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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.
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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.
Click image
for larger view
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
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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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