A humanized system for pharmacologic control of gene expression

Abstract

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.

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

“…3,[25][26][27][28] Some major advantages of the Tet-on system compared to the other gene regulation systems are a well-known safety profile, easy availability and good tissue penetration of the regulating drug dox. Bacterial origin of the Tet-on elements assures minimal interference with endogenous physiological processes of the eukaryotic cells.…”

Employment of the mdr1 promoter for the chemotherapy-inducible expression of therapeutic genes in cancer gene therapy

“…11,12 Therefore, we proposed that a gene therapy such as hormones, growth factors, but also by therapeutic approach combining chemotherapy and induced TNF modalities which are part of cancer management. 3,4 expression controlled by a drug-responsive promoter It is well accepted and corroborated in clinical practice might increase toxicity towards transduced cancer cells that the combination of different cancer treatment ( Figure 1). modalities such as chemotherapy, radiotherapy or…”

“…Supernatants were harvested 1, 2, 3, l-glutamine (GIBCO). 4,5,6,24,48,72,96 and 120 h after drug addition for ELISA. The inducing effects of the drugs on TNF Construction of the retroviral vector pM3mdr-p-hTNF To create a drug-inducible retroviral vector the Moloney expression were determined on both, mRNA and protein level by TNF-specific RT-PCR and TNF-specific ELISA.…”

“…These gene products are used erthermia. [3][4][5][6] Such studies have indicated that application either to induce tumor cell cytotoxicity for the elimination of a certain tumor treatment not only induced expression of malignant cells or to act as immune modulators. 1 In of the therapeutic gene, but also improves activity of the this context many attempts have been made to improve inducing treatment and the therapeutic gene product as the localized expression of therapeutic genes in tumors it had been shown for the X-ray-induced early growth using targeted vector systems for gene delivery or by response gene (EGR-1) promoter-driven tumor necrosis employing specific promoters/enhancers limiting factor alpha (TNF) expression in radiated tumors.…”

Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives

“…Many proteins have more narrow therapeutic windows, and overproduction could result in toxicity. Several regulation systems, including rapamycin, 40,41 mifepristone, 42 ecdysone 43 and tetracycline (tet) 44,45 have been developed to control transgene expression in vivo. Rapamycinmediated regulation of erythropoietin (Epo) expression has been demonstrated in rats and in one non-human primate following subretinal injection of two rAAV vectors, one encoding for the transactivator, the other for Epo.…”