Abstract
BackgroundThe in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models.ResultsIn this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression.ConclusionsThis study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.
Highlights
The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models
The hallmark of this system is the efficient transgene silencing in the OFF-state, while addition of the inducer drug allowed for robust activation of transgene expression in vitro and in vivo [10]
We report that sustained transgene regulation requires matrix attachment regions (MAR) epigenetic regulatory sequences, and we show that these elements favor stable and long-term expression but that they promote the chromosomal integration of the transgenes in vivo
Summary
The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. One of the favorite systems is based on the bacterial tetracycline-repressed TetR protein of E. coli [1,2] It relies on inducer compounds with a proven history of medical safety, i.e. tetracycline and derivatives, which regulate the ability of engineered TetR protein derivatives to bind particular DNA sequences inserted in target promoters. With this system, regulation of the mouse erythropoietin gene has been achieved in mice muscles over periods of several months using non-viral or viral vectors. The hallmark of this system is the efficient transgene silencing in the OFF-state, while addition of the inducer drug allowed for robust activation of transgene expression in vitro and in vivo [10]
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