a GH = Growth Hormone; Epo = erythropoietin; E1a, Elb = adenovirus early genes; Ins = insulin.

a GH = Growth Hormone; Epo = erythropoietin; E1a, Elb = adenovirus early genes; Ins = insulin.

trol of the dimerizer system. After a single IV or oral administration, peak transgene levels were reached after 24 h, maintained for the following 48 h, and then started to decline with a half-life of one day (108). Considering that the half-life of GH is only minutes and that of rapamycin is 4.5 h, this extended kinetic of expression is probably due to the rapamycin-mediated complex between the chimeric DBD and AD being highly stable for several hours in the cell nuclei. As a consequence, transcription shut-off in response to drug withdrawal is a slow process, which depends on degradation rate of the two transcription factors via the proteasome pathway. The intrinsic inertia of the dimerizer system may thus render daily drug administration superfluous to keeping the system in the on state. On the other hand, this also precludes prompt silencing of the system by drug withdrawal if and when required.

Rapamycin is only partially orally bioavailable (115). Maximal activation is observed at 10-fold higher doses after oral delivery than by parenteral administration and, while the ED50 derived from intravenous administration is slightly above 1 mg/kg, that derived from oral dosing is 9-10 mg/kg. This explains why in the majority of published studies the ligand was administered by intraperitoneal injection (107,108,110-113). Indeed, at the common doses of 1-2 mg/ kg used in small and large animal models, transgene expression is already half-maximally activated after parenteral injection of rapamycin, while only marginally induced when the drug is given orally (108). Highly immunosuppressive oral doses would thus be required to reach therapeutic levels of the target transgene in humans, thus making this version of the system impractical for clinical applications. These high concentrations of the ligand might also interfere with the physiological functions of FKBP, such as calcium channel modulation and cardiac development (116,117). The potential solution to these issues is to use rapamycin analogs that maintain the capacity to heterodimerize DBD-FKBP and FRB-AD but have lost their immunosuppressive properties.

Immunosuppression by rapamycin is due to the inhibition of FRAP enzymatic activity (118). The 3-dimensional structure of the ternary complex of FKBP, rapamycin, and FRAP is known, and has helped redesign the binding interface of rapamycin with FRAP (119). Bulky substituents were introduced in the C16 position of rapamycin, which abolished binding to wt FRAP and, as a consequence, its immunosuppressant properties. Likewise, compensatory mutations were obtained by genetic selection in yeast of a FRAP mutant library. These restored binding of a C16 substituted nonimmunosuppressant rapalog called Rap*, carrying a methallyl substituent (Fig. 11) (120). A triple substitution mutant, T2098L/W2101F/ K2095P, called FRB*, was shown to efficiently and specifically bind the FKBP-Rap* complex. When the FRB*-p65 fusion was used in transfected cells as a replacement of the original FRB-p65 component, it induced target gene expression in the presence of Rap* at an EC50 below 10 nM.

Other rapalogs substituted in the C7 position have been obtained (121). Although their structure has not yet been disclosed, they are assumed to act in combination with a single aminoacid-substituted FRB T2098L. This preferred FRB variant (called FRAPL) and some of its corresponding rapalogs AP22565 and AP21967 have been shown to efficiently induce gene expression in cell lines transduced with a retroviral vector or in vivo in tumors injected with conditionally replicating adenoviral vectors (103,114). Based on these results, it would seem the solution to immunosuppression at hand. However, it has to be kept in mind that changes to the structure of rapamycin may modify its pharmacology and drug metabolism. Until now, no transcription-specific rapalog has been reported with a defined oral bioavailability and acceptable pharmacokinetics.

F. Perspectives

This class of transcriptional activators and their related ligands are of relatively new conception but have quickly acquired importance as highly promising for in vivo gene therapy applications and future clinical development. The system embodies most of the properties required, i.e., low basal activity in the absence of the ligand, high dynamic range, and full reversibility of the transcription switch upon ligand withdrawal. Its level of characterization in in vivo preclinical models is fairly advanced, and the intrinsic flexibility and modularity of its components offer opportunities for future evolution.

