Tightly regulated, helper-dependent adenoviral vector-mediated transgene expression in vitro and in vivo

XIONG; GOVERDHANA; CURTIN; PUNTEL; GREENGOLD; BARCIA; ZIRGER; SOFFER; LOWENSTEIN; CASTRO

San Diego, California, USA

Congreso; American Society of Gene Therapy; 2004

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatableexpression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In thepresent work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent(TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. Weconstructed two HC-Ad vectors encoding
-galactosidase (
-gal) driven by a TetOn system containing thertTASsM2 transactivator and the tTSKid repressor under the control of the murine cytomegalovirus (mCMV)(HC-Ad-mTetON-
-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-
-Gal). Expression wastightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basallevels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgeneexpression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter.HC-Ad-mTetON-
-Gal yielded the highest transgene expression levels and expressed in both neuronsand astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested theinducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized againstadenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-
-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectorsdisplay two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness evenin the presence of prior immunization against adenovirus.