Anu-Maaria Sandmair

Thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses.

Herpes simplex virus thymidine kinase (HSV tk) gene therapy combined with ganciclovir (GCV) medication is a potential new method for the treatment of malignant glioma. We have used both retrovirus-packaging cells (PA317/tk) and adenoviruses (Adv/tk) for gene therapy for malignant glioma. Retrovirus-packaging cells were used for eight tumors in seven patients and adenoviruses were used for seven tumors in seven patients. As a control group, seven tumors in seven patients were transduced with lacZ marker gene 4-5 days before tumor resection. Safety and efficacy of the gene therapy were studied with clinical evaluation, blood and urine samples, MRI follow-up, and survival of the patients. Four patients with adenovirus injections had a significant increase in anti-adenovirus antibodies and two of them had a short-term fever reaction. Frequency of epileptic seizures increased in two patients. No other adverse events possibly related to gene therapy were detected. In the retrovirus group, all treated gliomas showed progression by MRI at the 3-month time point, whereas three of the seven patients treated with Adv/tk remained stable (p < 0.05). Mean survival times for retrovirus, adenovirus, and control groups were 7.4, 15.0, and 8. 3 months, respectively. The difference in the survival times between the adenovirus and retrovirus groups was significant (p < 0.012). It is concluded that HSV tk gene therapy is safe and well tolerated. On the basis of these results further trials are justified, especially with adenovirus vectors.

1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: Implications for the in vivo detection of apoptosis

1 H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: Implications for the in vivo detection of apoptosis

Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.

Herpes simplex virus thymidine kinase (HSV-tk) gene transfer and ganciclovir (GCV) administration have been suggested for the treatment of malignant gliomas. To understand tissue responses and possible ways to improve the treatment effect, we studied tumor growth, tissue reactions, and survival time after HSV-tk/GCV treatment in a syngeneic BT4C rat glioma model by mixing various ratios of stably transfected HSV-tk-expressing BT4C-tk glioma cells with wild-type BT4C glioma cells (percentage of BT4C-tk cells: 0%, 1%, 10%, 30%, 50%, and 100%), followed by injection into BDIX rat brains (n = 79). With the exception of some animals with end-stage tumors, very little astroglia or microglia reactivity was detected in the wild-type tumors as analyzed by immunocytochemistry using glial fibrillary acid protein (GFAP)-, vimentin-, human histocompatibility leukocyte antigen-DR-, OX-42-, and CD68-specific monoclonal antibodies. After 14 days of GCV treatment, tumors induced with ≥10% BT4C-tk cells showed a significant reduction in tumor size (P < .05) and prolonged survival time (P < .01). Astrogliosis, as indicated by a strong GFAP and vimentin immunoreactivity, was seen in the tumor scar area. GFAP and vimentin reactivity was already present after the GCV treatment in tumors induced with 1% BT4C-tk cells. Much less human histocompatibility leukocyte antigen-DR-positive microglia was seen in the treated animals, indicating low microglia reactivity and immunoactivation against the tumor. However, GCV-treated tumors were positive for apoptosis, indicating that apoptosis is an important mechanism for cell death in the BT4C-tk glioma model. Our results suggest that ≥10% transfection efficiency is required for a successful reduction in BT4C glioma tumor size with HSV-tk/GCV treatment in vivo. Tissue reactions after 14 days of GCV treatment are characterized by astrogliosis and apoptosis, whereas microglia response and immunoactivation of the brain cells appear to play a minor role. Stimulation of the microglia response by gene transfer or other means might improve the efficacy of the HSV-tk/GCV treatment in vivo.

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy in BT4C rat glioma model.

