947. Gene Directed Enzyme/Prodrug Therapy of Human Glioma Xenografts Using Mutant Escherichia coli Cytosine Deaminase

Combined treatment using suicide gene therapy and radiation therapy has the potential to become a powerful new method of cancer therapy. We have developed a non-replicative adenoviral vector encoding a mutant bacterial cytosine deaminase (bCD) gene harboring substitution of an alanine (A) for the aspartic acid (D) at position 314 in the CD protein (AdCD-D314A) which has a higher affinity for 5-fluorocytosine (5-FC) than wild type bCD (CDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdCD-D314A with the prodrug 5-FC and radiation treatment (RT) against human glioma. The results of CD enzyme activity assays showed that the conversion of 3H-5-FC to 3H-5-FU was elevated 183.3, 205.6 and 102.2-fold in D54MG, U87MG and U251MG human glioma cells, respectively, for cells infected with 2 multiplicity of infection (MOI) of AdCD-D314A compared to AdCDwt. AdCD-D314A infection also resulted in increased 5-FC-mediated cell killing. The 50% inhibitory concentration produced by 5-FC decreased by 7.8-fold for D54MG, 32.9-fold for U87MG, and 8.1-fold for U251MG cells infected with 10 MOI of AdCD-D341A in comparison with AdCDwt. Animal studies showed significant inhibition of tumor growth of D54MG glioma subcutaneous xenografts by the combination of AdCD-D314A/5-FC with RT as compared with either agent alone, or with AdCDwt/5-FC plus RT. The results demonstrate that the combination of AdCD-D314A/5-FC with radiation produces markedly increased cytotoxicity in cancer cells in vitro and in vivo compared to AdCDwt. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for glioma therapy.