Tetracycline-inducible transgene expression mediated by a single AAV vector

Abstract

Regulated gene delivery systems are usually made of two elements: an inducible promoter and a transactivator. In order to optimize gene delivery and regulation, a single viral vector ensuring adequate stoichiometry of the two elements is required. However, efficient regulation is hampered by interferences between the inducible promoter and (i) the promoter used to express the transactivator and/or (ii) promoter/enhancer elements present in the viral vector backbone. We describe a single AAV vector in which transcription of both the reverse tetracycline transactivator (rtTA) and the transgene is initiated from a bidirectional tetracycline-responsive promoter and terminated at bidirectional SV40 polyadenylation sites flanking both ITRs. Up to 50-fold induction of gene expression in human tumor cell lines and 100-fold in primary cultures of rat Schwann cells was demonstrated. In addition an 80-fold induction in vivo in the rat brain has been obtained. In vitro, the autoregulatory vector exhibits an induced expression level superior to that obtained using the constitutive CMV promoter. Although extinction of the transgene after removal of tetracycline was rapid (less than 3 days), inducibility after addition of tetracycline was slow (about 14 days). This kinetics is suitable for therapeutic gene expression in slowly progressive diseases while allowing rapid switch-off in case of undesirable effects. As compared to previously described autoregulatory tet-repressible (tetOFF) AAV vectors, the tet-inducible (tetON) vector prevents chronic antibiotic administration in the uninduced state.

Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS

“…Temporal regulation via rapamycin [59] and tetracycline [18] -based expression systems may provide useful tools.…”

Production and Titering of Recombinant Adeno-associated Viral Vectors

“…rAAVs can be combined with Cre-transgenic mice or cell-type specific promoters to achieve circuit-and cell-type-specific gene expression, permitting genetic manipulations with high spatial resolution 2,[19][20][21] , while fitting rAAVs with e.g. the tet system adds temporal control over virus-mediated gene expression 22,23 .These examples illustrate the enormous combinatorial potential of these vectors and predict a rapid increase in rAAV-based studies over the years to come.…”

Gene therapy for optic nerve disease

“…In the case of tetracycline-inducible promoters, the signal is either the introduction or the withdrawal of a particular antibiotic. 8,9 Such technologies allow more control over the whole process of gene therapy, but what is the potential application to the treatment of ocular disease in general and optic nerve diseases in particular?…”