Use of Lentiviral Particles As a Cell Membrane-Based mFasL Delivery System for In Vivo Treatment of Inflammatory Arthritis

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.

Keywords: FasL; apoptosis; caspases; lentiviral particles; rheumatoid arthritis.

Figure 1

FasL expression at the surface of lentiviral particles (LVP). (A) HEK293T cells transfected with GPI-linked FasL or control plasmid were analyzed by fluorescence microscopy and Western blot and (B) by flow cytometry using specific anti-mFasL antibodies. GFP expression was analyzed as a control of cell transfection. One experiment is shown of five performed. The gray bell-shaped curve indicates detection in untransfected HEK293T cells. (C) Flow cytometry analysis of control and mFasL expression in LVP coupled to latex beads. A representative experiment is shown of five performed. (D) Western blot analysis of mFasL- and control LVP using specific anti-GFP and -mFasL antibodies. A representative experiment is shown of more than five performed.

Figure 2

FasL-LVP mediated induction of cell death. (A) Flow cytometry analysis of Fas (CD95) expression in BaF/3; L1.2 cells and murine thymocytes. An experiment is shown of three performed. (B) Cell cycle analysis using propidium iodide (PI) incorporation and flow cytometry analysis of BaF/3 cells treated with crosslinked soluble FasL (sFasL), mFasL, and control lentiviral particles (LVP) for 6 h. Gates defining the different cell cycle phases are indicated. The figure shows one experiment of more than five performed. The percentages of cells in each cell cycle phase as well as those for cell apoptosis are shown. (C) Cell cycle analysis using PI incorporation and flow cytometry analysis of murine thymocytes, untreated or pretreated with Z-VAD and untreated or treated with crosslinked sFasL, mFasL, or control LVP for 6 h. The figure shows the percentage of apoptotic cells; data are shown as mean ± SD of six independent experiments (***p < 0.0001). (D) Apoptosis of murine thymocytes, BaF/3, and L1.2 cells treated with FasL- or control LVP (4 h). The number of early apoptotic (AV+/PI−) and necrotic/late apoptotic (AV+/PI+) cells was expressed as a percentage of total cells. Results of three independent experiments are shown as mean ± SD (***p < 0.0001).

Figure 3

Induction of caspase processing and cytochrome C release by FasL-LVP. (A) Western blot analysis of caspase 3 processing in murine thymocytes, alone or pretreated with Z-VAD and untreated or treated with crosslinked soluble FasL (sasL), FasL- and control lentiviral particles (LVP) for 6 h, using specific antibodies. An experiment is shown of three performed. (B). Western blot of caspase 9 processing in murine thymocytes as in panel (A). An experiment is shown of three performed. (C) Immunofluorescence analysis of cytochrome c release using specific antibodies and murine thymocytes treated with FasL- or control LVP for 6 h. One experiment is shown of five performed.

Figure 4

Effect of local administration of FasL-LVP in a murine model of arthritis. (A) Evolution of the paw swelling score after treatment with FasL- or control LVP (Cs_t), expressed as a percentage of the initial score (Cs₀) (n = 12; **p < 0.001, *p < 0.05). (B) Levels of total anti-collagen II IgG (micrograms per milliliter) were measured in mouse sera by ELISA at the end of the experiment (day 14 posttreatment). (C). Eosin–thiazin staining of representative sections of ankle joints of the mice in panel (A) at day 14 posttreatment with lentiviral particles (LVP) (left) or FasL-LVP (right). The figure shows decreased cell infiltration (a), bone erosion (b), and cartilage destruction (c) in FasL-LVP-treated mice. Original magnification 10×. (D) Levels of total anti-LVP IgG were measured in mouse sera by ELISA on days 0 and 14 after LVP and FasL-LVP treatments.