Regulatable systems: applications in gene therapy and replicating viruses (2000)

Effective gene-based therapies require efficient delivery of therapeutic genes to targeted mammalian cells as well as regulatable gene expression. Advances in gene therapy have mainly focused on the development of viral systems — adenovirus, retrovirus, adeno-associated virus, herpes virus, autonomous parvoviruses — and nonviral systems, such as direct injection of naked or conjugated DNA, for delivering transgenes into mammalian cells. Most of the currently applied gene delivery systems rely on strong viral promoters to drive high levels of expression in a wide range of tissues, although promoters from the cellular genes for phosphoglycerate kinase, actin and histones, have also been used with varying degrees of success. These promoters exhibit constitutive expression of proteins, which can cause cellular toxicity and may select for the downregulation of the effector systems.

Recent approaches to gene therapy have emphasized the need for gene delivery vectors that can efficiently introduce and control expression of foreign genes in a dose-dependent and reversible manner. Indeed, in certain disorders, only a specific range or dose of the therapeutic protein will achieve a successful outcome (1–3).

In clinical applications, it will be beneficial to regulate the transgene expression in order to maintain protein concentrations within the therapeutic window and to optimize efficacy in response to the evolving nature of the disease. A regulatable system would be of value in modifying specific therapies if it could offer tight regulation in response to pharmacologic agents that can be safely and repetitively administered. For cancer gene therapy, therapeutic gene products that would be particularly valuable to control in this way include cytokines (see Agha-Mohammadi and Lotze, this Perspective Series, ref. 4), prodrug activating enzymes (see Springer and Niculescu-Duvaz, this Perspective Series, ref. 5), ribozymes, antibodies, tumoricidal genes, or antisense oligonucleotides. Regulatable systems also have great utility in controlling the expression of the vector delivering the therapeutic gene. If replicating viruses are to be used to deliver such genes or to lyse tumors directly, regulation of viral early promoters may be required to control the rate of viral replication and enhance the safety of the recombinant virus.

First-generation regulatable systems, based on naturally occurring inducible promoters, generally suffer from high basal expression of the utilized promoter, weak induction of transgene expression, and reliance on inducible agents that exert pleiotropic effects on mammalian cells. Chimeric regulatable systems, devised to overcome these limitations, incorporate various prokaryotic and eukaryotic elements and offer greater specificity than can be achieved using natural inducible promoters. Transactivators in these chimeric systems are designed to interact specifically with sequences engineered into the vector. Recently developed chimeric systems are regulated by tetracycline (6), the progesterone antagonist RU486 (7), the insect hormone ecdysone (8), or rapamycin (FK506) (9). These drugs or hormones (or their analogs) act on modular transactivators composed of natural or mutant ligand binding domains and intrinsic or extrinsic DNA binding and transcriptional activation domains.