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: 6522938
• 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/6522938
• 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、神经胶质瘤和星形细胞瘤， 是毁灭性的恶性肿瘤，占美国所有癌症的 2.3% 死亡人数，是儿童第二常见的实体瘤。恶性 脑肿瘤对目前的治疗反应不佳，平均存活率为 尽管接受治疗仍不到一年，由于神经胶质瘤的侵袭性， 特别是多形性胶质母细胞瘤（GBM），局部抗癌策略， 例如手术切除，也无法充分阻止疾病。神经胶质瘤 选择多种肿瘤抑制基因的基因失活，包括 p53 (>60%)、PTEN (>75%)、p16/p I4ARF (50%)、pRB (30 %），以及 p27 Cdk 抑制剂的表观遗传下调。 抗癌疗法的一个中心假说认为，肿瘤的替代 恶性细胞中的抑制基因功能将导致特定的死亡或 癌细胞凋亡，同时不伤害周围的正常组织。 事实上，肿瘤细胞正在经历持续的 DNA 损伤，因此， 野生型p53在神经胶质瘤中的腺病毒表达的具体结果 神经胶质瘤肿瘤细胞的凋亡。我们建议通过以下方式检验这一假设 产生可转导的肿瘤抑制蛋白。 我的实验室进一步开发了蛋白质转导的方法。 含有 N' 末端的重组细菌表达融合蛋白 HIV TAT 的蛋白质转导结构域可 100% 快速转导 细胞。使用这种方法，我们已经生成并转换了 60 多个 15-120 kDa 的 TAT 融合蛋白。最近，我们展示了能力 TAT-B-gal 蛋白转导到大多数（如果不是全部）细胞和组织中 体内小鼠模型，包括跨越血脑屏障。因此，在 原理和实践，所有哺乳动物细胞类型都对蛋白质敏感 转导。 我们建议测试杀伤的抗癌有效性和特异性 通过可转导的肿瘤抑制蛋白在小鼠模型中诱导神经胶质瘤肿瘤，即 TAT-ARF 和 TAT-p53，并通过可转导的促凋亡病毒蛋白， TAT-凋亡素。 TAT 融合蛋白将在体外进行分析和优化 然后在裸鼠和 de 中针对异种移植颅内神经胶质瘤进行测试 B8 转基因 ras about2" 小鼠中 novo 衍生的星形细胞瘤。此外， 量化小鼠模型中的蛋白质转导潜力，我们打算分析 TAT-报告基因融合蛋白的转导和重折叠率，包括 TAT-B-gal 和 TAT-TK。 TAT-TK 活性也将通过以下方式进行体内监测： 使用 '8F-氟更昔洛韦作为正电子发射器的 microPET 成像。