Flow cytometric assessment of transduction efficiency and cytotoxicity of herpes simplex virus type 1-based amplicon vectors

Background: In this study, we compared herpes simplex virus type 1 (HSV-1) amplicon vector stocks prepared by transient cotransfection with two differ ent BAG-cloned packaging-defective HSV-1 helper genomes, fHSV Delta pac Del ta 27 and fHSV Delta pac, with respect to transduction efficiency and cytot oxicity. Both fHSV Delta pac Delta 27 and fHSV Delta pac are packaging defe ctive because the pac signals have been deleted; fHSV Delta pac Delta 27 co ntains an additional deletion in the HSV-1 ICP27 gene, which increases the safety of the system. Methods: HSV-1 amplicon pHSVGFP under the control of the HSV-1 immediate-ea rly (IE) 4/5 promotor was packaged into virus pat-tides by transient cotran sfection with either fHSV Delta pac Delta 27 or fHSV Delta pac DNA. Culture s were infected with the two different vector stocks and examined under the fluorescence microscope and analyzed by flow cytometry over a 5-day period to assess transduction efficiency and cytotoxicity. Results: Both vector stocks, pHSVGFP{fHSV Delta pac Delta 27} and pHSVGFP{f HSV Delta pac}, efficiently transduced the target cells. Interestingly, the highest mean fluorescence intensities were measured at 1 day after infecti on, whereas the number of GFP-fluorcscent cells reached a peak at day 3 aft er infection. At day 3 after infection, a slight increase in the number of dead cells was observed in those cultures transduced with high doses of vec tor stock. Between days 3 and 4 after infection, the number of dead cells i ncreased dramatically in all the cultures, transduced and nontransduced. On ly the cultures infected with a high dose of pHSVGFP{fHSV Delta pac} displa yed a significant further increase in the number of dead cells between days 4 and 5 postinfection. Conclusions: Flow cytometry allowed comparison of transduction efficiency a nd cytotoxicity mediated by the two different amplicon vector stocks. Cultu res infected with pHSVGFP{fHSV Delta pac Delta 27} were more viable than th ose infected with pHSVGFP{fHSV Delta pac}(P < 0.05). The practical implicat ions of this study are at the level of vector design. Flow cytometry has pr oven a fast and reliable approach to assess the quality of potential gene t ransfer vectors prior to their use in (pre) clinical trials. Cytometry 44:9 3-99, 2001. (C) 2001 Wiley-Liss, Inc.