548 Differential Regulation of Multiple Transgenes by Alternative Splicing Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S210 GENE REGULAT[.]
GENE REGULATION II inducible AAV2/9 vector expressing ffLuc; b) the AAV2/9 vector expressing transcription factor; and c) the constitutive AAV2/9 vector expressing ffLuc (used to assess the maximally achievable ffLuc expression) Twenty-one days after vector injection, the degree of gene expression ‘leakage’ of the inducible AAV vector system as well as the level of ffLuc in mice injected with the constitutive AAV2/9 vector were assessed Briefly, luciferin was delivered intranasally and using the Xenogen IVIS imaging system all control and vectortreated mice were imaged Only mice injected with the constitutive AAV2/9 vector expressed ffLuc (∼1.6 x106 photons/seconds; p/s) at levels significantly above baseline (103 p/s) No “leakage” of ffLuc from the inducible AAV vector system was observed All mice were then injected intraperitoneally with 1mg/kg rapamycin and ffLuc expression was monitored daily (for up to days) Twenty-four hours later we observed a ∼1000-fold increase in ffLuc expression in the nasal airways of mice injected with the inducible AAV vector system Indeed the level of ffLuc expression (∼1.6 x106 p/s) was statistically no different to that conferred by the constitutive AAV2/9 vector (P< 0.05, ANOVA, SNK test) ffLuc expression for the control groups remained similar to baseline We observed a modest increase in ffLuc expression in those mice injected with the constitutive AAV vector The induction was repeated three weeks later and similar levels of increase in ffLuc expression were observed No adverse events to either of the two rapamycin inductions were observed in any of the treated mice Here we demonstrate the utility of an AAV-based inducible vector system designed to regulate gene expression in airway epithelium in vivo, which is particularly applicable to disease states that require short-term high levels of gene expression 546 Development and Characterization of a New Tet-ON AAV Vector for Tightly Controlled Transgene Expression Davide Gianni,1 Nancy H Tran,2 Dimphna H Meijer,2 Miguel Sena-Esteves.1 Neurology and Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 2Neurology and Neuroscience Center, Massachussetts General Hospital, Charlestown, MA The regulation of transgene expression is a critical aspect for gene therapy-based therapeutic approaches In particular this aspect is of primary importance when the genes encoded in the vectors can potentially interfere with the cell metabolism and survival Tetracycline (Tet)-mediated regulation of gene expression has been extensively investigated and several generations of Tet-responsive systems have been developed However most of the systems in the context of viral vectors are affected by significant basal expression in the non-induced state (OFF state) Here we describe a new AAV vector system (TetD-AAV) carrying two different Tet-responsive elements (rtTA2s–M2 activator and tTSKid repressor) in cis with the transgene in order to achieve low background expression in OFF state and sustained expression during induction The in vitro characterization of gene expression regulation was performed with a TetD-AAV2 vector encoding Gaussia luciferase (Gluc) as reporter gene The vector was tested on several cell lines including 293T, U87, GL261, SH-S5Y5 and normal human astrocytes transduced at vector doses of 1e5 genome copies (gc)/cell Gluc assays in the supernatant of transduced cells showed low Gluc levels in the OFF state and expression levels in the ON state comparable to those obtained with an AAV vector encoding Gluc under a CBA promoter and also carrying a WPRE element This resulted in induction ratios of 400- to 1200-fold depending on the cell line with maximum transgene expression observed at a doxycycline (Dox) concentration of µg/ml Additional in vitro characterization was performed replacing Gluc with human (h) or murine (m) interferon b (IFN-b) in the vector and verifying the effects of these cytokines on the growth kinetics of glioblastoma cell lines U87 and GL261 in culture Our results clearly showed significant inhibition S210 of tumor cell growth in the presence of Dox (p