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Generation of an HVS-based episomally maintained gene delivery system for reprogramming adult somatic cells

Brown, Hannah Frances (2012) Generation of an HVS-based episomally maintained gene delivery system for reprogramming adult somatic cells. PhD thesis, University of Leeds.

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Abstract

iPSC technology has the potential to generate patient specific pluripotent cells for use in stem cell therapies and disease modelling. However, current reprogramming methods utilise retroviral vectors, which integrate into the host cell genome disrupting normal gene function. Transient gene delivery methods have been investigated as safer alternatives but demonstrate poor reprogramming efficiency. Therefore, there is a requirement for iPSC gene-delivery vectors which are capable of providing prolonged transgene delivery without integrating into the host cell genome. Herpesvirus saimiri (HVS) is a prototype member of the gamma-2 Herpesviridae, and is capable of persisting as a non-integrated episome in dividing and differentiating cell populations, providing sustained transgene expression. Therefore, this thesis has explored the potential of HVS-based vectors for iPSC generation. This thesis focuses on the generation of three HVS-based vectors expressing the iPSC reprogramming transgenes, Oct4, Lin28 and Nanog. The potential of these HVS-iPSC vectors to reprogram both primary and cancerous cells has been investigated. Human primary Neural Stem Cells demonstrated cytopathology upon transduction with HVS-based vectors, indicating the need for further improvements to the biosafety of these vectors before they are suitable for the generation of clinical grade iPSCs. However, reprogramming attempts utilising the Ewing‟s sarcoma cell line, A673 cells, successfully generated induced pluripotent cancer stem cell (iPC)-like colonies upon transduction with all three recombinant vectors. Results from detailed analysis of these colonies suggest some form of reprogramming has taken place, albeit incomplete, as indicated by elevated expression of Oct4, Rex1 and Klf4; in addition to positive alkaline phosphatase staining and SSEA4 expression. Furthermore, these iPC-like colonies were capable of differentiation down the ectodermal lineage, as evidenced by upregulation of MSX1, MAP2, and Nestin. In conclusion, this thesis has demonstrated the potential of HVS-based reprogramming vectors through the generation of A673-iPCs.

Item Type: Thesis (PhD)
ISBN: 978-0-85731-329-4
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Identification Number/EthosID: uk.bl.ethos.581602
Depositing User: Repository Administrator
Date Deposited: 06 Sep 2013 12:42
Last Modified: 07 Mar 2014 11:28
URI: http://etheses.whiterose.ac.uk/id/eprint/4445

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