NON-LEAKY, REGULATED GUTLESS ADENOVIRAL VECTORS MEDIATED INDUCIBLE TRANSGENE EXPRESSION IN THE BRAIN IN VIVO AND IN DOG AND HUMAN GLIOMA CELLS

XIONG; CANDOLFI; KROEGER; PUNTEL; PALMER; NG; LOWENSTEIN; CASTRO

Dallas, Texas, USA

Congreso; Society for Neuro-Oncology; 2007

ET-30. NON-LEAKY, REGULATED GUTLESS ADENOVIRALVECTORS MEDIATED INDUCIBLE TRANSGENEEXPRESSION IN THE BRAIN IN VIVO AND IN DOG ANDHUMAN GLIOMA CELLSWeidong Xiong,1 Marianela Candolfi,1 Kurt Kroeger,1 Mariana Puntel,1Donna Palmer,2 Phillip Ng,2 Pedro Lowenstein,1 and Maria Castro1;1Gene Therapeutics Research Institute, Cedars-Sinai Medical Center andUCLA, Los Angeles, CA, USA; 2Department of Molecular and HumanGenetics, Baylor College of Medicine, Houston, TX, USAIn view of recent advances in gene therapy technologies, the use of regulatable,non-leaky expression systems is becoming a critical requisite to safeand effective clinical gene therapy. Glioblastoma multiforme (GBM) is a devastatingbrain tumor for which there is no cure. Although serotype 5 adenoviral(Ads) have been used successfully in clinical trials for GBM (Immonenet al., Mol Ther., 2004), anti-viral vector immune responses limit the clinicalefficacy of first generation Ads. In the present work, we have engineered anovel regulatable, non-leaky system consisting of a tetracycline-dependentreverse-transactivator, rtTA2S-M2, in combination with Tet-repressor,tTSKid, under the control of the murine cytomegalovirus (mCMV) promoter,encoded within high capacity, helper-dependent adenovirus vectors(HC-Adv). We developed two HC-Adv encoding the b-galactosidase(HC-Ad-mTetON-bgal) and the therapeutic transgene, i.e., human solubleFlt3L (HC-Ad-mTetON-hsFlt3L), under the control of the regulatableTetON system driven by the mCMV promoter. We assessed b-Galactosidase(bgal) expression from HC-Ad-mTetON-bgal within the brain. HC-AdmTetON-bgal vector was injected into the striatum at 1 3 107 blue formingunits (BFU). Twenty-fours prior to injection, the rats were given drinkingwater with 2 mg/ml doxycycline (DOX). At the appropriate time points,rats were perfused and brains were homogenized for bgal enzyme activityassay, or post-fixed in 4% PFA for immunohistochemistry. Our in vivo datademonstrate that stringent regulation kinetics of bgal expression with colocalizationof bgal within MAP-2 (neurons) and GFAP (astrocytes) immunoreactivecells in the striatum. Non-leaky, regulated, long-term transductionin the brain was attained even in the presence of a systemic immuneresponse to Ads as would be encountered in the clinic. We also assessedinducibility, leakiness of expression of our HC-Ad in mouse GL26 cells,rat CNS1 cells, J3T dog glioma cells, established human glioma cell lines(U251, U87), and primary glioma cell cultures from intra-operative biopsies(IN859, IN2045) infected with either HC-Ad-mTetON-bgal or HC-AdmTetON-hsFlt3L (5,000VP/cell) and incubated with or without 1 mg/mlDOX for 72 h. Transgene expression levels were determined by immunocytochemistryand bgal enzymatic activity assay, or hsFlt3L concentration(ELISA). High level transgene expression was achieved in all the humanglioma cells, and in rat CNS-1 cells. The expression of Flt3L was tightlyregulated by DOX. hsFlt3L expressed from HC-mTetON-hsFlt3L was efficientlyreleased by all the GBM cells. The levels of hsFlt3L in the presenceof DOX were around 5–60 nmol/ml, while in the absence of DOX theywere undetectable (p , 0.001). Our results in vivo and in vitro demonstratethat the rtTA2S-M2 transactivator in conjunction with IRES and tTSkidtranscriptional silencer displays strong and stringent induction kinetics withnegligible basal activity in the uninduced state. Taken together, our datastrongly support the use of tightly regulated high-capacity adenoviral vectorsin gene therapy approaches for the treatment of human GBM. Supported byNIH/NINDS Grant 1R01 NS44556.01, Minority Supplement NS445561;1R21-NSO54143.01; 1UO1 NS052465.01to M.G.C.; NIH/NINDS Grants1 RO1 NS 054193.01; RO1NS 42893.01; U54 NS045309–01, and 1R21NS047298-01 to P.R.L.