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In vivo gene delivery
 

Our laboratory has also developed methods for extracellular delivery of non-viral vectors. It has been demonstrated by multiple labs that injection of naked DNA into muscle results in high level expression of gene product. More recently, electroporation has been coupled with the direct injection approach in muscle and liver of living animals and levels of expression have been shown to increase up to a thousand-fold. Using electroporation, we too have demonstrated gene transfer and expression in the muscle, cornea, and lungs of mice as well as in the vasculature and lungs of rats. We have designed a unique electrode system for the vasculature that allows us to use limiting amounts of DNA in the transfer process, yet yields nanogram amounts of gene product per mm of treated vessel:

 
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All cell layers of the vasculature express gene product, and we are now in the process of developing ways to restrict expression to certain cell types. This should be achieved by use of our different cell-specific DNA nuclear import sequences. Ultimately, these approaches should be testable in larger animal and human systems. The exciting thing about this technique and our new electrode design is that gene transfer can be completed within 1 minute per vessel with no resulting trauma or ischemia. Thus, its transition to human use is likely. An alternative use of this technology that is equally intriguing to us is to ask questions of basic cell biology in the context of the living organism.

 
   

In collaboration with Joseph Benoit (University of North Dakota), we are beginning to look at cyclic nucleotide signal transduction pathways in smooth muscle and endothelial cells in vivo. This is one area that has great potential, but which has not yet been tapped. In the case of the lung, we have achieved high level gene expression in all cell types, including airway smooth muscle and epithelial cells, and alveolar epithelial cells as well. The technique is non-invasive and causes no trauma, so we hope that one day soon, clinical applications will follow.

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Gene therapy represents the greatest hope for treatment and prevention of atherosclerosis and other proliferative diseases of the vasculature, since it can be highly cell-specific, mimics or restores normal in vivo function, and can be permanent or transient depending on vector design. Currently, a number of gene delivery systems for use in vivo are being studied, but as yet their low efficiency in gene transfer and lack of

   
cell-specific targeting and expression are major limitations. We are in a unique position to explore new methods for increased gene transfer. By combining our technology to target vectors to specific types of dividing and non-dividing cells with our recent electroporation designs and results, we can begin to target desired genes to desired cells with specificity and efficiency. Finally, the use of vectors containing such DNA targeting sequences will ensure safety since nuclear import and resulting gene expression will occur only in target cells.

One additional devastating disease that we are targeting is asthma. Apart from the inflammatory aspects of the disease, two of the hallmarks of asthma are airway hyperresonsiveness and remodeling, both of which are mediated by the airway smooth muscle. Thus, the smooth muscle is a major target for new treatments for asthma. As for proliferative diseases of the vasculature, we are combining our use of cell-specific DNA nuclear import sequences with in vivo gene delivery using electroporation to try to develop new ways to overcome this disease. Hopefully, our studies will open new avenues of research and therapeutics.
     
 
   
   
   
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