Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Director, Institute of Applied Biochemistry, Yagi Memorial Park, Mitake, Gifu 505-01, Japan
As an application of gene therapy by means of liposomes, we first conducted animal experiments to cure glioma produced in the brain of nude mice by intracranial injection of a cell suspension of human glioma cell line U251-SP. A human beta-interferon gene inserted into a simian virus 40-derived expression vector (pSV2IFN-beta) was encapsulated into the liposomes. Taking into account the results of the treatment of glioma transplanted subcutaneously into the back of nude mice (3), we injected the liposome-encapsulated gene (0.5 ug DNA and 30 nmol lipids) into the site of the brain where the glioma cells had been transplanted, once every second day for a total of six injections, starting at various times after the transplantation of the glioma. As a control experiment, the same amount of liposomes containing no plasmids was injected into the glioma of the brain of control animals in the same way as was done in the main experiment. On the 31st day after the transplantation of the cells, the animals were killed, and the tumour was examined by both the naked eye and light microscopic observation.
The transplanted glioma cells disappeared completely from all brains of 7 animals when the treatment was started either 1 day or 3 days after the transplantation of the glioma. While, in control animals injected with empty liposomes the tumour grew; and all of the animals died over a period of 30-54 days after the transplantation of the glioma. When the interval between the transplantation of the glioma cells and the start of the treatment was lengthened, the rate of disappearance of the tumour decreased. When the treatment was started 7 days after transplantation, the complete disappearance of the tumour was seen in 5 of 7 mice. Figure 1 shows typical histological features of the brains from experimental animals on the 31st day after the transplantation of the glioma. Even when the treatment was started 9 days after the transplantation of the glioma cells, the complete disappearance was seen in 2 of 7 mice, and the size of the tumour found in the other 5 animals was smaller than that in control animals injected with empty liposomes. Even though the tumour did not disappear completely, tumour-bearing animals that had been treated as mentioned above survived for a longer time than control ones. The presently observed effect can be ascribed mainly to the effect of endogenously produced human beta-interferon on glioma cells, as demonstrated in our earlier experiment using cultured glioma cells (4).
To check for possible side effects of the liposome-encapsulated plasmids, we injected such plasmids of the same amount as used in the above treatment into the brain of nude mice that had received no tumour transplant. Neither death nor irregular behaviour of animals was found. Following the treatment, human beta-interferon was not detected in the brain by either immunochemical assay or Western blot analysis. No significant histological change was found either.
From these results, we are now convinced that this treatment of experimental glioma can be applied clinically and that gene transfer by means of liposomes is a useful technique for gene therapy. Recently, we submitted an application of this method for gene therapy to the Ethics Committee of Nagoya University School of Medicine, and it is now under investigation. I hope that it will be approved soon so that it may be applied clinically to cure patients suffering from this formidable neoplasm.
1. Koshizaka, T., Hayashi, Y., & Yagi, K. (1989) Novel liposomes for efficient transfection of beta-galactosidase into COS-1 cells. J. Clin. Biochem. Nutr. 7: 185-192.
2. Yagi, K., Noda, H., Kurono, M., & Ohishi, N. (1993) Efficient gene transfer with less cytotoxicity by means of cationic multilamellar liposomes. Biochem. Biophys. Res. Commun. 196: 1042-1048.
3. Yoshida, J., Mizuno, M., & Yagi, K. (1992) Antitumor effect of endogenous human beta-interferon on malignant glioma and augmentation of the effect by tumor necrosis factor-alpha. J. Clin. Biochem. Nutr. 11: 123-128.
4. Yoshida, J., Mizuno, M., & Yagi, K. (1992) Cytoxicity of human beta-interferon produced in human glioma cells transfected with its gene by means of liposomes. Biochem. International 28: 1055-1061.