In Vivo Directed Evolution of Adeno-Associated Virus Vectors for Glioblastoma Multiforme Tumor-Initiating Cells

总结 神经胶质瘤是成人最常见的脑肿瘤，由于神经胶质细胞的异常生长和侵袭而发生。 星形胶质细胞肿瘤细胞即使采用积极的治疗，生存率也很低，这是由于 治疗抗性肿瘤起始细胞（TIC），具有高度迁移性和侵袭性，因此使 手术切除肿瘤是不可能的针对神经胶质瘤肿瘤细胞和TICs的工程疗法 可以提高疗效，并因此延长患有这种疾病的患者的临床存活时间。 疾病因此，本建议集中于胶质母细胞瘤基因治疗策略的发展 多形性胶质瘤（GBM）是一种侵袭性形式的胶质瘤，其基于靶向GBM肿瘤细胞和TIC。 腺相关病毒（Adeno-associated virus，AAV）是一种安全、有前途的基因载体 应用.然而，一般的病毒载体，特别是AAV，不显示强的内在细胞向性 对于中枢神经系统（CNS）中的神经胶质瘤细胞，此外，它们经历了许多递送， 全身递送至临床GBM的转运屏障，包括生物分布至CNS、血脑 屏障、实质内和肿瘤内转运至原发性和弥漫性继发性肿瘤。因此 非常需要开发能够全身递送并且能够克服 这些交付障碍。 我们提出改造AAV的外壳蛋白以靶向递送至神经胶质瘤肿瘤细胞和TIC， 大大提高了递送效率并减少了任何生物脱靶效应。我们假设AAV 定向进化，我们最初开发并成功用于增强病毒载体的策略， 特性，可以实施以在体内工程化AAV载体，用于增强的和潜在的选择性向性 GBM肿瘤细胞和TIC。具体来说，我们建议利用（1）基于 异种移植原代培养的患者来源的GBM TIC，其准确地代表GBM的标志， (2)高度多样性的AAV载体文库，和（3）复杂的定向进化策略，其包括 对可定位于CNS和GBM肿瘤的病毒颗粒具有严格的体内选择性 细胞和TIC。我们已经成功地从第一轮进化中恢复了病毒基因组， 这一战略的潜力。我们还建议通过研究来表征所得到的工程化AAV载体。 它们的向性和生物分布、临床实施的基本基因递送性质。 此外，我们建议通过递送两种重组腺相关病毒来评估工程化腺相关病毒的治疗潜力。 有希望的治疗基因，可以阻止肿瘤的进展，延长我们的动物模型的生存期， 或者为未来癌症免疫疗法的探索提供希望。这种分子病毒学蛋白质 因此，精确的动物模型将使工程化的增强 用于治疗多形性胶质母细胞瘤的遗传传递系统，以及将来可能用于治疗其他胶质母细胞瘤的遗传传递系统。 癌的

Summary Glioma, the most common brain tumor in adults, develops as a result of aberrant growth and invasion of astrocytic tumor cells. Even with aggressive treatment, survival is very poor and is attributed to the presence of therapy-resistant tumor-initiating cells (TICs), which are highly migratory and invasive and thus render complete surgical tumor removal impossible. Engineering therapies that target glioma tumor cells and TICs may enable enhanced efficacy and as a result longer clinical survival times in patients afflicted with this disease. Accordingly, this proposal is focused on the development of gene therapy strategies for glioblastoma multiforme (GBM), an aggressive form of glioma, based on the targeting of GBM tumor cells and TICs. Adeno-associated virus (AAV) has emerged as a safe and promising vector for gene delivery applications. However, viral vectors in general, and AAV in particular, do not display strong intrinsic cell tropism for glioma cells in the central nervous system (CNS), and in addition they experience a number of delivery and transport barriers for systemic delivery to clinical GBM, including biodistribution to the CNS, the blood brain barrier, and intraparenchymal and intratumoral transport to the primary and diffuse secondary tumors. Thus, it is highly desirable to develop vectors that can be systemically delivered and that are capable of overcoming these delivery barriers. We propose to engineer the coat proteins of AAV to target delivery to glioma tumor cells and TICs to greatly enhance delivery efficiency and reduce any biological off-target effects. We hypothesize that AAV directed evolution, a strategy we originally developed and have successfully employed to enhance viral vector properties, can be implemented to engineer AAV vectors in vivo for enhanced and potentially selective tropism for GBM tumor cells and TICs. Specifically, we propose to harness (1) a mouse model based on the xenografting of primary cultured, patient-derived GBM TICs that accurately represents the hallmarks of GBM, (2) highly diverse AAV vector libraries, and (3) a sophisticated directed evolution strategy that includes a stringent in vivo selection selective for viral particles that can localize to the CNS and transduce GBM tumor cells and TICs. We have successfully recovered viral genomes from the first round of evolution, highlighting the potential of this strategy. We also propose to characterize the resulting engineered AAV vectors by studying their tropism and biodistribution, essential gene delivery properties for clinical implementation. Furthermore, we propose to evaluate the therapeutic potential of engineered AAVs by delivering two promising therapeutic genes that can hamper tumor progression and extend the survival of our animal models, or that offer promise in future exploration of cancer immunotherapies. This blend of molecular virology, protein engineering, and a translationally accurate animal model will therefore enable the engineering of enhanced genetic delivery systems for the treatment of glioblastoma multiforme and in the future potentially other cancers.