Viral Gene Therapy For Non-Muscle Invasive Bladder Cancer.

In the UK approximately 12,000 new cases of bladder cancer are diagnosed every year. Approximately 80% of these cases are non muscle invasive bladder cancer (NMIBC) defined as stage Ta-T1, grade 1-3 and carcinoma in situ. Despite current operative and postoperative treatment options there is recurrence rate of 15-61% and significant risk of progression (1-17%). Gene therapy strategies have been developed as a therapeutic approach for a number of malignances. Gene transfer by viral vectors takes the advantage of the natural ability of viruses to enter cells and express transgenes by the infected cells. OncoVexGAL/CD is a modified oncolytic herpes simplex virus, where the virus is deleted for ICP34,5 region to provide tumour selective replication and also deleted for the ICP47 region to increase antigen presentation and the antitumor immune response. The OncoVexGALV/CD virus carries a highly potent prodrug activating gene for suicide gene therapy (Fcy:fur gene = yeast cytosine deaminase / uracil phospho-ribosyltransferase enzyme gene, the enzyme which converts 5-fluorocytosine (5-FC) to active 5-fluorouracil (5-FU)) and a fusogenic glycoprotein gene from gibbon ape leukaemia virus (GALV). OncoVexGALV/CD has shown enhanced local tumour control by combining its oncolytic activity with the effect of the carried pro-drug activating and a fusogenic gene. Effective use (enhanced cell killing and tumour shrinkage) of OncoVexGALV/CD vector has been previously described in vitro and in vivo other tumour sites but not the bladder. A version of this virus expressing GM-CSF has shown promising results in Phase I and II clinical trials. The present thesis aimed to test in vitro and in vivo the OncoVexGALV/CD as an intravesical therapy for NMIBC and to test the efficacy of the virus in combination with current chemotherapies. In vitro tumour cell killing by OncoVexGALV/CD was assessed by Fusion/Prodrug MTS assays and staining by Crystal Violet. Treatment of seven human bladder carcinoma cell lines with the virus resulted in higher tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. For the in vivo tests we have developed a rat orthotopic bladder tumour model using acid and alkali to damage the bladder mucosa and using AY-27 HVEM cells for implantation. We decided to determine the presence of tumour by quantitative reverse transcription polymerase chain reaction (QRT-PCR). However while we obtained positive signals on bladder tissue, signals using urine samples were insignificant. In vivo efficacy of the OncoVexGALV/CD treatment was studied on the orthotopic bladder tumour model, where the results showed enhanced local tumour control and higher body weights in the presence of both OncoVexGALV/CD and prodrug when compared to control (No virus + prodrug) or virus alone. The effect of OncoVexGALV/CD treatment in combination with chemotherapies (mitomycin, cisplatin, gemcitabine) on cell proliferation was assessed by calculating combination index values using CalcuSyn software. The coadministration of OncoVexGALV/CD and mitomycin showed synergistic, whereas the coadministration with cisplatin or gemcitabine showed antagonistic effect in vitro. In conclusion our results on human bladder carcinoma cell lines and rat ortotopic bladder tumour model indicate that OncoVexGALV/CD may improve local tumour control within the bladder and it can be used in combination with mitomycin. It is hoped that this will lead to new therapeutic options for bladder cancer