Regulated, High Capacity Adenoviral Vectors Mediated Long-Term Gene Expression in the Brain Even in the Presence of a Peripheral Immune Response to Adenovirus

XIONG; GOVERDHANA; CURTIN; BARCIA; ZIRGER; KING; SCIASCIA; PUNTEL; CANDOLFI; LOWENSTEIN; CASTRO

New Orleans, Lousiana, USA

Congreso; American Society of Gene Therapy; 2005

Regulated, High Capacity AdenoviralVectors Mediated Long-Term Gene Expression inthe Brain Even in the Presence of a PeripheralImmune Response to AdenovirusWeidong Xiong,1 Shyam Goverdhana,1 James F. Curtin,1 CarlosBarcia-Gonzalez,1 Jeffrey M. Zirger,1 Gwendalyn D. King,1Sandra A. Sciascia,1 Mariana Puntel,1 Marianela Candolfi,1 DonnaPalmer,3 Philip Ng,3 Pedro R. Lowenstein,1,2 Maria G. Castro.1,21Gene Therapeutics Research Institute, Cedars-Sinai MedicalCenter, Los Angeles, CA; 2Department of Medicine andDepartment of Molecular and Medical Pharmacology, DavidGeffen School of Medicine, University of California, Los Angeles,CA; 3Department of Molecular and Human Genetics, BaylorCollege of Medicine, Houston, TX.For gene therapy of chronic neurological disorders, vector systemsshould allow long-term transduction of brain cells in the completeabsence of undesirable long-term side-effects. We have engineered anovel regulatable system consisting of a tetracycline-dependentreverse-transactivator, rtTA2S-M2, in combination with Tetrepressor,tTSKid, under the control of either the major immediateearly human cytomegalovirus (hCMV) promoter or murine (mCMV)promoter, encoded within high capacity, helper-dependentadenovirus vectors (HC-Adv). We assessed inducibility, leakinessand time course of expression in-vitro and in-vivo within the centralnervous system (CNS). We also assessed cell-type specificexpression within neurons and glial cells, and the efficiency of theregulatable vectors in the presence of a systemic immune responseto adenovirus. HC-Adv vectors, i.e., STK120mBM [pSTK120m(TRE-β-Gal-pA)-(mCMV pr.-rtTA2S-M2-IRES-tTSKid-pA)-Kanamycin] and STK120hBM [pSTK120.1 (TRE-β-Gal-pA)-(hCMV pr.-rtTA2S-M2-IRES-tTSKid-pA)-Kanamycin] wereinjected into the striatum of rat brains at 1x107 blue forming units.Twenty-four hours prior to injection, the rats were given drinkingwater with 2 mg/ml doxycycline (Dox) and 1% sucrose, or 1%sucrose only. At the appropriate time points, rats were perfusedand brains were post-fixed in 4% PFA for 3 days and vibratomesectioned for immunohistochemistry. Assessment of transgeneexpression levels in vivo revealed higher potency of the mCMVdriven TetOn switch when compared to the hCMV driven switch inthe presence of Dox. Both mCMV and hCMV encoded regulatableswitches produced negligible gene expression levels in the absenceof Dox. Our results demonstrate that the rtTA2S-M2 transactivatorin conjunction with IRES and tTSKid transcriptional silencer displaysstrong and stringent induction kinetics with negligible basal activityin the uninduced state; in vivo data show stringent regulation kineticsof β-Galactosidase expression with co-localization of β-Galactosidase within MAP-2 (neurons) and GFAP (astrocytes)immunoreactive cells in the striatum; expression of β-Galactosidasewas dependent on the concentration of Dox and the time after HCAdvadministration. Regulated, long-term transduction in the CNSwas attained even in the presence of a systemic immune response toadenovirus as could be encountered in human patients, making HCAdvvectors very attractive for CNS gene therapy