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For the Love of Blood
By Bijal P. Trivedi

Featured Article.

On August 13, 1904, the day after the birth of Alexis, son of Tsarina Alexandra and Tsar Nicholas II, the baby Tsarevich began to bleed profusely from the navel. While the bleeding eventually stopped, hereafter his parents watched helplessly as their son developed grotesque bruises after bumping himself or falling. During one eleven-day bleeding spell Alexandra, an obsessively religious woman, summoned the monk Rasputin, reputed to be a healer and mystic, to cure Alexis. Two days after Rasputin's visit Alexis gradually stopped bleeding. Rasputin won the admiration of the Tsarina, became a cherished political advisor, and the rest is history.

Hemophilia, the rare bleeding disorder that plagued English, Spanish, German, and Russian royal families and triggered the events leading to the fall of the Tsarist Empire, is now poised to assume landmark importance in gene-based medicine. Effective treatment of this disease with the delivery of healthy genes could open the floodgates of genetic medicine and allow other more complex disorders to be treated using similar technology. Preliminary results from clinical trials look promising and gene therapy researchers hope that treating hemophilia will boost public confidence in the field, which plummeted after the death of 18-year-old Jesse Gelsinger, the first patient to die in a gene therapy clinical trial in September last year.

The theory behind gene therapy is deceptively straightforward. Use a gene instead of a drug to treat disease by giving it to a patient whose copy has been lost or damaged.

Hemophiliacs bleed spontaneously because they carry mutations in genes required for blood to clot; lack of Factor VIII or Factor IX causes hemophilia A or hemophilia B, respectively. By giving patients the gene for Factor VIII or IX the cells can produce the clotting protein.

Treating severe hemophilia can cost up to $100,000 per year

Mark Kay, a Stanford researcher, began working on hemophilia B ten years ago. For Kay, it was not the hemophilia that held allure, but rather that it was a simple disease and therefore a good candidate for gene therapy. "If we couldn't treat hemophilia by gene therapy other diseases would be tougher. And this is tougher to achieve than I thought," says Kay.

To researchers, treating hemophilia appears uncomplicated. The clotting proteins, which are normally produced by the liver, can be made by almost any tissue and find their way into the blood. No specific concentration is required. Virtually any increase in protein levels is therapeutic, and levels as low as six percent of normal can stop most bleeding. There are both dog and mouse models of hemophilia, bearing a tight resemblance to the human disease, which are invaluable to researchers like Kay for testing potential therapies. And it is easy to tell whether the therapy is working—the bleeding stops or it doesn't.

“Cure” is an unpopular word

The apparent simplicity of the disease has drawn many researchers and companies into a race to develop safe gene-based medicine. At a meeting of the American Society of Gene Therapy in Denver from May 31 to June 4, there were 29 presentations concerned with using gene therapy for hemophilia. Today three companies—Avigen, Inc., TransKaryotic Therapeutics, Inc., and Chiron Corporation—with different strategies for gene delivery are in the midst of Phase I clinical trials designed to see whether the treatment is safe. Phase II trials determine if the treatment is effective.

Kay and Katherine High, of the University of Pennsylvania School of Medicine, were competitors until two and a half years ago, when together they teamed up with Avigen for clinical trials, which began about a year ago. The trials focused on High's approach that uses adeno-associated virus (AAV) to deliver the Factor IX gene into muscle cells.

Gene therapy for hemophilia B: Avigenís Approach

1. Viral genes are removed from AAV 2. A gene for blood clotting protein Factor IX is placed inside the empty AAV 3. The Factor IX-bearing viruses are injected into the thigh muscle 4. The viruses infect muscle cells, and the gene begins to produce the Factor IX protein that allows the blood to clot.

The therapy is administered to the patients by injecting a solution containing the virus and the gene into the thigh muscle. A low dose requires about 20 injections, says Catherine Manno, who is conducting the clinical trials at the Children's Hospital of Philadelphia. When the virus infects the muscle cells it releases the DNA into the nucleus where the Factor IX gene produces protein that flows into the blood.

Kay and High's Phase I trials have already shown some therapeutic benefits. Low doses of AAV, which were administered solely to test for toxic side effects unexpectedly produced measurable amounts of the Factor IX in two of three patients. Results for the three patients who received medium doses have been obtained but are unpublished, and high dose studies will begin this summer.

