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

多形性胶质母细胞瘤肿瘤起始细胞腺相关病毒载体的体内定向进化

基本信息

批准号：
9353802
负责人：
DAVID V SCHAFFER
金额：
$ 22.46万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-16 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9353802
关键词：
Address Adult Anatomy Animal Model Animals Apoptosis Astrocytoma Biodistribution Biological Blood - brain barrier anatomy Brain Neoplasms Cancer Model Capsid Proteins Cells Clinic Clinical Clinical Trials Dependovirus Development Diagnosis Diffuse Directed Molecular Evolution Disease Distal Engineering Essential Genes Evolution Excision Familial Lipoprotein Lipase Deficiency Future Gene Delivery Gene Library Generations Genetic Genetic Enhancement Glioblastoma Glioma Growth Hemophilia A Herpesviridae Human Immunocompromised Host In Vitro Individual Injectable Injection of therapeutic agent Intervention Leber&apos s amaurosis Libraries Malignant Neoplasms Mediating Medical Mendelian disorder Modeling Molecular Molecular Virology Mus Neoplasm Metastasis Neuraxis Oncolytic Operative Surgical Procedures Patients Pre-Clinical Model Primary Brain Neoplasms Property Protein Engineering Radiation Recovery Safety Series Suicide System Therapeutic Time Tissues Treatment Efficacy Tropism Tumor Initiators Vaccinia Variant Viral Viral Genome Viral Vector Virus Work Xenograft Model Xenograft procedure adeno-associated viral vector antiangiogenesis therapy base brain parenchyma cancer immunotherapy cancer therapy chemotherapy clinical translation conventional therapy experience extracellular gene therapy immune checkpoint blockade improved in vivo in vivo Model mouse model neoplastic cell next generation novel novel strategies outcome forecast particle response success targeted delivery targeted treatment therapeutic gene therapeutic target therapy resistant trafficking transgene expression tumor tumor growth tumor progression vector virtual

项目摘要

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.

概括神经胶质瘤是成人最常见的脑肿瘤，是由于神经胶质细胞异常生长和侵袭而形成的。星形胶质细胞肿瘤细胞。即使采取积极的治疗，生存率也很差，这归因于存在耐药肿瘤起始细胞 (TIC) 具有高度迁移性和侵袭性，因此导致完全手术切除肿瘤是不可能的。针对神经胶质瘤肿瘤细胞和 TIC 的工程疗法可能会提高疗效，从而延长患有此病的患者的临床生存时间疾病。因此，该提案的重点是开发胶质母细胞瘤的基因治疗策略多形性（GBM），一种侵袭性神经胶质瘤，基于 GBM 肿瘤细胞和 TIC 的靶向。腺相关病毒（AAV）已成为一种安全且有前途的基因传递载体应用程序。然而，一般病毒载体，特别是 AAV，并不表现出强烈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，此外它们还经历了许多传递和全身递送至临床 GBM 的运输障碍，包括生物分布至中枢神经系统、血脑屏障，以及实质内和瘤内运输到原发性和弥漫性继发性肿瘤。因此，它非常需要开发可以系统递送并且能够克服的载体这些交付障碍。我们建议对 AAV 的外壳蛋白进行改造，以靶向递送至神经胶质瘤细胞和 TIC，从而大大提高递送效率并减少任何生物脱靶效应。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，可用于在体内设计 AAV 载体，以增强和潜在的选择性趋向性用于 GBM 肿瘤细胞和 TIC。具体来说，我们建议利用（1）基于原代培养的、源自患者的 GBM TIC 的异种移植可准确代表 GBM 的特征， (2) 高度多样化的 AAV 载体库，以及 (3) 复杂的定向进化策略，其中包括对可定位至 CNS 并转导 GBM 肿瘤的病毒颗粒进行严格的体内选择细胞和 TIC。我们已经成功地从第一轮进化中恢复了病毒基因组，这凸显了该策略的潜力。我们还建议通过研究来表征由此产生的工程 AAV 载体它们的趋向性和生物分布，以及临床实施所必需的基因传递特性。此外，我们建议通过提供两种方法来评估工程 AAV 的治疗潜力有前景的治疗基因可以阻碍肿瘤进展并延长动物模型的生存期，或者为未来探索癌症免疫疗法带来希望。这种融合了分子病毒学、蛋白质因此，翻译精确的动物模型将能够实现增强的工程设计用于治疗多形性胶质母细胞瘤以及未来潜在的其他治疗的基因传递系统癌症。