Second Generation Bio-Lentivir Packaging Cell Line for Facile Lentivirus Production

该项目旨在利用分子生物学来构建设计病毒，这些病毒在结构上适合于简单的亲和纯化，因此更容易制造。基因医学网站显示，病毒目前被用于所有基因治疗试验的三分之二，其中逆转录病毒和慢病毒约占40%。色谱法优选用于载体纯化，离子交换已用于1期临床试验的慢病毒载体的多步纯化。蛋白质生产中的优先权表明，这种低特异性方法现在几乎完全被亲和方法取代，这种偏好可能会出现在病毒生产中，以利用亲和色谱的分辨率和简单性。认识到这一点，我们设计了一种新的包装细胞系Bio-293 T，该细胞系通过代谢产生亲和标记的慢病毒，并且我们已经证明了亲和捕获对病毒浓缩提供的巨大效率节省和灵活性。第一代Bio-Lentivir包装细胞系突出了传统基质用于病毒亲和纯化的不足，优化病毒包膜上亲和标签密度以实现有效回收的迫切需要，以及新型大孔吸附剂提供简单单步处理的潜力。它还表明，生物素标记的慢病毒可以与链霉亲和素顺磁性颗粒复合，导致最有效的分离和浓缩方法，但慢病毒载体。然而，大多数临床应用需要游离病毒，对于游离病毒，在没有病毒洗脱的情况下使用顺磁性纳米颗粒捕获方法是不合适的，对于这种高亲和力系统，这导致低的工艺产率。根据我们的概念验证，因此需要第二代Bio-Lentivir包装细胞系，旨在克服这些限制，并充分利用高效生产高滴度，高纯度，临床级逆转录病毒和慢病毒基因转移载体的潜力。该项目的产出将是新的His标签/Bio-Lentivir包装细胞系，用于病毒纯化的新吸附材料和整合的病毒生产方案。这些目标源于Kings和剑桥团队成功开发Bio-293 T包装细胞系的联合经验，并将通过将分子生物学、载体设计和包装细胞系构建（Kings）的专业知识与生物材料、病毒和亲和处理专业知识（剑桥）结合起来加以解决。

The project seeks to exploit molecular biology to construct designer viruses that are structurally adapted for simple affinity purification and hence easier manufacture. The Gene Medicine web site shows that viruses are currently used in two-thirds of all gene therapy trials, with retro- and lentiviruses constituting ~40% of these. Chromatography is preferred for vector purification and ion-exchange has been used in the multi-step purification of a lentiviral vector for a Phase 1 clinical trial. Precedence in protein manufacturing shows that such low specificity processes are now almost entirely superseded by affinity processes and this preference will likely emerge in virus manufacturing in order to exploit the resolution and simplicity of affinity chromatography. Recognising this we engineered a novel packaging cell line, Bio-293T, that metabolically produces an affinity-tagged lentivirus and we have demonstrated the enormous efficiency savings and flexibility offered by affinity capture for virus concentration. This first generation Bio-Lentivir packaging cell line has highlighted the inadequacy of conventional matrices for virus affinity purification, the critical need to optimise affinity-tag density on the viral envelope for efficient recovery and the potential of novel macroporous adsorbents to provide simple, single-step processing. It has also shown that biotin-tagged lentivirus can be complexed with streptavidin paramagnetic particles, resulting in the most efficient isolation and concentration method yet described for lentiviral vectors. However, most clinical applications require free virus, for which use the paramagnetic nanoparticle capture approach is unsuitable without virus elution, which, for this high affinity system leads to low process yields. Following our proof-of-concept there is consequently a need for a second generation Bio-Lentivir packaging cell line designed to ovecome these limitations and fully exploit the potential for the efficient production of high titre, highly purified, clinical grade retroviral and lentiviral gene transfer vectors.The project outputs will be novel His-tag/Bio-Lentivir packaging cell lines, new adsorbent materials for virus purification and an integrated virus production scheme. These objectives stem from the joint experience of the Kings and Cambridge teams on the successful development of the Bio-293T packaging cell line and will be addressed by bringing together expertise in molecular biology, vector design and packaging cell line construction (at Kings) with bio-materials, virus and affinity processing expertise (at Cambridge).