title: Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.
abstract: BACKGROUND
  Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes.
  
  
  METHODS
  We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration.
  
  
  RESULTS
  For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels.
  
  
  CONCLUSIONS
  Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.
authors.affiliation: UPMC/CNRS UMR 7087, Bat CERVI, Hôpital Pitié-Salpêtrière, 83 bd de l'hôpital, 75651 Paris cedex 13, France.
authors.forename: Franck P
authors.initials: FP
authors.lastname: Dupuy
authors.forename: Enguerran
authors.initials: E
authors.lastname: Mouly
authors.forename: Mariana
authors.initials: M
authors.lastname: Mesel-Lemoine
authors.forename: Céline
authors.initials: C
authors.lastname: Morel
authors.forename: Julien
authors.initials: J
authors.lastname: Abriol
authors.forename: Mustapha
authors.initials: M
authors.lastname: Cherai
authors.forename: Claude
authors.initials: C
authors.lastname: Baillou
authors.forename: Didier
authors.initials: D
authors.lastname: Nègre
authors.forename: François-Loïc
authors.initials: FL
authors.lastname: Cosset
authors.forename: David
authors.initials: D
authors.lastname: Klatzmann
authors.forename: François M
authors.initials: FM
authors.lastname: Lemoine
mesh_terms: D018952:Antigens, CD34 / Q000235:genetics; D003713:Dendritic Cells; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D013601:T-Lymphocytes; D018800:Thy-1 Antigens / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection
chemical_list: D018952:Antigens, CD34; D018800:Thy-1 Antigens; D049452:Green Fluorescent Proteins