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 eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible 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 other self-contained 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 neuroblastoma cell line. The regulatory characteristics of the plasmids 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 autoregulatory 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 requirements 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.
Gould
D J
DJ
Bone and Joint Research Unit, St. Bartholomew's and Royal London School of Medicine and Dentistry, Charterhouse Square, Queen Mary, University of London, London ECIM 6BQ, UK.
Berenstein
M
M
Dreja
H
H
Ledda
F
F
Podhajcer
O L
OL
Chernajovsky
Y
Y
D000818:Animals; D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D002460:Cell Line; D004318:Doxycycline / Q000627:therapeutic use*; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008811:Mice, Inbred DBA; D010957:Plasmids; D011951:Receptors, Complement / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000379:methods*
D000900:Anti-Bacterial Agents; D008164:Luminescent Proteins; D011951:Receptors, Complement; D049452:Green Fluorescent Proteins; D008156:Luciferases; D004318:Doxycycline