We are currently interested in developing gene therapy for the eradication of solid tumours using GDEPT approaches. The therapeutic gene employed in our company is human cytochrome P450 2B6 (CYP2B6) which encodes a prodrug metabolising enzyme. Cyclophosphamide is one such prodrug and the active metabolite phosphamide mustard subsequently produced crosslinks DNA causing DNA damage and subsequent cell death. A novel retroviral vector encoding P450 2B6 (MetXia-P450) was constructed and used to transduce human tumour cell lines HT29 and T47D. MetXia-P450 transduction sensitises these cells to the anti-proliferative or cytotoxic effect of the prodrug cyclophosphamide. The results from a range of biochemical and cellular biological assays demonstrate inhibition on cell cycle progression and DNA synthesis and a reduction in clonogenic survival in cells transduced with MetXia-P450, in response to cyclophosphamide. Cytotoxic activity was accompanied by a significant bystander effect that was even more evident in 3-dimensional multicellular spheroid models than monolayers suggesting that this may be a more appropriate model for assessing the GDEPT approach. We have applied this approach in an active gene therapy setting using established subcutaneous tumour xenografts. Here we show for the first time the efficacy of P450-based GDEPT strategy mediated by direct intratumoral injection of the therapeutic retroviral vector. MetXia-P450 is being evaluated in phase I/II clinical trials in breast and ovarian cancer.