Sami Loimas

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.

Evaluation of recombinant alphaviruses as vectors in gene therapy

Alphavirus vectors based on Sindbis virus and Semliki Forest virus (SFV) were characterized as potential gene transfer vectors. Initial studies were performed using vectors engineered to transfer either lacZ or green fluorescent protein (GFP). High levels of gene transfer were achieved in human primary fibroblasts, BHK and 293T cells, with low levels of transduction observed in more than 20 other target cells. Alphavirus-based expression was generally very high, but transient in every cell type. Replication-competent alphavirus was never detected in SFV preparations but could be produced by Sindbis-based vectors at a frequency of up to 3 × 10−3 infectious units per ml. We constructed a human clotting factor IX (hFIX) cDNA-containing Sindbis virus and compared it with hFIX cDNA-harboring adenoviral and retroviral vectors. In most cases, hFIX levels obtained with Sindbis vector were initially at least an order of magnitude higher than those obtained with other viral vectors. These data demonstrate that alphavirus vectors compare favorably with adenovirus vectors as systems to promote high-level transient gene expression and should be considered as an alternative vector for gene transfer and potential gene therapy studies.

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.

Evaluation of herpes simplex thymidine kinase mediated gene therapy in experimental pancreatic cancer

Despite of recent improvements in the treatment of many malignant diseases, pancreatic ductal adenocarcinoma is still a disease with an extremely poor prognosis with current modes of treatment. Gene therapy has been suggested as a novel approach also against pancreatic cancer. Previous studies have been carried out predominantly with immunodeficient animal models and with tumors growing in environments other than the pancreas. We have attempted to mimic a more clinically relevant situation and investigated suicide gene therapy strategy for experimental pancreatic cancer in animals with an intact immune system.

Human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase-mediated suicide gene therapy.

To evaluate human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase (HSV-TK)–mediated gene therapy, we tested the utility of different viral vectors on three human cell lines DU-145, LNCaP, and PC-3. Our viral vectors were carrying a fusion gene of HSV-TK and green fluorescent protein for accurate determination of the gene transfer rate and its contribution to the treatment outcome in each case. We observed that adenoviral and lentiviral vectors were efficient vehicles for all the cell lines, whereas Semliki Forest virus and Sindbis virus vectors yielded only a few percent of transgene-positive cells. Despite sufficient gene transfer rates (25–45%) in the ganciclovir (GCV) sensitivity experiment, only DU-145 cells were efficiently destroyed under clinically relevant GCV concentrations. This was shown to be due to low level of “bystander effect” in PC-3 and LNCaP cells. Our data demonstrate that human prostate tumors can be good targets for adenovirus- or lentivirus-mediated HSV-TK/GCV gene therapy, but each tumor should be investigated for gene transfer rate and bystander effect to warrant a sufficient treatment result. Cancer Gene Therapy (2001) 8, 137–144

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.

VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct

VP22 is a herpes simplex virus type 1 (HSV‐1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22‐Tk‐GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo.

Azaserine-Induced Rat Pancreas Tumor Model with Transplantable Cultured Cells

The purpose of this study was to describe the inoculation technique and patterns of growth as well as to characterize typical histological features of Lewis rat subcutaneous and intrapancreatic tumors, induced by inoculation of cultured pancreatic cancer cells (DSL-6A/C1). Subcutaneous inoculation of cultured cells produced a solid tumor that was a locally invasive, well- to moderately differentiated ductal adenocarcinoma. Tumor take was 100% in animals 5 weeks of age; tumor growth was consistent and predictable and a tumor volume of −1 cm3 was reached in 8 weeks. After intrapancreatic transplantation the tumors showed the same histological features as subcutaneous tumors. During inoculation carcinoma cells easily spread around the injected area, and after 2 weeks both pancreatic tumors and superficially infiltrating carcinomas were found in the liver and spleen and around the peritoneum. Tumor take was 60% and tumor growth was somewhat indefinite and unpredictable in the pancreas. However, by reducing the injected carcinoma cell volume and solving the technical problems, 100% tumor take was achieved. The tumor volume reached 2 mm3 during 2 weeks and larger tumors showed a tendency for invasion. According to our results, subcutaneous as well as intrapancreatic tumor induction with cultured cells offers a model for pancreatic cancer studies.

The growth pattern and microvasculature of pancreatic tumours induced with cultured carcinoma cells

Pancreatic cancer is one of the most frustrating problems in gastroenterological surgery, since there is little we can do to improve the survival of patients with current treatment strategies. If one is to elucidate factors related to carcinogenesis, tumour biology, diagnostics and new treatment modalities of this malignant disease, then it is essential to develop a suitable animal model. In the present study we investigated rat pancreatic tumour growth after intrapancreatic injection of cultured pancreatic carcinoma cells (DSL-6A/C1), originally derived from an azaserine-induced tumour, as well as the features of tumour microcirculation using the microangiography technique. After intrapancreatic inoculation, tumours were detected in 64% of animals. A 1 cm3 tumour volume was reached within 20 weeks after inoculation. The tumours were ductal adenocarcinomas. Larger tumours showed invasive growth and spreading into the surrounding tissues, mainly into spleen and peritoneum. Microangiography revealed that the pancreatic tumours had an irregular and scanty vessel network and there were avascular areas in the center of the tumour. The area between normal pancreas and the induced tumour had dense vascularization. Intrapancreatic tumour induction with cultured pancreatic carcinoma cells produced a solid and uniformly growing tumour in Lewis rats and it thus provides a possible model for pancreatic cancer studies.