Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors.

Transfer of a herpes simplex virus-derived thymidine kinase (HSV-tk) gene into brain tumor cells and subsequent ganciclovir (GCV) treatment has been shown by others to be an effective treatment in rats with intracerebrally inoculated 9L gliosarcomas. Mechanism of action and reproducibility are, however, still a matter of debate. We have used the same model to test the therapeutic effects of both retrovirus- and adenovirus-mediated transfer of the HSV-tk gene followed by GCV treatment. Survival time of rats with intracerebral 9L tumors was significantly prolonged after a single administration of adenovirus carrying a HSV-tk gene as compared to controls. Retrovirus-mediated gene transfer also resulted in significantly prolonged survival time when recombinant retrovirus-producing cells were transplanted. Direct injection of the recombinant retrovirus, HSV-tk-expressing cells, virus-producing cells without GCV administration and recombinant retrovirus-lacZ or interleukin-2 (IL-2)-producing cells did not result in tumor cell kill. In the present study, no significant difference in survival of 9L brain tumor carrying rats was found after treatment with adenovirus as compared to retrovirus-mediated HSV-tk-mediated gene transfer and subsequent GCV treatment.

Amphetamine acts as a channel blocker of the acetylcholine receptor.

Non-competitive inhibitors (NCIs) of the nicotinic receptors (AChR) comprise a wide range of compounds. The chemical scaffold of amphetamine is similar to those of some NCIs. We investigated the effects of amphetamine (1-100 microM) on the muscle AChR by recording single-channel currents. The drug reduces the duration of the open state in a concentration-dependent manner and causes the appearance of brief closings, resembling the action of open-channel blockers. The forward rate constant for the blocking process is of the order of 10(7) M(-1) s(-1) and the blocking process is voltage dependent. The results are consistent with the steric block of the open channel as the primary action of amphetamine. At high drug concentrations the mechanism of inhibition deviates from that of classical open-channel blockers.

Preclinical testing of recombinant adenoviral herpes simplex virus-thymidine kinase gene therapy for central nervous system malignancies.

OBJECTIVES: Adenoviral gene transfer and killing efficiency using the thymidine kinase (TK)/ganciclovir (GCV) mechanism was evaluated in human cancer cells occurring as central nervous system tumors. The effectiveness of this approach was tested in vitro and in experimental models for brain tumor and leptomeningeal metastases in rats in vivo. Recombinant adenoviruses with different promoters were compared. METHODS: Adenoviral vectors harboring a marker (lacZ) or a TK gene were constructed. Transcription of genes was under the control of either the adenovirus Type 2 major late promoter (MLP) or the human cytomegalovirus (CMV) immediate early gene promoter. lacZ expression and GCV killing efficiency after TK gene transfer in several human tumor cells was evaluated in vitro. The 9L rat brain tumor and leptomeningeal metastases models were used to determine the effectiveness of adeno-TK and subsequent GCV treatment in vivo. MLP and CMV containing adenoviral vectors were compared. RESULTS: Gene expression and the killing of tumor cells were very efficient in all human tumor cell lines tested. The adenovirus containing the CMV promoter showed cytopathic effects in cultured tumor cells at high multiplicity of infections but also greater cell killing efficiency after TK/GCV treatment, as compared to the MLP promoter. Although both the MLP and CMV vectors showed a significant dose-dependent therapeutic effect, animals treated with recombinant adenovirus containing the CMV promoter showed significantly longer survival time (brain tumors) or symptom-free periods (leptomeningeal metastases). CONCLUSION: This study demonstrates the therapeutic efficiency and feasibility of the TK/GCV approach in experimental brain tumors and leptomeningeal metastases. It also demonstrates that the promoter driving the transgene in an adenoviral vector influences toxicity and efficiency of treatment.

Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene.

Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local treatment for malignant mesothelioma.

IL-1/IL-3 gene therapy of non-small cell lung cancer (NSCLC) in rats using 'cracked' adenoproducer cells.

Cytokine gene therapy was studied in established L42 tumours in syngeneic rats. L42 is a transplantable non-immunogenic non-small cell lung cancer (NSCLC). Genes coding for human interleukin-1 alpha and for rat interleukin-3 beta were transferred by injecting producer cells of recombinant adenovirus vectors into the tumour in attempts to achieve high concentrations of the cytokines inside the tumor without systemic toxicity. Limited tumour growth delay was obtained with viable producer cells. For logistic reasons stocks of pooled frozen producer cells allowed intensive treatment of groups of tumour bearing rats. The cells were lysed by thawing before administration. Ten daily injections of such 'cracked' producer cells induced reproducible tumour responses. These were due to local release of cytokines, not to systemic effects. Growth retardation also occurred in contralateral tumours which were not injected. When rats carrying established tumours were vaccinated with lysates of tumours collected during treatment with 'cracked' producer cells, significant tumour growth retardation was obtained. We speculate that both cytokines, if produced at sufficiently high concentrations in tumours, induce inflammation which in turn initiates an immune response against tumours growing at a distant site. These findings seem to justify further exploration of IL-1 and IL-3 gene transfer for the treatment of cancers.

Intra-CSF administered recombinant adenovirus causes an immune response-mediated toxicity.

High doses of adenotk were injected into the cerebrospinal fluid of rats and nonhuman primates (Macaca mulatta). Vector administration was followed by ganciclovir administration for 14 days. Despite the absence of clinical symptoms, analysis of the cerebrospinal fluid (CSF) and histopathological examination of the central nervous system (CNS) of the monkeys (3 weeks after vector injection) were consistent with a viral meningitis. Immunohistochemical analysis of the inflammatory infiltrates in the monkeys revealed the presence of T and B lymphocytes, indicating a combined cellular and humoral immune response to the vector. This latter was supported by the finding of intrathecal anti-adenovirus antibody synthesis. Rats receiving high intrathecal adenotk doses showed a transient and dose-dependent clinical toxicity consisting of lethargy, hyperemic eyes and weight loss. Histopathological examination of the meninges showed a shift from polymorphonuclear infiltrates during the first post-injection days to clusters of mononuclear cells after 7 days. Acute toxicity is probably related to the early, innate immune response to the vector. In a separate experiment, high levels of IL-8 and IL-6, were measured during the first 2-3 post-injection days in the CSF of two monkeys which received intrathecal adenoLacZ. Therefore, these cytokines seem to play an important role in initiating the nonspecific immune response. In one monkey which received adenotk, recombinant adenovirus was cultured from serum samples obtained at the 7th post-injection day. At this time-point, no vector could be isolated from CSF samples. Based on these preclinical data, we recommend careful dose finding for clinical studies that aim to treat patients with leptomeningeal metastases.