Highly suppressible expression of single-chain interleukin-12 by doxycycline following adenoviral infection with a single-vector Tet-regulatory system.

Abstract

Adenoviral vectors have been shown to efficiently transfer DNA into a wide variety of eukaryotic cells in vitro and in vivo. However, the therapeutic benefit of this approach is limited by severe side effects as a result of uncontrolled transgene expression. A bi-directional promoter that controls the desired transgene as well as a tetracycline-suppressible transactivator (tTA) was cloned into the E1-region of E1-deleted recombinant adenoviral vectors. Autoregulation within this construct was obtained by tTA expression under control of the operator, to which tTA binds in the absence of tetracycline. Consequently, binding of tetracycline to tTA results in downregulation of tTA as well as the co-expressed transgene in the infected cell. We were able to suppress luciferase-reporter gene expression by up to 16 000-fold in the presence of doxycycline (dox, 2 micro g/ml). Under control of this tetracycline-regulated system, single-chain interleukin-12 (scIL12) was expressed. Adenovirally mediated expression of this potentially lethal cytokine with strong activation of antitumoral immune response was downregulated by up to 6000-fold in the presence of dox. Subsequently, this downregulation also resulted in a highly significant reduction of interferon-gamma secretion by stimulated splenocytes. These mainly contribute to the toxicity of this immunotherapeutic approach. With expression levels exceeding those of the cytomegalovirus (CMV) promoter in almost all cell lines tested, these new vectors will also contribute to the safety of adenoviral approaches by controlled expression without compromising on maximum expression levels.