GENE THERAPY FOR NEUROBLASTOMA

神经母细胞瘤的基因治疗

• 批准号: 12671735
• 负责人: YOSHIDA Hideo
• 金额: $ 1.79万
• 依托单位: CHIBA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671735/
• 关键词: NEUROBLASTOMA; IMMUNOGENE THERAPY; SUCIDE GENE THERAPY; サイトカイン; Midkine; 遺伝子治療

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 … More (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. Less

神经母细胞瘤是儿童最常见的实体瘤之一，尽管神经母细胞瘤的治疗取得了进展，但晚期疾病儿童的预后仍然很差，应考虑寻找新的治疗策略，例如基因治疗。为此，我们研究了神经母细胞瘤的免疫基因疗法和通过转移自杀基因的前药激活疗法。1) 免疫基因疗法：我们研究了工程化的小鼠神经母细胞瘤细胞（C-1300）的抗肿瘤作用逆转录病毒转导人白细胞介素 2 (IL-2) 或鼠粒细胞巨噬细胞集落刺激因子 (GM-CSF) 的细胞产生多种细胞因子，但转导鼠白细胞介素 4 (IL-4) 基因的细胞不会失去致瘤性。在同源 A/J 小鼠中，尽管体外增殖率没有变化，但在裸鼠中没有观察到任何上述转导细胞的致瘤性丧失，但用野生型进行的攻击则不然。 3Y822D 细胞（具有相同遗传背景的纤维肉瘤）在拒绝 IL-2 或 GM-CSF 产生者的小鼠中不会形成肿瘤，与那些产生 IL-2 或 GM-CSF 产生者的静脉注射相比，形成的转移明显减少。此外，即使在注射前静脉注射野生型细胞，也观察到皮下接种IL-2或GM-CSF产生者的肝转移显着减少。此外，将IL-2或GM-CSF产生剂注射到已建立的肿瘤野生型细胞中抑制了随后的肿瘤生长。这些结果共同表明IL-2和GM-CSF基因转导的神经母细胞瘤细胞具有有效的作用。通过诱导T细胞依赖性和肿瘤特异性保护性免疫来发挥体内抗肿瘤抗转移作用。2)自杀基因治疗：肿瘤细胞中自杀基因的选择性表达应该对肿瘤的基因启动子区域产生优先的细胞毒性作用。在肿瘤中表达，但在正常组织中不表达，可用于自杀基因 (MK) 的肿瘤特异性转录，Midkine 是一种生长/分化因子，主要在各种类型的人类肿瘤中表达。 5' 侧翼，2.3 kb。 MK 基因的基因组区域显示以顺式作用方式驱动神经母细胞瘤细胞系中报告基因的转录，调节单纯疱疹病毒胸苷激酶 (HSV-TK) 基因的表达。 MK启动子的控制赋予MK阳性肿瘤细胞对更昔洛韦(GCV)的敏感性增加。将GCV注射到植入了在MK启动子控制下表达HSV-TK基因的MK阳性肿瘤细胞的裸鼠中。通过使用假定的顺式作用MK启动子，可以抑制随后的肿瘤生长。这些结果表明，实验室实验为实施人类临床提供了强有力的科学依据。神经母细胞瘤基因治疗试验较少。