Control of Therapeutic Gene Expression
Control of Therapeutic Gene Expression
Delivering functional genes into cells to replace mutated genes, an approach known as gene therapy, holds the potential for treating many types of diseases. Effective gene-based therapies require efficient delivery of therapeutic genes to targeted mammalian cells as well as regulatable gene expression. To date, various viral vector-based gene expression systems and improved gene regulatory systems have been developed to facilitate and control tightly therapeutic gene expression. These transcriptional regulatory systems have been encoded within several viral vectors to control gene expression and further improve the kinetics of gene regulation. Among the existing inducible transcriptional gene regulatory systems, the Tetracycline-based (Tet-based) regulatable system is the most widely used tool for controlling mammalian gene expression due to its various advantages.
Tet-based Regulatable System
The Tet-based regulatable system has been successfully used to control the expression of numerous genes in cells and organisms. There are two variants: the Tet-Off system is the first developed system, and the second is the Tet-On system, which has become increasingly popular. The Tet-Off system is negatively controlled and relies on tetracycline to inactivate expression, whereas the Tet-On system relies on tetracycline to activate gene expression. The Tet-Off system works by switching gene expression on and off in the absence or presence of the inducer, respectively.
In the off situation, that is, in the presence of tetracycline derivative antibiotic, tetracycline inducers such as doxycycline (Dox) bind to the transactivator (tTA), preventing it from binding to the tetO elements within the tetracycline-response element (TRE), thereby blocking promoter activation and subsequent gene expression. In the other situation, i.e., upon removal or absence of inducer, tTA binds to tetO sequences within the TRE, thus activating TRE and inducing gene expression (Fig.1 A). In the Tet-On regulatory switch, the absence of Dox results in the inability of tTA to bind to the tetO sequences inside the TRE, subsequently blocking promoter activation and subsequent gene expression. The presence of Dox causes it to bind to tTA and then the tTA/Dox complex binds to the tetO elements, allowing promoter activation and gene expression (Fig.1 B).
Figure 1. Tetracycline-based regulatable system. (A) Tet-Off regulatable switch. (B) Tet-On regulatable switch. (Goverdhana, 2005)
Advantages of Tet-based Regulatable System
Based on the characteristics of tTA-tetO binding and the properties of the tetracycline family of drugs, the Tet-based regulatable system offers many advantages that make it particularly attractive for controlling gene expression in vitro and in vivo.
- First, it is non-toxic to mammalian cells and exhibits no pleiotropic consequences for other cellular metabolic pathways. Secondly, the pharmacokinetics, pharmacodynamics and side effects of tetracycline and its derivatives are well known.
- Furthermore, the system can be switched off and then switched on within 72 hours when using oxytetracycline.
- Finally, it allows for exclusive control of two gene activities within the same cells at different concentrations of Dox.
Tet-based Regulatable System with Viral Vector
The tet-based regulatable system can be encoded within the lentivirus vectors (LV), adeno-associated viruses (AAV) vectors, adenovirus vectors (AdV), and herpes simplex virus (HSV) vectors for gene expression control.
- Control gene expression from AdV
Currently, gene regulation in vivo has been successfully achieved using AdV and Tet-based adjustable systems for specific cell types. For example, Smith-Arica and co-workers have established effective Dox dependent transcriptional regulation and cell-type-specific transcriptional targeting, producing successful regulatable transgenesis in cell lines, primary brain cultures, and localized regions within the rat brain. Besides, the researchers combined the Tet-On transactivator and repressor system with the high-capacity AdV to study the dynamic regulation of gene transfer and expression in vivo, and found that the maximum induction was generated in the on state and the minimum gene expression leakage was in the off state.
- Control gene expression from LV
LV has been shown to transduce and drive gene expression in many mammalian cells and animal models. At present, the Tet-On- and Tet-Off-based regulatable systems have been used to engineer lentiviruses and successfully regulated gene expression in 293 cells, the central nervous system (CNS) cells, and a rat model for Huntington disease.
- Control gene expression from AAV vectors
One of the first successful applications to achieve the regulation of gene expression within AAV was the effective regulation of green fluorescent protein (GFP) based on Tet-On. Two rAAV vectors, one engineered with a GFP-encoding cassette and the other with the silencer flanked tTA Tet-On transactivator, were constructed. Sub-retinal injection of these engineered rAAV particles into rats produced tightly regulated GFP expression.
- Control gene expression from HSV vectors
HSV vectors have been used in CNS gene therapy due to their ability to persist in a latent state in neurons. A number of studies generated successful and efficient regulatable genes from HSV-1-derived amplicon vectors engineered with the Tet-Off regulatable system. For example, researchers reported that HSV vectors based on the Tet-Off system effectively regulated gene expression in mouse brains.
Reference
- Goverdhana, S.; et al. (2005). Regulatable gene expression systems for gene therapy applications: progress and future challenges. Molecular Therapy. 12(2):189-211.
