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Adenoviral vector which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system
1Department of Laboratory Medicine, University of California, San Francisco, USA.
Abstract:
Fas ligand (FasL) is a member of the tumor necrosis family and when bound to its receptor, Fas, induces apoptosis. It plays important roles in immune response, degenerative and lymphoproliferative diseases, development and tumorigenesis. It is also involved in generation of immune privilege sites in the eye and testis. Harnessing the power of this molecule is expected to lead to a powerful chemotherapeutic. We describe the construction and characterization of replication-deficient adenoviral vectors that express a fusion of murine FasL and green fluorescent protein (GFP). FasL-GFP retains full activity of wild-type FasL, at the same time allowing for easy visualization and quantification in both living and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control of expression is achieved by creating a novel 'double recombinant' Ad vector, in which the tet-responsive element and the transactivator element are built into the opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently regulated by tetracycline or its derivatives in a dose-dependent manner. The vector was able to deliver FasL-GFP gene to cells in vitro efficiently, and the expression level and function of the fusion protein was modulated by the concentration of doxycycline. This regulation allows us to produce high titers of the vector by inhibiting FasL expression in an apoptosis-resistant cell line. Induction of apoptosis was demonstrated in all cell lines tested. These results indicate that our vector is a potentially valuable tool for FasL-based gene therapy of cancer and for the study of FasL/Fas-mediated apoptosis and immune privilege.
Insights
Researchers developed a novel adenoviral vector expressing FasL-GFP for cancer gene therapy. This system allows precise control over FasL-GFP expression, enabling effective apoptosis induction and study of immune privilege.
Area of Science:
- Molecular Biology
- Gene Therapy
- Immunology
Background:
- Fas ligand (FasL) is a key mediator of apoptosis, crucial in immune regulation and disease.
- FasL's role extends to immune privilege sites, development, and tumorigenesis.
- Targeting FasL offers potential for novel cancer chemotherapeutics.
Purpose of the Study:
- To construct and characterize replication-deficient adenoviral vectors expressing a fusion of murine FasL and green fluorescent protein (FasL-GFP).
- To establish a tetracycline-regulated system for precise control of FasL-GFP expression.
- To evaluate the vector's efficacy in gene delivery, protein expression modulation, and apoptosis induction.
Main Methods:
- Construction of a novel 'double recombinant' adenoviral vector with a tetracycline-regulated gene expression system.
- Fusion of murine FasL with green fluorescent protein (GFP) to allow visualization and quantification.
- In vitro testing of gene delivery efficiency, dose-dependent expression modulation by doxycycline, and apoptosis induction in various cell lines.
Main Results:
- The FasL-GFP fusion protein retained full activity of wild-type FasL.
- Efficient gene delivery and dose-dependent modulation of FasL-GFP expression and function were achieved using doxycycline.
- The vector successfully induced apoptosis in all tested cell lines.
- High vector titers were obtained by inhibiting FasL expression in apoptosis-resistant cells.
Conclusions:
- The developed adenoviral vector is a valuable tool for FasL-based cancer gene therapy.
- The system facilitates the study of FasL/Fas-mediated apoptosis and immune privilege.
- Precise, regulatable control of FasL expression is achievable, enhancing therapeutic potential.