Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo

Abstract

Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2 S -M2 and the other has a rtTA responsive promoter driving the expression of b-galactosidase gene (lacZ). The rtTA2 S -M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

“…Recently, it has been demonstrated that amino acid substitutions in rtTA have greatly improved the transcriptional activity and Dox-sensitivity of the protein (Zhou et al, 2007). In addition, modifications in the TRE-CMV promoter have been continued to further reduce the background activity of the Tet-on system (Koponen et al, 2003). Thus, a combination of our zebrafish line with an effective TRE-CMV promoter is required for functional analysis of certain genes.…”

Retrovirus-mediated tk gene therapy of implanted human breast cancer in nude mice under the regulation of Tet-On

“…While a dose-response relationship exists in chemotherapy, it may be possible that this relationship reaches a plateau at a higher dose due to limitations of normal organ toxicity. The Tet-On system, which uses an E. coli gene regulatory system, 11 appears to fulfill the criteria for clinical application with several advantages: 6,7,11,26 (1) gene expression is easily regulated by administration of Dox; (2) Dox is minimally toxic and has been widely used as an antibiotic; and (3) Dox acts specifically on the target gene and does not activate other Results in this report suggest that the HSVtk/Tet-On/ GCV system is a feasible method in the treatment of breast cancer and other solid tumors. Our data show that there is an apparent dose-dependent relationship between HSVtk gene expression and Dox concentration in infected MCF-7 cells when the concentration of GCV is at 1 mg/ ml.…”

“…This result requires the therapeutic gene expression to be regulated tightly in administration, induction, and termination. 7 To achieve this set of conditions, several inducible eukaryotic gene promoters have been used to deliver genes in a regulated manner. The promoter inducers include steroid hormones, oxygen, heavy metals, and physical stimulus (e.g.…”

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…In fact, although the binary strategy imposes the necessity that each target cell will have to be efficiently transduced by two vectors, it seems, however, to be less leaky by comparison with the single-based system, which therefore is better suited to the desired induction parameters. A more advanced tetracycline regulation design has recently been applied in the LV field context where the use of a codon-optimized tetracycline transactivator has been shown to significantly reduce the leakiness in different cell types [138,141].…”