New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo

Abstract

Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available. Gene Therapy (2005) 12, 504-511.

Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses

“…In vitro assays determined that in the absence of Dox, the silencer inhibits basal levels of gene expression from the tetracycline-inducible promoter even further (43). It has also been determined that the silencer reduces gene expression down to background levels in the absence of Dox in in vivo models (31,40,44,65). Finally, the transcriptional repressor enhances transactivator expression levels by inhibiting its degradation through the ubiquitindependent proteasomal degradation pathway (25).…”

“…Regulatable switches have been previously engineered into several vector systems, i.e., lentivirus (13,23,38,59), retrovirus (19), adeno-associated virus (10,11,20,27,33,40,44), firstgeneration adenovirus (11,27,33,44,47,48), herpesvirus (18), and high-capacity adenoviral vectors (5,6,45,57,60,64). Wang et al determined that an inducible system within a high-capacity adenovirus (HC-Ad) led to prolonged periods of regulatable transgene expression in the liver (60).…”

Improving the Titer of Recombinant Adenovirus by Suppressing Problematic Transgene Transcription During Packaging

“…Both the tTS coding sequence and the TRE (or TRE MIN ) were obtained commercially but were modified and used in a novel way that was not the original intent of the manufacturer (10,11). Although tTS has been used previously to reduce leakiness of tetracycline-inducible promoters (12), to our knowledge this is the first instance of tTS being used to increase viral titer from a constitutively active promoter/enhancer.…”

Regulatable Gene Expression Systems for Gene Therapy

“…tTS Kid totally abrogated basal transgene leak in the absence of doxycycline, without impairing the ability of this inducer to stimulate rtTA-mediated IL-13 expression. To optimize delivery of this activation/repression system, all components were incorporated in single adenoviral or episomally replicating vectors that performed well both in vitro and in vivo (Salucci et al, 2002;Mizuguchi et al, 2003;Bornkamm et al, 2005: Rubinchik et al, 2005Xiong et al, 2006). However, inclusion in tetracycline-regulated gene switches of tTS was also reported to result in reduced activation of transgenes (Lamartina et al, 2003).…”