Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.
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.
Dupuy
Franck P
FP
UPMC/CNRS UMR 7087, Bat CERVI, Hôpital Pitié-Salpêtrière, 83 bd de l'hôpital, 75651 Paris cedex 13, France.
Mouly
Enguerran
E
Mesel-Lemoine
Mariana
M
Morel
Céline
C
Abriol
Julien
J
Cherai
Mustapha
M
Baillou
Claude
C
Nègre
Didier
D
Cosset
François-Loïc
FL
Klatzmann
David
D
Lemoine
François M
FM
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
D018952:Antigens, CD34; D018800:Thy-1 Antigens; D049452:Green Fluorescent Proteins