Regulation of Transgene Expression Using an Inducible System for Improved Safety of Intervertebral Disc Gene Therapy

Abstract

Human nucleus pulposus cells (NPCs) were transduced with an adenoviral vector that expresses Fas Ligand (FasL) and green fluorescent protein (GFP) under the control of a tetracycline-regulated gene expression system to test the transgene control. To describe the application of a Tet-off gene regulation system for intervertebral disc (IVD) gene therapy. Gene therapy has proven its ability to beneficially modulate the biologic processes of the IVD cells in vitro and in vivo. However, we have observed that expression of transgenic growth factors outside the IVD in the event of a misdirected injection has potentially detrimental consequences (e.g., toxicity). To date, a safety system that allows the control transgene expression has not been produced for intradiscal gene therapy. Human NPCs were transduced with Ad/FasL-GFPTET, at 0, 50, 100, and 200 MOI. After 1 day (time 0) cells were cultured in the presence of tetracycline (1, 10, 100 mg/L) for 3 days, and then tetracycline was withdrawn. The transgene expression was evaluated either daily by flow cytometry (from time 0 to day 6) or by imaging the GFP signal (time 0, day 3 and day 9). NPC expression of GFP 1 day after transduction was proportional to the MOI used. GFP expression was decreased after 3 days of tetracycline administration at all concentrations used. The expression of GFP recovered after removal of tetracycline. The transgene expressed by the transduced NPC was efficiently regulated by inclusion of tetracycline in culture media. The presence of tetracycline turns off the protein expression and the subsequent absence allows it to recover again, demonstrating the ability to control gene expression in NPCs. Therefore, we propose a Tet-off inducible system as an efficient tool for modulating the transgene expression to avoid the toxicity that could result from a missed injection.