Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector

Abstract

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycinresponsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.S ystems for regulating gene expression by means of cellpermeant inducing agents have many applications in the study of gene function. The ability to vary expression levels of a gene product at will and to monitor effects on cells or whole animals can give powerful insights into its biological role. Regulation also facilitates the study of genes encoding products whose constitutive expression would be detrimental or lethal to cells. Finally, systems for the small molecule control of gene expression are applicable to gene therapies that require intermittent therapeutic protein delivery or delivery at levels that fall within a narrow therapeutic window.Several methods for the small molecule control of gene expression have been described including those induced by tetracycline, antiprogestins, and ecdysone (1). We have described a transcriptional switch based on rapamycin, a small molecule natural product that mediates the formation of heterodimers between the immunophilin FK506-binding protein (FKBP) and the lipid kinase homolog FRAP (2-5). By fusing FKBP domains to a DNA-binding domain called ZFHD1 and the rapamycin-binding domain of FRAP to a transcriptional activation domain from the p65 subunit of human NF-B, reconstitution of a functional transcription factor and expression of a target gene can be made dependent on the presence of rapamycin (4). This system has been shown to regulate t...

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.…”

A novel conditional Akt ‘survival switch’ reversibly protects cells from apoptosis

“…To improve the CID-responsiveness of iAkt, we utilized novel, high specificity heterodimerizing ligands, called rapalogs, based on the immunosuppressant drug, rapamycin. 35 Otherwise bioinert, rapalogs can reversibly crosslink proteins fused to the proline isomerase, FKBP12, with FRB 1 a 90 amino acid rapamycin-binding domain excised from the S6 kinase kinase, FRAP/mTOR, containing L2098 to confer specificity. While rapalogs bind with high affinity to FRB 1 binding to endogenous FRAP is sterically prevented.…”

“…37,38 More recently, 'rapalogs' have been developed that are modified to greatly reduce binding, and therefore bioactivity, to endogenous FRAP, but are able to bind with high affinity to modified FRB 1 (T2098L). 35 As shown in Figure 1a, the heterodimeric rapalog/CID HED can effect the crosslinking of FRB 1 and FKPB12 (called F). In these experiments, we used the non-toxic variant of FKBP12, F pk (FKBP12(G89P,190K)), to eliminate background toxicity.…”

Characterisation of systemic dissemination of nonreplicating adenoviral vectors from tumours in local gene delivery

“…To reduce transgene expression in normal tissues, unique promoters have been integrated into viral vectors. These promoters can be activated only by specific stimuli in tumours that are either endogenous or applied externally (Vaupel et al, 1989;Gossen and Bujard, 1992;Garver et al, 1994;Harris et al, 1994;Hallahan et al, 1995;Kumagai et al, 1996;Jaggar et al, 1997;Jenster et al, 1997;Blackburn et al, 1998;Chung et al, 1999;Abdul-Ghani et al, 2000;Huang et al, 2000;Koshikawa et al, 2000;Pollock et al, 2000;Spitz et al, 2000). However, the control of transgene expression will not reduce the acute toxicity in normal tissues caused by viral proteins.…”