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In Situ Retroviral-mediated Gene Transfer for the Treatment of Brain Tumors in Rats

Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke Clinical Center [Z. R., S. W., E. H. O.], and Metabolism Branch, National Cancer Institute [K. W. C., R. M. B.], NIH, Bethesda, Maryland 20892

Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells.

Rats inoculated with 4 x 10⁴ 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (ß-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3–5 x 10⁶ thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10⁶) resulted in a lower frequency of tumor regression (5 of 13 rats).

To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10⁶ ß-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-ß-D-galactopyranaside staining revealed maximal staining of ß-galactosidase within the tumor 7–14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranaside positive. It is probable that destruction of this local vasculature with ganciclovir therapy also contributes to the efficacy of tumor regression.

Our results substantiate the feasibility of this approach for the treatment of malignant brain tumors in humans.

¹ To whom requests for reprints should be addressed, at Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke Clinical Center, NIH, Building 10, Room 5D37, 9000 Rockville Pike, Bethesda, MD 20892.

Received 6/12/92. Accepted 10/21/92.

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