Vector Development and Production Core

载体开发和生产核心在这项临床基因治疗提案中发挥着关键作用。 与生产足够数量的慢病毒载体相关的挑战一直是一个主要的 阻碍了它们的临床应用，尽管该领域多年来已经意识到这些载体提供 显著的临床优势。我们的矢量核心为希望实现以下目标的研究人员提供了两大要素 在临床上使用慢病毒载体：最先进的GMP生产设施，配备齐全和积极的 生产慢病毒载体，以及利用在日本开发的稳定细胞系的新型载体生产系统 核心，能够产生超过100升的高滴度(&gt；10[7]tu/ml)、自灭活的临床慢病毒 在一次生产过程中将上清液转化为载体。我们计划生产至少三个临床批次的慢病毒 2011年前的载体，在开发和临床测试时将产生更多的载体 就会显示出来。第一个将是CL20i4-EF1a-Hy{c}opt载体，用于所述的SCID-X1临床试验 项目3“X连锁严重联合免疫缺陷的基因治疗”。生物反应器的生产 这种来自Master Cell Bank生产者克隆的载体已经在进行中，并且是一种经过临床认证的产品 预计将于2009年年中投入使用。其他表达γ-珠蛋白的载体(在项目1中描述， “通过提高胎儿血红蛋白来进行镰状细胞疾病的基因治疗”)或黄蜂(在 项目2，Wiskott-Aldrich综合征的基因治疗的发展，预计将完成临床 分别在2009年底和2010年底投产。除了临床病媒生产服务外， 载体核心实验室将继续开发我们稳定的生产者细胞系统，以促进快速 用新设计的载体高效获得克隆的生产细胞系。最后，就像三个人一样 本计划项目提交中建议的研究项目将涉及临床基因治疗 慢病毒载体，媒介核心将协调对患者样本的关键分析以进行媒介整合 站点分布。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，以 使用圣犹大新的大规模并行测序仪器促进样品分析， 并扩展我们的生物信息能力，以管理这项技术产生的大型数据集 随后，矢量核心将利用我们修改后的技术来执行FDA推荐的 定期对所有处理过的患者组织样本进行克隆性分析，并将整合部位入库 数据集，以便于在不同时间点、患者、载体和临床试验之间进行比较分析。

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-EF1a-hY{c}OPT 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.