Transduction of Tumor Suppressor Proteins into Gliomas

将肿瘤抑制蛋白转导至神经胶质瘤

• 批准号: 6482108
• 负责人: STEVEN F DOWDY
• 金额: $ 24.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-05 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6482108
• 关键词: athymic mouse; chimeric proteins; gene mutation; gene therapy; genetically modified animals; glioma; nonhuman therapy evaluation; p53 gene /protein; pharmacokinetics; protein structure function; protein transport; tumor suppressor proteins

DESCRIPTION: (provided by applicant) Brain tumors, gliomas and astrocytomas, are devastating malignancies that account for 2.3 percent of all US cancer deaths and represent the second most common solid tumor of children. Malignant brain tumors respond poorly to current therapies with a mean survival rate of less than one year despite treatment, Due to the invasive nature of gliomas, particularly glioblastoma multiformes (GBM), localized anti-cancer strategies, such as surgical removal, also fail to adequately halt the disease. Gliomas select for genetic inactivation of multiple tumor suppressor genes, including p53 (>60 percent), PTEN (>75 percent), p16/p I4ARF (50 percent), pRB (30 percent), and epigenetic down-regulation of the p27 Cdk inhibitor. A central hypothesis of anti-cancer therapies holds that replacement of tumor suppressor gene functions in malignant cells will result in specific death or apoptosis of the cancer cell while sparing the surrounding normal tissue. Indeed, tumor cells are undergoing continuous DNA damage and therefore, adenovirus expression of wild type p53 in gliomas by results in specific apoptosis to the glioma tumor cells. We propose to test this hypothesis by generating transducible tumor suppressor proteins. My laboratory has further developed the methodology of protein transduction. Recombinant, bacterially expressed fusion proteins containing an N' terminal protein transduction domain from HIV TAT rapidly transduce into 100 percent of cells. Using this methodology, we have generated and transduced over 60 TAT-fusion proteins from 15-120 kDa. Recently, we have demonstrated the ability of TAT-B-gal protein to transduce into most, if not all, cells and tissues of mouse models in vivo, including across the blood-brain barrier. Thus, in principle and practice, all mammalian cell types are susceptible to protein transduction. We propose to test the anti-cancer effectiveness and specificity of killing glioma tumors in mouse models by transducible tumor suppressor proteins, namely TAT-ARF and TAT-p53, and by a transducible pro-apoptotic viral protein, TAT-Apoptin. TAT-fusion proteins will be analyzed and optimized in vitro and then tested against xenograft intracranial glioma tumors in nude mice and in de novo derived astrocytomas in B8 transgenic ras about2" mice. In addition, to quantify protein transduction potential in mouse models, we intend to analyze the transduction and refolding rates of TAT-reporter fusion proteins, including TAT-B-gal and TAT-TK. TAT-TK activity will also be monitored in vivo by microPET imaging using '8F-fluoroganciclovir as a positron emitter.

描述：（由申请人提供）脑肿瘤，神经胶质瘤和星形胶质细胞瘤， 是毁灭性的恶性肿瘤，占美国癌症所有癌症的2.3％ 死亡，代表了儿童的第二大常见实体瘤。恶性 脑肿瘤对当前疗法的反应不佳，平均存活率为 尽管治疗了不到一年，但由于神经胶质瘤的侵入性性质 特别是胶质母细胞瘤多形式（GBM），局部抗癌策略， 例如手术清除，也无法充分停止该疾病。神经胶质瘤 选择多种肿瘤抑制基因的遗传失活，包括 P53（> 60％），PTEN（> 75％），P16/P I4ARF（50％），PRB（30 百分比）和p27 CDK抑制剂的表观遗传下调。 抗癌疗法的中心假设认为替代肿瘤 恶性细胞中抑制基因的功能将导致特定死亡或 癌细胞的细胞凋亡在保留周围正常组织的同时。 实际上，肿瘤细胞正在受到连续的DNA损伤，因此， 通过特异性的腺病毒在神经胶质瘤中野生型p53的表达 对神经胶质瘤肿瘤细胞的细胞凋亡。我们建议通过 产生可渗透肿瘤抑制蛋白。 我的实验室进一步开发了蛋白质转导的方法。 重组，细菌表达的融合蛋白，含有N'末端 蛋白质的转导域从HIV TAT迅速转移到100％ 细胞。使用这种方法，我们已经生成并转导了60多个 15-120 kDa的TAT融合蛋白。最近，我们证明了能力 tat-b-gal蛋白的转导至大多数（如果不是全部）的细胞和组织 小鼠模型在体内，包括跨血脑屏障。因此，在 原理和实践，所有哺乳动物细胞类型都容易受到蛋白质的影响 转导。 我们建议测试杀戮的抗癌有效性和特异性 可抑制肿瘤蛋白的小鼠模型中的神经瘤肿瘤，即 tat-arf和tat-p53，以及通过可渗透促凋亡的病毒蛋白 tat-apoptin。 TAT融合蛋白将在体外进行分析和优化，并且 然后对裸鼠和DE中的异种移植颅内神经胶质瘤肿瘤进行测试 Novo在B8转基因RAS中衍生的星形胶质细胞瘤约2英寸小鼠。此外， 量化小鼠模型中的蛋白质转导潜力，我们打算分析 TAT-Reporter融合蛋白的转导和转移速率，包括 tat-b-gal和tat-tk。 TAT-TK活动也将通过体内监测 使用'8F-Fluoroganciclovir作为正电子发射极的微型网络成像。