GENE THERAPY FOR NEUROBLASTOMA

• Project/Area Number: 12671735
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Pediatric surgery
• Research Institution: CHIBA UNIVERSITY
• Principal Investigator: YOSHIDA Hideo CHIBA UNIVERCITY, GRADUATE SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 大学院・医学研究院, 助教授 (60210712)
• Co-Investigator(Kenkyū-buntansha): KOUCHI Katsunori CHIBA UNIVERSITY, UNIVERSITY HOSPITAL, Assistant, 医学部付属病院, 助手 (40312938) ; TAGAWA Masatoshi CHIBA CANCER CENTER, RESEARCH INSTITUTE, DIVISION CHIEF, 研究局, 部長 (20171572) ; MATSUNAGA Tadashi CHIBA UNIVERCITY, UNIVERSITY HOSPITAL, Lecturer, 医学部付属病院, 講師 (80302561)
• Project Period (FY): 2000 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥2,800,000 (Direct Cost: ¥2,800,000); Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000); Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
• Keywords: NEUROBLASTOMA / IMMUNOGENE THERAPY / SUCIDE GENE THERAPY / サイトカイン / Midkine / 遺伝子治療

Research Abstract

Neuroblastoma is one of the most common solid tumors in children. Despite recent advances in the treatment of neuroblastoma, the prognosis in children with advanced-stage disease remains very poor. The search for novel therapeutic strategies such as gene therapy should be taken into account. For that purpose, we studied immunogene therapy and prodrug activation therapy by the transfer suicide genes for neuroblastoma.
1) Immunogene therapy : We studied the antitumor effects of murine neuroblastoma cells (C-1300) engineered to produce several kinds of cytokine. Retrovirally transduced cells with human interleukin-2 (IL-2) or murine granulocyte macrophage-ccolony stimulating factor (GM-CSF), but not with murine interleukin-4 (IL-4) gene lost their tumorgenicity in syngenic A/J mice, although in vitro proliferation rate was unchanged. The loss of tumorgenicity was not observed in nude mice for any of the above transduced cells. Subsequent challenges with wild-type, but not with 3Y822D cells (fibrosarcoma of the same genetic background), did not form tumors in the mice which had rejected IL-2 or GM-CSF producers. Intravenous administration of IL-2 or GM-CSF producers formed significantry less metastasis compared with those of wild-type cells or IL-4 producers. Moreover, significant reduction in liver metastasis by the subcutaneous inoculation of either IL-2 or GM-CSF producers was observed even when intravenous administration of wild-type cells preceded the subcutaneous inoculation. In addition, injection of IL-2 or GM-CSF producers into an established tumor wild-type cells inhibited the subsequent tumor growth. These results collectively indicate that IL-2 and GM-CSF gene-transduced neuroblastoma cells have potent in vivo antitumor antimetastatic effects by inducing T-cell dependent and tumor-specific protective immunity.
2) Suicide gene therapy : A selective expression of suicide gene in tumor cells should produce a preferential cytotoxic effect on tumors. Promoter regions of a gene that is expressed in tumors but not in normal tissues can be useful for tumor-specific transcription of a suicide gene. Midkine (MK), a growth/differentiation factor, is expressed predominantly in various types of human tumors. The 5'-flanking, 2.3 kb genomic region of the MK gene was shown to drive the transcription of a reporter gene in the neuroblastoma cell lines in a cis acting manner. Regulated expression of the herpes simplex virus-thymidine kinase (HSV-TK) gene under the control of the MK promoter conferred increased sensitivity to ganciclovir (GCV) on MK-positive tumor cells. Administration of GCV into nude mice that were implanted with MK-positive tumor cells that expressed the HSV-TK gene under the control of the MK promoter could suppress the subsequent tumor growth. Expression of the therapeutic genes restricted to tumors can be achieved by the use of the putative cis-acting MK promoter.
These results indicate that the labolatory experiments provided a strong scientific rationale for implementing human clinical trials of gene therapy for neuroblastoma.