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Binding, internalization, and transgene expression of an adenoviral vector retargeted to HER3/4 Open Access


Other title
Gene Therapy
Breast Cancer
Type of item
Degree grantor
University of Alberta
Author or creator
MacLeod, Sheena H
Supervisor and department
Hitt, Mary (Oncology)
Examining committee member and department
Tikoo, Suresh (Veterinary Microbiology)
Hendzel, Michael (Oncology)
Wang, Zhixiang (Medical Genetics)
Hugh, Judith (Oncology)
Department of Oncology
Experimental Oncology
Date accepted
Graduation date
Doctor of Philosophy
Degree level
Adenoviruses (Ads) have been well studied for use in cancer gene therapy. However, low levels of the primary receptor, coxsackie-adenovirus receptor (CAR), in tumor cells has been shown to be a factor in low transgene expression. To increase Ad infection of breast cancer cells we constructed a human Ad5 targeted to HER3/4 receptors by the insertion of the HER3/4 ligand, the HRG EGFlike domain. These growth factor receptors are overexpressed on breast cancer, as well as other cancer cells. Here, we have shown higher transgene expression levels after infection of breast cancer cells expressing high levels of HER3/4 by the modified virus, compared to the wild-type binding virus. Furthermore, we have shown expanded tropism of the modified virus to Chinese hamster ovary cells that are refractory to infection by the wild-type binding virus. Competition with either the HRG EGF-like domain or soluble Ad virus fiber knob supported these results. However, gene transfer to a breast cancer xenograft model was not improved by the addition of the heregulin (HRG) EGF-like domain. We compared binding and internalization of the modified virus to that of the wildtype binding virus. As expected, the wild-type virus bound and was taken up into CAR+ cells within 10 min. The modified virus was similar in CAR+ cell lines. Surprisingly, in CAR- cells, very little binding or internalization of the modified virus was detected within 10 min. When re-assessed under stringent conditions used in binding and internalization assays, there was no detectable reporter gene expression after infection of CAR- cells with either virus. Moreover, fluorescence microscopy demonstrated that longer incubation times increased internalization of the modified virus into CAR- cells, consistent with the original transgene expression assays. Thus, the modified virus internalization into CAR- cells appears to be delayed compared to internalization of the wild-type binding virus. We have shown differences in binding, internalization, and gene expression after modification of Ad to bind to HER3/4, in addition to CAR. Further study and modifications of this vector should result in an effective gene therapy vector for breast or other cancers.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
S MacLeod, M Elgadi, G Bossi, U Sankar, A Pisio, K Agopsowicz, D Sharon, F Graham, and MM Hitt, HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice. Cancer Gene Therapy (2012) 19, 888-898, doi:10.1038/cgt.2012.79

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