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，这种设计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，目前三分之二的基因治疗试验使用病毒，其中复古病毒和慢病毒占40%。色谱法是载体纯化的首选方法，离子交换法已用于慢病毒载体的多步骤纯化，用于一期临床试验。蛋白质制造的先例表明，这种低特异性的过程现在几乎完全被亲和过程所取代，这种偏好可能会出现在病毒制造中，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-293T，通过代谢产生亲和力标记的慢病毒，我们已经证明了亲和力捕获为病毒浓缩提供的巨大效率节约和灵活性。第一代Bio-Lentivir包装细胞系强调了传统基质在病毒亲和纯化方面的不足，优化病毒包膜上的亲和标签密度以实现有效回收的关键需求，以及新型大孔吸附剂提供简单、单步处理的潜力。研究还表明，生物素标记的慢病毒可以与链亲和素顺磁颗粒络合，从而产生迄今为止最有效的慢病毒载体分离和浓缩方法。然而，大多数临床应用需要游离病毒，因此使用顺磁性纳米颗粒捕获方法不适合没有病毒洗脱，这对于这种高亲和力系统导致低工艺收率。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-tag/Bio-Lentivir包装细胞系、用于病毒纯化的新吸附材料和综合病毒生产方案。这些目标源于金斯大学和剑桥大学团队在成功开发Bio-293T包装细胞系方面的共同经验，并将通过将分子生物学、载体设计和包装细胞系构建（金斯大学）方面的专业知识与生物材料、病毒和亲和处理专业知识（剑桥大学）结合起来来实现。

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 Precedence in protein manufacturing shows that such low specificity processes are now almost entirely superseded by affinity processes and this preference will likely emerge in virus manufacturing in order to exploit the resolution and simplicity of affinity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 overcome 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).