunel University London
Current Institution: Brunel University
Date of Award: 2016
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VSV-G has been used for several years to pseudotype reteroviral and lentiviral vectors to increase the range of cell types that these vectors can be targeted to as well as increasing transfection efficiency and serum resistance. It has previously been shown that purified VSV-G protein can be added to several types of non-viral complexes to produce these same advantages. VSV-G therefore holds great potential in gene therapy for both viral and non-viral vectors. Due to the cellular toxicity of VSV-G in mammalian cells VSV-G pseudotyped viral vectors are generally produced from transiently transfected cells which greatly limit the scale of viral production. VSV-G for non-viral vectors is also limited in the same manner but also suffer from expensive and time consuming methods to purify the VSV-G from the expression media. To address these problems with production we attempted to generate strains of the yeast Saccharomyces cerevisiae that can produce VSV-G pseudotyped lentivirus and VSV-G protein from inducible integrated vectors. We theorised that the cell wall of Saccharomyces cerevisiae would prevent syncytia and cellular toxicity of VSV-G during production, allowing the continuous production of virus or protein. In this report we show that this new production method allows us to produce and purify VSV-G from yeast using simple and scalable methods and that this produces a greater enhancement of transfection efficiency than mammalian derived VSV-G. However we were not able to demonstrate the production of VSV-G pseudotyped virus, seemingly due to the genotoxic effects of viral integrase.
Supervisor: Themis, M. ; Parris, C. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available