Despite a roller-coaster ride of ups and downs during the past 15 years, gene therapy has continued to attract many of the world's brightest scientists. They are tantalized by the enormous potential that replacing missing genes or disabling defective ones offers for curing diseases of many kinds.
One group, consisting of researchers from the University of Wisconsin Medical School, the Waisman Center at UW-Madison and Mirus Bio Corporation of Madison, Wis., now reports a critical advance relating to one of the most fundamental and challenging problems of gene therapy: how to safely and effectively get therapeutic DNA inside cells.
The Wisconsin scientists have discovered a remarkably simple solution. They used a system that is virtually the same as administering an IV (intravenous injection) to inject genes and proteins into the limb veins of laboratory animals of varying sizes. The genetic material easily found its way to muscle cells, where it functioned as it should for an extended period of time.
"I think this is going to change everything relating to gene therapy for muscle problems and other disorders," says Jon Wolff, a gene therapy expert who is a UW Medical School pediatrics and medical genetics professor based at the Waisman Center. "Our non-viral, vein method is a clinically viable procedure that lets us safely, effectively and repeatedly deliver DNA to muscle cells. We hope that the next step will be a clinical trial in humans."
Wolff conducted the research with colleagues at Mirus, a biotechnology company he created to investigate the gene delivery problem. He will be describing the work on June 3 at the annual meeting of the American Society for Gene Therapy in Minneapolis, and a report will appear in a coming issue of Molecular Therapy. The research has exciting near-term implications for muscle and blood vessel disorders in particular.
Duchenne's muscular dystrophy, for example, is a genetic disease characterized by a lack of muscle-maintaining protein called dystrophin. Inserting genes that produce dystrophin into muscle cells could override the defect, scientists theorize, ensuring that the muscles with the normal gene would not succumb to wasting. Similarly, the vein technique can be useful in treating peripheral arterial occlusive disease, often a complication of diabetes. The disorder results in damaged arteries and, frequently, the subsequent amputation of toes.
What's more, Wolff says, with refinements the technique has the potential to be used for liver diseases such as hepatitis, cirrhosis and PKU (phenylketonuria).
In the experiments, the scientists did not use viruses to carry genes inside cells, a path many other groups have taken. Instead, they used "naked" DNA, an approach Wolff has pioneered. Naked DNA poses fewer immune issues because, unlike viruses, it does not contain a protein coat (hence the term "naked"), which means it cannot move freely from cell to cell and integrate into the chromosome. As a result, naked DNA does not cause antibody responses or genetic reactions that can render the procedure harmful.
Researchers rapidly injected "reporter genes" into a vein in laboratory animals. Under a microscope, these genes brightly indicate gene expression. A tourniquet high on the leg helped keep the injected solution from leaving the limb.
"Delivering genes through the vascular system lets us take advantage of the access blood vessels have - through the capillaries that sprout from them - to tissue cells," Wolff says, adding that muscle tissue is rich with capillaries. Rapid injection forced the solution out of the veins into capillaries and then muscle tissue.
The injections yielded substantial, stable levels of gene activity throughout the leg muscles in healthy animals, with minimal side effects. "We detected gene expression in all leg muscle groups, and the DNA stayed in muscle cells indefinitely," notes Wolff.
In addition, the scientists were able to perform multiple injections without damaging the veins. "The ability to do repeated injections has important implications for muscle diseases since to cure them, a high percentage of therapeutic cells must be introduced," he says.
The researchers also found that they could use the technique to successfully administer therapeutically important genes and proteins. When they injected dystrophin into mice that lacked it, the protein remained in muscle cells for at least six months. Similar lasting power occurred with the injection of erythropoietin, which stimulates red blood cell production.
Furthermore, in an ancillary study, the researchers learned that the technique could be used effectively to introduce molecules that inhibit - rather than promote - gene expression, a powerful new procedure called RNA interference.
"This could be very useful if you want to down-regulate a protein that's causing a muscle disorder, such as with myotonic dystrophy," says Wolff.
In the late 1980s, Wolff and his UW-Madison colleagues surprised the scientific world with their discovery that they could get genes to express in muscle cells simply by injecting naked DNA into rodent muscle. The Wisconsin Alumni Research Foundation (WARF) licensed the technology to Vical, a California biotechnology company.
Once Wolff created Mirus, a local company, he and his colleagues turned their attention to the vascular system, a conduit to multiple leg and arm muscles they felt would work more efficiently than direct injection into muscle. WARF licensed the vascular technique to Mirus, which now holds the patent and continues to commercialize the technique.
In their first studies, the researchers focused on arteries, but then began to concentrate on veins. "Injecting any substance into arteries carries a degree of risk since, unlike veins, only one artery feeds a whole limb," notes Wolff.
In a related procedure, they experienced excellent results with high-pressure injection of genes into the tail veins of rodents, a technique that yielded extensive gene expression in the animals' livers.
"We think the genes traveled from the capillaries through the relatively large holes that exist in liver cells," Wolff says, adding that the technique has become a successful research tool for many laboratories around the world.
"For delivering genes to limb muscles, the vein approach is so simple," he says. "We never expected it to work so well."
Collaborating on the study were James Hagstrom, Julia Hegge, Mark Nobel, David Lewis and Hans Herweijer, from Mirus Bio; and Guofeng Zhang and Vladimir Budker, from the Waisman Center.