GENE THERAPY FOR NEUROBLASTOMA

神经母细胞瘤是儿童最常见的实体肿瘤之一。尽管最近神经母细胞瘤的治疗取得了进展，但患有晚期疾病的儿童的预后仍然很差。应该考虑寻找新的治疗策略，如基因治疗。为此，我们研究了利用自杀基因转移治疗神经母细胞瘤的免疫基因疗法和药物前激活疗法。1)免疫基因治疗：我们研究了通过改造产生多种细胞因子的小鼠神经母细胞瘤细胞（C-1300）的抗肿瘤作用。转染人白细胞介素-2 （IL-2）或小鼠粒细胞巨噬细胞集落刺激因子（GM-CSF）而不转染小鼠白细胞介素-4 （IL-4）基因的逆转录病毒转导细胞在同基因A/J小鼠中失去了致瘤性，尽管体外增殖率不变。在裸鼠中没有观察到任何上述转导细胞的致瘤性丧失。随后使用野生型，而不是3Y822D细胞…More（具有相同遗传背景的纤维肉瘤），在拒绝IL-2或GM-CSF产生者的小鼠中没有形成肿瘤。与野生型细胞或IL-4产生细胞相比，静脉注射IL-2或GM-CSF产生细胞形成的转移明显减少。此外，即使在皮下接种前静脉注射野生型细胞，皮下接种IL-2或GM-CSF产生物也能显著减少肝转移。此外，将IL-2或GM-CSF生产者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通过诱导t细胞依赖性和肿瘤特异性保护性免疫，在体内具有强大的抗肿瘤抗转移作用。2)自杀基因治疗：自杀基因在肿瘤细胞中选择性表达，对肿瘤产生优先的细胞毒作用。在肿瘤中表达但在正常组织中不表达的基因的启动子区域可能对自杀基因的肿瘤特异性转录有用。Midkine （MK）是一种生长/分化因子，主要在各种类型的人类肿瘤中表达。在神经母细胞瘤细胞系中，MK基因的5'侧，2.3 kb基因组区域以顺式作用方式驱动报告基因的转录。在MK启动子的控制下，单纯疱疹病毒胸苷激酶（HSV-TK）基因的表达受到调节，使MK阳性肿瘤细胞对更昔洛韦（GCV）的敏感性增加。在MK启动子控制下，将GCV注入表达HSV-TK基因的MK阳性肿瘤细胞的裸鼠体内，可以抑制随后的肿瘤生长。通过使用假定的顺式作用的MK启动子，可以实现肿瘤限制治疗基因的表达。这些结果表明，实验室实验为神经母细胞瘤基因治疗的人体临床试验提供了强有力的科学依据。少

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