The potential advantage offered by gene therapy over conventional medicine for the treatment of several diseases is unquestionable. It is nonetheless also clear that efficacy, and more importantly safety, remain the main issues to be solved before this technology can be adopted routinely as a standard therapeutic practice (1,2). At the time of writing this article, beginning in 2003, clinical efficacy of gene transfer has been convincingly demonstrated only for Severe Combined Immune Deficiency (SCID). In contrast, safety concerns have been raised as a consequence of adverse side effects in several clinical trials (3-8). Therefore, the major challenge still remains the development of vectors characterized by maximum transduction efficiency combined with minimal toxicity (9,10). In current years the continuous application of ''good basic science'' to the gene therapy field has been directed at fulfilling these two main goals. With the decline of the initial concept of a ''universal vector'' for all diseases, the emerging scenario is to generate a large repertoire of safe vectors, each suited to target a given tissue or cell type, or tailored to treat a specific clinical condition. (11-13). Based on these premises, any work directed at developing systems, which allow to control and finely tune the expression of therapeutic gene(s), must be taken into serious consideration.
Until now, clinical trials of somatic gene therapy have made exclusive use of constructs where transgenes are delivered under the control of potent and constitutively active promoters. In these applications the risk of toxic effects caused by overproduction of the therapeutic proteins was negligible for a variety of reasons, related partly to the features of the vector used and in part to the nature of the target disease.
In some cases short-term transgene expression was expected because the vector, for example 1st generation adenovirus, was not capable per se of long-term gene expression (2). In others, such as naked DNA delivered to muscle, it was known that gene transfer was not efficient enough to elicit too high levels of the transgene product (14). When cancer or other acute diseases were targeted, prolonged treatment was not required, and the main goal was to maximize gene transcription and protein production for a short period of time (15). Finally, in clinical trials of chronic diseases, where long-term gene expression was required, gene therapy was directed at curing clinical conditions in which the expressed proteins had large therapeutic indexes (TIs). Examples of this are coagulation factor IX for Hemophilia B, IL-2 receptor gamma-chain or adenosine deaminase for SCID, and CFTR for Cystic Fibrosis (16-18).
Constitutive promoters, however, have limited applications in gene therapy. The main reason is that they cannot be used for the delivery of proteins with small TI. In practical terms, several diseases could benefit from the delivery of genes whose activity must be kept within a narrow therapeutic window. Good examples are disease caused by protein hormone deficiencies, such as anemia or pituitary dwarfism, or those that require treatment with soluble receptors, cytokines and antibodies (19). These clinical conditions are currently treated by repeated administration of recombinant proteins, but less frequent or one-time delivery of the gene coding for the therapeutic protein would indeed represent a more cost-effective and successful approach (14). For these cases, regulating transgene expression would be crucial to maintaining the circulating levels of the protein within a well-defined therapeutic window, thereby preventing toxicity. Regulated gene tran scription would also allow therapy to be modulated in response to disease evolution, which varies greatly from patient to patient, and in the individual response to the therapy itself. Finally, the possibility to terminate and resume therapy (i.e., stop and restart transgene production) at will, would not only allow therapy to be halted in the presence of adverse side-effects, but would also enhance the flexibility of a gene-based approach by enabling combinations with conventional therapeutic modalities.
In addition, it has to be taken into account that vector improvements will probably increase efficiency and longevity of gene transfer over the next year. As a consequence, the expression levels of proteins with large therapeutic indexes will also need to be controlled. In the long term, therefore, it is not premature to think that regulating therapeutic gene expression will become an indispensable mechanism in broadening the application range of gene therapy and increasing both clinical efficacy and safety of the majority applications.
