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We Are Not Alone:
Harnessing the Invisible World of Microbes Within Us
New approach to gene therapy uses symbiotic microbes to deliver genes
By Bijal P. Trivedi

Featured article.

Americans are compulsive about eradicating bacteria. They obsessively spray their homes, load their soaps with anti-microbial chemicals and insist upon antibiotics for almost any ailment. There are even fibers containing antibacterial agents for the ultimate purpose of creating germ-free socks and other apparel! But attitudes toward microbes may change as scientists explore a new route for gene therapy by using bacteria to deliver therapeutic genes.

The idea is to take microbes already at home in the human body and endow them with therapeutic genes. When the microbes are returned to the body they revert to their symbiotic existence, but they also make a therapeutic protein for the treatment of a disease. In this way the bacterium serves as a microscopic drug factory. Some recent successes embracing this approach suggest these medicinal microbes may have a future.

A) Antifungal drugs for treating yeast infections delivered using a gel or lotion must be reapplied hourly or daily.
B) Colonizing the mucous membrane with S. gordonii, which has been modified to produce anti-fungal toxins, allows continuous local production of the drug for several weeks.

Flemish scientists are treating intestinal inflammation in mice using a bacterium that has been modified to produce anti-inflammatory proteins. Italian researchers have created a bacterium that secretes an antibiotic-like molecule that effectively treats yeast infections in rats. And in the U.S., a small start-up company, Symbiontics, Inc. is exploring the potential of Leishmania, a microbe that lies nestled in immune cells called macrophages, to treat lysosomal storage diseases.

"Our idea was to piggyback on something that already lives in the body instead of using foreign objects like viruses or stem cells to introduce healthy genes," says Dennis Vaccaro, founder of Symbiontics, Inc., in Wellesley, Massachusetts.

The advantages to using a microbe inhabiting the human body are two-fold. Microbes that have co-evolved with humans don't trigger immune reactions. Thus using them to smuggle therapeutic genes is a stealthy way to avoid the immune system. The other benefit is that by incorporating the therapeutic gene into the bacterium, it does the work of producing the drug.

Luciano Polonelli, of Università degli Studi di Parma, in Italy, and colleagues harnessed Streptococcus gordonii—a harmless, naturally occurring bacterium that colonizes mucous membranes in the human—to produce an antibody that kills Candida albicans. The C. albicans fungus is frequently the cause of human mucosal infection. It causes chronic and acute vaginal yeast infections in women and oral thrush in HIV patients.

"It's a very new approach," says Polonelli. "This is the first time a recombinant human commensal bacterium has been used to secrete an antibody."

Manufacturing this particular antibody in the same manner as other protein therapeutics is difficult. It is particularly unstable and degrades quickly at body temperature which is why it can't be synthesized and added to a lotion. On-site production by a therapeutic microbe overcomes this problem.

Symbiotic microbes living in the mucosal layer could deliver and produce vaccines against sexually transmitted diseases

Polonelli's team created a strain of S. gordonii carrying the antibody-producing gene. The antibody is designed to mimic the activity of a potent anti-microbial toxin from another fungus, Pichia anomala, and can kill a broad range of organisms.

"We show that 3 to 4 weeks after introduction of antibody-producing S. gordonii the Candida infection is gone and the natural physiology has been restored," says Polonelli. The rat model of vaginal yeast infection is very similar to human infection.

One of the largest obstacles to mucosal antibodies has been high cost and limited production capacity. Using a modified symbiotic bacterium like S. gordonii, which has been approved by the US Food and Drug Administration, to deliver therapeutic antibodies mucosally overcomes this limitation.

The antibody has a wide spectrum of antibiotic activity and can also kill Pneumocystis carinii and multi-drug-resistant strains of Mycobacterium tuberculosis, but these infections would require another route of delivery, says Polonelli. This technology could be applicable to infections on all mucosal surfaces, but the current method using S. gordonii is most efficient for treating mucosal infections in the mouth, gut and vagina.

Polonelli and colleagues are investigating the use of S. gordonii for vaccine delivery to mucous membranes. Currently there are no vaccines for preventing sexually transmitted diseases. Polonelli proposes that the bacteria could be used to deliver and produce vaccine antigens directly on the mucosal surface.

Whereas Polonelli's team decided to work with a bacterium that is already a thriving member of the body's bacterial community, Lothar Steidler of Ghent University and Flanders Interuniversity Institute for Biotechnology, in Gent, Belgium, and colleagues modified a common bacterium used in food fermentation. They are now using the microbe to treat inflammatory bowel disease (IBD), a chronic inflammation of the small and large intestine.

Steidler's laboratory specializes in the production of cytokines, proteins produced by immune cells. The cytokine interleukin-10, which reduces inflammation, has been approved for treating IBD, but is difficult and expensive to produce. One difficulty is that many strains of bacteria are unable to produce cytokines—the modified cells simply cease to grow. But this is not the case with Lactococcus lactis.

