Until the height of the AIDS crisis in the 1980s, few Americans were aware of hemophilia, a genetic disorder that prevents blood from clotting efficiently and causes its victims to bleed in their muscles and joints. Then Ryan White, a 16-year-old from Kokomo, Ind., contracted the HIV virus when he received a transfusion of contaminated blood plasma intended to treat his hemophilia. Thanks to extensive media coverage, White’s story raised the public’s consciousness of both diseases.
Blood transfusions are infinitely safer than they were 20 years ago, largely because of improved screening at blood banks and advanced purification processes. But each time a hemophiliac receives a plasma treatment, he or she still has a small chance of being exposed to HIV, hepatitis C and other blood-borne diseases. While safety has improved, the ability to obtain enough protein to effectively treat the disease has not. Thus, blood-based treatments are limited and expensive. That’s a serious obstacle, especially in underdeveloped countries where 80 percent of the world’s hemophiliacs live.
Since 1987, William Velander, chair of chemical & biomolecular engineering, has been researching safer, low-cost treatments for hemophilia. His efforts have resulted in genetically engineered pigs with the potential to produce large amounts of Factor VIII and Factor IX in their milk. Factors VIII and IX are blood proteins produced in the liver that are genetically deficient in people with hemophilia type A and type B, respectively.
Velander, the D.R. Voelte and N.A. Keegan Endowed Chair in Engineering, and a team of researchers will test the pig-derived Factor IX coagulant in hemophiliac dogs during the next two years. So far, the tests in vitro and in hemophiliac mice have been highly successful, and Velander believes clinical trials in humans will begin within five years.
An unconventional solution
Velander, whose background is in biochemistry and biomolecular engineering, described his journey into hemophilia research as serendipitous. He formed a close relationship with coagulation science specialists during his undergraduate and postgraduate studies and became fascinated with the idea of combining engineering and medical knowledge to treat blood disorders.
He began his research as a professor at Virginia Tech University. Velander was assisted by Kevin Van Cott, a doctoral student from Purdue University who is now an associate professor of chemical & biomolecular engineering at the University of Nebraska–Lincoln. Velander and Van Cott formed a partnership with the American Red Cross to produce genetically engineered versions of anti-hemophilic factors VIII and IX. While research for Factor IX is three years ahead of research for Factor VIII, a collaboration with University of Michigan researchers Randall Kaufman and Steven Pipe has accelerated the progress on a therapy for hemophilia type A.
Velander’s first step was to isolate the hemophilic factor in human blood. At first, researchers believed they could use a monoclonal antibody to capture the protein in human blood, and then purify it to eliminate viruses. However, they decided that although the process was safe, the supply would still be inadequate. Using a genetically engineered animal cell was the best way to increase the abundance of Factor IX.
They had to find the perfect animal—and the perfect cell. Velander said researchers considered several criteria: the animal needed the ability to produce complex proteins because Factor VIII Factor IX are two of the most complex proteins known; the animal’s biochemistry had to be similar to humans’; the cell had to be stable and prodigious in its production setting; and the molecules needed a long circulation lifetime once inserted into the body.
In the 1980s, advances in breast cancer research proved that mammals’ mammary glands make and secrete large volumes of protein. Researchers at universities in the United States, the Netherlands and Scotland were engineering transgenic cows, sheep and goats and using the milk to produce therapeutic proteins.
Velander considered using traditional dairy livestock, but found that ruminants’ mammary glands placed a problematic molecular signature on proteins made in milk. Any protein bearing this signature wouldn’t survive in the human body long enough to be effective, Velander said.
His decision to obtain milk proteins from a pig was unorthodox. However, Velander said, the biochemistry of pigs is the closest to our own. Pigs produce less milk than most dairy animals, but that doesn’t matter. “Pig proteins are so concentrated and potent that only a tiny amount needs to be injected in the body to work,” he said.
