HIGH-PRESSURE DELIVERY IMPROVES GENE TRANSFER TO MUSCLE

Even Naked Genes Get In

"Intravascular delivery of DNA is very promising," says Jon Wolff of the Department of Pediatrics and Medical Genetics of the University of Wisconsin at Madison. Since the early 1990s, Wolff, a physician and longtime MDA grantee, has been pursuing methods of delivering DNA (genes) to muscle tissue without the use of viruses.

Most gene therapy strategies involve viruses or parts of viruses to deliver the genes, which, while effective, may be hazardous. Now, Wolff's laboratory has produced evidence that DNA alone -- so-called "naked DNA" -- can enter muscle fibers at high levels (approaching those achieved with viral delivery), if the DNA is delivered under pressure with nearby blood vessels tied off.

He found that when he injected naked DNA at high pressure via an artery into the leg muscles of adult rats, the amount reaching the muscle was high enough to be considered therapeutic, much higher than when he injected naked DNA directly into the rats' muscles.

"The results demonstrate that the intra-arterial delivery of DNA to muscle can be greatly enhanced when injected rapidly, in a large volume, and with all blood vessels leading into and out of the hindlimb occluded [blocked]," according to the paper, published in the February issue of Gene Therapy. Using a drug that dilates blood vessels and an enzyme that breaks down a membrane in the vessels helped even more, but these agents weren't deemed necessary.

Wolff says further studies will be needed to determine the relevance of the findings to humans and whether these techniques are safe. If the approach can be used, it could improve delivery of any kind of gene therapy agent, including genes inside viruses, should viruses turn out to be necessary. "The delivery method applies to any type of vector [delivery vehicle]," Wolff says. "It might obviate the need for intramuscular injections in muscle-based gene therapy."

Although lots of combinations of genes and vectors are in the pipeline (see "Utrophin Strategies,"), the need for multiple, and possibly repeated, muscle injections, is one problem most gene therapy experts haven't tackled.

CLUE TO HEART PROBLEMS IN EMERY-DREIFUSS MD

Researchers See Future for Gene Therapy

The protein that's abnormal or deficient in Emery-Dreifuss muscular dystrophy (EDMD) is known as emerin, made from a gene located on the X chromosome that was identified by an Italian research group in 1994. Since then, studies of emerin have found that it's in many tissues and that it's particularly plentiful in heart and skeletal muscles. Its location has been traced to the nucleus of the cells. Little is known about emerin's functions, but EDMD involves progressive muscle weakness and wasting, joint contractures (frozen joints) and, unlike most muscle diseases, very serious problems with the electrical system of the heart, known as cardiac conduction block. The heartbeat may be slowed or irregular.

Now, another Italian study, published in the December 1997 issue of Human Molecular Genetics, shows emerin isn't confined to the nucleus in heart muscle cells; it's also found in specialized structures known as "intercalated discs" (see illustration, below). These discs are involved in transmitting signals from cell to cell.

The muscle protein emerin, which is abnormal or deficient in Emery-Dreifuss muscular dystrophy, has been found in the intercalated discs of heart muscle cells. These discs, and the emerin in them, normally help conduct signals from cell to cell and help regulate the heartbeat.

MDA grantee Stephen Warren, a molecular biologist, is studying the cellular aspects of EDMD at Emory University School of Medicine in Atlanta. "One part of Emery-Dreifuss dystrophy is that heart involvement is one of the more significant aspects of the disease. But when the gene was found, it wasn't immediately apparent how this protein influenced the heart," he says. "This finding of it in the intercalated discs is important because it gives us some clue as to what may be going awry when you don't have the protein around. This is a new way to think about the problem."

Neurologist Alan Pestronk, an MDA grantee studying the clinical aspects of EDMD at Washington University School of Medicine in St. Louis, where he also directs the MDA clinic, was similarly impressed with the finding. "It puts together a previously unexplained piece of the puzzle," he notes, explaining that the intercalated discs allow cells in the heart to stick together and communicate with each other, and that loss of a protein in them could explain the conduction block in Emery-Dreifuss dystrophy.

"For the heart to function, an impulse has to start in a small area and spread over the heart," Pestronk said. "If that's disrupted, the heart can't beat at normal speed or in a coordinated way."

Current treatment for the cardiac conduction block in EDMD is an electronic pacemaker, which works fairly well, both researchers say. But gene therapy, especially for the heart problems associated with this disorder, may be a future possibility. Warren's laboratory is working on an animal model of EDMD in which they plan to test gene therapy strategies. Pestronk says the emerin gene, which is relatively small, might be easy to work with.

"If it becomes feasible to transfer genes, all you'd have to do to correct the heart problem is get it into the heart," he says, which would be easier than delivery to the entire body.

CLINICAL TRIALS STILL OPEN IN ROCHESTER

Two MDA-sponsored clinical trials, one in Duchenne muscular dystrophy (DMD) and another in facioscapulohumeral muscular dystrophy (FSHD), are still open at the University of Rochester in Rochester, N.Y.

A trial of the drug oxandrolone (Oxandrin), an anabolic steroid, for DMD, needs boys age 5 to 10 with that diagnosis who haven't been taking steroids or other therapeutic agents. The study will last six months and will involve inpatient and outpatient visits. It will test the safety of oxandrolone and its effectiveness in increasing strength or slowing strength decline. In a pilot trial of 10 boys, small improvements in strength were found with this drug. Call Shree Pandya, study coordinator, at (716) 275-1005.

A trial of the drug albuterol (Proventil), a beta receptor agonist, also needs participants for the Rochester center. Albuterol has been shown experimentally to increase muscle bulk and strength in people without muscular dystrophy, and preliminary results in a small trial of those with FSHD suggest it would have similar benefits for those with this disorder. Participants must have a diagnosis of FSHD, be between 18 and 60 years old, and have no history of heart disease or high blood pressure. The study will last a year and will involve inpatient and outpatient visits. Call Lynn Cos, study coordinator, at (716) 275-7680.

To keep up with news about clinical trials, check the MDA Web site at www.mda.org.