Then as now, a major obstacle to successful gene therapy was the difficulty of getting a chosen gene into the desired tissue. Like many other researchers, we selected a virus as our delivery vehicle, or vector, because viruses are skilled at smuggling genes into cells. They survive and propagate by tricking the cells of a host organism into bringing the virus inside, rather like a biological Trojan horse. Once within the nucleus of a host cell, the virus uses the cellular machinery to replicate its genes and produce proteins. Gene therapists capitalize on this ability by loading a synthetic gene into the virus and removing any genes the virus could use to cause disease or to replicate itself. We selected a tiny virus called adeno-associated virus (AAV) as our vector, in part because it infects human muscle readily but does not cause any known disease.
We modified it with a synthetic gene that would produce IGF-I only in skeletal muscle and began by trying it out in normal mice. After injecting this AAV-IGF-I combination into young mice, we saw that the muscles' overall size and the rate at which they grew were 15 to 30 percent greater than normal, even though the mice were sedentary. Further, when we injected the gene into the muscles of middle-aged mice and then allowed them to reach old age, their muscles did not get any weaker.
To further evaluate this approach and its safety, Rosenthal created mice genetically engineered to overproduce IGF-I throughout their skeletal muscle. Encouragingly, they developed normally except for having skeletal muscles that ranged from 20 to 50 percent larger than those of regular mice. As these transgenic mice aged, their muscles retained a regenerative capacity typical of younger animals. Equally important, their IGF-I levels were elevated only in the muscles, not in the bloodstream, an important distinction because high circulating levels of IGF-I can cause cardiac problems and increase cancer risk. Subsequent experiments showed that IGF-I overproduction hastens muscle repair, even in mice with a severe form of muscular dystrophy.
Raising local IGF-I production allows us to achieve a central goal of gene therapy to combat muscle-wasting diseases: breaking the close connection between muscle use and its size. Simulating the results of muscle exercise in this manner also has obvious appeal to the elite athlete. Indeed, the rate of muscle growth in young sedentary animals suggested that this treatment could also be used to genetically enhance performance of healthy muscle. Recently my laboratory worked with an exercise physiology group headed by Roger P. Farrar of the University of Texas at Austin to test this theory.
We injected AAV-IGF-I into the muscle in just one leg of each of our lab rats and then subjected the animals to an eight-week weight-training protocol. At the end of the training, the AAV-IGF-I-injected muscles had gained nearly twice as much strength as the uninjected legs in the same animals. After training stopped, the injected muscles lost strength much more slowly than the unenhanced muscle. Even in sedentary rats, AAV-IGF-I provided a 15 percent strength increase, similar to what we saw in the earlier mouse experiments.
We plan to continue our studies of IGF-I gene therapy in dogs because the golden retriever breed is susceptible to a particularly severe form of muscular dystrophy. We will also do parallel studies in healthy dogs to further test the effects and safety of inducing IGF-I overproduction. It is a potent growth and signaling factor, to which tumors also respond.
Safety concerns as well as unresolved questions about whether it is better to deliver AAV in humans through the bloodstream or by direct injection into muscle mean that approved gene therapy treatments using AAV-IGF-I could be as much as a decade away. In the shorter term, human trials of gene transfer to replace the dystrophin gene are already in planning stages, and the Muscular Dystrophy Association will soon begin a clinical trial of IGF-I injections to treat myotonic dystrophy, a condition that causes prolonged muscle contraction and, hence, damage.
