Ecdysoneresponsive Regulatory System

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A. General Principles

The steroid hormone ecdysone, which is normally found in mammals, plays a fundamental role during Drosophila molting and metamorphosis. Pulses of 20-hydroxyecdysone mediate that cascade of morphological modification, which results in degeneration of larval tissues and development of adult structures (162). Ecdysone acts by stimulating a transcription factor, the ecdysone receptor (EcR). The EcR is a member of the nuclear receptor superfamily, and its modular organization resembles that of steroid receptors (163). The functional EcR is a noncovalent heterodimer with the insect protein ultraspira-cle (USP): the two proteins share the multidomain organization of the nuclear receptor family. USP is an obligatory heter-odimeric partner of RcR, required for both ligand and DNA binding (164). EcR carries a transcriptional silencer in its li-gand-binding domain, which acts by recruiting corepressors such as N-CoR and SMRT (165,166). Binding of the cognate ligand induces a conformational change that makes the core-pressor dissociate from the receptor and recruits activators, which ultimately leads to transcriptional activation of genes under EcR control. Also, USP contains a ligand-binding domain, but the natural ligand is unknown: interestingly, recent evidence suggests the USP ligand-binding pocket may be locked in an inactive conformation (167,168).

The mammalian counterpart of USP is the retinoid-X-re-ceptor (RxR), a member of the nuclear receptor family, whose natural ligand is the 9-ds-retinoic acid (9cRA), a vitamin A metabolite, which binds to and activates RxR (169). RxR forms homodimers as well as heterodimers with several hormone and orphan receptors. The vitamin D receptor (VDR), the peroxisome proliferator activated receptor gamma (PPAR-7), the hepatocyte nuclear factor 4 (HNF4), the retinoic acid receptor (RAR) and the thyroid hormone receptor (TR) are among the normal heterodimeric partners of RxR (169). RxR heterodimerize also with the EcR, albeit much less efficiently than USP (170,171).

B. Transcription Factors

In pioneering experiments, insect responsiveness to ecdysone or its analog muristerone A (Fig. 16), was recreated in cells by cotransfecting Drosophila EcR and USP with an EcR-re-sponsive gene. However, in this configuration, muristerone A stimulated transgene expression only to a very limited extent (164,172).

Several modifications were introduced to enhance the dynamic range of the system generating a system schematically represented on Fig. 17. The N-terminal activation domain of EcR was substituted with the VP16 activation domain, thus obtaining a chimeric activator called VpEcR, and the natural heterodimeric partner USP was substituted with RxR. This heterodimer stimulated gene expression up to 1000-fold in cultured CV1 cells (173). Since the mammalian farnesoid-X-receptor (FxR) can weakly activate transcription from EcR-responsive elements, using a mutant ECR DBD capable of binding a synthetic responsive element further increased the specificity of gene regulation. The mutant activator and the synthetic sequence were called VgEcR and E/GRE element, respectively (173). Although FxR can activate a promoter containing the wild-type EcR element, it cannot bind the E/GRE site. The salient attribute of this system was its low basal activity, which is probably due to the natural repressing capability of the EcR (see above) in the absence of the ligand. The VgEcR/rRxR system is now sold commercially and is widely for conditional gene expression in transfected cells.

A modified system, called RheoSwitch, has been recently described. It relies on the coexpression in target cells of GAL4-EcR, which consists of the fusion of the GAL DBD with the EcR of the lepidopteran Choristoneura occidentalis, and VP16-RxR, a fusion between RxR and the VP16 activation domain (174). In stable transfectants, RheoSwitch enabled up to 500-and 100-fold induction of integrated SEAP and luciferase genes, respectively. In this case, GS-E was used as an inducer (Fig. 16). GS-E is a member of a family of diacylhydrazines

Figure 16 Chemical structure of ecdysone-dependent regulatory system inducers. (A) Plant-derived inducers (Muristerone A and Ponasterone A); (B) synthetic dibenzoylhydrazine compounds (GS-E, Tebufenozide).

that have been found to act as nonsteroidal ecdysone mimics that can function as gene inducers (see below).