Using microbes to deliver genes is a new idea that affordably connects genomics with the patient; the drug is free because the microbe is the manufacturing plant

Steidler's team found that L. lactis is one of the few types of bacteria able to produce IL-10 and remain healthy. The finding led the researchers to wonder whether simply administering IL-10-producing bacteria could treat mice with either genetic or chemically induced chronic colitis. Currently, the usefulness of IL-10 is limited because systemic delivery of the protein causes a suppression of the entire immune system. Local delivery requires injections and enemas.

"L. lactis is used to produce dairy products, like hard Dutch cheeses," says Steidler. "It's also used to prepare fermented meats and vegetables, it's something we probably eat everyday and it is regarded as safe by the FDA."

IBD—which includes ulcerative colitis and Crohn's disease—is common in western society and affects about one in one thousand individuals. The intestinal inflammation can cause diarrhea, abdominal cramps, weight loss, and nausea and can recur throughout life. In severe cases, malnutrition and dehydration due to poor absorption of nutrients in the gut, can lead to death.

The drug-producing bacteria were added to a watery slurry and fed to the mice. Mice lacking both copies of the IL-10 gene are genetically prone to developing colitis by the time they are eight weeks old. Steidler's team found that a single dose of IL-10-producing L. lactis prevented colitis in the mice. The mice had slight inflammation of the intestinal lining, but were not significantly different from healthy mice. By contrast, untreated mice developed a severe inflammation. The treatment was also effective in mice with chemically induced colitis.

A) The thick band of purple shows inflammation of the intestinal lining in a mouse genetically prone to colitis.
B) After four weeks of daily treatments with the IL-10-producing L. lactis the inflammation has disappeared and the lining of the intestine resembles that of a healthy mouse.

Symbiontics is focusing on the treatment of lysosomal storage diseases of which there are about 50, including Gaucher disease, Fabry, and TaySachs. Vaccaro's team intends to use Leishmania, a microbe that lives quietly inside the lysosomes of macrophages, to treat these disorders.

A lysosome is like the digestive system for the cell. It is a bubble filled with an acidic soup of enzymes that degrades sugars, proteins and other large molecules. In lysosomal storage disorders some of these enzymes are malformed or absent, which causes a dangerous buildup of certain chemicals. Vaccaro's intention is to modify Leishmania by adding the gene to produce normal versions of the enzymes in order to correct the disease.

"Leishmania is the perfect organism, because it targets the correct cell and the correct compartment," says Vaccaro.

Genome projects are rapidly providing a catalog of all the genes, but what is lacking are vehicles to deliver healthy genes to people whose copies are missing or mutated, says Vaccaro. "Using symbiotic microbes to deliver genes is a new idea that connects genomics with the patient in an affordable way. Here is a delivery device that provides the drug for free because the microbe is also the manufacturing plant," says Vaccaro.

"Protozoa live in just about every ecological niche in the body, which makes them ideal for targeting a range of diseases," says Vaccaro. There are about 30 types of protozoa that can infect humans. They have sophisticated genomes and can evade the immune system and live in the body for decades without symptoms. The other advantage to using protozoa is that, like human cells, they are eucaryotic, which means they are able to modify proteins in ways that bacteria cannot.

One of the major advantages of this technology is that it is reversible and, unlike viral and stem cell gene therapy, there is no mutagenic potential. The therapeutic protein is produced only as long as the microbe remains in the individual's body.

Symbiontics has now engineered up to 10 strains of Leishmania, each of which produces and secretes a different human protein. The researchers are currently testing the microbes in animal models.

Some scientists estimate there are as many microbes living in the body as there are human cells, many of which we can't live without, says Vaccaro. He believes that a better understanding of these microbes may enable humans to ride the coat tails of a few for medicinal purposes. "One of the next big genome-size type projects would be to define all the microorganisms in the human body and understand the discussions between host and microbes," he says.

In a report recently published in Science, researchers have examined the genetic interactions between mice and a numerous bacterial inhabitant, Bacteroides thetaiotaomicron (B. theta). The bacterium is also a prominent microbe in humans.

Using gene chips containing about 25,000 mouse genes and a laser technique that enables isolation of single cells, researcher Jeffrey Gordon, of Washington University School of Medicine, in St. Louis, Missouri, and colleagues studied the effect of B. theta on mouse gene activity.

"What was most surprising was the number of normal functions affected by a single organism," says Gordon. B. theta affected genes involved in the absorption of sugars and fats, blood vessel formation, intestinal development, the metabolism of toxic compounds, and maintenance of the mucosal barrier of the intestine.

For years scientists have speculated about the great importance of symbiotic microbes living within us. Now, with DNA microarrays and the laser technology we can explore the scope of their influence, says Gordon. "But we must have a better understanding of how much human gene activity is modulated by our microbial partners before converting some of them into drug factories for therapeutic purposes," he added.

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Hooper, L.V. et al. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291, 881-884 (February 2, 2001).
Vaccaro, D.E. Symbiosis therapy: the potential of using human protozoa for molecular therapy. Mol Ther 2, 535-538 (December 2000).
Beninati, C. et al. Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria. Nat Biotechnol 18, 1060-1064 (October 2000).
Steidler, L. et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352-1355 (August 25, 2000).

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