LV vectors provide powerful tools for transgene delivery into dividing as well as nondividing cells and for long-term transgene expression in target cells in vitro
and in vivo
. Currently, the use of LV vectors is commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis have emerged [1
]. LV vectors are also being pursued in a number of clinical trials (see http://www.gemcris.od.nih.gov/
). Despite significant progress, the production and concentration of high-titer, high-quality LV vectors for preclinical studies in animal models is still cumbersome and costly [2
The production of LV vectors is typically carried out using transient transfection approaches involving tissue culture dishes or flasks [3
], cell factories [4
], or stirred-tank bioreactors [7
]. These protocols are cumbersome to scale up (dishes, flasks) or technically challenging (cell factories, bioreactors), preventing their routine use in a standard laboratory setting.
Typical LV vector titers involving the vesicular stomatitis virus (VSV) G glycoprotein range from 106
]. Higher titers can be achieved by physical concentration [2
], including ultracentrifugation [3
], or filtration approaches such as ultrafiltration [3
], and diafiltration [4
]. Vector production for large-scale in vivo
applications in animal models requiring high-titer LV vector stocks is challenging due to the lack of simple procedures allowing rapid processing of large volumes of LV vector-containing cell culture supernatants. The traditional ultracentrifugation-based methods are limited in terms of their capacity to handle large volumes, thus making this procedure extremely tedious. Filtration approaches such as diafiltration are well suited for processing large volumes of vector supernatants. However, they are difficult to implement in a standard laboratory setting. Thus, there is an emerging need for simple and less laborious procedures that result in a rapid reduction of the volume of the cell culture supernatant to be processed without the need for a centrifugation step.
One problem with the centrifugation and filtration-based methods outlined above is that cell-derived components are concentrated along with the vector particles. These have the potential to cause immune and inflammatory responses [15
]. For example, concentrated VSV-G-pseudotyped LV vector preparations were shown to be contaminated with tubovesicular structures of cellular origin which carried nucleic acids, including the plasmid DNAs that were used to generate the LV vector stocks. DNA carried by these tubovesicular structures acted as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production [16
]. Thus, additional steps including chromatography-based methods such as anion exchange chromatography are needed in order to reduce host cell and cell culture-derived contaminants from LV vector preparations. Methods based on anion exchange column chromatography of LV vectors pseudotyped with VSV-G [17
] or the baculovirus GP64 glycoprotein were previously described [19
]. However, the yields and purity of the LV vector stocks obtained in this way were not reported.
In an attempt to simplify the production and concentration of LV vectors and to make this approach more reproducible and cost-effective for preclinical studies in animals, we have worked out a facile LV vector production system based on HYPERFlasks. We also implemented a straightforward concentration procedure based on Mustang Q anion exchange membrane chromatography. Mustang Q anion exchange-based chromatography protocols for concentrating/purifying LV vectors were previously reported [4
]. Such vector preparations displayed reduced toxicity compared to vectors concentrated using ultracentrifugation [20
], as well as enhanced purity [4