This fall, pending approval from the Food and Drug Administration (FDA), MDA-supported investigators at Ohio State University in Columbus and the University of Michigan in Ann Arbor will begin a very limited human trial of gene therapy in Duchenne muscular dystrophy (DMD). Starting this spring, 12 patients will be followed to establish baseline measurements, which will be important for comparison once the gene transfers begin.

Dr. Jerry Mendell at Ohio State and Jeffrey Chamberlain at Michigan are co-investigators on the project. Mendell, a neurologist whose career has focused on neuromuscular disease, is a long-time MDA research grantee as well as director of the Neuromuscular Center at OSU and co-director of the MDA clinic at OSU. He has conducted other clinical trials in DMD.

Chamberlain, a molecular biologist, is among the world's foremost gene therapy researchers. He has systematically studied gene replacement in the mdx mouse (an animal model of DMD) to determine which vectors (carriers for the gene) and which portions of the therapeutic gene are necessary to potentially reverse the symptoms of muscular dystrophy.

The phase 1 studies will probably begin in the fall at OSU, and will include 5-, 6- and 7-year-old boys with DMD. The gene to be injected is a dystrophin gene, which codes for the dystrophin protein, the muscle protein missing in children with this disorder. The researchers will deliver the gene using a highly modified adenovirus as a vector. Chamberlain developed this vector, known as a "gutted" adeno-virus, with MDA support.

The dystrophin gene will be a full-length gene, which is thought to have a better chance of correcting the muscle degeneration seen in the disease than various dystrophin "minigenes" researchers have developed in recent years. The highly modified adenovirus, which lacks all viral genes, has room to carry the whole dystrophin gene. The so-called gutted virus is also expected to cause minimal adverse responses from the body's immune system. Immune responses have been a problem in animal studies that used adeno-viruses that more closely resemble actual viruses.

However, the children will take the immunosuppressant drug FK506 (tacrolimus, Prograf), a fairly new immunosuppressant that has shown much promise in animal models of gene transfer, for 12 weeks.

INTRAMUSCULAR INJECTIONS INTO BICEPS

Doctors will give each child in the study injections into the biceps muscle (upper arm muscle) of each arm. One injection will contain the gene in the gutted viral construct, and the other will be an injection of the same fluid without the gene. Neither the doctors nor the patients will know which arm got which injection, although, for safety reasons, a research pharmacist and one doctor not involved in the evaluation procedure will know.

The major goal of the 24-week study is to establish the safety of the gene transfer procedure. Investigators will carefully follow the children for signs of immune-system rejection and will measure strength in the muscles receiving the injections. The study will also ensure that no harmful effects are caused by FK506.

FREQUENT EVALUATIONS

The plan calls for evaluations for safety and effectiveness at weeks 1, 2, 4, 6, 8, 12, 16, 20 and 24. These will include physical and laboratory evaluations for signs of immunologic rejection of the gene transfer and for any complications associated with the drug FK506. The doctors will take monthly blood samples to detect any antibodies (signs of an immune response) to the transferred dystrophin protein or to any viral proteins that may be produced.

The children's biceps muscle strength will be tested as well as strength in 32 other muscle groups, to determine whether FK506 has any effect (positive or negative) on muscle.

Four of the 12 boys will undergo muscle biopsies of both their biceps muscles at four weeks; another four at eight weeks; and the final four at 12 weeks. Muscle tissue will be analyzed for the presence of dystrophin.

PHASES 2 AND 3

Later phases of the trial will depend in part on the outcome of phase 1. Right now, plans are to test an alternative genetic "promoter," or chemical "on switch," and to test an alternative gene. Another aspect of the later part of the study will be to see what happens when FK506 treatment is discontinued.

A total of 36 boys will be tested over a three-year period.

TIME TO MOVE FORWARD

"Animal trials, especially those in mice and dogs with dystrophin deficiency, have only addressed some, not all, of the variables that confound successful gene therapy," Mendell says. "The immune systems of these animals differ significantly from those of patients with Duchenne dystrophy, and there are compelling reasons to address the challenge of gene transfer in human clinical trials.

"Duchenne dystrophy is a fatal disease of childhood and severely diminishes quality of life," Mendell added. "The sooner we can begin to unravel the questions surrounding gene therapy in a clinical setting, the more potential we have to help these patients."

MONKEY TESTS PAVE WAY FOR HUMAN TRIALS

As Mendell's group gets under way with baseline measurements for clinical trials, the Institute for Human Gene Therapy (IHGT) at the University of Pennsylvania in Philadelphia will begin rapid testing of several vectors and gene constructs in rhesus monkeys and other animals.

The IHGT at Penn is headed by MDA grantee Dr. James Wilson, a world-renowned expert in gene therapy. The IHGT is also developing gene therapies for cystic fibrosis, cancer, a genetic form of elevated serum cholesterol, hemophilia and other disorders. Wilson recently received a $3.2 million grant from MDA, the largest in the Association's history, to rapidly and systematically develop the best possible gene therapy systems for treating muscular dystrophy. The first muscular dystrophies the group will address will be Duchenne and limb-girdle.

The Penn group may start its own clinical trials as early as September, pending FDA approval. Long-time MDA grantee Dr. Kenneth Fischbeck at Penn will manage the clinical aspects of these studies. Fischbeck is a neurologist specializing in both patient care and research in neuromuscular disorders.

This winter, IHGT researchers began injecting six rhesus monkeys with adenoviral vectors and a vector known as the adeno-associated virus, a virus that's much smaller than the adeno-virus but which doesn't seem to attract the attention of the immune system or cause any disease. The vectors carry a gene for human growth hormone. This gene was chosen as a test, or marker, gene, because it's easy to measure the protein it produces in a blood test, while measuring muscle proteins generally requires a biopsy.

The team plans to use the adeno-associated virus to carry one of the limb-girdle genes in a future clinical trial, but it's too small to carry a full-length dystrophin gene. They plan to test different versions of the adeno-virus vector for the dystrophin gene, including the fully deleted vector.

Another novel strategy they're considering is splitting the large dystrophin gene into two sections, each carried by the small adeno-associated virus.