Finding the golden genesBy Patrick Barry Web edition : Wednesday, August 13th, 2008
Advances in gene therapy could tempt some athletes to enhance their genetic makeup, leading some researchers to work on detection methods just in case. Click here to see an animation of how one gene therapy can enhance endurance. This month — 8/8/08, to be precise — the curtain rose on what many experts believe could prove to be the first genetically modified Olympics. For the unscrupulous or overdriven Olympic athlete, the banned practice of “doping” by taking hormones or other drugs to enhance athletic prowess may seem so last century. The next thing in doping is more profound and more dangerous. It’s called gene doping: permanently inserting strength- or endurance-boosting genes into DNA. “Once you put that gene in, it’s there for the rest of that person’s life,” says Larry Bowers, a clinical chemist at the U.S. Anti-Doping Agency in Colorado Springs, Colo. “You can’t go back and fish it out.” Scientists developed the technology behind gene doping as a promising way to treat genetic diseases such as sickle-cell anemia and the “bubble boy” immune deficiency syndrome. This experimental medical technology — called gene therapy — has begun to emerge from the pall of early failures and fatalities in clinical trials. As gene therapy begins to enjoy some preliminary successes, scientists at the World Anti-Doping Agency, which oversees drug testing for the Olympics, have started to worry that dopers might now see abuse of gene therapy in sport as a viable option, though the practice was banned by WADA in 2003. “Gene therapy has now broken out from what seemed to be too little progress and has now shown real therapies for a couple diseases, and more coming,” says Theodore Friedmann, a gene therapy expert at the University of California, San Diego and chairman of WADA’s panel on gene doping. While gene therapy research has begun making great strides, the science of detecting illicit use of gene therapy in sport is only now finding its legs. To confront the perceived inevitability of gene doping, Friedmann and other scientists have started in recent years to explore the problem of detecting whether an athlete has inserted a foreign gene — an extra copy that may be indistinguishable from the natural genes — into his or her DNA. It’s proving to be a formidable challenge. Genetic makeup varies from person to person, and world-class athletes are bound to have some natural genetic endowments that other people lack. Somehow, gene-doping tests must distinguish between natural genetic variation among individuals and genes inserted artificially — and the distinction must stand up in court. Scientists are fighting genetics with genetics, so to speak, enlisting the latest technologies for gene sequencing or for profiling the activity of proteins to find the telltale signs of gene doping. Some techniques attempt the daunting search for the foreign gene itself, like looking for a strand of hay in an enormous haystack. But new research could also lead to an easier and more foolproof approach: detecting the characteristic ways that an inserted gene affects an athlete’s body as a whole. Resurgence of gene therapy In 1999, 18-year-old Jesse Gelsinger died during a gene therapy trial for a rare liver disease. Investigators later attributed his death to a violent immune reaction to the delivery virus rather than to the therapeutic gene. His death was a major setback for the field. It also may have scared away early would-be gene dopers. In recent years, safety and efficacy of gene therapy have shown signs of progress in numerous clinical trials for conditions ranging from early-onset vision loss to erectile dysfunction. As scientists develop ways to use safer, weaker viruses for delivery, and as gene therapies wind their way through clinical trials, athletes and coaches might start to see gene doping as even more viable than they already do. In the courtroom during the 2006 trial of Thomas Springstein, a German track coach accused of giving performance-enhancing drugs to high-school–age female runners, prosecutors read aloud an e-mail Springstein had written that would shock the sports world. “The new Repoxygen is hard to get,” the e-mail read, according to press reports. “Please give me new instructions soon so that I can order the product before Christmas.” Repoxygen isn’t merely another doping drug such as a hormone or the latest designer steroid — it’s an experimental virus designed to deliver a therapeutic gene and insert it into a person’s DNA. British pharmaceutical company Oxford BioMedica developed Repoxygen in 2002 as a treatment for severe anemia. The therapy “infects” patients with a harmless virus carrying a modified gene that encodes erythropoietin, a protein that boosts red blood cell production. This protein, often called EPO, is itself a favorite among dopers seeking to increase their oxygen capacity, and hence their endurance. Viruses have the natural ability to inject genetic material into their host’s DNA. The host’s cells can translate that gene into active proteins as if the foreign gene were the cells’ own. So by delivering the gene for EPO within a virus, Repoxygen could potentially increase the amounts of EPO protein — and the change would be permanent. Athletes might also be tempted by perhaps the most tantalizing gene therapy experiment of all: the “mighty mouse.” In 1998, H. Lee Sweeney and his colleagues at the University of Pennsylvania School of Medicine injected mice with a virus carrying a gene that boosted production of insulin-like growth factor 1, or IGF-1, a protein that regulates muscle growth. As a result, the mice had 15 percent more muscle mass and were 14 percent stronger than untreated mice — without ever having exercised. The treatment also prevented the decline of muscle mass as the mice grew older. Other genetic paths to increase muscle strength and volume could include the gene for human growth hormone or segments of DNA that block a protein called myostatin, which normally limits muscle growth. Endurance might also be boosted by the gene encoding a protein called peroxisome proliferator-activated receptor delta, or PPAR-delta. Mice engineered to have extra copies of this gene hopped onto a treadmill and, without ever having trained, ran about twice as far as unaltered mice. The extra PPAR-delta improved the ability of the mice’s muscles to use fat molecules for energy, and it shifted the animals’ ratio of muscle fiber types from fast-twitch toward slow-twitch fibers — a change that would improve muscle endurance in people as well. Ronald Evans and his colleagues at the Salk Institute for Biological Studies in La Jolla, Calif., published the research in 2004. Since then, Evans says, he has been routinely approached by curious coaches and athletes. “I’ve had athletes come to my lectures and go to the microphone and say, ‘If I took this drug, would it work with EPO and growth hormone?’ I mean, they would ask this publicly,” Evans says. “Based on athletes I’ve talked with, I’d say that it’s a reasonable possibility that gene doping will be used in this Olympics, and I think there’s a very high probability that it will be used in the next Olympics,” he says. Elusive signs Around the time that Evans was announcing his “marathon mouse” results, WADA kicked off a funding program to focus scientific research on strategies for detecting gene doping. “A key part of our project is to try to define what we call signatures of doping,” says Olivier Rabin, a biomedical engineer and director of science for WADA. “We are looking at the impact of those kinds of genetic manipulations at different levels.” The first and most obvious approach is simply to look for the inserted gene among the roughly 6 billion “letters” of genetic code in both sets of a person’s chromosomes. For clinical gene therapy trials, finding the inserted gene is fairly easy. Scientists know the exact sequence of the gene they inserted, and often they know where on the person’s chromosomes the gene should have ended up. Standard DNA sequencing techniques can reveal the genetic code for that region on the chromosomes, and the unique sequence of the inserted gene will be in plain view. With gene doping, the situation is much trickier. “In sport, you don’t know where that gene will be put, what virus was used or even what particular variety of gene was used,” Friedmann says. “You don’t have the advantage of knowing where to look and for what, so the argument is to look everywhere.” Another difficulty is that copies of the foreign gene wouldn’t be in all of a person’s cells. The gene-carrying viruses selectively target certain tissues such as muscle or liver (the liver helps to regulate muscle metabolism). Some blood cells might also take in the viruses’ genetic payloads, but it’s questionable whether a standard blood sample from an athlete would contain the gene. Instead, anti-doping officials would have to sample muscle tissue directly using punch biopsies, a procedure that is mildly painful. “No one’s expecting that an athlete will agree to a muscle biopsy,” Friedmann says. “That’s a nonstarter.” Still, direct detection of inserted genes could work in some cases. Evans points out that an artificially inserted gene for PPAR-delta would be much smaller than the natural gene. That’s because the natural gene is far too big to hitch a ride on the carrier virus. Fitting the gene onto a virus means only a trimmed down version of the gene can be used. This distinctive genetic pattern would only exist in a person who had undergone gene doping. In other cases, genes would end up in tissues where they’re not normally active, making detection more straightforward. For example, the liver and kidneys normally produce the protein EPO, which makes red blood cells, but gene doping could deliver the EPO-coding gene directly to muscle tissues. The trick, then, is to find a noninvasive way to detect where EPO production is occurring inside the body. One solution is to use medical imaging techniques such as PET scans. In research funded by WADA, Jordi Segura and his colleagues at the Municipal Institute for Medical Research in Barcelona, Spain, attached slightly radioactive “flags” to molecules made during EPO production. A standard PET scan can spot this radioactivity, revealing where EPO was being made in the bodies of mice injected with gene-doping viruses, the team reported in the October 2007 Therapeutic Drug Monitoring. The researchers showed that production of EPO in muscle tissue was a telltale sign of gene doping. With radioactivity that is relatively mild, the labels are routinely used in medical imaging to diagnose diseases and don’t pose a significant hazard. But Friedmann notes that asking athletes to undergo such a procedure could be controversial. Detection by proxy Another approach is to look for signs of the viral “infection,” rather than for the gene itself. Even a weakened virus could trigger a mild, and specific, immune reaction that might show up in a blood test. Perhaps the greatest challenge facing this method is that viruses aren’t the only way to deliver a gene into a doper’s body. “The reality is that you can just inject naked DNA directly into tissues” with a syringe, Evans says. “Direct injection could be more local and harder to detect.” This relatively crude way to insert a gene won’t spread the gene as widely through a person’s body as viruses injected into the bloodstream would. But many cells near the site of injection could take in the gene, perhaps enough to improve athletic performance. Microscopic, synthetic spheres of fat molecules called liposomes can also shuttle doping genes into the body. To prevent dopers from evading detection by simply changing delivery vehicles, scientists are also exploring a third approach to developing tests: proteomics, the detailed study of all the proteins in the human body. Regardless of the vehicle used, adding a new gene to the body’s tightly woven web of interacting genes and proteins will cause ripples of change to spread throughout that web. “There will be a body-wide response no matter what gene you use or where in the body you put it,” Friedmann says, “and those changes can be used as a signature of doping.” Painful biopsies wouldn’t be required. Because the cascade of changes in protein activity would be widespread, anti-doping officials could test using blood, urine, hair or even sweat. Tools developed for the burgeoning fields of genomics and proteomics allow scientists to see the activity levels of thousands of genes or proteins simultaneously. In preliminary unpublished experiments, Friedmann and his colleagues injected a type of muscle cell with the gene for IGF-1. Activity of hundreds of genes changed as a result, including a boost in the activity of genes that control production of cholesterol, steroids and fatty acids. All of these changes might be detectable with simple blood tests. WADA is funding half a dozen or so ongoing studies on this proteome-based detection strategy, but research in this area is still at an early stage. “There’s good reason to think that’s likely to work, and a number of labs are having some nice results,” Friedmann says. As for whether any tests for gene doping will be ready in time for the Beijing Olympics, anti-doping authorities aren’t giving away many hints that might help dopers evade detection. “We never say when our tests are going to be in place,” WADA’s Rabin says. Even if detection methods do lag behind the games, dopers may want to think twice before assuming they’re in the clear, Friedmann notes. “With stored [blood and urine] samples, one always has the option of going back some months or years later and checking again with the newest tests.” Just in case the dangers of tampering with a person’s genetic makeup weren’t enough of a disincentive.