In particular, zinc-finger technology has made such significant progress over the past years that it is now possible to engineer zinc-finger domains targeted to any desired DNA sequence (122). These novel polypeptides can be used to activate or block gene expression when targeted to open chroma-tin domains of resident genes. By using appropriately designed zinc fingers, it may thus be possible to generate dimerizer-regulatory systems capable of specifically targeting and activating promoters of endogenous genes. This approach worked for VEGF gene regulation in a stably transfected cell line (123). Rapamycin- or rapalog-inducible endogenous VEGF gene expression has been shown to be tight and rapid, of magnitude similar or superior to that achieved by the natural stimulus hypoxia, and accompanied by the simultaneous expression of the whole repertoire of VEGF splice variants. Although in its initial phase, this strategy has great potential for gene therapy (see also steroid-regulated systems).

The exclusive use of human components to assemble these two hybrid proteins should significantly decrease the possibility of immune response, but might not completely eliminate it. In fact, each polypeptide contains one or two novel junction epitopes that can be presented in the context of MHC class-I molecules and recognized as nonself, depending on the hap-lotype in which they are expressed. In theory, our greater knowledge of the immune system may permit these epitopes to be redesigned in order to reduce their possibility for presentation (124).

Of importance are the efforts of synthetic chemistry to expand the set of available heterodimerizers. Examples in this direction are the creation of fusions between methotrexate and a synthetic FKBP ligand or between methotrexate and dexamethasone, which are capable of triggering the formation of heterodimerizers between DHFR and FKBP12 or between DHFR and the glucocorticoid receptor respectively (125,126). The extensive use of combinatorial chemistry, coupled with molecular modeling and protein design, will expand the number of reagents in the following years. We can expect to see a proliferation of dimerizer-regulated systems, which will significantly differ according to the type of inducer used. Demonstration of their feasibility in cell cultures will be merely the first and easiest step. This will have to be followed by the demonstration of suitable pharmacological properties and lack of toxic effects in vivo. As with every drug discovery project, these new chemical entities will have to pass the severe examination of in vivo pharmacology, safety assessment, and drug metabolism before becoming suitable for transferring to a clinical setting.


Steroid receptors are natural examples of ligand-dependent transcription factors but cannot be used for gene therapy in their native form because they lack the required specificity, i.e., would also activate endogenous genes. Hence, by taking advantage of the modular nature of steroid receptors, in which DNA-binding, transactivation and ligand-binding functions reside in distinct domains, regulatory switches have been developed (127). Domain-swapping experiments have demonstrated the independent character of these domains. Heterolo-gous proteins can be rendered hormone responsive by fusing them with the hormone-binding domain (HBD) of steroid receptors (128). In the absence of the ligand, the fusion protein is bound to the heat shock protein 90 complex (HSP90) through the HBD: in this state the fusion protein is inactive, probably due to steric hindrance and/or maintenance in an inactive conformation. Upon binding of the specific ligand to the HBD, the fusion protein is released from the HSP90 complex and becomes active.

The number of proteins adaptable to this approach is continuously expanding and includes kinases, recombinase, integrases, and oncogenes (129). This strategy has also been applied to generate hormone-dependent transcription factors. In pioneering works, the HBD of the estrogen receptor (ER) was fused to the DNA-binding domain of the yeast transcription factor GAL4 and to the Herpes simplex virus transactivation domain VP16 (GAL4-ER/HBD-VP16). This chimeric protein was able to stimulate transcription from artificial promoters containing GAL4-responsive elements in an Estradiol (E2)-dependent manner in cultured cells (130). This activator is predicted to be highly specific in terms of transgene transcription, because it only recognizes promoters containing the 17-mer GAL4 DNA-binding sequence that is not present in the mammalian genome. However, chimeric transactivators that carry natural HBDs of steroid receptors cannot be used in gene therapy applications because their activity would be severely influenced by endogenous steroids.

