Oncolytic adenovirus vectors for nitroreductase suicide gene therapy of prostate cancer

摘要

Prostate cancer is the most common male cancer in the UK and USA, with a 1/13 chance of diagnosis and a 1/30 lifetime risk of death from the disease. Current treatment options include radiotherapy, surgery and hormone therapy, however 1/3 patients escape from all therapies and novel therapies are urgently required for this patient group. The University of Birmingham gene therapy group constructed two oncolytic adenovirus vectors, CRAd-NTR and vNR6, both of which contained the E1B-55K deletion and expressed the transgene nitroreductase for combined oncolytic virotherapy and enzyme/prodrug gene therapy. The latter of these two vectors, vNR6, expressing nitroreductase from the pIX virus promoter demonstrated the greatest cytotoxicity at low virus concentrations however also showed some lytic activity to non-transformed human fibroblasts. Our collaborators at the Institut Català d'Oncologia designed a panel of oncolytic adenovirus vectors with the E1A CR2 24 deletion and the E1A promoter replaced by an insulated E2F-1 promoter. The latest two in this series of vectors, termed ICOVIR-5 and ICOVIR-7, provide potential oncolytic backbones for the introduction of the therapeutic transgene nitroreductase. The aim of this thesis was therefore to ‘arm’ the ICOVIR based vectors with nitroreductase for combined oncolytic virotherapy and enzyme/prodrug therapy. At the beginning of this study no reports were published with either ICOVIR-5 or ICOVIR-7 based vectors. It was therefore first decided to construct both vectors expressing the marker transgene eGFP. These vectors were characterised in terms of cytotoxicity, transgene expression, DNA replication and E1A expression. Furthermore, these vectors were compared to the vNR3, an E1B-55K deleted virus similar to vNR6, but the eGFP ORF replacing that of pIX. The ICOVIR-7 based vectors were identified as being the most tumour selective vectors and demonstrated no cytotoxicity to non-transformed human fibroblasts, and were therefore chosen for the introduction of the therapeutic transgenes. The new ICOVIR-7 based vectors were constructed to express either wildtype, double mutant or triple mutant nitroreductase. Double and triple mutant nitroreductase are two previously characterised mutant nitroreductases, which show enhanced catalytic activity for the prodrug CB1954. The new nitroreductase expressing ICOVIR-7 vectors were characterised in terms of virus mediated cytotoxicity, tumour selectivity, E1A and NTR expression and cytotoxicity with the prodrug CB1954. One vector, expressing double mutant nitroreductase, showed the highest tumour selectivity and greatest combined cytotoxicity with the prodrug CB1954. Furthermore, this vector showed greater tumour selectivity and combined cytotoxicity than the E1B-55K deleted vector vNR6.