Impact of retroviral vector components stoichiometry on packaging cell lines: Effects on productivity and vector quality

The productivity of retroviral vector producer cell lines is currently low; this can be attributed partly to disruption of the viral components stoichiometry originated by the dissociation of gag-pol, env, and viral genome. This paper addresses the impact of viral components stoichiometry on cell productivity and vector quality (vector stability and transduction efficiency). A strategy is proposed for the development of packaging cell lines allowing for stoichiometric optimization of viral components. This strategy is based on the introduction of a transgene in the first step of cell line development, thus making it possible to eliminate transgene limitations and therefore exploring maximal infectious productivity based on the levels of expression of Gag-Pol and envelope. The transgene can subsequently be exchanged for a gene of interest, using site-specific flipase/flipase recombination target (Flp/FRT) cassette replacement. It was observed that Gag-Pol controlled the order of magnitude of infectious titer: a small, 2-fold variation in its expression could result in a 10- to 100-fold improvement in infectious titer; the RNA transgene and envelope expression limitations had a lower impact. The ratios of retroviral components strongly affected cell productivity but did not affect vector stability; however, transduction efficiencies were affected by an imbalanced ratio of the components, resulting in the production of higher amounts of defective virus. Thus, stoichiometric optimization of viral components not only improves cell productivity but also increases vector quality.