Adenovirus (Adv)-mediated herpes simplex virus thymidine kinase (adv/tk) gene therapy combined with ganciclovir (GCV) medication is a promising approach for the treatment of malignant glioma. However, optimal administration and the effect of possible adjuvant treatments have not been fully examined. In the present study, we examined the efficacy of adv/tk/GCV gene therapy in a syngeneic BT4C rat malignant glioma model, either as a single administration or given as three injections during three consecutive days. The effect of combined adv-mediated macrophage colony-stimulating factor (MCSF) and adv/tk gene transfer was also studied. BT4C malignant glioma cells were injected into the right corpus callosum of BDIX rats (n=112). Before gene therapy, the presence of tumors was verified by MRI. The rats were divided into eight groups as follows: group I (n=20) received a single adv/tk gene transfer (total dose 4×108 pfu) and GCV treatment for 14 days; group II (n=5) received the same gene transfer without GCV; group III (n=28) received three adv/tk injections (total dose 4×108 pfu) on three consecutive days and GCV for 14 days; group IV (n=5) received three similar adv/tk injections without GCV medication; group V (n=13) received three adv/MCSF injections (total dose 2×108 pfu) on three consecutive days and GCV medication; group VI (n=12) received three adv/tk and adv/MCSF (total dose 6×108 pfu) injections on three consecutive days followed by GCV medication; and group VII (n=12) the same treatment without GCV. Group VIII (n=17) consisted of wild-type BT4C malignant glioma tumors without any treatment. Treatment effect and tissue responses were characterized by general histology, immunohistochemistry, MRI, and survival of the study groups. The best treatment effect and survival was found in rats treated with adv/tk gene transfer once a day for three consecutive days (P<.05). No improvement of the treatment effect was seen after the combined adv/tk and adv/MCSF gene transfer compared with the repeated adv/tk gene transfer. The results show that 20% of the rats can be cured (survival >6 months) after optimized adv/tk gene therapy. It is concluded that repeated intratumoral administration of adv/tk is a promising approach for the treatment of malignant glioma tumors in vivo.

Low Efficacy of Gene Therapy for Rat BT4C Malignant Glioma Using Intra-Tumoural Transduction with Thymidine Kinase Retrovirus Packaging Cell Injections and Ganciclovir Treatment

Summary Background. The purpose of this study was to test the use of Herpes Simplex virus thymidine kinase (HSVtk) retrovirus packaging cell injections in the treatment of malignant brain tumours. Methods. Therapeutic effect and tissue responses were examined in vivo in a syngeneic BT4C rat glioma model after HSVtk-producing PA317 packaging cell injections and intraperitoneal ganciclovir (GCV) medication. MRI was used to visualise the tumours before and after the treatment. Immunohistochemical stainings were performed to study astroglia and microglia responses and apoptosis-mediated cell death. Results. The results suggest that only a limited treatment effect can be achieved with HSVtk packaging cell injections with no prolonged survival rates. Histological examination showed a strong astroglia response but only a modest microglia response after the treatment. HSVtk and GCV-induced cell death was at least partially mediated by apoptosis. It is concluded that HSVtk packaging cell injections and GCV treatment do not lead to eradication of malignant cells in a syngeneic BT4C rat glioma model. The lack of efficacy is most likely due to low gene transfer efficiency and limited life span of the injected packaging cells inside the tumours. Conclusions. Improvements in gene transfer efficiency, and stimulation of immunoresponse against tumour cells might lead to a more effective therapeutic response in vivo.

ADENOVIRUS-MEDIATED HERPES SIMPLEX THYMIDINE KINASE GENE THERAPY FOR BRAIN TUMORS

Adenovirus-mediated HSVtk gene therapy has been shown to be eefective for the treatment of malignant glioma when the gene transfer efficiency is high. Adenoviruses are currently the most promising vectors for gene therapy of malignant glioma especially because of their high efficiency of gene transfer. Experiences from preclinical in vitro and in vivo studies are promising and in the near future the first reports of clinical phase I and II studies should be available. These results will significantly advance our understanding about the efficacy of adenovirus-mediated gene transfers in the treament of malignant glioma in man.