Ligand-Dependent Regulation of Vascular Endothelial Growth Factor and Erythropoietin Expression by a Plasmid-Based Autoinducible GeneSwitch System

Abstract

We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate the expression of genes for two therapeutic proteins: vascular endothelial growth factor (VEGF) and erythropoietin. The GeneSwitch system consisted of two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene. When the constitutive CMV promoter of the GeneSwitch plasmid was replaced by an autoinducible promoter consisting of four copies of GAL4 DNA binding sites linked to a minimal thymidine kinase promoter, the tightness of transgene regulation was improved by an order of magnitude. Quantitative RT-PCR analysis of GeneSwitch mRNA confirmed that the autoinducible promoter was responsive to mifepristone. We demonstrated the ability of the improved GeneSwitch system to regulate the expression of VEGF or erythropoietin in a biologically relevant manner after delivery of plasmids to the hind-limb muscle of adult mice. This ability of the autoinducible GeneSwitch system to regulate the expression of therapeutic proteins in mice indicates its potential for use in human gene therapy applications.

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer

“…45,46 The properties of the mifepristone-regulated system have been investigated in transgenic animals and naked DNA plasmid in muscles. [47][48][49][50] Burcin et al 45 have incorporated this system into a gutless adenovirus with a human growth hormone target gene. They showed that background transcription was undetectable in vitro and in vivo and that over 50 days' human growth hormone production could be cycled on and off 3 times or maintained at steady state levels by delivery of the inducer.…”

Therapy of cancer by cytokines mediated by gene therapy approach

“…In the mifepristone system, drug-regulated transcription is achieved by fusing a heterologous DNA binding domain (DBD) of yeast GAL4 protein and activation domain (AD) of VP-16 or NF-κB p65 proteins to a mutant human progesterone receptor that is unaffected by endogenous hormones but is activated by synthetic antiprogestins, at doses sufficiently low to avoid side-effects in human [43]. The properties of the mifepristone-regulated system have been investigated in transgenic animals and naked DNA plasmid in muscles [44]. Burcin et al [43] have incorporated this system into a gutless adenovirus with a human growth hormone (hGH) target gene.…”

Gene therapy progress and prospects: transcription regulatory systems

“…The latest version of the system, which is now commercialized with the registered trademark GeneSwitch s , strongly induces transcription, responds to very low (10 À10 M) concentrations of RU486 but may display a significant basal activity. 26,27 RU486 inducible expression of transgenes has been documented in animal models of gene therapy. A suboptimal version of the activator enabled long-term regulation of the human growth hormone (hGH) expression upon delivery in mice liver with a helperdependent adenoviral vector.…”

“…This displays a lower basal activity and a higher foldinduction; however, maximum expression levels are lower than those obtained from more conventional constructs. 27 A further improved version of transactivator, called GS4, has been recently described: it carries a shorter version of the GAL4 DBD and has a lower basal activity as a consequence of the reduced capability to homodimerize in the absence of the ligand.…”