B

ch3 och3 ch3 ch3

Figure 11 Chemical structure of dimerizer-dependent system inducers. (A) Rapamycin, the more commonly used inducer of the system; (B) Rap*, nonimmunosuppressive rapamycin-derivative.

Figure 12 Dimerizer-dependent system. One component is a DNA binding domain consisting of a composite zinc-finger homeodomain (ZFHD1) fused to three FKBP repeats. The second component is an activation domain fused C-terminally to the FRB domain. The two fusion proteins can be joined by heterodimerizer drugs, thus generating a composite transactivator that activates a target promoter consisting of 12 ZFHD1-binding sites.

Figure 12 Dimerizer-dependent system. One component is a DNA binding domain consisting of a composite zinc-finger homeodomain (ZFHD1) fused to three FKBP repeats. The second component is an activation domain fused C-terminally to the FRB domain. The two fusion proteins can be joined by heterodimerizer drugs, thus generating a composite transactivator that activates a target promoter consisting of 12 ZFHD1-binding sites.

commonly the minimal cytomegalovirus immediate early promoter (101), or the minimal IL-2 promoter (107).

The upstream element is the binding site for ZFDH1. As described above, ZFDH1 is a synthetic DBD generated by protein design. Its consensus DNA-binding site (DBS), 5'-TAATTANGGGNG-3', has been derived by selection from a random pool of oligonucleotides (100). The particular juxta position of its two components, the two zinc fingers from Zif268 and the POU domain of Oct-1, allows ZFDH1 to selectively bind the sequence 5'-TAATGATGGGCG-3' with a dissociation constant of 8.4 x 10~10 M, an affinity at least 30fold higher than that of either Zif268 or Oct-1 for the same sequence. Hence, the ZFDH1 DBS is not recognized by endogenous transcription factors: multimers of this DBS linked to a minimal promoter and to a reporter gene, when transfected in cells, do not give rise to detectable reporter gene expression (101). The most commonly used promoter configuration contains 12 tandem copies of the ZFDH1 DBS (Z12).

D. Dimerizer-regulated System for Gene Therapy Application

In general, dimerizer-based systems display a very low basal activity in the absence of the inducer drug. The main reason for this is the physical separation of the DBD from the AD in two distinct fusion proteins that cannot interact in the absence of ligand. This tight regulation was constantly observed in cells after transient or stable transfection, and also when stably transfected cells were implanted in vivo at various dosages (101,108). The tightness of the system was demonstrated by efficient generation of stable cell lines carrying an induci-ble highly toxic gene (103).

Since basal transcription ranges from low to undetectable, induction ratios in the presence of rapamycin are very high (up to 1000-fold) in cultured cells. The dose-response curve with the standard system, i.e., ZF3/S1, reaches maximal induction in stably transfected cells at approx. 50 nM rapamycin with an EC50 of 6-8 nM. Maximal gene expression levels are comparable to those obtained with constitutive strong promoters like the CMV promoter/enhancer (107). However, in a comparative study with the rtTA2-M2-based tet-on system, the rapamycin system displayed lower leakiness but also lower maximum induced activity after transient transfection (109).

The dimerizer-regulated switch enabled tight control of gene expression also when delivered in various animal models of gene therapy using viral vectors (mainly AAV), moving from small animals to nonhuman primates (Table 2). When delivered by AAV vectors in mouse muscle, the system enabled long-term and strictly rapamycin-dependent control of GH and Epo (107,110). Importantly, varying dosage of the inducer drug modulated gene expression (107,110). Inducible expression was also demonstrated in mouse eye and liver following AAV and adenoviral gene transfer (111,112).

Similar studies were also performed in nonhuman primates: of particular relevance was the demonstration that a regulated macaque Epo gene delivered with AAV vectors in the muscle of rhesus macaques could be repeatedly induced upon systemic drug delivery (110). Inducibility of transgene expression was not maintained over a prolonged period of time in all animals: out of six primates tested in total, three underwent expression shut-off. However, it has been recently reported in an extended trial involving eight animals injected with an improved system that all of them show persistent expression (92). This is a preliminary indication that the shut-off observed in the first study may not be due to immunologi-cal response against the activator but, possibly, to decreased expression of the two transactivators below a therapeutic threshold.

Further confirmation of this system reliability comes from the inducibility of Epo expression in the anterior chamber of macaque eye after subretinal injection of AAV vectors (112).

A recent report described use of the rapamycin-dependent system to control replication of adenoviral vectors in cancer cells. Conditionally replicating adenoviral vectors have been recently introduced in clinical trials with promising results (113). These vectors are designed to undergo replication and spread only to cancer cells. For this purpose, intratumoral, loco-regional, or also systemic delivery are being investigated. However, safety concerns have raised the interest in pharmacological regulation of replicating adenoviral vectors. A recent study has shown that it is possible to engineer a conditionally replicating adeno in which the E1a gene is put under the control of a minimal promoter with 12 ZFDH1 sites (114). Direct coinjection in tumor cells of this virus with a first generation adeno expressing the two hybrid transcriptional activators ZH3 and S1 leads to rapamycin-inducible adenovirus replication (114). Although therapeutic efficacy in a xenograft model has not yet been convincingly shown, and the current system requires two complementary adenoviruses, a refined version in which all these elements are contained in a single vectors can be envisaged as a new weapon in the fight against cancer.

E. Inducer Drugs

Rapamycin is the principal ligand used to stimulate the dimer-izer-based regulatory system, which indeed is commonly known as the ''rapamycin system.'' Its pharmacological and pharmacodynamic properties are well known (115). It is orally bioavailable (see below) and has a half-life of 4.5 h. A fairly extensive characterization of the kinetics of transgene induc-tion-deinduction in vivo was performed in mice after IM implantation of stable clones expressing the hGH under the con

Table 2 Dimerizer-dependent System: In Vivo Studies in Animal Models of Gene Therapy

Transactivator

Transgenea

Vector

Tissue

Species

Refs.

ZF3 + FRAP-S1

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