Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.

Cancer remains one of the leading causes of death worldwide. Conventional therapeutic strategies usually offer limited specificity, resulting in severe side effects and toxicity to normal tissues. Targeted cancer therapy, on the other hand, can improve the therapeutic potential of anti-cancer agents and decrease unwanted side effects. Targeted applications of cytolethal bacterial toxins have been found to be especially useful for the specific eradication of cancer cells. Targeting is either mediated by peptides or by protein-targeting moieties, such as antibodies, antibody fragments, cell-penetrating peptides (CPPs), growth factors, or cytokines. Together with a toxin domain, these molecules are more commonly referred to as immunotoxins. Targeting can also be achieved through gene delivery and cell-specific expression of a toxin. Of the available cytolethal toxins, diphtheria toxin (DT) is one of the most frequently used for these strategies. Of the many DT-based therapeutic strategies investigated to date, two immunotoxins, OntakTM and TagraxofuspTM, have gained FDA approval for clinical application. Despite some success with immunotoxins, suicide-gene therapy strategies, whereby controlled tumor-specific expression of DT is used for the eradication of malignant cells, are gaining prominence. The first part of this review focuses on DT-based immunotoxins, and it then discusses recent developments in tumor-specific expression of DT.