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: 6233010
• 负责人: Neil Josephson
• 金额: $ 12.03万
• 依托单位: UNIVERSITY OF WASHINGTON
• 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/6233010
• 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.

描述（根据应用程序改编） 该研究项目旨在为申请人尼尔·约瑟夫森（Neil Josephson）提供 在逆转录病毒载体发展和基因转移领域进行培训 进入造血干细胞。约瑟夫森博士是董事会认证的血液学家 对干细胞疾病和基因疗法感兴趣。干细胞基因 治疗提供了治疗遗传疾病（例如镰状细胞）的承诺 贫血和丘脑贫血。它也可能在获得的治疗中发挥作用 癌症和艾滋病毒等疾病。这项有希望的技术尚未产生 临床结果，因为电流逆转录病毒载体无法有效地转移 基因进入造血干细胞。本申请中提出的工作将 基于人泡沫开发和测试新的逆转录病毒基因转移系统 病毒（HFV）。 HFV矢量具有许多素质，使他们成为了好候选人 用于干细胞基因转移。它们是非致病性的，有一个广泛的宿主 范围，可以传递静态细胞。 AIM 1专注于向量发展。 使用当前可用的HFV矢量骨架，新结构将是 由各种不同的内部启动子和报告基因产生。 当前的HFV矢量生产方法依赖于向量的瞬时转染 构造并产生被毒素污染的粗股。密度 将研究用于净化HFV股票的离心技术。到 允许更轻松，更纯净的矢量生产HFV包装线将是 发达。 AIM 2着眼于HFV向量转导人的能力 造血细胞。感染的多样性和长度的影响 将探索在造血细胞转导中接触载体库存。 还将探讨细胞周期在转导效率中的作用。 发现最有效地转导祖细胞的条件将 应用于标记人类多能重生细胞的研究 点数/SCID异种移植模型。 AIM 3概述将尽力而为 AIM 1中产生的HFV矢量和工作的最佳转导协议 在AIM 2中，将它们应用于非人类灵长类动物的临床前标记研究。 非人类灵长类动物是与人类最相似的动物模型。 因此，必须使用此模型来测试功效和 HFV向量的安全性在将其应用于临床研究之前。大多数灵长类动物 被囚禁的被囚禁感染了非常非常非常 类似于HFV。 HFV载体转导的动物中SFV的存在可能 使标记和毒性结果的解释复杂化。因此，在 将探索对HFV对SFV影响的体外分析。转导协议 从AIM 2的研究中将应用于标记非人类灵长类动物的研究 造血祖细胞。一旦最佳转导协议已 确定的，将进行体内移植和标记研究。 将遵循明显的动物，以进行转导的细胞。 评估报告基因表达和前病毒拷贝数。动物会 评估转导的任何潜在毒性作用和 移植。