Abstract
During budding, lentiviral particles (LVP) incorporate cell membrane proteins in the viral envelope. We explored the possibility of harnessing this process to generate LVP-expressing membrane proteins of therapeutic interest and studied the potential of these tools to treat different pathologies. Fas-mediated apoptosis is central to the maintenance of T cell homeostasis and prevention of autoimmune processes. We prepared LVP that express murine FasL on their surface. Our data indicate that mFasL-bearing LVP induce caspase 3 and 9 processing, cytochrome C release, and significantly more cell death than control LVP in vitro. This cytotoxicity is blocked by the caspase inhibitor Z-VAD. Analysis of the application of these reagents for the treatment of inflammatory arthritis in vivo suggests that FasL-expressing LVP could be useful for therapy in autoimmune diseases such as rheumatoid arthritis, where there is an excess of Fas-expressing activated T cells in the joint. LVP could be a vehicle not only for mFasL but also for other membrane-bound proteins that maintain their native conformation and might mediate biological activities.
Highlights
Lentiviral vectors are very widely used in biological research, functional genomics, and gene therapy since they can mediate gene transfer into dividing and non-dividing cells both ex vivo and in vivo
Transfection efficiency was controlled by assessing GFP expression via fluorescence microscopy, Western blot and flow cytometry, whereas flow cytometry and Western blot analysis using anti-FasL mAb confirmed specific expression of FasL in cells transfected with FasLGPI-LVTHM/GFP (Figures 1A,B)
Flow cytometry analysis of lentiviral particles (LVP), isolated by centrifugation through a sucrose pellet, using specific mAb showed that FasL could be detected on the surface of LVP obtained from cells transfected with FasL-GPI (FasL-LVP) (Figure 1C, lower panel), but not on LVP obtained from HEK293T cells transfected with a control vector (LVP) (Figure 1C, upper panel)
Summary
Lentiviral vectors are very widely used in biological research, functional genomics, and gene therapy since they can mediate gene transfer into dividing and non-dividing cells both ex vivo and in vivo. Several examples of their use to correct genetic defects in have been described in both human disease [1,2,3] and animal models [4,5,6,7]. We addressed the hypothesis that the LVP-Expressing FasL Reduce Joint Inflammation controlled incorporation of specific membrane glycoproteins into LVP could be utilized to generate nanoparticles able to modulate immune system function
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