"The important message based on these six patients is that so far the therapy is safe, well tolerated, and without any adverse side effects," says Manno.

When asked whether the patients in the trial are obtaining curative levels of Factor IX, Manno tenses. Gene therapists don't like to use the word "cure" when discussing final clinical outcomes of their work. "Cure means treatment for life. This would be a little to good to be true. A more realistic goal would be effective treatment," says Savio Woo, president of the American Society of Gene Therapy.

"For a man with severe hemophilia, who had less than one percent of Factor IX, having five percent of normal Factor IX levels would be an enormous improvement in his day-to-day life—he might even consider himself cured," says Manno.

The severity of hemophilia is correlated with the amount of Factor IX in the blood. Among severe hemophiliacs, spontaneous bleeding occurs frequently in internal organs and the brain. Frequent bleeding in the joints and muscles releases enzymes that corrode the bones, cartilage and nerves causing excruciating pain. People with a more moderate form of the disease have between one and five percent of clotting proteins and are protected from spontaneous bleeding. Those with five to twenty percent of Factor IX have a mild form of hemophilia that rarely interferes with daily life.

Severe hemophilia is currently treated with two injections of clotting proteins a week, which can cost up to $100,000 per year. Supplying the body with a healthy gene would allow it to make its own clotting factor.

Some question whether viruses are safe

While collaborating on these trials Kay believes placing the virus into the muscles may not be the most effective way to deliver the gene. When AAV is delivered to liver cells, the healthy gene actually incorporates itself into the DNA of the chromosome, according to Kay.

"If you have a gene that integrates into the chromosome, then you have the possibility the gene will produce Factor IX for the life of the patient," says Kay. It is still unclear what happens when the gene is delivered in the muscle.

Kay also believes that liver cells are more easily infected with AAV, suggesting that lower doses can be used. This lessens the chance of alerting the immune system to launch a defense against the virus. Kay expects that clinical trials for AAV delivery of Factor IX to the liver will begin late this year.

Kay has taken an unusually aggressive approach to battling hemophilia. Unlike most of his colleagues he has launched a comprehensive assault on the disease trying a slew of stealthy vehicles to encase the healthy genes and get them into the cells.

After years of research, clinical benefits are finally emerging

What makes Kay's approach unusual is that he is willing to try almost everything. "I'm not prejudiced against any strategy or virus. I just want to use what I think will work—and I've been complemented and criticized for this," he says. He has experimented with five types of viruses to determine which is best able to smuggle the genes past the immune system and into the cells. He has also been trying to inject the desired DNA directly into the blood without enveloping them in a virus.

Kay's search to find the most efficient and stealthy virus addresses two of the major hurdles facing gene therapy researchers. The first challenge is to get enough of the healthy gene into the patient to induce a therapeutic effect. The other issue is getting genes into cells without the immune system attacking either the virus, or the protein produced by the new gene. If the immune system destroys the new therapeutic protein, the patient will not benefit from the gene therapy. Targeting genes to specific tissues and regulating the quantity of protein produced are problems that are also pending.

Researchers at Chiron and TransKaryotic Therapeutics are also progressing with clinical trials, but for treating hemophilia A. The Chiron study began in June of 1999 and has delivered a virus containing the Factor VIII into 10 patients. At this point no side effects have been observed.

TransKaryotic is also delivering Factor VIII but without using viruses. "We feel that a non-viral strategy may be safer," says David Roth, of Beth Israel Deaconess Medical Center, in Boston, and principal investigator for the clinical trial. Viruses have been known to cause severe immune responses in patients and viruses randomly incorporating the gene anywhere on a chromosome can lead to problems, says Roth.

TransKaryotics's approach is to remove fibroblast cells from the patient, introduce the new gene, choose a cell where the gene is incorporated in a safe position on the chromosome, replicate it, and then inject the cells back into the patient's skin. In November, TransKaryotic reported that six patients had been enrolled in the study, and had been treated without significant side effects. Since then, more patients have been enrolled, and the study continues to have a good safety record. "I'm reluctant to jump on a podium and hype it up, but so far I'm happy with the way the study has gone," says Roth.

"For gene therapy researchers this is more than just a treatment for hemophilia, it is proof of a concept. After all the years of research we are finally seeing some clinical benefits emerging," says Woo.

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