Targeting glioblastoma stem-like cells with custom-designed viral vectors

胶质母细胞瘤(GBM)是最致命的成人脑癌，中位生存期为15个月。 尽管有激进的标准放化疗。GBM是由GBM干细胞样细胞(GSCs)形成的，GSCs是一种主要的 是肿瘤复发的贡献者，也是治疗发展的天然焦点。主要有两个原因 治疗失败的原因：1)肿瘤内和肿瘤间高度的细胞和分子异质性 具有不同遗传决定因素的多个亚克隆；2)GSC显示多个冗余信号 需要同时针对重叠路径的路径。我们已经发明了生物上的 验证了一种集成大规模基因的新型串联计算平台GeneRep-nSCORE 带有基因组变化的表达谱，以识别常见的创始改变或掌握 跨越大量(如果不是全部)的GSC在GBM肿瘤内和跨GBM肿瘤之间的GSC亚克隆。我们发现了这样的 GCSs中四个共同的主调节器的核心集，是临床开发的杰出目标。 这四个因子的表达足以将正常星形胶质细胞重新编程为GSCs，而它们的 在所有八种患者来源的品系中，枯竭都深刻地破坏了GSCs，从而导致了体内肿瘤的发展 到目前为止，研究了不同遗传和分子背景的GSCs。 该应用程序目标是开发一组定制的基于腺相关病毒(AAV)的基因 针对所有GSC的工具(第一阶段)，目的是将目标构建交付给 耗尽四种常见的导致GSCs恶性转化和增殖的主调控因子 (第二阶段)。这一建议的具体目标是：(1)使用定向进化和现有的组合 AAV衣壳文库，并首次引入了图书馆试剂的动态给药模式 在肿瘤进展的时间过程中，极大地增加识别新的AAV变异的可能性 在患者来源的异种移植模型(PDX)中特别针对慢周期和快周期的GSC(第一阶段)； 设计和验证一组AAV载体，表达靶向GSC核心主调控子的shRNA，以 确定临床前试验的线索；(3)优化病毒传递方式、药代动力学和 铅靶向AAV治疗正常和PDX的药效学参数及安全性和毒性 以及(Iv)基于这些结果、工具和创建的基本DMPK数据，进行临床前疗效 铅靶向AAV盒单独或与标准品联用治疗PDX的研究 化疗和放射治疗(第二阶段)，为患者临床试验的研究性新药应用做准备 与GBM合作，并用于这项新技术的商业开发。

Glioblastoma (GBM) is the most lethal form of adult brain cancers with a median survival of <15 months despite aggressive standard chemoradiation. GBM are formed by GBM stem-like cells (GSCs) - a major contributor to tumor recurrence and a natural focus for therapeutic development. There are two main reasons responsible for treatment failure: 1) high intra- and inter-tumor cellular and molecular heterogeneity with multiple subclones possessing distinct genetic determinants; 2) GSCs exhibit multiple redundant signaling pathways requiring simultaneous targeting of overlapping pathways. We have invented and biologically validated a novel tandem computational platform, GeneRep-nSCORE that integrates large-scale gene expression profiles with genomic changes to identify common founding alterations or master regulators of GSCs that span a large number, if not all, GSC subclones within and across GBM tumors. We discovered such a core set of four common master regulators in GCSs that are outstanding targets for clinical development. Expression of these four factors was sufficient to reprogram normal astrocytes to GSCs, whereas their depletion profoundly abrogated GSCs, and thus tumor development in vivo, in all eight lines of patient-derived GSCs of varied genetic and molecular backgrounds examined to date. The goal of this application is to develop a customized set of Adeno-associated virus (AAV)-based genetic tools to target the whole spectrum of GSCs (Phase I) for the purpose of delivering targeting constructs to deplete the four common master regulators responsible for malignant transformation and proliferation in GSCs (Phase II). The specific objectives of this proposal are: (i) using directed evolution and available combinatorial AAV capsid library, and for the first time, introducing a dynamic mode of administration of a library reagent over the time course of tumor progression, to greatly increase the probability of identifying novel AAV variants specifically targeting slow-, and fast-cycling GSCs in patient-derived xenograft models (PDX) (Phase I); (ii) To design and validate a panel of AAV vectors that express shRNAs targeting core master regulators of GSCs to identify leads for preclinical testing; (iii) To optimize modes of viral delivery, pharmacokinetics and pharmacodynamics parameters, and safety and toxicity in normal and PDX treated with lead targeting AAV cassettes; and (iv) Based on these results, tools and basic DMPK data created, to conduct preclinical efficacy studies in PDX treated with lead targeting AAV cassettes either alone or in combination with standard chemoradiotherapy (Phase II) to prepare for an investigative new drug application for clinical testing in patients with GBM, and for commercial development of this novel technology.