1. Introduction
Since most genetic disorders result from deregulated transcription or mutated genes, current gene therapy initiatives focus on complementation of genetic defects using adjustable expression of therapeutic transgenes to ensure precise titration into the therapeutic window, coordination with patient-specific disease dynamics, and termination of therapeutic interventions. Ideal conditional therapeutic transcription interventions require complex systems that (1) are of heterologous origin to ensure an interference-free operation, (2) enable seamless integration into regulatory networks of target cells, (3) exhibit low immunogenicity, (4) provide high-level expression under induced and low basal expression under repressed conditions, (5) show adjustability to intermediate levels over a wide range of inducer concentrations, (6) are responsive to a bioavailable inducer including clinically licensed small-molecule drugs, other inert molecules, or any specific well-tolerated physical condition, (7) exhibit high compatibility with approved viral and nonviral gene transfer technologies, (8) support configurations to restrict interventions to specific tissues or disease foci, and (9) are amenable to compact genetic design to limit pleiotropic effects associated with repeated molecular intervention on the patient’s chromosome.
Generic design concepts of most advanced transcription control systems include artificial transcription-modulating (fusion) proteins that bind or assemble at specific target promoters in a small molecule-dependent manner or at specific physical conditions.
2. Antibiotic-responsive gene regulation systems
Antibiotic-responsive gene regulation systems are derived from prokaryotic antibiotic response regulators evolved to coordinate resistance to specific classes of antibiotics. A protein represses a resistance gene until its release from the target promoter following binding of a specific antibiotic. Such antibiotic-responsive protein-DNA interactions have successfully been assembled in three different configurations for conditional transgene transcription in mammalian cells: (1) Fusion of the bacterial antibiotic resistance gene repressor to a generic transactivation domain creates an antibiotic-dependent transactivator, which, in the absence of regulating antibiotics, binds and activates chimeric promoters containing transactivator-specific (tandem) operator modules 5′ of a minimal eukaryotic promoter (Gossen and Bujard, 1992; Fussenegger etal., 2000a; Weber etal., 2002). (2) The aforementioned transactivator can be mutated to enable reverse antibiotic-dependent binding, resulting in dose-dependent transgene induction in the presence of regulating antibiotics (Gossen etal., 1995). (3) The bacterial antibiotic resistance gene repressor bound to its cognate operator may repress transcription from 5′-located mammalian promoters. The antibiotic-induced repressor release then correlates with increased transgene transcription (Fussenegger et al., 2000a; Yao et al., 1998; Weber et al., 2002). Three different transcription control systems responsive to tetracyclines (Gossen and Bujard, 1992), streptogramins (Fussenegger etal., 2000b), and macrolides (Weber et al., 2002) have been developed. Antibiotic-responsive gene regulation systems comply with ideal systems at a high standard. Yet, potential immunogenicity of bacterial components, tissue-specific accumulation, and/or promotion of antibiotic resistance will remain ongoing challenges for clinical implementation (Darteil et al., 2002).
3. Hormone-inducible gene expression
Lipophilic hormones are key players of intercellular communication in higher eukaryotes. They freely cross cell membranes, bind intracellular receptors, and modulate target gene expression following nuclear translocation. The generic design concept for hormone-inducible gene regulation systems consists of fusing the hormone-binding domain of a hormone receptor to a heterologous DNA-binding module and optionally to a transactivation/transsilencing moiety. This chimeric hormone receptor will initiate/repress transcription from target promoters equipped with DNA-binding domain-specific operator modules in the presence of regulating hormones. The use of human hormone receptor mutants specific for endogenous hormone agonists is expected to enable increased immunocompatibility without interfering with endogenous hormone regulatory networks. Yet, some hormone agonists exhibit a major clinical impact. Three hormone-inducible transcription control systems responsive to estrogen (Braselmann et al., 1993), the progesterone antagonist mifepristone (Wang et al., 1994), and the insect moulting hormone ecdysone (No et al., 1996) have been constructed, and are continuously improved for human compatibility.
4. Dimerizer-regulated gene expression
Chemically induced dimerization (CID) is a phenomenon by which two proteins (hetero)dimerize in the presence of a molecule. The most prominent heterodimerizer is the immunosuppressive agent rapamycin, which unites FKBP (FK506-binding protein) and FRAP (FKBP rapamycin-associated protein), thus impairing cell cycle regulatory networks involved in T cell expansion. Rapamycin-inducible heterodimerization of FKBP fused to artificial DNA-binding domains and FRAP to a transactivation domain reconstitutes a chimeric transactivator that initiates transcription from target promoters containing specific operator modules. In order to alleviate cosuppression of the immune system when rapamycin-based CID transcription control is in action, protein engineering initiatives to alter FKBP and FRAB’s specificities for a nontoxic, clinically inert molecule are promising and may secure a clinical future of this technology (Pollock and Clackson, 2002).
