Gene Therapy (2000) 7, 2061–2070  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications DJ Gould 1 , M Berenstein 2 , H Dreja 1 , F Ledda 2 , OL Podhajcer 2 and Y Chernajovsky 1 1 Bone and Joint Research Unit, St. Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary, University of London, London, UK; and 2 Fundacion Campomar Conicet, Faculty of Exact and Natural Sciences, University of Buenos Aires, Argentina The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible sys- tem in self-contained retroviral and plasmid vectors. Regu- lated reporter gene expression from the autoregulatory plas- mid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-con- tained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule, truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblas- toma cell line. The regulatory characteristics of the plasmids Keywords: rtTA; reverse tetracycline transactivator; autoregulatory; gene therapy Introduction Regulating expression of therapeutic genes is a prerequi- site to the clinical application of gene therapy to the treat- ment of chronic conditions with relapsing symptoms, such as rheumatoid arthritis and multiple sclerosis. Restricting expression of therapeutics in these conditions to periods when relapses occur will prevent potential side-effects from long-term expression of therapeutic proteins. The tetracycline system for regulated gene expression was originally developed as a two vector ‘off’ system in which a chimeric tetracycline transactivator (tTA), com- posed of tetR and VP16 is constitutively expressed from one vector, and binds to a tetracycline operon (tetO) con- taining promoter in a second vector inducing gene expression. 1 When tetracycline is added it binds tTA and induces a conformational change which prevents it from binding to the tetO and thus switches off gene expression. The ‘off’ system displays regulated expression in excess of 1000-fold when the two vectors are stably transfected into HeLa cells. Several studies Correspondence: Y Chernajovsky, Bone and Joint Research Unit, St Bar- tholomew’s and Royal London School of Medicine and Dentistry, Char- terhouse Square, Queen Mary, University of London, London, EC1M 6BQ, UK Received 18 February 2000; accepted 29 September 2000 were shown to be optimal for gene therapy applications, as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulat- ory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the require- ments for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis, where novel protein therapeutics and combination therapies are needed. Gene Therapy (2000) 7, 2061–2070. have since reported adaptations of the original tetracyc- line ‘off’ system which facilitate application in gene ther- apy settings. One variation involves combining the components of the two vectors in a single self-contained vector, which avoids the need to deliver two vectors to a single cell, this combination has been reported with both retroviral 2,3 and plasmid vectors. 4 Another modification has been expressing tTA under the control of a tetO pro- moter. In this autoregulatory format the tTA regulates its own expression, in the absence of tetracycline the tTA is able to interact with the tetO promoter and can therefore up-regulate its own expression in a positive feedback loop. When tetracycline is added to the system the tTA can no longer interact with the promoter and so tTA expression is down-regulated. The advantage of auto- regulated expression of tTA is low level expression of tTA in the presence of tetracycline, which should reduce toxic effects of constitutively expressed tTA, and higher levels of inducible gene expression when tetracycline is removed. This autoregulation has been adopted in both two vectors 5 and self-contained vectors. 4,6 Gossen and co-workers 7 later developed a two-vector tetracycline ‘on’ system. Mutation of the tTA produced the reverse tetracycline transactivator (rtTA) which induces gene expression in the presence of doxycycline (Dox). 7 Modifications of the ‘on’ system include the con- struction of a self-contained single bacterial plasmid, 8 a retroviral vector 9 and an autoregulatory bicistronic plas-