BioWorld International Correspondent

LONDON - The first-ever clinical trial to test whether live, genetically modified bacteria can help to treat disease in humans has just begun in the Netherlands.

Twelve patients with Crohn's disease are receiving enteric-coated capsules to deliver bacteria normally used in cheese-making to their intestines. The modified microorganisms will, in this case, deliver the anti-inflammatory cytokine interleukin-10 to the intestinal mucosa.

The Dutch government gave permission for the trial to go ahead after the researchers who developed the therapy devised a way of ensuring the genetically modified bacteria would not be able to survive outside the body. Nevertheless, the patients will have to stay in secure accommodations until they stop excreting bacteria with any trace of the transgene.

The researchers, led by Lothar Steidler, then professor of biotechnology at the University of Ghent in Belgium, describe the strategy that allows the modified bacteria to survive only while in the gut in a paper in Nature Biotechnology (June 15 online publication) titled "Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin-10."

Steidler, who is currently based at the University of Cork in Ireland, told BioWorld International: "We removed a gene that is essential for this bacterium to survive outside of the body, and replaced it with the gene for IL-10. This technology has a vast area of potential applications, because it can be used to deliver any therapeutic protein that can be given through the mucosal route."

Steidler and his colleagues began working several years ago on developing a way of delivering therapeutic proteins to the mucosal surfaces of the body, such as the lungs and the gut. For their vector, they focused on bacteria of the species Lactococcus lactis, which has been used to make cheese for thousands of years (including today's mozzarella and Gouda) without any apparent harmful effects on those who consume it.

For their therapeutic protein, they selected IL-10, which damps down the excessive inflammatory immune response that is responsible for the symptoms of Crohn's disease.

Steidler told BioWorld International, "Crohn's disease is an ideal disease to try to develop new therapies for, because most of the existing treatments have severe side effects and are very expensive, as well as not always being effective."

Crohn's disease is also common. An estimated 2.5 people per 1,000 in Western Europe, Scandinavia, North America and Australia suffer from inflammatory bowel diseases, about 90 percent of them with Crohn's disease. The disease is debilitating and disabling, as those with it have up to 30 bowel movements a day, and frequently develop malnutrition and anemia. About two-thirds eventually are treated with surgery, but the disease can be fatal if left untreated. Treatment costs per patient are about €16,000 a year, and the peak age of onset is falling, so many patients are now diagnosed in their teens.

The Belgian group took the gene for IL-10, engineered it on a plasmid into L. lactis and were able to show that the bacteria made biologically active IL-10. "We also inoculated this strain into a mouse model of inflammatory bowel disease, and saw, to our great happiness, that the animals were cured of their bowel inflammation," Steidler said. That work was published in Science in 2000.

"Colleagues at the Academisch Medisch Centrum in Amsterdam, led by Professor Sander Van Deventer, wanted us to pursue this approach to develop a therapy for humans, but we knew that it was a legitimate concern that the plasmid, or even the genetically modified organism itself, could escape and could survive in the environment," Steidler added. "So, when we applied to the Dutch government, we had a debate with the regulatory authorities about how we could tackle this problem."

The solution they fixed on was to exchange a gene essential to the survival of L. lactis with the gene for IL-10. That had the advantage that the transgene, rather than being on a plasmid, would be embedded in the chromosome of the bacterium and therefore less easily transferred to other bacteria.

The essential gene that they replaced was one encoding an enzyme called thymidylate synthase. This adds a methyl group to uracil or uridine in order to make thymidine or thymine. The bacterium has no alternative way of making thymidine, so unless thymidine or thymine are present in its environment (as they are in the gut, for example), it cannot make DNA. In the absence of thymidine, the bacterium switches on a suicide program and dies.

Experiments carried out by the Belgian group on pigs, reported in Nature Biotechnology, showed that the genetically modified L. lactis was able to produce human IL-10 in the animals' bodies. The authors wrote, "When deprived of thymidine or thymine [on leaving the animals' bodies], its viability fell by several orders of magnitude, essentially preventing its accumulation in the environment."

"This is a neat closed system," Steidler concluded. "The genetically modified organism dies once it is outside the body, and even if someone were to accidentally ingest some of the transgenic bacteria, the acid of the stomach and the bile will ensure that it is destroyed."