Novel Lentiviral Packaging Systems

新型慢病毒包装系统

• 批准号: 7079435
• 负责人: Philippe Leboulch
• 金额: $ 34.43万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7079435
• 关键词: CD34 molecule; Lentivirus; SCID mouse; biotechnology; gene delivery system; gene therapy; globin; hematopoietic stem cells; immunomagnetic separation; membrane proteins; technology /technique development; transfection /expression vector; virion

DESCRIPTION (provided by applicant): Our long-term goal is the development of novel gene transfer strategies that are both safe and effective for the gene therapy of genetic diseases. Lentiviral vectors offer unique advantages for both ex-vivo and in-vivo gene transfer, because they can provide long-term gene expression of complex genetic structures even in non-dividing cells. However, important safety concerns and manufacturing hurdles remain. A considerable hazard is the contamination with a replication-competent Lentivirus (RCL), which can lead to spreading cytopathic effects and oncogenesis by iterative insertional mutagenesis. Large-scale manufacturing is preempted by (i) the unavailability of GMP-grade, high-titer, stable lentiviral packaging cell-lines and (ii) the inadequacy of current methods for vector particle concentration and purification. On the basis of extensive preliminary results, this proposal will attempt to remedy these important issues. In Specific Aim 1, we will focus on the development and characterization of a novel supersplit, transient lentiviral packaging system, based on 7 non-overlapping plasmids, that makes it virtually impossible to generate an RCL or pre-RCL, while affording very high viral titers. This advance is made possible by separate VPR-mediated virion tethering of the viral protease. In Specific Aim 2, we are testing the hypothesis that lentiviral vector particles can be effectively magnetized by clinical-grade super-paramagnetic nanoparticles to allow unparalleled large-scale concentration and purification of virions. The underlying principle is to express the human CD4 or CD34 transmembrane proteins in packaging cells, which are efficiently presented at the surface of virions upon budding, to allow specific binding of virions to magnetic particles covalently labeled with anti-CD4 or anti-CD34 antibodies. This approach also increases cell transduction rates in-vitro by opposing Brownian movement in a directional magnetic field. In Specific Aim 3, we will build step-by-step under GMP conditions, supersplit, inducible, stably transfected lentiviral packaging cell-lines based on 293 cells to yield reproducibly high viral titers free of detectable RCL. CD4 or CD34 will be constitutively expressed in some of the cell-lines to permit subsequent magnetization of the virions. The aforementioned approaches will be ultimately evaluated by ex-vivo transfer of a complex beta-globin gene in hematopoietic stem cells of mice and humans followed by transplantation in a thalassemia mouse model and in SCIDINOD mice, respectively.

描述（由申请人提供）：我们的长期目标是开发新的基因转移策略，对遗传疾病的基因治疗既安全又有效。慢病毒载体为离体和体内基因转移提供了独特的优势，因为它们甚至可以在非分裂细胞中提供复杂遗传结构的长期基因表达。然而，重要的安全问题和制造障碍仍然存在。一个相当大的危险是具有复制能力的慢病毒（RCL）的污染，它可以通过迭代插入诱变导致扩散细胞病变效应和肿瘤发生。由于 (i) 无法获得 GMP 级、高滴度、稳定的慢病毒包装细胞系，以及 (ii) 当前载体颗粒浓缩和纯化方法的不足，大规模生产被抢占先机。在广泛的初步结果的基础上，该提案将尝试解决这些重要问题。在具体目标 1 中，我们将重点开发和表征一种新型超分裂、瞬时慢病毒包装系统，该系统基于 7 个非重叠质粒，这使得几乎不可能生成 RCL 或前 RCL，同时提供非常高的病毒滴度。这一进展是通过单独的 VPR 介导的病毒蛋白酶病毒体束缚而实现的。在具体目标 2 中，我们正在测试以下假设：慢病毒载体颗粒可以被临床级超顺磁纳米颗粒有效磁化，从而实现无与伦比的大规模病毒粒子浓缩和纯化。基本原理是在包装细胞中表达人类CD4或CD34跨膜蛋白，这些蛋白在出芽时有效地呈现在病毒粒子的表面，从而使病毒粒子与共价标记有抗CD4或抗CD34抗体的磁性颗粒特异性结合。这种方法还通过定向磁场中的反向布朗运动来增加体外细胞转导率。在具体目标3中，我们将在GMP条件下逐步构建基于293细胞的超分裂、可诱导、稳定转染的慢病毒包装细胞系，以产生可重复的高病毒滴度，且不含可检测到的RCL。 CD4 或 CD34 将在一些细胞系中组成型表达，以允许病毒颗粒随后磁化。上述方法最终将通过在小鼠和人类的造血干细胞中体外转移复杂的β-珠蛋白基因，然后分别移植到地中海贫血小鼠模型和SCIDINOD小鼠中来进行评估。