Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines.

Lentiviral vectors (LV) represent a key tool for gene and cell therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field toward developing suspension processes that are conducive to large-scale production. This study describes a LV production strategy using a stable inducible producer cell line. The HEK293 cell line employed grows in suspension, thus offering direct scalability, and produces a green fluorescent protein (GFP)-expressing lentiviral vector in the 106 transduction units (TU)/mL range without optimization. The stable producer cell line, called clone 92, was derived by stable transfection from a packaging cell line with a plasmid encoding the transgene GFP. The packaging cell line expresses all the other necessary components to produce LV upon induction with cumate and doxycycline. First, the study demonstrated that LV production using clone 92 is scalable from 20 mL shake flasks to 3 L bioreactors. Next, two strategies were developed for high-yield LV production in perfusion mode using acoustic cell filter technology in 1-3 L bioreactors. The first approach uses a basal commercial medium and perfusion mode both pre- and post-induction for increasing cell density and LV recovery. The second approach makes use of a fortified medium formulation to achieve target cell density for induction in batch mode, followed by perfusion mode after induction. Using these perfusion-based strategies, the titer was improved to 3.2 × 107 TU/mL. As a result, cumulative functional LV titers were increased by up to 15-fold compared to batch mode, reaching a cumulative total yield of 8 × 1010 TU/L of bioreactor culture. This approach is easily amenable to large-scale production and commercial manufacturing.

Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells.

The adenovirus major late promoter (MLP) and its translational regulator - the tripartite leader (TPL) sequence - can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie) promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO) cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds) than translation (11 folds). Even with adenovirus infection to provide additional elements (in trans), CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.