Vector Development and Production Core

载体开发和生产核心在这一临床基因治疗方案中起着关键作用。尽管该领域多年来一直意识到慢病毒载体具有显著的临床优势，但与生产足够数量的慢病毒载体相关的挑战一直是其临床应用的主要障碍。我们的Vector Core为希望在临床中使用慢病毒载体的研究人员提供了两个主要元素：一个最先进的GMP生产设施，配备充足的人员并积极生产慢病毒载体，以及一个使用核心开发的稳定细胞系的新型载体生产系统，能够在一次生产中生产超过100升的高滴度bb10 ^7 tu/ml)，自我灭活的临床慢病毒载体上清液。我们计划在2011年之前生产至少三个慢病毒载体的临床批次，并随着它们的开发和临床测试的需要而生产更多的载体。第一个将是CL20i4-EF1α -hYcOPT载体，用于项目3“x连锁严重联合免疫缺陷的基因治疗”中描述的SCID-X1临床试验。该载体的生物反应器生产工作已经在进行中，一种经临床认证的产品预计将于2009年年中投入使用。其他表达y-珠蛋白（项目1“通过增强胎儿血红蛋白的镰状细胞病基因治疗”）或WASp（项目2“Wiskott-Aldrich综合征基因治疗的发展”）的载体预计将分别在2009年底和2010年底完成临床生产。除了临床病媒生产服务外，病媒核心实验室将继续发展稳定的生产细胞系统，以促进使用新设计的病媒克隆生产细胞系的快速有效衍生。最后，由于本项目提交的所有三个拟议研究项目都涉及慢病毒载体的临床基因治疗，因此Vector Core将协调患者样本的关键分析，以进行载体整合位点分布。我们将首先修改目前在我们的研究实验室中使用的已建立的PCR技术

The Vector Development and Production Core occupies a pivotal role for this clinical gene therapy proposal. The challenges associated with production of adequate quantities of lentiviral vectors has been a major hurdle to their clinical application, even though the field has been aware for years that these vectors provide significant clinical advantages. Our Vector Core contributes two major elements to researchers wishing to use lentiviral vectors in the clinic: a state-of-the-art GMP production facility, fully staffed and actively producing lentiviral vectors, and a novel vector production system using stable cell lines developed in the Core and capable of producing over 100 liters of high titer >10^7 tu/ml), self-inactivating, clinical lentiviral vector supernatants in a single production run. We plan to produce at least three clinical batches of lentiviral vectors before 2011, with additional vectors being produced as they are developed and clinical testing becomes indicated. The first will be the CL20i4-EF1α -hYcOPT vector for the SCID-X1 clinical trial described in the project 3, "Gene Therapy for X-Linked Severe Combined Immunodeficiency". Bioreactor production of this vector from a Master Cell Banked producer clone is already in progress, and a clinically certified product is expected to be ready for use by mid-2009. Other vectors expressing y-globin (described in project 1, "Gene Therapy of Sickle Cell Disease through Enhancement of Fetal Hemoglobin") or WASp (described in project 2, "Development of Gene Therapy for Wiskott-Aldrich Syndrome", are predicted to complete clinical production by the end of 2009 and 2010, respectively. In addition to the clinical vector production services, the Vector Core Laboratory will continue developing our stable producer cell system to facilitate the rapid and efficient derivation of cloned producer cell lines with newly designed vectors. Finally, as all three proposed research projects in this Program Project submission will involve clinical gene therapy with lentiviral vectors, the Vector Core will coordinate the critical analysis of patient samples for vector integration site distribution. We will first modify established PCR techniques currently in use in our research labs to facilitate sample analysis using the new massively parallel sequencing instrumentation available at St. Jude, and expand on our bioinformatic capability to manage the large data sets this technology generates Subsequently, the Vector Core will utilize our modified techniques to perform the FDA-recommended clonality assays on all treated patient tissue samples at regular intervals, and warehouse the integration site data sets to facilitate comparative analysis between different timepoints, patients, vectors, and clinical trials.