BRIC: Packaging cell lines for inherently manufacturable viral vectors

目前在临床基因治疗试验中测试的载体类型绝大多数是病毒，而这些复古病毒和慢病毒数量最多。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已经解决了前者的问题，并导致了一种新型慢病毒载体包装细胞系的发展，在这种细胞系中，可制造性被构建到包装细胞的基因组中，并在随后产生的病毒表面共同表达。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，我们对新型色谱技术和顺磁颗粒的初步工作为一种实用而有效的替代繁琐的超离心浓缩技术奠定了基础。对于慢病毒载体，我们设计了一种新的生产细胞类型，该细胞使用VSV-G或MLV两性亲和性包膜，提供适合于各种慢病毒载体的生物素标签。我们已经证明，这些生物慢病毒载体以正常的方式产生，只需要在培养基中存在生物素就可以表现出它们对链霉亲和素的亲和力。因此，载体可保留在链霉亲和素顺磁镁球上以供感染，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伪类型，使生产和浓度提高几个数量级。使用这种可扩展的方案，我们在3小时内浓缩了超过4500倍的慢病毒，并提供了VSV-G和MLV两性包膜假型的滴度，均为1010 IU/ml。然而，这些病毒不容易从吸附剂中洗脱出来，需要在包装细胞的生长培养基中添加生物素。本研究的目的是在慢病毒表面表达替代的去硫代生物素配体，这样可以更容易地从吸附剂中洗脱，从而获得更高的工艺产率，并且避免了在生长培养基中添加亲和配体结合前体。

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.