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<15个月 尽管进行了标准化放疗GBM由GBM干细胞样细胞（GSC）形成，GBM干细胞样细胞（GSC）是GBM的主要来源。 肿瘤复发的贡献者和治疗发展的自然焦点。主要有两个原因 导致治疗失败的原因：1）高的肿瘤内和肿瘤间细胞和分子异质性， 具有不同遗传决定子的多个亚克隆; 2）GSC表现出多个冗余信号传导 需要同时靶向重叠途径的途径。我们发明了生物学上 验证了一种新的串联计算平台，GeneRep-nSCORE，整合大规模基因 表达谱与基因组变化，以确定共同的基础改变或主调节因子， GSC跨越GBM肿瘤内和跨GBM肿瘤的大量（如果不是全部）GSC亚克隆。我们发现， GCS中的四种常见主调节剂的核心组是临床开发的突出目标。 这四种因子的表达足以将正常星形胶质细胞重编程为GSC，而它们的表达则不足以将正常星形胶质细胞重编程为GSC。 在所有八个患者来源的细胞系中， 迄今为止检查的不同遗传和分子背景的GSC。 本申请的目标是开发一套定制的基于腺相关病毒（AAV）的遗传修饰的基因。 用于靶向GSC的整个谱的工具（阶段I），目的是将靶向构建体递送至 耗尽负责GSC恶性转化和增殖的四种常见主调节因子 （第二阶段）。该建议的具体目标是：（i）使用定向进化和可用的组合 AAV衣壳库，并首次引入库试剂的动态施用模式 随着肿瘤进展的时间推移，大大增加识别新型腺相关病毒变体的可能性 在患者来源的异种移植模型（PDX）中特异性靶向慢循环和快循环GSC（I期）;（ii） 设计并验证一组AAV载体，其表达靶向GSC的核心主调节子的shRNA， 确定临床前测试的线索;（iii）优化病毒递送、药代动力学和 药效学参数，以及正常和用铅靶向AAV治疗的PDX的安全性和毒性 试剂卡;以及（iv）基于这些结果，创建工具和基本DMPK数据，以进行临床前疗效 在用单独的铅靶向AAV盒或与标准AAV盒组合处理的PDX中的研究 放化疗（II期），为研究性新药申请做准备，用于患者临床试验 与GBM，并为这种新技术的商业发展。

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.