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Title: The development of an adenovirus vector system to study virus entry and genetic modification of immune cells
Author: Findlay, James Sorley
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Extensive in vitro studies have shown that adenovirus (Ad) entry occurs in a two-step process. However, distribution in vivo appears to be affected by host factors such as blood coagulation Factor X (FX). The intravenous introduction of Ad vectors, such as Ad5 (which uses the coxsackie and adenovirus receptor, CAR, for attachment) or Ad5F35 (which uses CD46 for attachment), will result in an interaction between lymphocytes, blood coagulation factors and the vectors. It was found that Ad5F35, but not Ad5, could transduce primary lymphocytes. The presence of FX resulted in a significant reduction in Ad5F35 transduction of primary lymphocytes and lymphoid cell lines; the presence of FX also resulted in a significant reduction in Ad5•EGFP transduction of lymphoid cells. To understand the role of heparan sulphate proteoglycans (HSPGs) in this process, the effect of FX on adenovirus attachment and transduction of a panel of cells with varying HSPG, CAR and CD46 expression were analysed. The presence of FX, in a CAR-independent manner, enhanced Ad5•EGFP binding to and transduction of only those cells that expressed HSPGs. In the absence of HSPGs, FX had no effect on Ad5-EGFP binding but caused a significant reduction in Ad5•EGFP transduction. The presence of FX also enhanced Ad5F35-EGFP binding to cells which expressed HSPGs. In the absence of HSPG, FX reduced Ad5F35•EGFP attachment irrespective of the cell-surface expression of CD46. FX had different effects on Ad5F35•EGFP transduction which appear to be linked to the presence of HSPGs and CD46. In the presence of HSPGs, but in the absence of CD46, FX enhanced Ad5F35•EGFP transduction. However, in cells which expressed CD46 but not HSPGs, or those which expressed neither, FX reduced Ad5F35 transduction. Therefore the presence of CAR, CD46 and HSPGs appear to affect the outcome of the FX •mediated interaction. The ability of Ad5F35, but not Ad5, to transduce primary lymphocytes suggested that, in order to manipulate primary lymphocytes, the Ad5-based vectors should be modified to use CD46. Therefore the AdZ•5•CY5 system was modified by replacement of the Ad5 fibre DNA sequence with that of Ad35 to create AdZ•5F35•CYS using recombination-mediated genetic engineering. To characterise the resulting virus, an EGFP cassette was cloned in place of the El region, the virus propagated and tested on CHO, CHO-CD46 and HeLa cells. AdZ•5F35•EGFP transduced CHO-CD46 and HeLa, but Ad5 only transduced HeLa cells. This indicated that AdZ-5F35•EGFP utilised CD46 for cell entry. Peripheral blood mononuclear cells (PBMCs) were isolated and transduced with either Ad5-EGFP or AdZ-5F35-EGFP. EGFP expression was only detected in the lymphocyte population transduced with AdZ-5F3S-EGFP. Therefore AdZ-SF3S-EGFP had the ability to transduce primary lymphocytes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available