Recombinant AAV is increasingly becoming the vector of choice for many gene therapy applications in the CNS, due to its lack of toxicity and high level of sustained expression. With recent improvements in the generation of pure, high titer vector stocks, the regulation of gene expression is now a key issue for successful translation of gene therapy-based treatments to the clinic. The level of the transgene protein may need to be maintained within a narrow therapeutic window for the successful treatment of human disease. The doxycycline responsive system directs a dose-responsive, tightly regulated level of gene expression and has been used successfully in transgenic mouse models. Here, we have optimized an autoregulatory, bidirectional doxycyline responsive cassette specifically for use in rAAV. We minimized the size of the cassette and decreased the basal leakiness of the system, leading to tight regulation in the rat