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

Madalina Dumitrescu,Lorand Savu,Ioana Georgeta Stancu,Violeta Georgeta Trusca,Attila Cristian Ratiu,Anca Violeta Gafencu,Maya Simionescu

doi:10.3390/ijms21176011

Madalina Dumitrescu, Lorand Savu + Show 5 more

Open Access

https://doi.org/10.3390/ijms21176011

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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.

Highlights

Fas ligand (FasL, CD95L) is a type-II transmembrane protein of approximately 40 kDa acting as an executioner of apoptosis within the tumor necrosis factor (TNF) superfamily
A series of studies showed that T regulatory cells decorated with FasL on their surface are effective immunomodulators that alleviate graft-versus-host disease, arrest progression of autoimmune insulitis in non-obese diabetic (NOD) mice, and ameliorate chronic colitis [7,12,13,14,15]
The sequence showed that the coding part of the minigene as well as cDNA represents a perfect match with the sequence deposited in the GenBank (No U58995.1) for BALB/c mice [21]

Summary

Introduction

Fas ligand (FasL, CD95L) is a type-II transmembrane protein of approximately 40 kDa acting as an executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. Numerous studies have attempted to harness this physiological mechanism of immune regulation for therapeutic purposes, with an unequivocal demonstration of the efficacy of FasL to selectively eliminate activated immune cells, primarily in autoimmune diseases. The FasL protein has been used to restrain inflammatory insulitis in models of autoimmune diabetes [7] and to induce immune privilege in grafted tissues and organs, such as pancreatic islet grafts [8,9]. Effective in suppression of immune activity, causing immune privilege, short-term therapeutic activity of proteins encoding FasL is not always sufficient in the induction of transplant tolerance and abrogation of autoimmune disorders, which are frequently broken by incidental infections and relapse of autoimmunity [18]

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