SECTION FOR STUDIES ON HOST-IMMUNE RESPONSE

16.SECTION FOR STUDIES ON HOST-IMMUNE RESPONSE

    The research in the Section for Studies on Host-immune Response has focused on the 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 an Adenovirus Vector

    The section is developing an adenovirus vector-based cDNA expression library to effectively identify genes from the aspects of specific biological function. Adenovirus vector has a characteristic of broad host range with high infectivity; this range is also seen in non-dividing cells. The system employs a method for generating the adenovirus vector, which constructs the full-length recombinant adenoviral DNA in vitro by Cre/lox recombination reaction. By the improvement of the gene transfer method, cloning of effective virus-producing 293 clones and use of adenoviral terminal protein, the system is expected to detect the expression of a gene at an abundance of lower than 0.003%. Furthermore, the library can produce more than 1,000 clones of adenoviral vectors per well in a 96 well microtiter plate, demonstrating that adenovirus vector-mediated high throughput gene analysis is available for a variety of purposes.
Polyethylenimine-Based Gene Delivery into Peritoneal Dissemination

    Although peritoneal dissemination of cancer cells often occurs at advanced stages of pancreatic, gastric or ovarian cancers, no effective therapy has been established. Cationic lipid-mediated gene transfer into peritoneal dissemination may offer the prospect of a safe therapy, but vector improvements are needed with regard to the efficiency and specificity of the gene transfer. Polyethylenimine (PEI) is the organic macromolecule with the highest cationic charge density potential. Intraperitoneal injection of plasmid: PEI complexes into mice was evaluated as a gene delivery system for peritoneal dissemination. PEI was more efficient than the cationic lipids such as DMRIE-C and DOGS, and the transgene was preferentially expressed in the tumors. A factor which may account for the apparent tumor-preference is that the peritoneum and underlying connective tissue may constitute a barrier against the entry of DNA:PEI complexes through the surface of normal organs. Although PCR analysis showed that the injected DNA was delivered to various organs, the distributed DNA became undetectable by 6 months after the gene transfer. There was no significant toxicity in the injected mice. The results demonstrated that the intraperitoneal injection of DNA: PEI is a promising delivery method to transduce a gene into disseminated cancer nodules in the peritoneal cavity.⁽²¹¹⁾
Gene Therapy for Cancer

    The expression of antisense K-ras RNA markedly inhibited the growth of pancreatic cancer cells in vitro and in vivo. This strategy was applied for colon cancer, because K-ras point mutation was detected in 40-50% of colon cancers. The infection of adenovirus vector expressing antisense K-ras RNA (AxCA-AS-K-ras) effectively inhibited the growth of colorectal cancer cell lines in vitro , whereas there was no growth inhibitory effect for normal cells such as hepatocytes. The intratumor infection of AxCA-AS-K-ras significantly suppressed the growth of the HCT-15 subcutaneous tumor. The adenovirus-mediated gene transfer of antisense K-ras construct may be a useful therapeutic strategy for colorectal cancer.
    Prostate cancer is one of the most prevalent cancers in many developed countries. Although androgen ablation has been the standard treatment for metastasized prostate cancer, prostate cancer cells eventually lose androgen dependency and become refractory to the conventional endocrine therapy. Androgen-independent prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cell. Although prostate specific antigen (PSA) and rat probasin (rPB) promoters have been investigated in the development of gene therapy targeted to prostate cancer, the promoters may not be able to target the AR-negative hormone refractory prostate cancer because promoters require the AR expression. The rPB promoter was modified so that it is activated by the retinoid-retinoid receptor complex in place of the androgen-AR complex. The modified promoter expressed transgenes in both androgen-dependent and -independent prostate cancer cells in response to retinoids. Since the growth inhibitory effect of retinoids per se has been documented in prostate cancer, the combination of retinioids and prostate specific gene therapy may be a reasonable and realistic option for the prostate cancer treatment.




16.SECTION FOR STUDIES ON HOST-IMMUNE RESPONSE



	The research in the Section for Studies on Host-immune Response has focused on the 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 an Adenovirus Vector The section is developing an adenovirus vector-based cDNA expression library to effectively identify genes from the aspects of specific biological function. Adenovirus vector has a characteristic of broad host range with high infectivity; this range is also seen in non-dividing cells. The system employs a method for generating the adenovirus vector, which constructs the full-length recombinant adenoviral DNA in vitro by Cre/lox recombination reaction. By the improvement of the gene transfer method, cloning of effective virus-producing 293 clones and use of adenoviral terminal protein, the system is expected to detect the expression of a gene at an abundance of lower than 0.003%. Furthermore, the library can produce more than 1,000 clones of adenoviral vectors per well in a 96 well microtiter plate, demonstrating that adenovirus vector-mediated high throughput gene analysis is available for a variety of purposes. Polyethylenimine-Based Gene Delivery into Peritoneal Dissemination Although peritoneal dissemination of cancer cells often occurs at advanced stages of pancreatic, gastric or ovarian cancers, no effective therapy has been established. Cationic lipid-mediated gene transfer into peritoneal dissemination may offer the prospect of a safe therapy, but vector improvements are needed with regard to the efficiency and specificity of the gene transfer. Polyethylenimine (PEI) is the organic macromolecule with the highest cationic charge density potential. Intraperitoneal injection of plasmid: PEI complexes into mice was evaluated as a gene delivery system for peritoneal dissemination. PEI was more efficient than the cationic lipids such as DMRIE-C and DOGS, and the transgene was preferentially expressed in the tumors. A factor which may account for the apparent tumor-preference is that the peritoneum and underlying connective tissue may constitute a barrier against the entry of DNA:PEI complexes through the surface of normal organs. Although PCR analysis showed that the injected DNA was delivered to various organs, the distributed DNA became undetectable by 6 months after the gene transfer. There was no significant toxicity in the injected mice. The results demonstrated that the intraperitoneal injection of DNA: PEI is a promising delivery method to transduce a gene into disseminated cancer nodules in the peritoneal cavity.⁽²¹¹⁾ Gene Therapy for Cancer The expression of antisense K-ras RNA markedly inhibited the growth of pancreatic cancer cells in vitro and in vivo. This strategy was applied for colon cancer, because K-ras point mutation was detected in 40-50% of colon cancers. The infection of adenovirus vector expressing antisense K-ras RNA (AxCA-AS-K-ras) effectively inhibited the growth of colorectal cancer cell lines in vitro , whereas there was no growth inhibitory effect for normal cells such as hepatocytes. The intratumor infection of AxCA-AS-K-ras significantly suppressed the growth of the HCT-15 subcutaneous tumor. The adenovirus-mediated gene transfer of antisense K-ras construct may be a useful therapeutic strategy for colorectal cancer. Prostate cancer is one of the most prevalent cancers in many developed countries. Although androgen ablation has been the standard treatment for metastasized prostate cancer, prostate cancer cells eventually lose androgen dependency and become refractory to the conventional endocrine therapy. Androgen-independent prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cell. Although prostate specific antigen (PSA) and rat probasin (rPB) promoters have been investigated in the development of gene therapy targeted to prostate cancer, the promoters may not be able to target the AR-negative hormone refractory prostate cancer because promoters require the AR expression. The rPB promoter was modified so that it is activated by the retinoid-retinoid receptor complex in place of the androgen-AR complex. The modified promoter expressed transgenes in both androgen-dependent and -independent prostate cancer cells in response to retinoids. Since the growth inhibitory effect of retinoids per se has been documented in prostate cancer, the combination of retinioids and prostate specific gene therapy may be a reasonable and realistic option for the prostate cancer treatment.