Novel Lentiviral Packaging Systems

新型慢病毒包装系统

基本信息

批准号：
6821822
负责人：
Philippe Leboulch
金额：
$ 35万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
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/6821822
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

项目摘要

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型基因转移策略，这些策略既安全又有效，既可以安全又有效。慢病毒载体为前体和体内基因转移提供了独特的优势，因为它们即使在非分散细胞中也可以提供复杂遗传结构的长期基因表达。但是，仍然存在重要的安全问题和制造障碍。具有相当大的危险是具有复制能力的慢病毒（RCL）的污染，这可以通过迭代插入诱变导致扩散的细胞病变作用和肿瘤发生。（i）GMP级，高速公路，稳定的慢病毒包装细胞线以及（ii）当前方法不足的矢量颗粒浓度和纯化的当前方法不足。根据广泛的初步结果，该提案将试图纠正这些重要问题。在特定的目标1中，我们将重点关注基于7种非重叠质粒的新型Supersplit，瞬态慢病毒包装系统的开发和表征，这几乎无法产生RCL或Pre-RCL，同时提供非常高的病毒性病毒滴度。通过单独的VPR介导的病毒蛋白酶的病毒率束缚，使这一进步成为可能。在特定的目标2中，我们正在测试以下假设：慢病毒载体颗粒可以通过临床级的超顺磁性纳米颗粒有效地磁化，以使无与伦比的大规模浓度和病毒体的纯化。基本原理是在包装细胞中表达人类CD4或CD34跨膜蛋白，在萌芽后有效地显示在病毒体表面，以允许病毒体与用抗CD4或抗CD34抗体共价标记的磁颗粒特定结合。这种方法还通过在方向性磁场中反对布朗运动来增加体外的细胞转导率。在特定的目标3中，我们将在GMP条件下逐步建立，诱导，稳定，稳定转染的慢病毒包装细胞线基于293个细胞，以产生不可检测的RCL的可重复的高病毒滴度。 CD4或CD34将在某些细胞线中组成性表达，以允许随后的病毒体磁化。上述方法最终将通过在小鼠和人类的造血干细胞中的复杂β-珠蛋白基因的外传递来评估，然后分别在Thalassexemia小鼠模型和Scidinod小鼠中进行移植。