EFFICIENT RETROVIRUS-MEDIATED CYTOKINE-GENE TRANSDUCTION OF PRIMARY-CULTURED HUMAN GLIOMA-CELLS FOR TUMOR VACCINATION THERAPY

In order to realize a novel vaccination treatment for malignant glioma s using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primar y cultured cells. We investigated the feasibility of preparing a gliom a vaccine through retrovirus-mediated gene transduction. Glioma cells were cultured primarily from surgically resected tumor tissues of six patients, We obtained more than 1000-fold proliferation of cultures wi thin eight weeks in all six cases. In vitro infection with a recombina nt retrovirus GKlacZ carrying an Escherichia coli beta-galactosidase m arker gene resulted in over 65% gene transfer to the primary cultured glioma cells. Further enrichment (similar to 90%) of transduced cells was possible by employing repeated infections or using vectors with ne o(r) marker gene. Two cytokine genes, granulocyte-macrophage colony-st imulating factor and interleukin-4, were introduced into glioma cells by sequential transduction with two single-expression GK vectors. The transduced glioma cells produced high levels of both cytokines. We als o evaluated simultaneous introduction of two genes with double-express ion GK vectors containing internal ribosomal entry site (IRES) or inte rnal promoter (PGK). Although the cells transduced with double-express ion vectors secreted both cytokines, the level of the gene product fol lowing IRES or PGK was 10 times lower than that of the upstream gene p roduct. The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation. Our data indicate the feasibility of retrovirus-mediated preparation of gene-modified tumor vaccines from clinical glioma materials, which could be useful for potentiating anti tumor immunity in glioma patients.