Thus, more recent studies have focused on the development of inducible chimeric activators equipped with mutant HBDs that do not interact with natural steroids but only bind to synthetic analogs. HBD-based chimeric transactivators that are specifically induced either by the antiprogestin RU486 or by the antiestrogen 4-hydroxytamoxifen (4-OHT) have been constructed and used to regulate transcription of target genes in animal models of gene therapy (Fig. 13). Here we describe the molecular architecture of these systems, summarize the results obtained in vivo, and discuss the potential of this type of switches for human gene therapy.

B. GeneSwitch: An RU486 Dependent Regulatory System

1. Transcription Factors

In 1992, BW. O'Malley and colleagues demonstrated that the c-terminus of the progesterone receptor (PR) is essential for its transcriptional activity upon interaction with progesterone (131). They also isolated a 42-amino acid C-terminal deletion mutant of the PR (called hPRB891) that no longer interacted with progesterone or other endogenous steroids, but retained its ability to bind progesterone antagonists, such as RU486, also known as mifepristone (Fig. 14) (131).

Based on this finding, the same group constructed a first version of an RU486-dependent transcription factor by fusing the HBD of hPRB891 (PR-HBD640-891) C-terminally to the GAL4 DNA binding domain (DBD). This activator, called pGL, only minimally activated transcription from reporter genes cloned downstream of 4 tandem repeats of GAL4 binding sites (132). A more potent activator was obtained when the VP16 transactivation domain (residues 411-487) was fused N-terminally to the GAL4 DBD. This activator, called GLVP (or GS 1.0), enabled from 10- to 50-fold induction of reporter genes in cultured cells (132). As expected, GLVP transcriptional activity was stimulated only by adding RU486 but not in the presence of natural progesterone or synthetic progesterone agonists.

A more potent activator, called GL914VPc' (or GS 2.0), was then generated by using a 19-amino-acid C-terminal deletion of the PR-HBD (aa 640-814) and by placing the VP16 activation domain at the C-terminus of the chimera (133). GS 2.0 induced gene expression in transiently transfected cells at 10-fold lower concentrations of RU486 than GS 1.0 (0.01 nM vs. 0.1 nM). However, GS 2.0 displayed a basal activity higher than that of GS 1.0, so that the net increase in induction was only 2-3 fold. By substituting the VP16 activation domain with that of the p65 subunit of human NF-kB protein, GLp65 activator (also called GS 3.0) was obtained, which was 2-fold less potent than GS 2.0, but displayed a lower basal activity in the uninduced state (78). Additional modifications were made by Valentis Inc., which now commercializes the system with the registered trademark ''GeneSwitch.'' The version called GLp 65.1 (or GS 3.1) is identical to GS 3.0 in terms of functionality and overall structure and only differs for the

Figure 13 Hormone Binding Domain (HBD)-based regulatory switches. The activator consists of a DNA binding domain (GAL4 or HNF1, but also zinc fingers) fused to a mutant HBD, unable to bind endogenous steroids, and to an activation domain, such as VP16 and p65. In the absence of the ligand, the protein is bound to the hsp90 (heat-shock protein 90) complex and therefore inactive. In the presence of the inducer drug (RU486 or 4-OHT), the activator is first released from hsp90, and then binds and activates the target promoter, which consists of multimeric DNA binding sites upstream of a minimal promoter sequence.

Figure 13 Hormone Binding Domain (HBD)-based regulatory switches. The activator consists of a DNA binding domain (GAL4 or HNF1, but also zinc fingers) fused to a mutant HBD, unable to bind endogenous steroids, and to an activation domain, such as VP16 and p65. In the absence of the ligand, the protein is bound to the hsp90 (heat-shock protein 90) complex and therefore inactive. In the presence of the inducer drug (RU486 or 4-OHT), the activator is first released from hsp90, and then binds and activates the target promoter, which consists of multimeric DNA binding sites upstream of a minimal promoter sequence.

shorter (3 aa long) linker between the PR-HBD and the p65 activation domain. GS 3.1 is responsive to RU486 concentrations ranging from 10-11 M to 10-8 M, with half-maximal activation occurring at 10-10 M (134). It is worth mentioning that a further version of this transactivator, called GS 4.0, has been described. It carries a shorter version of the GAL4 DBD and is claimed to have a lower basal activity as a consequence of the reduced capability to homodimerize in the absence of the ligand (135). However, no data have been published in support of this.

