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DURHAM, N.C. -- Duke University Medical Center researchers,
using a modified cold virus, have delivered a nitric oxide
producing gene to key liver cells in rats, reversing the major
complication of cirrhosis of the liver.

The strategy may eventually prove feasible for humans who
suffer from liver cirrhosis, a difficult-to-treat disease that
often leads to liver failure and death, said the researchers,
who published their study findings in the March 15 issue of the
Journal of Clinical
Investigation.

"To have a targeted therapy would be of immense help to
physicians who take care of these patients," said Dr. Don
Rockey, director of the Duke Liver Center. "Further studies
will determine whether or not this approach will be feasible in
humans."

Rockey's research was supported by grants from the National
Institutes of Health, and the American Digestive Health
Foundation.

The scientists took advantage of the fact that the
adenovirus, the common cold virus that is used in many gene
therapy experiments, likes to infect the liver. Because of
this, the researchers were able to use small amounts of the
modified adenovirus - not the larger amounts that have caused
some problems in some gene therapy trials.

The researchers inserted into to the adenovirus a gene that
produces nitric oxide, a chemical that can relax blood vessels
within the liver. One of the hallmarks of cirrhosis is portal
hypertension, which occurs when blood coursing from the gut to
the heart faces strong resistance as it travels through the
liver. Some of this resistance is caused by the shrinkage and
scarring of the liver caused by cirrhosis.

However, the contractility of these vessels is also
controlled by a balance of two different biochemical signals -
a family of proteins known as endothelins, which act to
constrict vessels, and nitric oxide, a naturally produced
chemical that can relax blood vessels. In livers injured by
cirrhosis, researchers have noticed an overproduction of
endothelins and a decrease in nitric oxide production.

"In our series of experiments, we were able to use the virus
as a 'shuttle' to carry a genetic 'cargo' directly to the
affected cells," Rockey said. "Once the virus infected the
target cells, the genes produced nitric oxide, which opened up
the vessels and significantly reduced the hypertension."

The adenovirus "vector" used by Rockey is the same one used
in many similar experiments across the country for different
diseases. The adenovirus has many advantages for gene therapy -
it can carry practically any size gene, it can infect virtually
all cells in the body, and it can be easily produced in large
quantities.

Over the years, gene therapy researchers have noted that the
adenovirus has an special affinity for the liver. The key,
however, was to infect the appropriate cells.

"While many different liver cell types became infected and
produced nitric oxide, the adenovirus vector was more effective
in infecting the cells that line the vessels than other cells
deeper in the vessels," Rockey said.

The target cells, including sinusoidal endothelial cells and
stellate cells, are highly specialized cells that appear only
in the liver and which line the tiny blood vessels that form a
vast network throughout the organ. They act as a buffer between
circulating blood and the hepatocytes, key liver cells that
produce as many as 5,000 different proteins.

"The gene was expressed to the highest degree in the
sinusoidal lining cells, a little less in the next layer of
cells, and even less in the hepatocytes," Rockey said,
explaining that as the virus moved from the blood through the
organ, it slowly lost its viral payload. However, enough nitric
oxide was produced by the cells - primarily the sinusoidal
lining cells -- to have a positive effect on the vessels,
Rockey said.

In the experimental model, the delivered genes keep
producing nitric oxide for about 10 days to two weeks. Part of
the continued research of the Rockey laboratory will be to find
ways to extend this period of gene expression.

Unlike other gene therapy experiments, Rockey's team did not
detect any signs that the rats' immune systems were mobilized
to fight off the virus.

Portal hypertension can be a devastating condition to treat,
Rockey said, because the continued pressure placed on the
circulatory system due to the resistance of blood flowing
through the liver can cause weaknesses and abnormalities in
blood vessels throughout the gastrointestinal and esophageal
tracts. Often, serious bleeding results when one these weakened
vessels burst.

"In humans, for example, about 30 percent of patients will
die in the hospital after the first bleeding complication from
portal hypertension," Rockey said. "The one-year mortality rate
after diagnosis is about 60 percent. This is a very difficult
condition to treat successfully."

So far, there is not an effective treatment for portal
hypertension. Drugs such as beta-blockers or nitrates can be
prescribed to improve control over vessel constriction, but
with limited success.

A newer surgical procedure, known as Transjugular
Intrahepatic Portal systemic Shunt (TIPS), is used to prop open
the portal vein with a shunt, but it is usually used as a
short-term fix for patients awaiting liver transplantation.
Other surgical procedures have not been very successful in
relieving the pressure, Rockey said.

Worldwide, cirrhosis is the 10th leading cause of death in
the world, with the primary causes being hepatitis C and
hepatitis B.