VgEcR/rRxR and RheoSwitch systems are based on the Drosophila EcR, which is a reluctant dimer partner for RxR. Since RxR is generally expressed at low levels, efficient EcR mediated stimulation can only be achieved by overexpressing RxR in the target cells (164,172). Overexpression of RxR in target cells is undesirable for human gene therapy (see next paragraph). Therefore, the recent finding that EcR from Bombyx mori (BeCR) dimerize much more efficiently with RxR is of particular interest (175). The determinants of this higher affinity map within the HBD and the hinge D domain of BEcR. As a consequence of this higher affinity dimerization, VBEcR (a fusion between BEcR and the VP16 activation domain) strongly stimulates transcription in the absence of cotransfected RxR. BecR is also more responsive than EcR to nonsteroidal agonists (175).

In a more advanced version, the properties of VgEcR and of BEcR were combined. The activation and DNA-binding domain of VgEcR were fused to the hinge region and the HBD of BEcR. This hybrid-receptor (called DB-EcR) thus recognizes the unnatural E/GRE responsive elements but does not require RxR to induce gene expression in mammalian cells and responds more efficiently to nonsteroidal agonists (176).

C. Responsive Promoters

Various EcR responsive promoters have been described that shares the same basic architecture. They usually consist of four EcR-responsive elements (or E/GRE sites, in the case of VgEcR and DB-EcR) cloned upstream of a variety of minimal promoters such as those derived from the Drosophila heat shock or timidine kinase genes (173,175). RheoSwitch recognizes multimeric GAL4 binding sites (174).

D. Ecdysone-regulated Systems for Gene Therapy Applications

The ecdysone system enables tight regulation of gene expression in cell culture and transgenic animals, but its potential for gene therapy applications has not been systematically explored yet.

As to the VgEcR/rRxR and RheoSwitch, several shortcomings may prevent their application in human gene therapy. Both systems are based on overexpression of wild-type RxR, which would thus be overrepresented in all endogenous RxR-containing receptors (see above). This poses a safety concern, considering the large number of metabolic pathways in which heterodimers containing RxR are involved. The recent finding

Figure 17 Ecdysone-responsive regulatory system. One component consists of the human retinoid X receptor (RxR) that carries a DNA-binding domain. The second component consists of a modified ecdysone receptor (EcR) that carries a DNA binding domain and is fused to a VP16 activation domain. Following administration of the drug, the modified EcR binds the ligand and forms a functional heterodimer with RxR. This heterodimer recognizes a specific DNA sequence and therefore activates promoters containing multimers of this sequence.

Figure 17 Ecdysone-responsive regulatory system. One component consists of the human retinoid X receptor (RxR) that carries a DNA-binding domain. The second component consists of a modified ecdysone receptor (EcR) that carries a DNA binding domain and is fused to a VP16 activation domain. Following administration of the drug, the modified EcR binds the ligand and forms a functional heterodimer with RxR. This heterodimer recognizes a specific DNA sequence and therefore activates promoters containing multimers of this sequence.

that RxR overexpression in cardiomyocyte caused dilated car-diomyopathy in mice further emphasizes this aspect (177). Similar concerns would also apply to USP, with the additional problem that this heterologous protein might prove strongly immunogenic in humans. Another issue is the fact that ligands for RxR can modulate the effect of EcR agonists on the RxR/ EcR heterodimers (171). This implies that activity of the RxR/ EcR receptor would not be controlled solely by the inducer drug but also by endogenous natural ligands for RxR, such as the natural agonist 9cRA. Notably, mutant RxR unable to bind the endogenous ligands cannot be used because they would probably also affect activity of all the heterodimeric receptor for RxR.

