title: Flow cytometric assessment of transduction efficiency and cytotoxicity of herpes simplex virus type 1-based amplicon vectors.
abstract: BACKGROUND
  In this study, we compared herpes simplex virus type 1 (HSV-1) amplicon vector stocks prepared by transient cotransfection with two different BAC-cloned packaging-defective HSV-1 helper genomes, fHSVDeltapacDelta27 and fHSVDeltapac, with respect to transduction efficiency and cytotoxicity. Both fHSVDeltapacDelta27 and fHSVDeltapac are packaging defective because the pac signals have been deleted; fHSVDeltapacDelta27 contains 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-early (IE) 4/5 promotor was packaged into virus particles by transient cotransfection with either fHSVDeltapacDelta27 or fHSVDeltapac DNA. Cultures 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[fHSVDeltapacDelta27] and pHSVGFP[fHSVDeltapac], efficiently transduced the target cells. Interestingly, the highest mean fluorescence intensities were measured at 1 day after infection, whereas the number of GFP-fluorescent cells reached a peak at day 3 after 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 vector stock. Between days 3 and 4 after infection, the number of dead cells increased dramatically in all the cultures, transduced and nontransduced. Only the cultures infected with a high dose of pHSVGFP[fHSVDeltapac] displayed a significant further increase in the number of dead cells between days 4 and 5 postinfection.
  
  
  CONCLUSIONS
  Flow cytometry allowed comparison of transduction efficiency and cytotoxicity mediated by the two different amplicon vector stocks. Cultures infected with pHSVGFP[fHSVDeltapacDelta27] were more viable than those infected with pHSVGFP[fHSVDeltapac](P < 0.05). The practical implications of this study are at the level of vector design. Flow cytometry has proven a fast and reliable approach to assess the quality of potential gene transfer vectors prior to their use in (pre) clinical trials.
authors.affiliation: Institute of Virology, University of Zurich, Zurich, Switzerland. nunerzr@mskcc.org
authors.forename: R
authors.initials: R
authors.lastname: Nunez
authors.forename: M
authors.initials: M
authors.lastname: Ackermann
authors.forename: Y
authors.initials: Y
authors.lastname: Saeki
authors.forename: A
authors.initials: A
authors.lastname: Chiocca
authors.forename: C
authors.initials: C
authors.lastname: Fraefel
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D014161:Transduction, Genetic / Q000379:methods*; D014709:Vero Cells