5. Systems of the future
Despite quantitative differences in regulation performance, most aforementioned transcription control systems qualify for clinical implementation. However, improvement of immunocompatibility and tolerability of inducers remain future challenges. Current systems based on prokaryotic antibiotic resistance operons promise specific regulation by clinically licensed molecules, but are compromised by the use of bacterial epitopes and long-term accumulation of antibiotics in various tissues of the body. Hormone- and CID-responsive transgene control systems can be optimally humanized, yet remain limited by pleiotropic and other side effects of their inducing agents. Even when using artificial molecule-protein interactions, immunocompatibility and side effects of designer molecules remain imminent concerns. On the way toward ideal transgene regulation modalities, a variety of strategies have been designed, which promise important improvements over existing technologies: (1) construction of transcription modulators responsive to clinically inert compounds, temperature, light, and dynamic electromagnetic fields, (2) systems responsive to specific nucleic acids, and (3) epigenetic gene networks.
Prokaryotes manage inter- and intrapopulation communication by quorum-sensing molecules, which bind to receptors in target cells and initiate specific regulon switches by modulating the receptors affinity to cognate promoters (Bassler, 2002). Systems derived from bacterial, quorum-sensing regulatory networks are expected to be of particular interest since many regulating molecules of commensal prokaryotic populations have a long history of human-prokaryotic coevolution. Following the generic design principle of antibiotic-adjustable transcription control modalities, bacterial cross talk systems responsive to butyrolactones have been successfully validated in mice without signs suggestive of inducer-related side effects (Weber et al., 2003b; Neddermann et al., 2003).
Throughout the development of eukaryotes, production of ribosomal proteins (rp) is regulated at the translational level. Translation control is mediated by a terminal oligopyrimidine element (TOP) present in the 5′ untranslated region of rp-encoding proteins. TOP elements adopt a translation-prohibitive secondary structure, which is resolved upon binding of a specific cellular nucleic acid-binding protein (CNBP). TOP-complementary nucleic acids interfere with the translation-promoting TOP-CNBP interaction and so repress top-tagged mRNAs in a dose-dependent manner (Schlatter and Fussenegger, 2003).
Two low-temperature-inducible mammalian gene regulation systems have been designed capitalizing on (1) a heat-labile alphaviral replicase that transcribes target genes driven by subgenomic promoters only at permissive temperatures (Boorsma et al., 2000) and (2) a thermosensor managing heat-shock response in Streptomyces albus (Weber et al., 2003a). Owing to dominant environmental conditions, clinical implementation of temperature-controlled expression technology would require local temperature control by a Pelletier element.
Light-inducible gene regulation may represent an alternative to temperature-based control. A recently discovered photosensitive plant protein heterodimerizes with a partner protein following exposure to the cofactor phynocynanobilin and transient red and far-red light pulses. Assembly of these heterodimerizing proteins analogous to CID systems enabled light-inducible transgene expression in yeast (Shimizu-Sato et al., 2002). Owing to limited tissue penetration, nonvisible electromagnetic fields have come into the limelight of the gene control community, but these systems are at best in the discovery stage: (1) several electromagnetic field response elements (EMRE) have been discovered in humans (Lin etal., 2001; Rubenstrunk et al., 2003) and (2) radio-frequency magnetic fields were shown to remotely control hybridization of oligonucleotides linked to gold nanoparticles (Hamad-Schifferli et al., 2002).
Throughout multicellular systems, cell identity is maintained by epigenetic regulation circuits, which imprint transient morphogen gradients during early embryogenesis by locking the transcriptome of adult cells in a cell phenotype-specific manner. By combining two repressors, which control each other’s expression, an epigenetic circuitry able to switch between two stable expression states by transient addition of two inducers has been pioneered (Kramer et al., 2004). Owing to transient administration of regulating agents, long-term side effects will be eliminated.
6. Conclusions
Much like drug dosing is the key parameter in modern molecular medicine since Paracelsus’ statement that “the dose makes the poison”, gene expression dosing will be of central importance for next-generation gene therapy and tissue engineering initiatives. Capitalizing on achievements accumulated for over a decade, conditional transcription control of therapeutic transgenes stands now on the threshold to a clinical reality. With the first transcription control units being assembled into regulatory gene networks and prototype epigenetic gene switches being designed for mammalian cells, the future for multigene-based therapeutic interventions in regulatory networks of patients’ cells has just begun.
