Novel Lentiviral Packaging Systems

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，这些策略对于遗传性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被具有复制能力的慢病毒（RCL）污染，这可能会通过迭代插入突变导致细胞病变效应和肿瘤发生。大规模生产被（i）GMP级、高滴度、稳定的慢病毒包装细胞系的不可用和（ii）目前用于载体颗粒浓缩和纯化的方法的不足所取代。根据广泛的初步结果，本建议将试图纠正这些重要问题。在特定目标1中，我们将专注于基于7个非重叠质粒的新型超分裂瞬时慢病毒包装系统的开发和表征，该系统使得几乎不可能产生RCL或前RCL，同时提供非常高的病毒滴度。这一进展是可能的，通过单独的VPR介导的病毒蛋白酶的病毒粒子拴系。在具体目标2中，我们正在测试慢病毒载体颗粒可以被临床级超顺磁性纳米颗粒有效磁化的假设，以实现病毒粒子的无与伦比的大规模浓缩和纯化。基本原理是在包装细胞中表达人CD 4或CD 34跨膜蛋白，其在出芽时有效地呈递在病毒体表面，以允许病毒体与用抗CD 4或抗CD 34抗体共价标记的磁性颗粒特异性结合。这种方法还通过在定向磁场中对抗布朗运动来增加体外细胞转导率。在特定目标3中，我们将在GMP条件下逐步构建基于293细胞的超分裂、诱导型、稳定转染的慢病毒包装细胞系，以产生不含可检测RCL的可重现高病毒滴度。CD 4或CD 34将在一些细胞系中组成型表达，以允许病毒粒子的后续磁化。上述方法将通过在小鼠和人的造血干细胞中离体转移复合β-珠蛋白基因，然后分别在地中海贫血小鼠模型和SCIDINOD小鼠中移植来最终评估。

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.