750. Coelectrotransfer to Spinal Cord of Three Plasmids Coding for Pain Killer Gene and Regulatory Factors of the Tetracycline-Inducible System To Achieve Tightly Regulated Expression, Inhibition of Thermal Hyperalgesia, and Mechanical Allodynia

Abstract

We describe an approach employing intrathecal plasmid electrotransfer to deliver secretable forms beta-endorphin, which can produce profound and long lasting analgesia in neuropathic rats. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. The regulation of beta-endorphin expression was first assessed in spinal neuronal culture, then we electrotranfected this plasmid into the spinal cord of mononeuropathic rats and evaluated the analgesic potential of this therapy in vivo by thermal and mechanical withdrawal latency. Intraperitoneal injections of doxycycline were made to elucidate the possible exogenous upregulation of transfected beta-endorphin gene expression in vivo. The levels of beta-endorphin were analysed by intrathecal microdialysis and RIA. We found that he expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) both in vitro and vivo. The beta-endorphin protein was secreted into CSF at a level of 45 pmol/L in dialysate, which was sufficient to inhibit neuropathic pain. In conclusion, we have shown that controllable expression of beta-endorphin can be obtained following intrathecal electrotransfer of a tetracycline-inducible, three-plasmid-based system. Beta-endorphin protein expression following intrathecal electrotransfer allweviates siatic nerve ligation-induced limb pain. Although physiological minute-to-minute beta-endorphin regulation may not be attained, this approach suffice as a sole or complementary treatment for chronic pain.