Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: Optimized design and procedures for highly efficient transduction compatible with clinical constraints

Abstract

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.

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination

“…As reported previously, 37 after 48 h of transduction with concentrated SIN Lenti-eGFP vector particles (MOI B10), B90% of the immature DCs expressed eGFP, irrespective of the purity of monocytes used to generate DC cultures. Furthermore, no significant decline in either eGFP expression or viability of eGFP + DCs was documented in lentivirally transduced immature DC cultures for up to 6 days after transduction, irrespective of the presence or absence of lymphocyte contaminants (Figure 3).…”

Immunization with Lentiviral Vector-Transduced Dendritic Cells Induces Strong and Long-Lasting T Cell Responses and Therapeutic Immunity

“…These results from our analysis of mouse DCs resemble those of previous studies focused on human CD34 ϩ -derived DCs (58), indicating that even though lentivirus could be imported into the nucleus through its unique nuclear import protein, cell proliferation still facilitated gene delivery and expression in DCs. It is important to note that others have reported that factors such as the use of 1) higher multiplicity of infection for transduction, 2) increased concentration of vector, 3) cocentrifugation of DCs and viral particles, and 4) repeat transduction can all increase DC transduction efficiency (36,59,60). This suggests that the potency of the transduced DCs used in our studies could be even further enhanced by optimizing DC transduction.…”

Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors

“…[8][9][10][11][12][13] Efficient gene delivery to human DC has been achieved with a variety of viral vectors derived from adenovirus, 8,14 adeno-associated virus, 13 onco-retrovirus, 10,11,15 pox viruses such as vaccinia, 9,16 and canary pox 17,18 and lentivirus. [19][20][21][22] Although adenovirus-and vaccinia virus-derived vectors result in efficient transduction and immune activation, they express viral proteins that evoke a potent antivector immune response, which might dampen the immune response against the tumor antigen in repeated vaccination regimens. Together with the fact that most humans have pre-existing immunity against these viruses, their applicability for DC vaccination is rather limited in their current form.…”