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: 6826231
• 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/6826231
• 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（改编自应用程序） 这个研究项目的目的是提供申请人，尼尔约瑟夫森， 在逆转录病毒载体开发和基因转移领域接受过培训 造血干细胞约瑟夫森医生是一位经过认证的血液学家 对干细胞疾病和基因治疗感兴趣干细胞基因 这种疗法有望治疗遗传性疾病， 贫血和地中海贫血。它也可能在治疗获得性 例如癌症和艾滋病。这项有前途的技术尚未取得成果 因为目前的逆转录病毒载体不能有效地转移 造血干细胞本申请中提出的工作将 开发和测试一种新的逆转录病毒基因转移系统， HFV病毒。HFV载体具有许多使它们成为免疫缺陷病毒的良好候选者的品质。 用于干细胞基因转移。它们不致病，宿主广泛 范围，并能刺激静止细胞。目标1侧重于病媒开发。 使用目前可用的HFV载体骨架，将构建新的构建体。 用多种不同的内部启动子和报道基因产生。 目前的HFV载体生产方法依赖于载体的瞬时转染 构建体并产生被毒素污染的粗制原液。密度 将研究用于纯化HFV原液的离心技术。到 允许更容易和更纯的载体生产HFV包装线将 开发目标2着眼于HFV载体感染人类的能力 造血细胞感染多重性和持续时间的影响 将探索暴露于载体储液对造血细胞转导的影响。 细胞周期在转导效率中的作用也将被探讨。 发现最有效地使祖细胞增殖的条件将 应用于人多能再生细胞的标记研究， NOD/SCID异种移植模型。目标3概述了将最好的 目的1中产生的HFV载体和来自工作的最佳转导方案 并将其应用于非人灵长类动物临床前标记研究。 非人类灵长类动物是与人类生物学最相似的动物模型。 因此，必须使用该模型来测试功效， 在将HFV载体应用于临床研究之前，应确保其安全性。大多数灵长 被关在笼子里的猴子感染了猿猴泡沫病毒（SFV）， 类似于HFV。在HFV载体转导的动物中SFV的存在可以 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 目标2中研究的结果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的转导方案已经确定， 确定后，将进行体内移植和标记研究。 通过以下方式跟踪标记动物是否存在转导细胞： 报告基因表达和前病毒拷贝数的评估。动物将 评估转导的任何潜在毒性作用， 移植