To be used in the context of human gene therapy, a transcription regulation system must precisely modulate target gene expression in response to administration or withdrawal of a specific external stimulus. Initial attempts to generate regulated gene expression systems were mainly based on using endogenous promoters and enhancer elements specifically responsive to environmental stimuli such as metal ions, heat, and oxygen tension (20). A detailed description of these systems is beyond the scope of this chapter, but it is worth remarking that these types of regulatory systems have severe drawbacks. The most important is that the inducer stimuli heavily interfere with the regulatory networks of the host and therefore display pleiotropic effects. Moreover, these systems display a relatively high basal activity in the uninduced state, promote only modest levels of induction, and frequently lose responsiveness to the inducer over time (20). These limitations obviously preclude the use of endogenous control systems in human gene therapy, and have been gradually superseded by more efficient approaches based on exogenous regulators.
Over the past decade, several artificial systems have been developed that enable regulable expression of the desired transgene in response to a small molecule ligand (21). In general, these systems are based on two components. The first is a chimeric transcription factor obtained by fusing a DNA-binding domain (DBD), (which usually does not bind endogenous cellular sequences), a transcription activation domain (AD), and a domain that interacts with a small molecular weight compound that acts as inducer drug. The second component is an artificial promoter consisting of multimeric binding sites for the DBD followed by a minimal promoter sequence containing a TATA box. The chimeric transcription factor is recruited to (or in some cases released from) the specific target promoter upon interaction with the exoge-
nously added drug: transcription of a transgene cloned downstream of this promoter can thus be modulated in vivo by systemic delivery or withdrawal of the inducer drug (Fig. 1).
To be suitable for human gene therapy, the ideal pharmacologically regulated system should fulfill several criteria listed below:
1. ON switches vs. OFF switches: the ligand should activate (ON switches) rather than inhibit (OFF switches) activity of the chimeric transcription factor. In practice, off switches are unlikely candidates for human gene therapy because of two major drawbacks: in order to fully repress an off-switch system, all intracellular molecules of the chimeric activators must theoretically be bound to the drug. This implies that prolonged exposure of patients to high doses of the drug would be required to silence the system in vivo, thus increasing the probability of drug-related side effects. Secondly, induction kinetics would be poor in the case of off switches and mainly determined by the rate of drug clearance (inactivation of the drug or its removal from body tissues).
2. Specificity: the system should not interfere with endogenous regulatory pathways. This means that the transcription factor should only activate the target promoter and the drug should be devoid of pleiotro-pic effects.
3. Bioavailability of the drug: the inducer drug should be orally bioavailable and capable of readily penetrating all tissues, as well as crossing the blood-brain barrier in cases of gene therapy to the CNS.
4. Drug safety: the inducer compound must have a safety profile compatible with prolonged therapeutic use in humans.
5. Reversibility: the system should be fully and rapidly reversible to enable prompt modification of the dosing regimen when required. In relation to this, it is important that the inducer drug is cleared from body tissues within a reasonable amount of time, thus enabling rapid switching from the on- to the off-state upon drug delivery and withdrawal, respectively.
6. Low basal activity: the system should be inactive in the absence of the drug when delivered with any type of vector of both viral and non viral origin. It is important that this tight control should be maintained at all vector doses and in every target tissue.
7. High degree of inducibility: the system should be induced over a wide dose range. In particular, strong induction should be obtained at relatively low (i.e., compatible with therapy in humans) drug concentrations.
8. Dose-dependence: a pre requisite for a fine modulation of protein production in vivo is that a precise correlation must exist between drug dosage and target gene expression level.
9. Low immunogenicity: to enable long-term applications, the chimeric transcriptional activator(s) should exhibit a low potential of eliciting an immune response in man.
Several ligand-dependent systems have been described in the past years, but only four of them have been extensively tested in animal models and are currently being refined for use in human applications. These four systems are regulated by a) the tetracycline (Tet) antibiotics or its analog doxycycline (Dox); b) chemical dimerizers such as the immunosuppres-sants rapamycin and its analogs; c) the synthetic steroid antag onists mifepristone (RU486) and tamoxifen (TAM); and d) insect steroid ecdysone or its analogs. In this chapter we will review in depth ligand-dependent gene regulation with particular emphasis on these four systems, and will discuss to what extent they come close the ideal features outlined above.
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