Glioblastoma multiforme (GBM) is the most aggressive brain tumor and kills through intracranial growth and spread. We have previously shown (i) the efficiency of the herpes simplex virus type 1 thymidine kinase (HSV1-TK) and systemic ganciclovir (GCV) in eradicating an experimental syngeneic glioma model, (ii) 2-3 fold higher astrocyte- and glioma-specific high-level expression from the powerful 1.4kb major immediate early routine cytomegalovirus promoter (mCMV) compared to the human CMV equivalent, and (iii) unexpected long-term presence (12 months) of HSV1-TK in the brain. Although the efficiency of HSV 1-TK and GCV has been shown in a large variety of experimental models, the clinical results, while encouraging, remain inconclusive. The main reason thought to underlie this difference is the low levels of HSV1-TK expression from currently available vectors. Our experiments will address this issue by vastly increasing therapeutic transgene expression (through the use of a novel promoter) and reducing the viral vector toxicity (through the use of novel safer vectors of reduced toxicity). These findings will have important clinical implications and provide a blueprint for the implementation and design of Phase I clinical trials of gene therapy for GBM. We will validate the efficiency of a novel, safe, high capacity, helper dependent adenoviral vector (HC-Ad) expressing HSV1-TK under the control of the powerful mCMV promoter in a clinically relevant syngeneic experimental glioma model. HSV1-TK induces glioma cell death by phosphorylating the prodrug GCV, and killing both transduced and adjacent non-transduced, actively dividing cells. Killing of non-transduced cells, the 'bystander effect', amplifies this strategy's efficiency through cell-cell diffusion of cytotoxic intermediates (e.g. phosphorylated GCV), release of pro-apoptotic molecules, and immune stimulation. We hypothesize that our novel anti-tumor strategy will deliver high intra- and peritumoral expression of the therapeutic transgene that, combined with systemic dosing of GCV, will lead to sustained and effective anti-tumor effect. Our long term aim is to translate this novel therapeutic approach into a Phase I clinical trial for GBM.