BRIC: Packaging cell lines for inherently manufacturable viral vectors

金砖四国：用于固有可制造病毒载体的包装细胞系

• 批准号: BB/E005853/1
• 负责人: Nigel Slater
• 金额: $ 29.39万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE005853%2F1
• 关键词: BRIC; Packaging; cell; lines; inherently

Viruses dominate overwhelmingly the types of vectors currently being tested in clinical gene therapy trials and of these retro- and lentiviruses are the most numerous. Until recently two technological problems have hampered progress in gene therapy; production of high titre clinical grade virus and efficient tissue specific targeting. Research at Cambridge and King's College London has addressed the former and led to the development of a novel lentiviral vector packaging cell line in which manufacturability is built into the genome of the packaging cell and co-expressed on the surface of the viruses produced thereafter. We initially used simple retroviral vectors, and latterly the more complex lentiviral vectors based on a core of HIV-1, and have developed strategies for increasing the titre by several orders of magnitude. This is an active area of research amongst which our preliminary work with novel chromatographic techniques and paramagnetic particles set the foundation for a practical and efficient alternative technique to cumbersome ultracentrifugal concentration. For lentiviral vectors we engineered a new producer cell type that provides a biotin tag amenable to various lentiviral vectors produced from these cells using either VSV-G or MLV amphotropic envelopes. We have shown that these bio-lentiviral vectors are produced in the normal manner and only require the presence of biotin in the culture medium to manifest their affinity for streptavidin. Vectors can thus be retained on streptavidin Paramagnetic Magnespheres for infection, or eluted from streptavidin adsorbents. This cell line allows the capture of multiple envelope pseudotypes of lentiviral or MLV derived vectors, enabling production and concentration to titres that are several orders of magnitude higher. Using this scalable protocol we have concentrated lentivirus in excess of 4500-fold in only 3 h and have provided titers for both VSV-G and MLV amphotropic envelope pseudotypes of 1010 IU/ml. However, these viruses could not be easily eluted from adsorbents and required the addition of biotin to the growth medium of the packaging cells. This proposal aims to express the alternative desthiobiotin ligand on the surface of lentiviruses in such a way that elution from adsorbents may be more readily preformed to give higher process yields and the addition of an affinity ligand binding precursor to growth medium is avoided.

目前在临床基因治疗试验中测试的载体类型中，病毒占据压倒性优势，而在这些逆转录病毒和慢病毒中，病毒数量最多。直到最近，两个技术问题阻碍了基因治疗的进展：生产高滴度临床级别病毒和有效的组织特异性靶向。剑桥大学和伦敦国王学院的研究解决了前者，并导致了一种新型慢病毒载体包装细胞系的开发，在该包装细胞系中，可制造性被构建在包装细胞的基因组中，并在此后产生的病毒的表面共表达。我们最初使用简单的逆转录病毒载体，后来使用基于HIV-1核心的更复杂的慢病毒载体，并开发了将滴度提高几个数量级的策略。这是一个活跃的研究领域，其中我们在新的层析技术和顺磁性粒子方面的初步工作为替代繁琐的超速离心浓缩的实用和有效的替代技术奠定了基础。对于慢病毒载体，我们设计了一种新的生产者细胞类型，它提供了一种生物素标签，可以通过使用VSV-G或MLV两性包膜从这些细胞产生各种慢病毒载体。我们已经证明了这些生物慢病毒载体是以正常方式产生的，并且只需要在培养基中存在生物素就可以显示它们对链霉亲和素的亲和力。因此，载体可以保留在链霉亲和素顺磁磁球上以备感染，或从链霉亲和素吸附剂中洗脱。该细胞系允许捕获慢病毒或MLV衍生载体的多种包膜假型，使生产和浓缩滴度能够高出几个数量级。使用这种可扩展的方法，我们仅在3h内就浓缩了4500倍以上的慢病毒，并提供了1010IU/ml的VSV-G和MLV两性包膜假型的滴度。然而，这些病毒不容易从吸附剂中洗脱，需要在包装细胞的生长介质中添加生物素。这一建议的目的是在慢病毒表面表达另一种脱硫生物素配体，这样可以更容易地从吸附剂中洗脱，从而获得更高的过程产率，并且避免在生长介质中添加亲和配体结合前体。

项目成果

Magnetic capture of superparamagnetic nanoparticles in a constant pressure microcapillary flow

• DOI: 10.1016/j.jmmm.2009.02.088
• 发表时间: 2009-05
• 期刊: Journal of Magnetism and Magnetic Materials
• 影响因子: 2.7
• 作者: N. Darton;B. Hallmark;T. A. James;Pulkit Agrawal;M. Mackley;N. Slater

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling.

通过二氨基壬酸介导的脱硫生物素标记恢复慢病毒的亲和力。

• DOI: 10.1016/j.jchromb.2010.05.019
• 发表时间: 2010
• 期刊: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
• 作者: Chen R