A novel doxycycline inducible autoregulatory plasmid which displays 'on'/'off' regulation suited to gene therapy applications

The development of transcriptionally controlled systems which function in e ukaryotic cells are important for achieving regulated gene expression in ge ne therapy. In this study we combined the components of the tetracycline-in ducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from othe r self-contained retroviral and plasmid vectors. Induction kinetics and exp ression levels of luciferase and the therapeutic molecule, truncated solubl e complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the pla smids were shown to be optimal for gene therapy applications, as there was a rapid reduction in expression levels following removal of Dox. Co-transfe ction of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of us ing this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregula tory plasmids display the requirements for gene Therapy applications in chr onic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis, where novel protein therapeutics and combination the rapies are needed.