Abstract
For gene therapy of chronic neurological disorders, vector systems should allow long-term transduction of brain cells in the complete absence of undesirable long-term side-effects. We have engineered a novel regulatable system consisting of a tetracycline-dependent reverse-transactivator, rtTA2 S -M2, in combination with Tet-repressor, tTSKid , under the control of either the major immediate early human cytomegalovirus (hCMV) promoter or murine (mCMV) promoter, encoded within high capacity, helper-dependent adenovirus vectors (HC-Adv). We assessed inducibility, leakiness and time course of expression in-vitro and in-vivo within the central nervous system (CNS). We also assessed cell-type specific expression within neurons and glial cells, and the efficiency of the regulatable vectors in the presence of a systemic immune response to adenovirus. HC-Adv vectors, i.e., STK120mBM [pSTK120m (TRE-β-Gal-pA)-(mCMV pr.-rtTA2 S -M2-IRES-tTSKid -pA)-Kanamycin] and STK120hBM [pSTK120.1 (TRE-β-Gal-pA)-(hCMV pr.-rtTA2S -M2-IRES-tTSKid -pA)-Kanamycin] were injected into the striatum of rat brains at 1×107 blue forming units. Twenty-four hours prior to injection, the rats were given drinking water with 2 mg/ml doxycycline (Dox) and 1% sucrose, or 1% sucrose only. At the appropriate time points, rats were perfused and brains were post-fixed in 4% PFA for 3 days and vibratome sectioned for immunohistochemistry. Assessment of transgene expression levels in vivo revealed higher potency of the mCMV driven TetOn switch when compared to the hCMV driven switch in the presence of Dox. Both mCMV and hCMV encoded regulatable switches produced negligible gene expression levels in the absence of Dox. Our results demonstrate that the rtTA2 S -M2 transactivator in conjunction with IRES and tTSKid transcriptional silencer displays strong and stringent induction kinetics with negligible basal activity in the uninduced state; in vivo data show stringent regulation kinetics of β-Galactosidase expression with co-localization of β-Galactosidase within MAP-2 (neurons) and GFAP (astrocytes) immunoreactive cells in the striatum; expression of β-Galactosidase was dependent on the concentration of Dox and the time after HC-Adv administration. Regulated, long-term transduction in the CNS was attained even in the presence of a systemic immune response to adenovirus as could be encountered in human patients, making HC-Adv vectors very attractive for CNS gene therapy.