As outlined in the preceding paragraph, DB-EcR does not require a codelivered dimerizing partner: its use is thus much more suitable for gene therapy applications. So far, this system has been delivered in vivo only by intramyocardial injection of recombinant adenovirus vectors in adult rats. In a first study, the VgEcR/rRxR and DB-EcR regulatory systems were inserted into two distinct Ad vectors (176). Each vector was then coinjected intramyocardially with a reporter Ad vector containing a luciferase gene under the control of an E/GRE-based promoter: rats were then injected intraperitoneally with 45 mg of GS-E (Fig. 16). Three days after, relative luciferase activity was measured: GS-E treatment caused a 40-fold induction of luciferase activity in rats injected with the DB-EcR regulatory system, while it was essentially ineffective in those injected with VgEcR/rRxR. This confirms that VgEcR/rRxR is inefficiently activated by GS-E-like molecules. Basal activities of the two systems were comparably low (176). In a related study, the DB-EcR regulatory system was delivered by adenoviral vectors to express a dominant negative form of a myocardial ion channel in a GS-E-dependent manner (178).

These studies indicate that DB-EcR may be useful for gene therapy applications, but they also highlight that the potency of the system is at this stage inadequate for most gene therapy applications and should therefore be increased. Moreover, the sequestration of endogenous RxR by DB-EcR could have deleterious effects, which may only become evident in long-term experiments. Additional work with different vectors and target tissues is required.

Finally, the presence of the nonhuman protein domains poses the usual concerns about the potential immunogenicity.

E. Inducer Drugs

Ecdysterone is not able to efficiently stimulate the ecdysone-responsive system in mammalian cells (171). More efficient analogs were isolated from plants, which protect themselves from insect feeding by producing substances that are toxic to insects. Ecdysteroids are among these chemicals: when insect larvae eat the leaves of the plant, they also ingest ecdysteroids, which stimulate the EcR to start an abnormal molting that leads to premature death (179). Ecdysteroids may represent up to 1% of the total dry weight of a plant (180).

The most efficient phytoecdysteroids isolated so far are Muristerone A (murA) and Ponasterone A (PonA) (Fig. 16). MurA was isolated in the early 1970s from the seeds of kala-dana, a rare plant native to the southern slopes of the Himalayas. It proved capable of stimulating the VgEcR/rRxR and related systems with an EC50 comprised between 0.5 and 1 uM (173). Difficulties in obtaining seeds from kaladana prompted investigators to identify inducers derived from other plants. Ponasterone A (ponA) was thus identified: it can be purified from the leaves of widespread diffused plants and is as potent as MurA (181). It is conceivable that other inducers will be isolated in the future.

Ecdysteroids are usually presented as safe compounds based on the consideration that humans eat large amounts of phytoecdysteroids (contained in vegetables) without apparent detrimental effect (180). Nonetheless, it is clear that in the light of their potential use in human gene therapy, more detailed toxicology data would be required. Oral bioavailability of ecdysteroids is also a matter of concern: PonA and MurA have always been administered to rodents by intraperitoneal or subcutaneous injections (173,176,178,181). The possibility also exists that patients harboring the ecdysone-receptor switch will have to reduce their oral intake of dietary phytoec-dysteroids to avoid nonspecific activation of the system.

A family of bisacylhydrazines has been identified that function as insecticides by mimicking the activity of ecdysone on the EcR (179). Not surprisingly, they have also been found to function as inducers of the ecdysoneswitch. These molecules, such as tebufenozide and GS-E, are less active than PonA on the VgEcR/rRxR system (Fig. 16) (181), but more potent on the DB-EcR switch (176). These compounds can be easily manufactured but show a very poor solubility: this limits their potential for in vivo applications (179,181). A recent study demonstrates that molecular modeling and site directed mutagenesis of the EcR generated a mutant EcR, which induces transcription of target genes in response to bisacylhydrazines but not to ecdysteroids (182). As outlined above, because of the dietary intake, the development of ecdy-steroid-insensitive regulatory switches might be necessary in humans. Therefore, albeit the clinical potential of bisacylhy-drazines remains to be established, more soluble molecules would represent an important starting point and prove a useful tool.

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