Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential

Abstract

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3′-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Enhanced Suppression of Immune Cells In Vitro by MSC Overexpressing FasL

“…The expression of FasL protein evaluated 48 h after transduction with an MLF4 antibody was detected in a small fraction (~2%) of naïve MSC and was present in 60% ± 17% of cells transduced with Ad-FasL-GFP ( Figure 1 H,I and Figure S3C ). Notably, in MSC expressing FasL protein, less than half of the cells co-expressed the GFP reporter molecule ( Figure 1 H,I), suggesting a possible partial silencing of the second CMV promoter, as previously reported [ 29 ]…”

“…For the expression of FasL, a truncated sequence of the gene was inserted in the adenoviral expression vector under control of the cytomegalovirus (CMV) promoter alongside green fluorescent protein (GFP) as a reporter protein ( Figure 1 A,B). The FasL gene was modified to reduce its size by exclusion of the second intron from the murine FasL gene, as recently described [ 29 ]. The optimization steps showed the effective infection of MSC at 150 transduction units (TU)/cell, which were used for both the control (Ad-GFP) and the FasL gene-carrying adenovirus (Ad-FasL-GFP) ( Figure 1 ).…”

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

“…The adenoviral system AdEasy developed by B. Vogelstein [ 31 ] was used to pack an adenovirus carrying cDNA encoding GFP protein (AdV) as we described before [ 32 ].…”

“…Here, we studied the transduction susceptibility of the MSC isolated from three mouse strains and we found major differences between the transduction yields obtained for each of them. For this, we used the adenoviral system AdEasy developed by B. Vogelstein [ 31 ] to pack an adenovirus carrying cDNA for GFP expression (AdV), as we previously described [ 32 ]. For titration, we employed a method based on flow cytometry that we previously described in detail [ 43 ].…”

“…The adenovirus was obtained and titrated as we previously described in detail [ 32 , 43 ]. Briefly, pAdTrack adenoviral vector containing DNA encoding GFP under CMV promoter (plasmid #16405, Addgene, Watertown, MA, USA) was linearized with PmeI and used to transform AdEasier-1 cells (BJ5183-bacteria containing the pAdEasy1 plasmid), a gift from Bert Vogelstein (#16399, Addgene Watertown, MA, USA).…”