GENE TRANSFER INTO STEM CELLS BY FOAMY VIRUS VECTORS

通过泡沫病毒载体将基因转移到干细胞中

• 批准号: 6684124
• 负责人: Neil Josephson
• 金额: $ 12.57万
• 依托单位: BLOODWORKS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6684124
• 关键词: NOD mouse; Retroviridae; SCID mouse; baboons; cell cycle; cord blood; gene delivery system; hematopoietic stem cells; human tissue; stem cell transplantation; technology /technique development; transfection /expression vector; xenotransplantation

DESCRIPTION (adapted from the application) This research project is designed to provide the applicant, Neil Josephson, with training in the areas of retroviral vector development and gene transfer into hematopoietic stem cells. Dr. Josephson is a board certified hematologist with an interest in stem cell disorders and gene therapy. Stem cell gene therapy offers the promise of treating hereditary disorders such as sickle cell anemia and thalassemia. It also may play a role in the therapy of acquired diseases such as cancer and HIV. This promising technology has not yet yielded clinical results because current retroviral vectors do not efficiently transfer genes into hematopoietic stem cells. The work proposed in this application will develop and test a new retroviral gene transfer system based on the human foamy virus (HFV). HFV vectors have many qualities that make them good candidates for use in stem cell gene transfer. They are non-pathogenic, have a wide host range, and can transduce quiescent cells. Aim 1 focuses on vector development. Using currently available HFV vector backbones, new constructs will be generated with a variety of different internal promoters and reporter genes. Current HFV vector production methods rely on transient transfection of vector constructs and yield a crude stock contaminated with toxins. Density centrifugation techniques for purifying HFV stocks will be investigated. To allow for easier and more pure vector production an HFV packaging line will be developed. Aim 2 looks at the ability of HFV vectors to transduce human hematopoietic cells. The impact of multiplicity of infection and length of exposure to vector stock on hematopoietic cell transduction will be explored. The role of cell cycle in transduction efficiency will also be explored. Conditions that are found to most efficiently transduce progenitor cells will be applied to marking studies of human pluripotent repopulating cells in the NOD/SCID xenotransplantation model. Aim 3 outlines work that will take the best HFV vectors produced in aim 1 and the optimal transduction protocols from work in aim 2 and apply them to a pre-clinical marking study of nonhuman primates. Non-human primates are the most biologically similar animal model to humans. Therefore, it is essential to use this model for testing the efficacy and safety of HFV vectors before applying them to clinical studies. Most primates kept in captivity are infected with the simian foamy virus (SFV) which is very similar to HFV. The presence of SFV in HFV vector transduced animals could complicate the interpretation of marking and toxicity results. Therefore, in vitro analysis of HFV effects on SFV will be explored. Transduction protocols from studies in aim 2 will be applied to marking studies of non-human primate hematopoietic progenitor cells. Once optimal transduction protocols have been determined, in vivo transplantation and marking studies will be performed. Marked animals will be followed for the presence of transduced cells by evaluation of reporter gene expression and proviral copy numbers. Animals will be evaluated for any potential toxic effects of the transduction and transplantation.

说明(改编自应用程序) 这项研究项目旨在为申请者尼尔·约瑟夫森提供 在逆转录病毒载体开发和基因转移领域接受培训 转化为造血干细胞。约瑟夫森博士是一名获得董事会认证的血液学家。 对干细胞疾病和基因治疗感兴趣。干细胞基因 治疗提供了治疗遗传性疾病的希望，如镰状细胞 贫血和地中海贫血。它也可能在获得性心脏病的治疗中发挥作用。 癌症和艾滋病毒等疾病。这项前景看好的技术尚未见效。 临床结果，因为目前的逆转录病毒载体不能有效地转移 基因转化为造血干细胞。本申请中提出的工作将 基于人泡沫的新型逆转录病毒基因转移系统的研制与试验 病毒(HFV)。肾综合征出血热病毒载体具有许多特性，使它们成为 用于干细胞基因转移。它们是非致病的，宿主广泛 范围，并可以转换静止的细胞。目标1侧重于病媒的发展。 使用目前可用的HFV载体骨架，新的结构将被 产生了各种不同的内部启动子和报告基因。 目前肾综合征出血热病毒载体的生产方法依赖于瞬时转染法 并产生一种被毒素污染的粗制原料。密度 将研究用于提纯HFV库存的离心法技术。至 考虑到更容易和更纯的媒介生产，HFV包装线将是 发展起来的。目标2着眼于HFV载体转导人类的能力 造血细胞。感染的多样性和持续时间的影响 将探索与载体有关的造血细胞转导。 还将探讨细胞周期在转导效率中的作用。 已发现的最有效转导祖细胞的条件将 应用于人类多能再生细胞的标记研究 NOD/SCID异种移植模型。目标3概述了将采取最佳措施的工作 在AIM 1中产生的HFV载体和Work中的最佳转导方案 并将其应用于非人类灵长类动物的临床前标记研究。 非人灵长类是与人类在生物学上最相似的动物模型。 因此，使用这一模型来检验药效和疗效是必要的。 在将HFV载体应用于临床研究之前，对其安全性进行评估。大多数灵长类动物 被关在笼子里的人感染了猿猴泡沫病毒(SFV)，这种病毒非常 类似于肾综合征出血热。在HFV载体转导的动物中存在SFV可能 使标记和毒性结果的解释复杂化。因此，在 将探索HFV对SFV影响的体外分析。转导方案 来自AIM 2的研究将应用于非人灵长类动物的标记研究 造血祖细胞。一旦最佳转导方案 确定后，将进行体内移植和标记研究。 标记的动物将被跟踪，以确定是否存在转导细胞 报告基因表达和前病毒拷贝数的评估。动物们会 对转导的任何潜在毒性影响进行评估，并 移植。