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SAN DIEGO—Twenty years after a mutation of the dystro-phin gene was implicated in Duchenne muscular dystrophy (DMD) – the most common dystrophy affecting boys – a pioneering gene therapy trial is underway. Lead researcher Jerry Mendell, MD, called the day in March when he treated the first patient “the most exhilarating day of my life.” Dr. Mendell spoke about the trial, and the history of gene therapy for muscular dystrophy, at the Frontiers of Neuroscience plenary session here at the AAN Annual Meeting in April.


All forms of gene therapy have had to cope with the challenges of maximizing delivery of the gene to the target tissue while minimizing an immune response to the gene vector, said Dr. Mendell, Director of the Center for Gene Therapy at the Columbus Children's Research Institute in Ohio. These challenges have been acute in DMD, because, at 2.6 million base pairs, dystrophin is the largest gene in the entire human genome. Even with all the introns removed, forming the cDNA, it is 11 thousand base pairs, too big for most viral vectors.

The size of the gene caused early researchers to focus almost exclusively on adenovirus – the only vector large enough to carry the big genetic payload. But early adenovirus vectors provoked a severe immune reaction. Attempts to modify the vector and reduce the immunogenicity were modestly successful in animal models, but gene expression remained short-lived. Then, in September 1999, a young asymptomatic man enrolled in a gene therapy trial of Batten disease died four days after receiving the adenovirus vector. In 2002, three boys whose gene therapy with a retrovirus had cured them of X-linked severe combined immunodeficiency developed leukemia, as a result of the random insertion of the therapeutic gene into a pro-leukemia gene. One has since died.

These tragedies put the spotlight on other delivery systems. One strategy, tried last year in France, delivers a “naked DNA” plasmid containing the dystrophin gene. Although there were no side effects, there was also limited and weak gene expression.


Adeno-associated virus (AAV) has long been an attractive vector for gene therapy. Unlike adenovirus, it does not cause any human disease and has low toxicity; unlike naked DNA, well-known cell receptors bind the virus for endocytosis; and unlike retrovirus, it does not require insertion into the chromosome to be expressed. However, it is much smaller than adenovirus, and can only carry a small payload.


Dr. Jerry Mendell said the investigators do not expect clinically meaningful results in the current pilot trial. The goal is to work together developing an effective and safe means for delivering the corrective gene through “regional vascular delivery,” before trying systemic delivery.

In 1999, Dr. Mendell used AAV for the first time to deliver the alpha-sarcoglycan gene for limb girdle muscular dystrophy. The cDNA for this gene was only 1,500 bases, “not even in the ballpark” of the dystrophin gene, he said. This trial was stopped, along with other gene therapy trials, by the death in the Batten disease trial.

But despite its early halt, the trial instituted several important innovations, and important lessons were learned. “One of the most important things we introduced was the randomized, double-blind trial structure,” he said. Virus was injected into one extensor digitorum brevis muscle, while the vehicle (but not empty vector) was injected into the other. Investigators also used electromyography under ultrasound guidance to find the muscle and track exactly where in the muscle the injection occurred, allowing them to biopsy the transfected tissue precisely at a later date. At six weeks, there was only limited expression of the alpha-sarcoglycan gene, but no toxicity and no inflammation.


“It was always our goal to transfer the dystrophin gene,” Dr, Mendell said. To make that possible with AAV, the gene had to be severely downsized, without preventing the resulting protein from doing its job at the muscle cell membrane. A serendipitous discovery in 1990 showed the way. Analyzing the dystrophin gene in a patient with the clinically milder Becker form of muscular dystrophy, researchers discovered that almost half the gene was missing – instead of the normal 20 central repeat units, this gene had only eight. Despite that, the protein apparently functioned well enough to allow the patient to continue to walk.

Experiments by University of Washington Neurology and Neurogenetics Professor Jeffrey Chamberlain, PhD, and others whittled the gene even further, to below 4,000 base pairs, small enough to fit into the AAV viral capsid. By injecting the viral capsid into the standard mouse model for DMD, researchers showed the force generated in transfected mice was less than normal, but better than with no treatment.


This paved the way for the current clinical trial and Dr. Mendell's “exhilarating day.” The trial began with one patient – six are planned – and is structured as a double-blind, randomized trial. Patients are all boys with DMD with mutations that completely eliminate gene expression. Patients will continue prednisone therapy, with an extra bolus dose at the time of injection, because of a study showing that immunomodulation enhances gene expression. “This sends an important message: We are in the learning phase,” Dr. Mendell said.

The gene is being injected into the biceps in this trial, with the hope that future trials may build on this to deliver functional benefit. “Clinically meaningful results are the target,” he said, but they are not expected in this trial. “We know that local injections result in no clinical improvement.”

Instead, future trials will need to deliver the gene to whole muscles, or even groups of muscles. “Our target clearly is that we will be able to do regional vascular delivery,” perhaps into the lower limbs, he said. This would precede attempts at systemic delivery.

In mice, femoral artery catheterization is being tried as a start. “We are definitely having success with this,” he said, although the mouse limb is obviously orders of magnitude smaller than the human. Success in this model would be followed by a scale-up trial in the golden retriever model of DMD.

Enhancement strategies being tried include altering the viral capsid to target other viral receptors on the muscle surface, and combination therapies, such as disrupting the myostatin signaling system, which limits muscle growth. “We can extend these findings to other forms of muscular dystrophy as well,” said Dr. Mendell, “and this is what makes the field so exciting.”


DMD investigator Tejvir Khurana, MD, PhD, is not surprised it has taken this long to begin these gene therapy trials. Even the conceptually simplest type of gene therapy – enzyme replacement for metabolic disorders – has been hard to achieve, and muscle delivery presents many more challenges. “There have been incremental advances at each stage. But part of the problem is that people's expectations are higher as soon as the gene is cloned – they think we have a cure,” said Dr. Khurana, Assistant Professor of Physiology at the Pennsylvania Muscle Institute at the University of Pennsylvania.

As for the future of gene therapy in Duchenne dystrophy, he said: “It's a hard question. I think we will end up with different treatments for different patients, based on their specific type of mutation. Some will be more amenable to pharmacologic therapies to encourage skipping of mutated exons, for example, while others may benefit from antisense oligonucleotide therapy – a newcomer on the scene that may selectively prevent production of defective proteins. It's not going to be one therapy fits all.”

Nonetheless, he said, viral-mediated gene transfer has the potential to benefit the vast majority of patients, if it can be developed for muscle-wide delivery. Scaling up to vascular injections will pose a new set of problems, though, even if the current trial establishes the proof of principle for AAV as the gene carrier. “I am guardedly optimistic,” said Dr. Khurana. “That's the best you can say about any trial.”


  • ✓ Dr. Jerry Mendell provided an overview of the challenges of gene therapy, and discussed a pilot clinical trial now underway to inject a corrective gene into the biceps of boys with Duchenne muscular dystrophy.