Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers

Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the c ytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plas mid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<10(5) expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an ef fort to enhance amplicon titers, we introduced in trans a genomic copy of t he virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- a nd BAG-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To furth er enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre -loading of packaging cells with VP16 led to an additional enhancement of a mplicon titers, an effect that did not occur in the absence of vhs. Increas ed helper virus-free amplicon titers resulting from these modifications wil l make in vivo transduction experiments more feasible.