2. GeneSwitch Responsive Promoters

The structure of the GeneSwitch responsive promoters has not been significantly modified over the years (135). Initial experiments in cultured cells demonstrated that the composition of the minimal promoter cloned downstream of the mul-

Figure 14 Chemical structure of RU486, inducer of the GeneSwitch system. RU486 (mifepristone) is a synthetic progesterone antagonist.

timeric GAL-binding sites significantly influences both the basal and maximal activity of the system. The minimal promoter that displayed the best compromise between basal activity and inducibility contained only the TATA box from the adenoviral major late promoter E1b. No further improvement in this direction has been reported following these early findings. In its most frequently used configuration, a GeneSwitch promoter consists of 6 tandem copies of GAL4 binding sites cloned upstream of the E1 TATA box (6 x GAL4-TATA)

3. GeneSwitch for Gene Therapy Applications

The efficiency of GeneSwitch in regulating transcription of target genes in vivo depends heavily on the delivery vectors used (Table 3). In a first study, a replication incompetent HSV vector carrying a CMV-driven GS 1.0 expression cassette and the p-gal gene under the control of a GAL responsive promoter was inoculated into rat hippocampus (136). Forty-eight hours postinjection, a 150-fold increased expression of p-gal was measured in brain extracts from rats treated with RU486. Minimal background p-gal expression was measured in untreated rats.

In another study using viral vectors, GS 3.0 under the control of the liver-specific transthyretin (TTR) promoter and a GAL4-responsive human growth hormone (hGH) cDNA were incorporated into the same helper-dependent adenoviral vector (78). Infection of cultured hepatoma cells and of mouse livers resulted in thousands-fold induction of hGH expression in rats treated with RU486. Following RU486 withdrawal, hGH expression returned to basal levels. hGH expression was repeatedly reinduced over a 9-month period, but the extent of induction progressively decreased (78,137). It was not established whether this decrease was caused by an immune response against hGH and/or GS 3.0, loss of vector DNA or transcriptional inactivation of TTR, and/or the GAL4-respon-sive promoter.

When delivered intramuscularly as plasmid DNA, GeneSwitch performed more poorly (Table 3). A 15-fold induction of SEAP gene expression was measured upon code-livery into mouse muscles of a CMV-driven GS 3.0 expression vector and a GAL4-responsive SEAP plasmid (134). Moreover, time-course experiments revealed that the leakiness increased over time, resulting in lower magnitude of induction at later time points. Interestingly, this poor induction level upon intramuscular delivery of plasmid DNA correlates with the finding that GeneSwitch displays a significant basal activity in transient transfection of several cell types, resulting in low induction ratios in the range of 10-40-fold (134). The relatively low potency of this system upon intramuscular plasmid injection was confirmed in experiments performed in rats (138).

Intramuscular delivery of GeneSwitch as a plasmid DNA may thus represent a valid therapeutic approach only in those cases where a low-fold induction is sufficient. This was demonstrated by a recent experiment in which intramuscular elec-troinjection of the GeneSwitch systems as plasmid DNA in SCID mice was used to regulate expression of the growth hormone-releasing hormone (GHRH) (Table 3). Prolonged treatment with RU486 led to a 1.1-1.7 rise in IGF-1 levels, presumably as a consequence of enhanced GHRH expression. This low-fold induction nonetheless provoked a significant increase in body weight, lean body mass, and bone mineral density with a concomitant decrease of fat mass (139).

In an attempt to increase the dynamic range of induction, an autoinducible GeneSwitch system was generated (140). In this system, the GS 3.1 transactivator was placed under the

Table 3 GeneSwitch System: In Vivo Studies in Animal Models of Gene Therapy







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