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

• Published in: International journal of molecular sciences 2020-08, Vol.21 (17), p.6011
• Main Authors: Dumitrescu, Madalina, Trusca, Violeta Georgeta, Savu, Lorand, Stancu, Ioana Georgeta, Ratiu, Attila Cristian, Simionescu, Maya, Gafencu, Anca Violeta
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; Adenoviridae - genetics; adenovirus; Animals; Apoptosis; Cattle; cDNA; Coculture Techniques; Endothelial Cells - cytology; Endothelial Cells - physiology; Fas Ligand Protein - genetics; Fas Ligand Protein - metabolism; FasL; Gene Transfer Techniques; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Hepatocytes - physiology; Humans; Introns; Mice; Mice, Inbred BALB C; Mice, Transgenic; minigene; Promoter Regions, Genetic; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Transgenes
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21176011

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 gene, named 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. 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.