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Foamy virus vector integration and application in gene therapy

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Title: Foamy virus vector integration and application in gene therapy
Authors: Sweeney, Nathan Paul
Item Type: Thesis or dissertation
Abstract: Foamy viruses (FVs) are unique ancient retroviruses that infect all non-human primates, but do not cause disease. We aimed to understand the FV pre-integration complex by isolating it from infected cells and characterising its protein constituents. Using a PCR to quantify integration in infected cells, we determined that integration occurs from 10 hours post-transduction. In synchronised cells, the peak of integration correlated well with cells passing through mitosis. However, we were unable to detect in vitro strand-transfer activity to indicate that active pre-integration complexes had been isolated. We conclude that FV pre-integration complexes are likely to be inactive in the conditions tested. A further aim was to optimise FV vectors for use in mesenchymal stem cells and test this vector in mouse models of sphingolipidoses, namely metachromatic leukodystrophy and Krabbe disease. We permitted transduction of cells at a high multiplicity of infection by exchanging the envelope from the prototype FV to that of the macaque. We tested various FV vectors in mesenchymal stem cells and determined that the non-toxic macaque envelope increased transduction efficiency from under 65% to over 95% in a single round of transduction. We achieved high and sustained transgene expression using the phosphoglycerate kinase promoter. Transduced MSCs delivered to the brains of the mouse model for metachromatic leukodystrophy caused only a modest improvement in sulphatide storage, the primary biochemical output for efficacy, although results are inconclusive. In the mouse model for Krabbe disease, transduced MSCs delivered to the brain or the peritoneum had no effect on disease progression. In conclusion, FV vectors are suited to gene therapy of MSCs since they offer the highest transduction efficiency from a single round of transduction, while MSC based gene therapy strategies for Krabbe disease or metachromatic leukodystrophy are unlikely to offer clinical benefit.
Content Version: Open Access
Issue Date: May-2015
Date Awarded: Aug-2015
URI: http://hdl.handle.net/10044/1/50704
DOI: https://doi.org/10.25560/50704
Supervisor: McClure, Myra
Sponsor/Funder: Wellcome Trust (London, England)
Funder's Grant Number: WMND_PS1706
Department: Department of Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses



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