D. Peng

Combined gene therapy with suicide gene and interleukin-12 is more efficient than therapy with one gene alone in a murine model of hepatocellular carcinoma.

BACKGROUND/AIMS Gene therapy has emerged as a new form of treatment for unresectable hepatocellular carcinoma (HCC). We evaluate here the effect of IL-12 and the suicide gene thymidine kinase as single agents and in combination to treat experimental liver cancer. METHODS Recombinant adenoviruses expressing mouse interleukin-12 (AdCMVIL-12) or thymidine kinase of herpes simplex virus (AdCMVtk) or lacZ reporter gene (AdCMVlacZ) were constructed. The efficacy of the treatment was evaluated in a murine HCC model based on subcutaneous implantation of liver tumor cells (BNL). RESULTS Transduction of BNL cells after in vitro infection with AdCMVlacZ was very low at multiplicity of infection (moi) of 100, whereas 10-15% of cells were transduced when using moi 1,000. Similarly, production of IL-12 was detectable only in BNL cells infected with AdCMVIL-12 at moi 1,000. In vitro infection of BNL cells with AdCMVIL-12 at moi 100 did not abrogate tumorigenicity, whereas moi 1,000 resulted in inhibition of tumor growth in all mice as well as in abrogation of tumor formation in 3 out of 8 animals. In vivo studies showed that intratumor injection of AdCMVIL-12 induced a dose-dependent effect on tumor regression. However, none of the animals exhibited complete tumor elimination with this treatment. We observed that suppression of tumor growth was more intense in animals treated with the combination of AdCMVIL-12 plus AdCMVtk than in animals which received AdCMVtk or AdCMVIL-12 alone. The combined treatment resulted in a significant increase in animal survival, and 25% of treated animals were free of tumor for over 100 days without recurrence of the disease. CONCLUSIONS Combination of AdCMVIL-12 and AdCMVtk is more efficient than either of the two vectors alone for the treatment of the murine model of HCC used in this study.

Transduction of hepatocellular carcinoma (HCC) using recombinant adeno-associated virus (rAAV): in vitro and in vivo effects of genotoxic agents

BACKGROUND/AIMS Adeno-associated virus (AAV) is an attractive tool for gene therapy. Here we investigated the in vitro and in vivo transduction of hepatocellular carcinoma (HCC) cells by an AAV vector and the efficacy of different strategies to enhance the transduction of the tumor. METHODS Transduction efficiency was determined by analyzing AAV-mediated beta-galactosidase gene (rAAV/lacZ) expression. RESULTS Adenovirus help or pretreatment of HCC cells with y-irradiation or with the topoisomerase inhibitor etoposide resulted in marked enhancement of cell transduction in vitro. In vivo studies in nude mice with subcutaneous HCC tumors showed that HCC cells were not transduced by AAV vector alone. However, co-infection of the tumor with adenovirus allowed an efficient expression of the reporter gene but only at the sites of vector injection. Previous gamma-irradiation of subcutaneous tumors with 1800 rad was able to improve transduction of HCC cells (up to 30%) using recombinant AAV. Continuous i.p. infusion of etoposide in buffalo rats harboring HCC tumors in the liver resulted in transduction of normal liver tissue and also of very small neoplastic lesions (<2 mm) but no transduction was observed in tumors bigger than 2 mm. To analyze this phenomenon we determined etoposide concentration in hepatic tissue. Our results revealed high concentrations of the drug in non-tumoral tissue but almost undetectable levels in big tumor nodules. CONCLUSIONS Our results indicate that while both radiotherapy and etoposide enhance transduction of tumor cells by rAAV in vitro, only radiotherapy increases tumor transduction in vivo. Our data suggest the existence of a barrier which limits in vivo the diffusion of chemotherapeutic agents to well-established HCC nodules.

Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone

To investigate the cooperative effect of B7-1 and IL-12 in the induction of antitumor activity, we have developed retroviral vectors encoding human B7-1, murine IL-12, or both B7-1 and IL-12 coordinately. Murine transformed liver cells (BNL) were engineered to stably express B7-1, IL-12, or both by infection with corresponding retroviruses. No tumor was observed in 20, 75, and 95% of mice receiving, respectively, B7-1-, IL-12-, and B7-1/IL-12-modified tumor cells after 250 days of inoculation. In contrast, injection of parental BNL or BNL/Neo cells resulted in lethal tumor progression in all mice. Protection against rechallenge with parental tumor cells was observed only in mice who had rejected BNL/IL-12, but not in animals that rejected BNL/B7-1 or BNL/B7-1-IL-12. Growth of parental tumor cells was significantly delayed by simultaneous injection in a distant site of irradiated tumor cells engineered to express IL-12 or both B7-1 and IL-12 but not B7-1 alone. BNL/B7-1 and BNL/B7-1-IL-12 showed similar efficacy in these experiments. Antitumor immunity induced by B7, with or without IL-12, was found to depend mainly on CD4+ T cells with a minor contribution of a non-T cell mechanism; whereas the effect of IL-12 was dependent on CD8+ T cells and on non-T cell effectors. Immunization of mice with IL-12-modified BNL cells induced secretion of a Thl pattern of cytokines while immunization with cells expressing both IL-12 and B7-1 resulted in inhibition of IFN-gamma production. Immunization with BNL/B7-1-IL-12 cells in the presence of anti-human B7-1 MAb resulted in restoration of IFN-gamma production to the levels found in animals injected with BNL/IL-12 cells. To summarize, in our model coexpression of B7-1 and IL-12 in tumor cells does not result in improved antitumoral activity as compared with expression of IL-12 alone. This may be related to the fact that B7-1 changes the mechanisms of antitumor immunity and inhibits IFN-gamma production induced by IL-12 in vivo.