This rapid and precise on/off switch regulate the expression of transgenes in mammalian cells

SUMMARY: Regulated expression of transgenes in mammalian cells is a valuable tool for both functional genomic studies and clinical applications. Attempts to control gene activity in eukaryotes with inducible expression systems have been made but often resulted in modest induction levels or in pleiotropic effects. A regulated mammalian transgene expression system was designed based on coumarin-switched dimerization of a chimeric transactivator.

APPLICATIONS: Tightly controlled regulation of gene expression in mammalian cells for functional analysis of cellular proteins and drug discovery applications.

Transient production of highly toxic proteins or proteins interfering with host cell functions.

Rapid and precise regulation of gene expression in vivo using low antibiotics concentration for gene therapy applications.

CONCEPT: Inducible mammalian expression systems that feature low levels of basal activity and rapid induction of gene expression in response to safe and reliable inducers are needed to control protein expression. A novel on/off switch that relies on the rapid and sensitive modulation of a chimeric transactivator by coumarins was developed to provide tightly controlled gene expression in mammalian cells. The transactivator comprises a repressor protein DNA binding domain (lambdaRdelta), a transcription activation domain (AD) and the bacterial DNA gyrase B subunit (GyrB). The interaction of coumermycin with GyrB induces homodimerization of the transactivator maintained at a low basal level by a relatively weak hybrid SV40-CMV promoter. This homodimer triggers transactivator self-expression through a positive regulatory feedback loop designed by insertion of operator sequences (4x lambda Op). In turn, transgene expression is triggered by the increased number of homodimers available for binding to the operator sequences (12x lambda Op) situated upstream of the modified CMV minipromoter that regulates it. Conversely, the expression system can be promptly and effectively switched off by addition of novobiocin, a competitive monomer of coumermycin that readily abolishes transactivator dimerization (see Figure).

FEATURES AND BENEFITS: High level of inductibility - Inducibility was improved through reduced basal expression and transactivator auto-amplification. The lambdaR moiety of the transactivator was mutated to reduce its intrinsic dimerization activity and consequently increase the coumermycin- induced/basal-level ratio of gene expression. As well, a positive regulatory feedback loop was used to increase the activation potency of the transactivator controlled by the relatively weak SV40 early promoter.

Readily transferable expression cassette - The chimeric transactivator and the coumarin-responsive transgene expression cassettes, included on one or two separate plasmids, can be moved to any commonly used vector for in vitro or in vivo delivery into cells or to a pharmaceutically suitable carrier for human administration.

Rapid and sensitive on/off switch - The coumarin-regulated expression system is induced by coumermycin concentrations in the nM range; this compares favorably to rapamycin and tetracycline-regulated systems. Conversely, novobiocin which also binds gyrB with high affinity effectively promotes dimer dissociation and shuts down the system in only a few hours. The very short half life of both compounds also contributes to the highly dynamic nature of the coumarin-regulated expression system.

Safety for gene therapy applications - The coumarin-based on/off switch is well adapted for the safe in vivo regulation of gene expression in mammalian cells. Both coumermycin and novobiocin have excellent pharmacokinetic properties, display no overt sign of toxicity, and have no know endogenous binding targets in mammalian cells.

Early Stage

A coumermycin/novobiocin-regulated gene expression system (NRC no. 11444).

Japan, Application number JP2006-515586 , Filed --

United States, Application number 60/477,055 , Filed 2003-06- 10

Patent Cooperation Treaty, Application number PCT/CA04/000854 , Filed 2004-06-09

European Common Market, Application number 04737796.5 , Filed 2004-06-09

Australia, Application number 20044245598 , Filed 2004-06-09

Canada, Application number 2,523,879 , Filed 2004-06-09

Licencing for R&D use and for biopharmaceutical production

Europe: 1631671; Australia: 20044245598; Netherlands : 1 631 671; Switzerlands: 1 631 671; Germany: 1 631 671; Great Britan: 1 631 671