1031. Improved Anti-Brain Tumor Efficacy of |[Delta]||[gamma]|134.5 HSV Expressing Cytosine Deaminase|[ast]|

134.5 Herpes Simplex Viruses (HSV) are non-neurovirulent genetic constructs that have been shown to grow conditionally in a wide variety of mouse and human tumor cells in vitro. Despite the fact that these replication competent HSV can readily kill susceptible tumor cells in vitro, intratumoral (i.t.) injection of these viruses in animals bearing intracranial (i.c.) or flank tumors has not uniformly inhibited tumor growth. To improve in vivo tumoricidal activity, we have inserted the bacterial gene for cytosine deaminase (CD) in the loci from which the 134.5 genes responsible for neurovirulence were deleted. This initial virus was designated HSV M012. CD efficiently converts the relatively non-toxic prodrug 5-fluorocytosine (5-FC) to the potently toxic 5-fluorouracil (5-FU) anti-metabolite. CD has been used in a wide variety of experimental and clinical gene therapy studies to eliminate tumor cells by impairing pyrimidine synthesis in both RNA and DNA pathways. Moreover, 5-FU has been proven to be a true radiation sensitizer, which offers the possibility of using combined modalities to eliminate tumors. A second HSV construct was made, M1012, which also carried an insertional deletion of the virus gene encoding for the large subunit of ribonucleotide reductase. The rationale was that virus-produced ribonucleotide reductase in infected cells may be able to incorporate 5-(F)UMP into virus RNA synthesis and impair virus replication. Both viruses were shown to infect and kill human tumor cells and produce detectable amounts of bioactive CD. Conversion of 5-FC to 5-FU by infected human glioma cells was quantified. In vitro, mouse Neuro-2A (N2A) cells are relatively resistant to killing by HSV at multiplicities of infection (MOI) ranging from 1 to 20. Addition of increasing concentrations of 5-FC (100-1,000 M) resulted in significant killing of N2A cells at progressively lower MOI. C-b.17 scid mice bearing i.c. gliomas induced by injection of 5105 U87MG human glioma cells were treated 7 days later by i.t. injection of 107 pfu of either HSV M012 or HSV M1012 or received an equivalent volume of saline. Two days later mice began a 7-day regimen of twice daily i.p. injections of 500 mg/kg 5-FC, then followed for survival. Mice receiving saline i.t. and 5-FC had a median survival time (MST) of 34 days, characteristic of this model. Those that received M012 i.t. followed by saline (MST = 43 days) or 5-FC (MST = 55 days) had significantly enhanced survivals (over saline +5-FC) but there were no long-term survivors. Likewise mice receiving M1012 i.t. followed by saline experienced increased survival (MST=53 days) but without any cures. However, mice receiving M1012 followed by 5-FC have yet to reach a median survival with over 66% long-term survivors at 210 days. These and other data not described suggest that the addition of prodrug therapy to virus-mediated oncolysis may afford a significant improvement over virus therapy alone. Further, this study suggests that HSV ribonucleotide reductase may exert a negative survival effect on the viral vector, limiting its potential efficacy as a gene therapy agent.