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Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential
Madalina Dumitrescu1, Violeta Georgeta Trusca1, Lorand Savu2
1Gene Regulation and Molecular Therapies Laboratory, Institute of Cellular Biology and Pathology "N. Simionescu", 8, B.P. Hasdeu Street, 050568 Bucharest, Romania.
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.
Insights
A novel adenovirus vector carrying a truncated FasL minigene (Ad-gFasL) efficiently induces FasL expression for apoptosis. This method shows faster mRNA and protein expression compared to traditional FasL cDNA vectors (Ad-cFasL).
Area of Science:
- Molecular Biology
- Immunology
- Gene Therapy
Background:
- Fas ligand (FasL) is a key mediator of apoptosis in the TNF superfamily.
- Adenovirus vectors offer strong, transient gene induction for therapeutic applications.
Purpose of the Study:
- To develop a more efficient adenovirus vector for inducing functional FasL expression.
- To compare the efficacy of a truncated FasL minigene (Ad-gFasL) with a FasL cDNA (Ad-cFasL) vector.
Main Methods:
- Construction of an adenovirus vector (Ad-gFasL) with a truncated FasL minigene, optimized for adenoviral genome size limitations.
- Transduction of hepatocytes and endothelial cells with Ad-gFasL and Ad-cFasL.
- Analysis of FasL mRNA and protein expression levels and kinetics.
- Co-culture experiments to assess the apoptotic effect of FasL-expressing cells.
Main Results:
- Ad-gFasL demonstrated significantly faster mRNA expression (10h vs 16h) and higher protein expression levels compared to Ad-cFasL.
- Detectable protein expression was achieved with Ad-gFasL at lower transfection units (0.5 TU/cell), while Ad-cFasL showed no detectable protein.
- FasL-expressing cells induced apoptosis in co-cultured A20 cells.
Conclusions:
- The Ad-gFasL vector is a superior tool for efficient and rapid induction of functional FasL expression.
- FasL-expressing cells hold potential for treating autoimmune diseases like graft-versus-host disease, chronic colitis, and type I diabetes.
