One approach to improve transduction efficiency is to increase the concentration of infectious virus particles. The concentration of virus particles in virus stocks can be increased by optimizing packaging cell culture conditions, concentrating the virus stocks after they are harvested from the packaging cells, and/or by altering the virus or packaging cell lines.
Significant increases in virus titer have been achieved by optimizing cell culture conditions, such as by reducing the ratio of culture medium to cell number. Packaging cells seeded in the extra capillary space of a hollow fiber bioreactor, and grown to densities (108 cells per milliliter) about 100-fold higher than normally achieved in conventional cell culture flasks, produced virus stocks with titers (2 x 107 particles per milliliter) 18-fold higher than the titers of virus stocks generated in T-flasks.
The cell culture incubation temperature can also be optimized to increase virus concentrations. Kotani et al. increased the concentration of virus particles nearly tenfold by lowering the incubation temperature of packaging cells from 37oC to 32oC. Another possibility is to increase the concentration of virus particles by physically concentrating the virus stocks, although doing so without losing infectivity has proven difficult. Standard techniques such as centrifugation and ultra filtration have failed.
More recently, tangential flow and hollow fiber filtration have been shown to increase virus concentrations more than 30-fold with minimal losses in viral infectivity. Synthetic approaches have also been used to increase virus concentrations, such as the production of chimerical virus particles (particles composed of structural proteins derived from two or more viruses) that are easy to concentrate without loss of infectivity, and the construction of packaging cell lines designed to optimize viral protein expression.
Using the latter approach, Cosset et al. transected human HT-1080 cells with gag-pol and env expression plasmids, each encoding a different selectable marker. The selectable markers were expressed by reinitiating of translation of the mRNA encoding the viral proteins, which ensured that only cells expressing all the viral proteins would survive incubation in selective culture medium.
Titers as high as 3 x 107 infectious particles per milliliter were achieved, much higher than titers generated by previous packaging cell lines (105 to 106 infectious particles per milliliter)