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Lipoplex-mediated gene transfer : Influence of selected chemotherapeutic agents on transgene expression

University of British Columbia

The ultimate goal of anticancer therapies should be the elimination and/or suppression of tumour cells. Lipid-based gene transfer systems have been employed in promoting transgene expression of exogenous DNA whose products are capable of performing these roles. Since improvements in antitumour efficacy may be obtained by combining gene therapy with other therapeutic modalities such as chemotherapy, it is pertinent to characterize the influence of conventional cytotoxic drugs upon transgene expression, achieved following use of a lipoplex-mediated gene transfer system. SKOV-3 human ovarian carcinoma and B16/BL6 murine melanoma cells were used as in vitro models to evaluate levels of transgene expression. Lipoplexes, [DODAC/DOPE (1:1 mol ratio) liposomes complexed with plasmid DNA containing the luciferase or chloramphenicol acetyltransferase reporter gene] were added to cells that had been pre-exposed for 24h to various cytotoxic drugs. Transgene expression levels were established 48h post-transfection. Two in vivo models were also established to evaluate whether chemotherapeutic agents would affect transgene expression following lipoplex-mediated gene transfer. C57BL/6 mice bearing B16/BL6 tumours were injected intraperitoneally with lipoplexes, three days after receiving an intravenous drug injection equivalent to the maximum therapeutic dose. The second model analyzed transgene expression in spleen and bone marrow samples from CD1 mice. Lipoplexes were intravenously administered to mice 3,7, 14 or 21 days after they had received an intravenous drug injection equivalent to the maximum therapeutic dose. In both models, transgene expression was determined 24h post-transfection. In vitro cytotoxicity data indicates that lipoplexes did not influence the sensitivity of SKOV-3 or B16/BL6 cells to cisplatin, doxorubicin, vincristine or bleomycin. Transgene expression levels increased, decreased or remained the same (relative to lipoplex only controls), depending upon the drug, its concentration and the cell type. Combining chemotherapy with an intraperitoneal model for lipoplex-mediated gene transfer did not affect transgene expression levels evaluated in B16/BL6 tumours. The in vivo intravenous model measuring spleen and bone marrow transfection levels showed that all selected drugs caused some inhibition of transgene expression. These results suggest that DNA damage is not a mechanism by which to increase gene transfer by lipoplexes, rather drug-induced alterations in cellular uptake and processing of lipoplexes are believed to be responsible for inducing changes (increases or decreases) in transgene expression.

Thesis/Dissertation

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2009-06-15

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http://hdl.handle.net/2429/9120

Pathology

University of British Columbia

UBCV

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