BRIC: Packaging cell lines for inherently manufacturable viral vectors

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.

It addresses Improved Downstream Processing for nanoscale vectors, transferring in-built manufacturability from laboratory to GMP practice. It provides virus manufacturers with tools to engineer viral envelopes in a way that uncertainties in downstream processing are avoided whilst overcoming problems of low titre that have hampered the field. These tools will bring increased predictability for bioprocessing, including improved scale-up and reproducibility and so enable increased speed to clinic and market.