Rabbit trail may lead to human gene therapy.

Rabbit trail may lead to human gene therapy

In a small hop toward a gene therapy for humans, scientists have temporarily abated an inherited, cholesterol-elevating disease in rabbits using transplants of genetically modified liver cells.

The disease, known as familial hypercholesterolemia, results from a genetic defect causing a lack of the cell receptors that normally bind to and mediate the breakdown of low-density lipoprotein (LDL) cholesterol. In the absence of these receptors, LDL cholesterol builds up in the bloodstream, leading to artherosclerosis and an increased risk of heart attack or stroke.

A severe form of the disease strikes one in every million people in the United States, most of whom die of a heart attack between age 5 and 30. At present, the only moderately successful treatment for these patients is liver transplantation. Physicians treat a milder form, affecting one in 500 Americans, with drugs that are only partially effective and have undersirable side effects.

Researchers led by James M. Wilson of the Howard Hughes Medical Institute at the University of Michigan in Ann Arbor are now testing a gene therapy on the Watanabe rabbit, which invariably suffers from familial hypercholesterolemia and provides "as good an animal model as they come," Wilson says. The scientists targeted hepatocytes -- cells of the liver, the sole organ that metabolizes and excretes cholesterol -- in severely affected rabbits. Using a retrovirus, thery inserted the normal human LDL-receptor gene into hepatocytes removed from three rabbits, then tranplanted the modified cells into seven recipient rabbits.

Although the genetically altered cells boosted LDL-receptor activity to only 1 to 3 percent of the normal level, they substantially reduced blood cholesterol levels, the researchers report in the November PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE (Vol. 87, No. 21). Before treatment, the recipient rabbits had an average cholesterol concentration of 600 milligrams per deciliter of blood. (Less than 90 is normal for these rabbits.) Afterward, their counts dropped to between 400 and 450--a decrease of roughly 30 percent. A control group of six Watanabe rabbits that received hepatocytes lacking the functional gene showed no significant changes.

Within two weeks, however, cholesterol counts in the "cured" rabbits returned to pretreatment levels, probably because the immune system rejected the modified cells, Wilson says. The researchers now seek to prolong the effect by injecting the altered hepatocytes into the same rabbits from which the cells were taken. "That way we can circumvent the problem of rejection based on different cells," he says.

If the revised approach works, the next steps will be to "make the efficiency of gene transfer a whole lot better" and to perfect the transplantation technique, says study coauthor J. Roy Chowdhury of the Albert Einstein College of Medicine in New York City.

"For sure, it has a great potential [for human gene therapy]," comments Barbara Obrepalska-Bielska, a biologist at Lehigh University in Bethlehem, Pa. "But our knowledge about all the immunological [aspects of such transplants in humans] is very small, so there will be a lot of technical problems."

To get an idea of how humans would react to injections of these genetically modified hepatocytes, scientists would first have to investigate the effects of transplanting normal, unaltered liver cells into people with familial hypercholesterolemia, Wilson adds. Indeed, that approach may offer a workable alternative to gene therapy. Researchers at the University of Illinois in Chicago have already shown that transferring normal hepatocytes from healthy New Zealand White rabbits into severely afflicted Watanabe rabbits can lower cholesterol levels -- but "it didn't work 100 percent," says study leader Raymond Pollak. After injecting each of four Watanabe rabbits with four doses of normal hepatocytes over a three-week period, his team observed temporary cholesterol reductions ranging from 32.8 to 82.7 percent and lasting for more than 18 weeks. The findings will appear in the February TRANSPLANT PROCEEDINGS.

While Pollak's results are encouraging, such transplants would require a continuing series of injections, whereas Chowdhury's group hopes that improvements in the gene therapy approach will eventually bring lasting results with just one or two injections of modified, self-proliferating cells. "Trying to introduce the gene would probably be the highest form of refinement [in treating the disorder]," Pollak say. "But the next best thing would be to transfer the normal hepatocyte."

When and if human gene therapy for this disease becomes reality--a prospect some scientists envision within the next decade -- it would benefit only those individuals whose high cholesterol results from familial hypercholesterolemia. Many different factors can elevate cholesterol levels, Chowdhury says, and unless a patient lacks the functional LDL receptor, gene-altered liver cells "won't help."