Novel Lentiviral Packaging Systems

描述(由申请人提供)：我们的长期目标是开发对遗传病的基因治疗既安全又有效的新的基因转移策略。慢病毒载体在体外和体内的基因转移方面都具有独特的优势，因为即使在未分裂的细胞中，它们也可以提供复杂遗传结构的长期基因表达。然而，重要的安全担忧和制造障碍依然存在。一个相当大的危险是复制能力慢病毒(RCL)的污染，它可以通过反复插入突变导致传播细胞病变和肿瘤形成。大规模生产由于(I)GMP级、高滴度、稳定的慢病毒包装细胞系的缺乏和(Ii)目前载体颗粒浓缩和纯化方法的不足而先发制人。在广泛的初步结果的基础上，本提案将试图纠正这些重要问题。在具体目标1中，我们将重点开发和鉴定一种基于7个非重叠质粒的新型超分裂瞬时慢病毒包装系统，该系统几乎不可能产生RCL或Pre-RCL，同时提供非常高的病毒滴度。这一进展是通过单独的VPR介导的病毒蛋白水解酶的病毒粒子连接来实现的。在具体目标2中，我们正在测试这样一个假设，即临床级别的超顺磁性纳米颗粒可以有效地磁化慢病毒载体颗粒，从而实现无与伦比的大规模病毒粒子浓缩和纯化。其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，以允许病毒粒子与共价标记的抗CD4或抗CD34抗体的磁性粒子特异性结合。这种方法还通过在定向磁场中反对布朗运动来提高体外细胞转运率。在具体目标3中，我们将在GMP条件下逐步建立以293细胞为基础的超分裂、可诱导、稳定转染的慢病毒包装细胞系，以产生可复制的高滴度的病毒，而不检测到RCL。CD_4或CD_(34)将在某些细胞系中有成分地表达，以允许病毒粒子随后的磁化。上述方法最终将通过将复杂的β-珠蛋白基因在小鼠和人类的造血干细胞中进行体外转移，然后分别移植到地中海贫血小鼠模型和SCIDINOD小鼠身上来进行评估。

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.