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Title: Anti-angiogenic gene therapy
Author: Hayes, E. A. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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The aim of this project was to assess a novel anti-angiogenic gene therapy in which a therapeutic gene is activated exclusively in proliferating endothelial cells using tissue-specific promoters and the Cre/IoxP recombination system. Adenoviruses and transgenic mice were generated in parallel to test individual components of the targeting system. One of the therapeutic effector strategies investigated was the herpes simplex virus-1 thymidine kinase (HSV-1TK)/ganciclovir (GCV)-mediated suicide system, which is reported to kill proliferating cells selectively. Administration of GCV to pTie2-TK transgenic mice expressing HSV-1 TK under the control of the endothelial cell-specific Tie2 promoter and to mice treated systemically with a pTie2-TK adenovirus was lethal, demonstrating that additional control was required to target exclusively proliferating endothelial cells. Intra-tumoural (i.t.) injection of pTie2-TK adenovirus resulted in tumour-restricted expression of HSV-1 TK and preliminary data demonstrated a trend towards a decrease in primary tumour growth following treatment of mice with i.t. pTie2 adenovirus and GCV. The tet Off system was investigated as a method to obtain conditional control of Cre recombinase expression. Despite showing tight regulation in vitro, this system did not result in complete silencing of transgene expression in vivo. Transgenic mice expressing tamoxifen (TMX)-regulated Cre recombinase under the control of the cell-cycle dependent Cyclin A promoter were also generated, but problems with TMX administration precluded determination of whether Cre recombinase was activated by TMX in these mice. However, conditional transgene activation was achieved in vivo by generating a pCycA-Cre adenovirus in which the Cyclin A promoter was used to drive expression of Cre recombinase. A pTie2-stuffer-TK transgenic mouse line was generated which expressed a Cre-activatable form of HSV-1 TK under the control of the Tie2 promoter. To target proliferating endothelial cells specifically, the pCycA-Cre adenovirus was used to activate HSV-1 TK in these transgenic mice. Preliminary data showed that there was a trend towards a decrease in primary tumour growth following treatment of pTie2-stuffer-TK transgenic mice with i.t. pCycA-Cre adenovirus and GCV. Transgenic mice expressing an alternative Cre-activated therapeutic gene, the pro-apoptotic gene Bax, under the control of the Tie2 promoter were generated by direct pronuclear injection and by site-specific transgene insertion into the hprt locus. pTie2-stuffer-Bax mice generated using the latter technique showed higher levels of Tie2 promoter-driven transgene expression than those made by pronuclear injection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available