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Gene Therapy Rescues Mutant Gene in Limb Girdle Muscular Dystrophy
Proof-of-Principle for Approach


Investigators reported the findings from a small trial that found gene therapy safe for three patients with limb-girdle muscular dystrophy.

Scientists at Ohio State University filled viruses with normal genes and injected them into a small muscle in the foot of six patients with limb-girdle muscular dystrophy (LGMD). In showing that the single injected muscle produced a missing protein called alpha-sarcoglycan, Jerry R. Mendell, MD, provided a much-needed nod in the direction of gene therapy for severe muscle diseases.

“It was a proof-of-principle experiment,” said Dr. Mendell, the lead author of the study in Annals of Neurology and a scientist who has spent decades working out the basics of gene therapy for muscular dystrophy (MD).

A decade ago, Dr. Mendell, director of the Center for Gene Therapy at the Research Institute at Nationwide Children's Hospital and professor of pediatrics and pathology at the Ohio State University (OSU) College of Medicine, and his colleagues were conducting the first gene therapy trial for LGMD that was cut short after the death of a patient enrolled in a different gene therapy study at the University of Pennsylvania. The young man's death shut the door on gene therapy for muscular dystrophy for many years.

Behind the scenes, Dr. Mendell continued to focus on the problems and forged ahead in the hope that viral delivery of genes did not pose a threat to humans, and that the genes would take up residence in a muscle and begin making the deficient protein. Patients with LGMD presented a good opportunity for gene therapy: the gene for alpha-sarcoglycan is small enough to fit inside an adeno-associated virus that is used as the viral vector to deliver the gene.

The current report — on the first three patients followed up to three months — is the first completed trial of a gene therapy for the disabling condition, said Dr. Mendell.Three other patients are being followed for six months to see if the gene is still expressing protein. So far, the scientists reported that it appears safe — no immunological reaction was observed — and the muscle is filled with the once-missing protein. That it may work is promising but the real challenge is to reach many muscles at the same time in an attempt to support the global problem of muscle weakness.

The three young boys in the study were all severely impaired and confined to wheelchairs. The study was designed to test whether gene therapy could be used to safely deliver protein to one muscle and inject a placebo in the same muscle of the contralateral foot. Only the muscle that held the human form of the normal gene packed in millions of viral particles showed evidence of the normal alpha sarcoglycan. Without this protein, the muscle loses integrity and weakens.

These studies are not designed to show improvement in muscle strength. It would be impossible given the small and singular muscle of the foot in patients who can no longer walk. But that the muscle is making protein — and the right protein that could protect the integrity of the muscle – is proof that it is a worthy approach.

Dr. Mendell is focusing next on how to inject the genes into the blood to reach groups of muscles at risk for weakness. “We can successfully deliver the gene though the circulation in the non-human primate and this establishes the gateway for a clinical trial with the potential to help patients with muscular dystrophy,” he said.

Dr. Mendell is hopeful. “In 1999, when we did our first trial in LGMD we did not have good gene expression,” he explained. “In contrast in the current study, we have robust expression.” He thinks the key difference is in the larger dose that was delivered — about ten times as much virus that contain the normal gene. The trick is to prevent the virus from replicating and have it benignly deliver the targeted gene. In Ohio, it seemed to work.


DR. JERRY R. MENDELL is focusing next on how to inject the gene into the blood to reach groups of muscles at risk for weakness.

The MDA clinic at Nationwide Children's Hospital has accommodated about 400 patients with LGMD and 20 patients have an alpha-sarcoglycan deficiency. They selected patients — ages 12–14 years old — for the study who had low titers to the common adeno-associated virus, the vector used for gene therapy. Two patients were tested at six weeks and one was studied three months after the injection of the virus into the foot muscle. At the three-month follow-up, the muscle had increased four- to five-fold over the baseline to near-normal expression of the protein. There was no immune response to protein or virus. The patient tested at three months had increased the size of the foot muscle fiber from 32 micrometers to 45. The researchers will continue to study gene expression and test for signs of a rogue immune response.

“We want long-term gene expression,” said Dr. Mendell. The three patients still undergoing testing will complete studies at six months post gene delivery.

Findings from these patients have helped the scientists move forward in monkeys to test the viability of delivering the virus through the circulation. So far, Dr. Mendell said, they have been successful at isolating the vasculature of the main thigh muscle (by using two blood pressure cuffs) as they inject the virus. “This will be important for making our case for vascular delivery of the therapy,” Dr. Mendell said. “We are confident we can get it to where we want it to go,” he added.

If they can show that the strength of the thigh muscle improves following gene therapy it would offer hope that patients may remain ambulatory longer than they would have without this therapy.

Gene therapy has been fraught with roadblocks. Dr. Mendell and his colleagues collaborated with colleagues at the University of North Carolina for another gene therapy experiment for the more common Duchenne muscular dystrophy. “We did not have success,” the Ohio neurologist said. “We ran into immune issues when transferring the gene.”

Eric Hoffman, PhD, director of the Research Center for Genetic Medicine at Children's National Medical Center in Washington, DC, said that these gene therapy trials face the same problems that scientists have grappled with for two decades. The immune system doesn't like foreign material and can wage an immune response against either the new protein or the virus used to carry the normal copy of a gene. There is also the hurdle of delivering the virus. “It's one thing to get it working in one muscle,” he said of the latest study. “It's another thing to deliver it systemically.”

He said that the promise of gene therapy remains elusive. “Gene therapy has been guilty of raising false hopes for 20 years.” He said the new drugs that alter expression of genes could revolutionize the field. Trials are underway in Duchenne muscular dystrophy using antisense oligonucleotide medicines that skip over the mutated gene. The result is production of the needed protein. Dr. Hoffman's study on dogs, published in the Annals of Neurology in January, led to clinical trials now underway in Europe. The drug latches on to RNA and restores the reading frame of the gene. The drug does not elicit an immune response. Other medications have been developed to suppress the stop codon and lead to normal gene expression.

“There are several promising gene therapies for MD,” said Valerie Cwik, MD, executive vice president for research and medical director for the Muscular Dystrophy Association. The association has funded Dr. Mendell's work and she is excited by the early results. “This is an important first step, demonstrating that it is feasible; that you can deliver it to muscle and get expression. But, she added, the study could not answer the question of whether the once-missing protein will make the muscle stronger.


• Mendell JR, Rodino-Klapac LR, Clark KR, et al. Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins. Ann Neurol 2009;66(3):290–297.
    • Yokota T, Lu QL, Hoffman, et al. Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Ann Neurol 2009;65(6):667–676.