ET-13RETROVIRAL REPLICATING VECTOR (RRV)-MEDIATED PRODRUG ACTIVATOR GENE THERAPY WITH CODON-OPTIMIZED NITROREDUCTASE IN EXPERIMENTAL GLIOMA

Our studies to date have demonstrated dramatic survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for prodrug activator gene therapy in a variety of preclinical cancer models. RRV-mediated gene therapy using yeast cytosine deaminase (CD) is under investigation in multi-center ascending dose trials in patients with recurrent high grade glioma in the United States. We have further developed an RRV encoding E.coli nitroreductase (NTR), a prodrug activator enzyme which converts CB1954 to a potent bifunctional alkylating agent. We constructed RRV encoding wild-type E.coli NTR genes (RRV-NfsA, RRV-NfsB) as well as NTR variants extensively modified to optimize human codon usage and vector stability (RRV-NAO, RRV-NBO). NTR transgene insertion did not affect vector replication, which resulted in increasing NTR expression over time in U87 human glioma cultures for all vectors, but sequence optimisation significantly increased genomic stability of the RRV-NAO and RRV-NBO vectors over serial passage. U87 cells fully transduced with the optimized vectors showed higher levels of NTR protein and increased levels of enzymatic activity compared with cells transduced with wild-type vectors. In vitro cytotoxicity was examined by MTS assay after CB1954 treatment of fully transduced U87 cells. Viability was reduced by >80% within 48 hrs in cells transduced with RRV-NAO, which showed the most potent cell killing efficiency and bystander effect among all vectors tested. Significant reduction in luminescence and inhibition of tumor growth was observed in subcutaneous U87-FLuc2 tumors initiated with 2% RRV-NAO transduction followed by intraperitoneal administration of CB1954. In intracerebral U87-FLuc2 orthotopic tumor models, stereotactic intratumoral injection of RRV-NAO and repeated cycles of prodrug treatment also resulted in significant luminescence reduction, and achieved prolonged survival benefit. These data indicate that we have been successful in developing an improved prodrug activator gene with therapeutic efficacy when delivered by RRV in experimental models of human glioma.