Abstract
Regulation of transgene expression in target cells represents a critical and challenging aspect of gene therapy. Recently, a two-plasmid tetracycline-inducible system was developed in which the tetracycline repressor (tetR) alone, rather than the tetR-VP16 fusion derivative, was shown to function as a potent trans-modulator of a second plasmid that contains two tandem repeats of the tetracycline operator (tetO) inserted between the TATA box and the transcription start site of the hCMV major immediate-early promoter. A technological advance in this area would be the development of a single autoregulatory cassette that incorporates both of these components into nonviral and viral gene transfer vectors. For the latter, an inducible lentiviral vector that is capable of temporal and quantitative control of gene expression in either dividing or nondividing cells is highly desirable. A one-piece inducible (1Pi) autoregulatory cassette was constructed to provide IRES-mediated translation of the tetR as well as tight control over the tetO unit preventing transcription initiation of the first cistron in the absence of the tetracycline. To increase efficiency of tetR-mediated repression, a nuclear localization signal was incorporated at the 3' end of the tetR gene. Regulation of gene expression at the transcriptional and protein level was analyzed in transient transfection experiments using plasmid DNA. Construction of a self-inactivating lentiviral vector containing this 1Pi cassette allowed the study of its long-term effectiveness in primary human cells. The 1Pi autoregulatory cassette when incorporated into plasmid DNA allows efficient control of the secretable hEGF as well as eGFP expression in a variety of cell types. Transient transfection studies demonstrated that the time course of repression is different for the 1Pi and two-plasmid system (2Pi). In the 2Pi system, greater repression is seen with the first 24-48 hr; however, by 72 hr, similar levels of repression with the 1Pi and 2Pi systems are obtained. This regulation is reached three times faster when the tetR is modified with a nuclear localization signal to direct nascent proteins into the nuclear compartment. In addition, stable transduction of human umbilical vein endothelial cells (HUVEC) with a self-inactivating lentiviral vector incorporating this single regulator cassette provided tetracycline-inducible control of gene expression that is not diminished over time and is completely reversible upon removal of tetracycline. These results suggest a model in which the 1Pi autoregulatory system reaches a steady state over time, the minimal amount of tetR produced by the basal activity of the CMV promoter and accumulated is adequate to replace the tetR that is lost over time. These studies also show that the inducible self-inactivating lentiviral vector can temporally and reversibly regulate transgene expression in HUVECs. The use of this transcriptional control unit in both nonviral and viral vector delivery systems will constitute an attractive technological advance for many gene therapy applications where temporal and quantitative control of gene expression is desired. The strengths and limitations of the 1Pi system are discussed.
Highlights
Inducible expression systems provide a valuable tool for the study of gene functions in prokaryotic and eukaryotic cells and are useful for investigations of developmental genes
Yao et al [4] demonstrated that the tetracycline repressor itself, rather than tetRmammalian cell transcription factor fusion derivatives described previously, can function as a powerful transcriptional modulator to regulate gene expression from the tetracycline operator-bearing hCMV major immediate-early promoter. Because this system employs tetR itself as transcriptional modulator, it avoids some of the potential pleiotropic and toxic effects that are sometimes seen with the chimeric transactivator used in the model system initially described by Gossen and Bujard [5], where regulation of transgene expression is achieved by an heptamerized tetO sequence fused to the hCMV minimal promoter and the hybrid transactivator between tetR and the transcription activation domain of herpes simplex virus protein, VP16 [5,6,7,8]
With tetR production and intracellular accumulation, transcriptional shut-off occurs in the absence of Tc through a high-affinity and effective interaction between dimers of tetR and two tandem tetO sequences located between the TATA box and transcription start site of the CMV promoter
Summary
Inducible expression systems provide a valuable tool for the study of gene functions in prokaryotic and eukaryotic cells and are useful for investigations of developmental genes. Yao et al [4] demonstrated that the tetracycline repressor (tetR) itself, rather than tetRmammalian cell transcription factor fusion derivatives described previously, can function as a powerful transcriptional modulator to regulate gene expression from the tetracycline operator (tetO)-bearing hCMV major immediate-early promoter. A modified version of this tetracycline-inducible system was used to create a single inducible cassette that provides simultaneous expression of both the target transgene and its modulator tetR under the tetO-containing hCMV major immediate-early promoter. A technological advance in this area would be the development of a single autoregulatory cassette that incorporates both of these components into nonviral and viral gene transfer vectors For the latter, an inducible lentiviral vector that is capable of temporal and quantitative control of gene expression in either dividing or nondividing cells is highly desirable. Construction of a selfinactivating lentiviral vector containing this 1Pi cassette allowed the study of its long-term effectiveness in primary human cells
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