16.SECTION FOR STUDIES ON HOST-IMMUNE RESPONSE
	The Section for Studies on Host-immune Response has focused on fundamental studies of gene and cell therapy for cancer and the development of novel vectors. The Section has been collaborating with the Genetics Division and the Central RI Laboratory in the field of gene therapy research. Development of Novel cDNA Selection Method Using Adenovirus Vector     The Section is developing an adenovirus vector-based cDNA expression library to effectively identify cancer antigens. Adenovirus vector has a characteristically broad host range with high infectivity, a range that is also seen in non-dividing cells. The system employs a novel adenoviral vector generation method, which constructs full-length adenoviral DNA in vitro using a Cre/lox recombination reaction. By improving the gene transfer method, cloning of high adenovirus productive 293 cells and use of adenoviral terminal protein, the system is expected to detect the expression of genes at levels as low as 0.01% in model experiments. Polyethylenimine-Based Gene Delivery into Peritoneal Dissemination     Safer and more efficient vectors are needed to develop gene therapy for peritoneal dissemination. Polyethylenimine (PEI) showed superior gene transfer efficiency in comparison with various liposomes in several pancreatic cancer cell lines in vitro. Next, to investigate whether an in vivo gene transfer could be effectively achieved by PEI, pancreatic cancer cells (AsPC-1) were inoculated into the peritoneal cavities of nude mice, and the luciferase expression plasmid was then injected intraperitoneally as a DNA-PEI complex. High luciferase activities were observed only in tumors on the mesentery and pancreas, and low activities were detected in some organs such as the spleen, stomach, and skeletal muscle. Other organs such as the brain, lung, heart, liver, kidney, testis, and small intestine showed no luciferase activity. The peritoneum and underlying connective tissue may constitute a barrier against the entry of DNA: PEI complex via the surfaces of organs. Although PCR analysis showed the injected DNA to be delivered to various organs, the distributed DNA became undetectable by 6 months after gene transfer. Blood chemistry and histological analysis showed no significant toxicity in the injected mice. The results demonstrate that intraperitoneal injection of DNA: PEI complexes is a promising delivery method for transducing a gene into disseminated cancer nodules in the peritoneal cavity. Gene Therapy for Colon Cancer using Antisense K-ras RNA     Previous reports from the Genetics Division have shown that the transduction of an antisense K-ras expression plasmid markedly inhibited the growth of pancreatic cancer cells in vitro and in vivo. The Section applied this strategy to gene therapy for colon cancer, because a K-ras point mutation was detected in approximately 40-50% of colon cancers, a frequency second to that of pancreatic cancer. The strong expression of antisense K-ras RNA by the adenovirus vector (AxCA-AS-K-ras) effectively inhibited cell growth of all of seven colorectal cancer cell lines in vitro. The status of the K-ras point mutation did not correlate with the growth suppressive effect of the antisense K-ras vector; both the K- ras-mutation-positive and -negative colorectal cancer cells showed suppressed growth. Interestingly, there was no growth inhibitory effect on normal cells such as hepatocytes and vascular endothelial cells. Next, to test in vivo efficacy, HCT-15 cells were inoculated subcutaneously into the left flank of SCID mice, and AxCA-AS-K-ras was injected intratumorally three times after the tumor mass was established. The injection of AxCA-AS-K-ras significantly suppressed growth of the HCT-15 subcutaneous tumor. This study shows that adenovirus-mediated in vivo gene transfer of an antisense K-ras construct may be a useful therapeutic strategy for colorectal cancer.