Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Abstract

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affilnity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a 18-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no .8-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of f8-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of 8-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, 18-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.The renal glomerulus is a microscopic vascular structure scattered throughout the cortex of the kidney. During the past decade, numerous studies have been done to elucidate molecular mechanisms of glomerular diseases. Among various approaches utilized, in vivo gene transfer technology would be one of the most powerful and promising strategies, since this allows us to modulate activity of a certain molecule within the glomerulus (1, 2). However, recent investigations have encountered some hurdles. Because of the microscopic size (100 jam in diameter) and number (3 x 104 to 1 X 106/kidney) of glomeruli, it is difficult to efficiently and site-specifically deliver foreign genes into the majority of the glomeruli. Conventional cationic liposomes or adenoviral vectors, the most popular in vivo gene transfer vectors, were found to be inefficient for this purpose (3,4). A modified liposome system could be useful (5), but exogenous gene expression has been limited to several days (6). To overcome these problems, I established a novel gene transfer system that specifically targets the glomerulus (7-9). In this method, the glomerular mesangial cell ...

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