GENETIC MODULATION OF HUMAN NEUROBLASTOMA

人类神经母细胞瘤的基因调控

基本信息

批准号：
2270033
负责人：
CHIN-TO FONG
金额：
$ 10.4万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 1998-08-31
项目状态：
已结题

项目摘要

Successful treatment of the childhood cancer neuroblastoma is difficult, particularly in patients with advanced stage disease. One novel treatment approach is genetic tumor modulation in which a "susceptibility gene" is introduced into tumor cells to increase their sensitivity to treatment. While this approach holds significant promise, it is unlikely that the introduction of a single gene will lead to eradication of all the cancer cells carrying the introduced gene due to occasional mutation or modification of the gene by the host cell. During the past six months, my laboratory has succeeded in transferring a number of yeast artificial chromosomes (YACs) into several human neuroblastoma cell lines by yeast spheroplast x neuroblastoma cell fusion. The main objective of this proposal is to develop a multimodal genetic therapy approach using the YAC vector, which has the unique ability of accommodating a large cloned insert. This will allow multiple copies of a single, or more, "susceptibility gene(s)" to be transferred into human neuroblastoma cells simultaneously such that the tumor cells become susceptible to multiple modalities of therapy. Using this gene transfer technique, I will explore the target-specificity for YAC transfer, and test whether YACs with a circular configuration are more like to be transferred. Furthermore, I will introduce one or more copies of the genes representing the herpes simplex virus thymidine kinase (HS-tk), a mouse histocompatibility antigen, H-2Kb, and the human deoxycytidine kinase (dCK) into human neuroblastoma cells through a YAC and determine whether these genes can be expressed simultaneously. These genes, when expressed, will impart to the neuroblastoma cells sensitivities to the anti-viral agent ganciclovir, anti-H-2Kb antibodies and complement, and the chemotherapeutic agent cytarabine. Hybrids carrying one or more copies of HS-tk gene(s) will be exposed to different concentrations of ganciclovir and the percentage of cell kill will be assessed. Similarly, cell hybrids carrying the H-2Kb antigen gene(s) will be exposed to cytotoxic antibodies against H-2Kb and those carrying the human dCK gene(s) will be exposed to varying concentrations of cytarabine and the percentage of cell kill will be quantitated for each treatment. In addition, hybrids containing multiple different genes, together with parental neuroblastoma control cells, will be exposed to these agent sequentially. The goal is to achieve 90% cell kill using each agent individually which will give an overall cell kill of >>99% per cycle of treatment. This will determine whether near-complete cell kill can be achieved using a combined regimen of sub-lethal level of each of these agents. Results from this study are expected to be directly applicable to designing new gene therapy approaches in cancer.

儿童癌癌神经母细胞瘤的成功治疗很困难，特别是在患有晚期疾病的患者中。一本小说治疗方法是遗传肿瘤调节，其中“敏感性基因“被引入肿瘤细胞，以提高其对治疗。尽管这种方法具有巨大的希望，但不太可能引入一个基因将导致根除所有基因由于偶尔突变而携带引入基因的癌细胞或通过宿主细胞修饰基因。在过去的六个几个月，我的实验室已经成功地转移了许多酵母人工染色体（YAC）成几个人类神经母细胞瘤细胞系通过酵母球体X神经母细胞瘤细胞融合。主要目标该建议是使用多模式基因治疗方法使用 YAC矢量，具有适应大型的独特能力克隆插入物。这将允许多个单个或更多的副本要转移到人神经母细胞瘤细胞中的“敏感性基因” 同时使肿瘤细胞易于多个治疗方式。使用此基因转移技术，我将探索目标特异性用于YAC转移，并测试具有圆形配置的YAC是否为更喜欢被转移。此外，我将介绍一个或多个代表单纯疱疹病毒胸苷的基因的副本激酶（HS-TK），小鼠组织相容性抗原，H-2KB和人类脱氧胞苷激酶（DCK）通过YAC进入人神经母细胞瘤细胞并确定这些基因是否可以同时表达。这些基因表示，将赋予神经母细胞瘤细胞对抗病毒剂Ganciclovir，抗H-2KB抗体的敏感性和补体和化学治疗剂细胞押滨。杂种携带一个或多个HS-TK基因的副本将暴露于不同的 Ganciclovir的浓度和细胞杀死的百分比将为评估。同样，携带H-2KB抗原基因的细胞杂种（S）将暴露于针对H-2KB的细胞毒性抗体和携带的细胞毒性抗体人DCK基因将暴露于不同浓度的细胞押滨和细胞杀伤的百分比将被定量治疗。另外，包含多个不同基因的杂种，与亲本神经母细胞瘤控制细胞一起，将暴露于这些试剂依次。目的是使用使用90％的细胞杀死每个代理人分别将使整体细胞杀死>> 99％治疗周期。这将确定几乎完整的细胞是否杀死可以使用每种次致命水平的组合方案来实现这些代理。预计这项研究的结果将直接适用于设计新的基因治疗方法。