Velander produced genetically engineered cells by inserting the human Factor IX gene into the animal cell. Figuring out how to reproduce the cell was a challenge. Reproducing cells in a stainless steel bioreactor prevented disease and contamination. However, Velander found that a bioreactor was 100 to 1,000 times less effective than cells living within tissue. This is because fewer cells can grow and receive nutrients in a culture setting compared with tissue.
Not a typical barnyard animal
Thus, Velander used the natural productivity of tissue as a bioreactor setting—in this case, the mammary gland of a transgenic pig. To produce such an animal, he inserts the Factor IX gene into several freshly fertilized one- or two-cell embryos. The embryos are transferred to a surrogate mother pig. Through its natural gene maintenance machinery, the cell slices the Factor IX gene into the pig’s chromosomes. The gene becomes a transgene, or a permanent part of the unborn pig’s heredity. Ten to 30 percent of a litter derived from these embryos is transgenic. Once a transgenic pig reaches maturity, they are bred with ordinary production pigs. After farrowing, the pigs are milked for two 50-day lactation cycles. Each sow typically yields 100 to 300 liters of milk annually.
The first pig able to produce Factor IX was born in 1994. Her milk contained about 75 times more Factor IX than human plasma. With optimization in molecular design of the transgene, recent pigs have produced more than 100 times the amount of Factor IX found in human plasma. Velander estimates that milk from just 100 to 200 transgenic pigs could produce enough Factor IX to meet the world’s demand for hemophilia type B therapy.
The first Factor VIII pig produced as much as 20 times the amount of the protein found in human blood. Newer versions of this pig, expected to become available within two years, should produce high levels of the protein needed for hemophilia type A treatment.
Velander acknowledges that the public is skeptical of the benefits of cloning and genetically modified animals. His pigs live in a secluded facility in the mountains of Virginia to shield them from disease. Visitors are required to shower and change into clean clothing before entering. “The animals are extraordinarily well taken care of and arguably have a higher quality of life than most people in this world,” he said. “Even activists have been generally agreeable that this kind of effort is a win-win for the animal and people.”
Tackling a global problem
About 100,000 people worldwide have hemophilia type B, and 500,000 have hemophilia type A. The World Federation of Hemophilia estimates that the real total may be two or three times higher, but many hemophiliacs die during infancy because they’re not properly diagnosed and treated. And while Americans have difficulty stomaching the cost of treatment, people in Third-World countries can’t afford treatment at all, Velander said. He estimates that his new drug would cost $2,000 to $10,000 annually. For a severe hemophiliac living in the United States, plasma treatments cost up to $200,000 annually. The cost of drug therapy is several times higher.
“While the U.S. can certainly benefit from this technology, it’s important to note that the biggest impact will be in lesser developed countries where 80 percent of the dire need exists,” Velander said. “This has the potential to give them access to sophisticated healthcare they don’t have now.”
Researchers will test the potent, pig-based coagulant in hemophiliac dogs, and Velander said the results in hemophiliac mice have been promising. In September 2005, UNL received a five-year grant in excess of $10 million from the National Institute of Health’s (NIH) National Heart, Lung and Blood Institute. The grant is being used to complete preclinical research in animals. If everything goes according to plan, Velander will seek approval from the federal Food and Drug Administration to do clinical evaluations in humans within five years.
UNL chemical & biomolecular engineering professors Van Cott and Michael Meagher and research assistant professor Todd Swanson are assisting Velander. Paul Monahan and Timothy Nichols from the University of North Carolina-Chapel Hill are leading the animal trials. Others involved in the grant are Stephan Abramson, LifeSci Partners of California; Julian Cooper, ProGenetics LLC of Virginia; and William Dernell and Mark Manning of Colorado State University.
The researchers also are studying new ways to administer the drug because chronic intravenous injections are dangerous, especially in young children and infants. He hopes the drug can be administered through the mouth, nose or trachea. This may be possible because of the abundance of medicine that transgenic pigs provide.
“This would be a welcomed miracle for the parents of hemophilic infants,” Velander said.