Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints.
Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector-mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75-85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14(+) precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.
Laboratory of Experimental Hematology, Bordet Institute, Brussels, Belgium.
Redouane
R
Rouas
Rathviro
R
Uch
Yvette
Y
Cleuter
François
F
Jordier
Claude
C
Bagnis
Patrice
P
Mannoni
Philippe
P
Lewalle
Philippe
P
Martiat
Anne
A
Van den Broeke
D017931:DNA Primers / Q000737:chemistry; D003713:Dendritic Cells / Q000378:metabolism* / Q000821:virology; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D018189:Immunomagnetic Separation; D016130:Immunophenotyping; D018664:Interleukin-12 / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D019076:Transgenes
D017931:DNA Primers; D018950:Lipopolysaccharide Receptors; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D018664:Interleukin-12