PURIFIED HERPES-SIMPLEX THYMIDINE KINASE RETROVECTOR(TM) PARTICLES .1. IN-VITRO CHARACTERIZATION, IN-SITU TRANSDUCTION EFFICIENCY, AND HISTOPATHOLOGICAL ANALYSES OF GENE THERAPY-TREATED BRAIN-TUMORS

Replication-defective, highly purified retroviral vectors (Retrovector TM), at titers of 10(8) colony forming units/mL, were prepared that co nferred either P-galactosidase or herpes simplex thymidine kinase (HSV -TK) activity. 9L gliosarcoma cells, transduced efficiently in vitro, were highly sensitive to ganciclovir (CCV). The mean frequency of in s itu transduction, measured by flow cytometry of single-cell tumor susp ensions isolated from rat brains, was 3.2 +/- 0.6%; similar assessment s were made by staining of P-galactosidase or by immunohistochemistry with anti-HSV-TK. In vitro HSV-TK-transduced and G418-selected 9L-TK g liosarcoma tumors treated with CCV were eradicated in approximately 53 % of the animals (10/19) at day 26, however, 89% (17/19) histologicall y showed <1% tumor volume. Histologic evaluation at day 26 of animals with established 9L tumors treated with intralesional injection of HSV -TK vector followed by CCV treatment showed that 29% (4/14) had no tum or; 50% (7/14) had <1% tumor volume. Regression of tumors proceeded ov er time since the complete response rate was increased at day 60. Neit her HSV-TK vector particles nor CCV alone altered the histological pro file of 9L tumors, but substantial numbers of CD4+ and CD8+ lymphocyte s infiltrated the tumors of animals treated with both. In cured animal s, the former tumor bed contained cell debris, immune cells, and fibro blasts and was without damage to adjacent brain. The efficacy of suici de gene therapy for rat gliosarcoma using highly purified virion vecto rs approaches that of packaging cell lines.