Now University of Florida researchers may be one step closer to finding a safe way to spur production of this missing protein in patients with the most common form of the hereditary bleeding disorder. Using a dormant strand of DNA that has quietly existed in fish for millions of years, the researchers replaced the faulty gene responsible for the disease in neonatal mice, according to findings published online this month in the journal Molecular Therapy.
"The degree to which these patients have problems from hemophilia stems from how much of this protein, factor VIII, is missing," said Bradley Fletcher, M.D., Ph.D., a UF assistant professor of pharmacology and one of the lead authors of the study. "If they have very low levels of it, they have lifelong problems of bleeding, but what's even more problematic for them is they bleed into their joints, knees, hips and ankles, which limits their mobility."
Although hemophiliac mice don't develop some of these more extreme symptoms of the disease, gene therapy prevented profuse bleeding in the animals, the findings show.
More than 18,000 Americans, nearly all men, have hemophilia A, the most common form of the disease, according to the Centers for Disease Control and Prevention. Currently, the only safe treatment for the disorder is a purified form of the protein, but it can cost patients thousands of dollars and its effects don't last long. Scientists have been trying to find a safe way to perform gene therapy in hemophilia patients for years, but problems with the viruses typically used to transport needed genes to their target destinations have stymied their success, Fletcher said.
Researchers usually hide corrective genes inside viruses, which then infect cells. Without the virus to act as a key, the gene would be unable to enter the cell. But viral gene therapy has been associated with medical complications, and a few human patients have died as a result.
Instead, UF researchers used a novel nonviral approach, employing a strand of DNA present in modern-day fish called a transposon to transport the gene directly into the DNA of the mice. Nonviral therapy is thought to be safer, Fletcher said.
Transposons have the natural ability to bounce to different positions in DNA, allowing them to chauffeur genes into the cell. The transposon UF researchers used is one of a few that work in mammals, but until University of Minnesota scientists discovered it in 1997, it had remained hidden in the DNA of fish like trout for 15 million years. Years of mutations in the genetic code had buried the transposon, silencing its ability to issue molecular marching orders.
Fletcher and researchers Li Liu, M.D., Ph.D., an adjunct postdoctoral associate in the department of pharmacology and therapeutics, and Cathryn Mah, a UF assistant professor of pediatric cellular and molecular therapy, used the transposon to inject the gene into endothelial cells, which line blood vessels and other parts of the body. This was unique, Fletcher said, because the liver is generally considered the body's powerhouse for producing the protein needed to keep hemophilia at bay. The study showed that these endothelial cells also can produce enough protein to correct the problem, he said.
"I think endothelial cells are potentially a very important cell to make factor VIII," said Katherine P. Ponder, M.D., an associate professor of medicine and a hemophilia researcher at Washington University in St. Louis. "They're a very attractive cell type to express it."
It also was the first time researchers attempted such an approach on an animal so young. In adult mice, the immune system normally views clotting protein as an invader and rejects it after traditional gene therapy. But UF researchers bypassed this immune respo nse by performing gene therapy on mice within 24 hours of their birth, when their immune systems were still naive and would accept the protein.
This type of approach cannot be used in human babies yet because doctors have no way of gauging how severe hemophilia is in newborns. Patients with mild disease will have fewer problems and the benefits of gene therapy may not outweigh risks to the baby, Fletcher said.
Ponder said she thinks the technique also may prove to share some of the same problems as viral-based gene therapy, potentially activating cells that cause cancer.
Now UF researchers are studying different ways to use the transposon and trying to find a way to overcome the immune attack when performing gene therapy on adult animals.
"I don't think the research is done," Mah said. "But this is definitely a step forward for hemophilia gene therapy."