Mar. 20, 2000 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.
Joining Rockey in the study were Qing Yu, Rong Shao, Hu Sheng Qian and Dr. Samuel George, all from Duke.
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