Pharmaceutical Research, Gene Technology, F Hoffmann-La Roche Ltd, 4070 Basel, Switzerland

Amplicon vectors incorporate genetic elements from Herpes simplex virus (HSV) in a plasmid form which is packaged into virions in the presence of a replication-defective helper virus. We constructed a new amplicon vector, pHermes-tet-lacZ, that carries the bacterial -galactosidase (lacZ) gene under the control of a minimal promoter preceded by a heptameric tetracycline operator. The minimal promoter element is activated by a tetracycline-responsive hybrid protein, the gene for which is also present in the vector. This amplicon was propagated in parallel in two different permissive cell lines, E5 and 2-2, in the presence of two helper viruses, d120 and 5dl1.2, respectively. The viral stocks produced were injected into the hippocampal region of the mouse brain, where strong localized expression of the transgene developed in the granular cell layer of the dentate gyrus with limited cytotoxicity. The transgene expression could be repressed by a factor of 10 after administration of tetracyclines. The repression level depended on the helper virus present in the injected viral stock. The in vivo regulation of transgene expression conferred by the tetracycline-responsive element improves the flexibility of amplicon vectors as tools for gene transfer into the brain.

Herpes simplex virus; amplicon vector; regulatable promoter; tetracycline; hippocampus