Vadalà Gianluca, Sowa Gwendolyn A, Smith Lauren, Hubert Mark G, Levicoff Eric A, Denaro Vincenzo, Gilbertson Lars G, Kang James D
Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Spine (Phila Pa 1976). 2007 Jun 1;32(13):1381-7. doi: 10.1097/BRS.0b013e3180601215.
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(time 0) cells were cultured in the presence of tetracycline (1, 10, 100 mg/L) for 3 days, and then tetracycline was withdrawn.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.
用人核髓核细胞（NPCs）转导一种腺病毒载体，该载体在四环素调控基因表达系统的控制下表达Fas配体（FasL）和绿色荧光蛋白（GFP），以测试转基因控制。
描述Tet-off基因调控系统在椎间盘（IVD）基因治疗中的应用。
基因治疗已证明其在体外和体内有益调节IVD细胞生物学过程的能力。然而，我们观察到，如果注射方向错误，IVD外转基因生长因子的表达可能会产生潜在的有害后果（如毒性）。迄今为止，尚未为椎间盘内基因治疗生产出一种能够控制转基因表达的安全系统。
用Ad/FasL-GFPTET以0、50、100和200的感染复数转导人NPCs。1天后（时间0），将细胞在四环素（1、10、100mg/L）存在的情况下培养3天，然后撤去四环素。通过流式细胞术每天（从时间0到第6天）或通过对GFP信号成像（时间0、第3天和第9天）评估转基因表达。
转导后1天NPCs中GFP的表达与所用的感染复数成正比。在使用的所有浓度下，四环素给药3天后GFP表达降低。去除四环素后GFP表达恢复。
转导的NPCs所表达的转基因通过在培养基中加入四环素得到有效调控。四环素的存在会关闭蛋白质表达，随后四环素的缺失又使其恢复，证明了在NPCs中控制基因表达的能力。因此，我们提出Tet-off诱导系统作为一种有效工具来调节转基因表达，以避免因注射失误可能导致的毒性。
