Advances in Duchenne Muscular Dystrophy Gene Therapy

Andrew Kilbarger (then 9 years old) from Lancaster, Ohio is participating in a gene therapy trial. MDA Publications/2006
Article Highlights:

Originally published in Quest magazine in September 2008, this article discusses progress to date in gene therapy (gene transfer) for Duchenne muscular dystrophy (DMD). The article reports:

  • Aged DMD mice benefited from new dystrophin genes.
  • The heart will likely have to be treated, not just the skeletal muscles, in DMD.
  • Heart health can benefit from gene transfer in DMD mice.
  • Severe DMD mice can benefit from gene therapy.
  • The immune system may pose obstacles to gene therapy.
by Margaret Wahl on September 1, 2008 - 10:07am

QUEST Vol. 15, No. 5

Real progress is being made in supplying functional dystrophin genes to treat Duchenne muscular dystrophy (DMD), a disease in which mutated dystrophin genes keep this critical protein from being produced in muscle fibers.

An MDA-supported clinical trial has shown the procedure to be safe. Higher doses will now be tested.

Jeffrey Chamberlain Jeffrey Chamberlain’s group found gene therapy effective even in aged DMD mice.

Meanwhile, investigators are testing various sizes of miniaturized or non-miniaturized dystrophin genes, tucked inside adenoviral (AV) or adeno-associated viral (AAV) transporters (“vectors”) or inside donated cells.

At the 11th annual meeting of the American Society of Gene Therapy, sponsored in part by MDA, which took place in Boston May 28-June 1, many sessions focused on the problem of overcoming potentially harmful responses of the immune system to the viral vectors, donated cells or even to the new dystrophin protein made from the transferred genes.

Gene therapy surprisingly effective in aged DMD mice

A research group coordinated by MDA grantee Jeffrey Chamberlain and including MDA grantee Paul Gregorevic, both at the University of Washington-Seattle, has shown that gene therapy using highly miniaturized dystrophin genes can be effective even in aged, dystrophin-deficient mice with a DMD-like disease.

The study, published in the April issue of Molecular Therapy, bodes well for the potential benefit of gene transfer strategies in older patients with DMD.

The investigators injected shortened versions of the dystrophin gene, which is flawed in DMD, into the tail veins of 20-month-old, dystrophin-deficient mice. (These mice normally live about 24 months.) They encased the so-called microdystrophin genes in modified type 6 adeno-associated viral vectors (AAV6), which are known to penetrate muscle tissue effectively.

Eighteen weeks after injection of the microdystrophin genes, the mice showed body-wide production of the dystrophin protein in their muscles, improved function of their respiratory and back leg muscles, and reduced muscle-fiber degeneration.

Treating skeletal muscles alone bad for hearts

Ever since gene therapy and other strategies for treating DMD have been seriously contemplated, doctors have wondered what might happen to patients if they became more active because of better skeletal muscle function but didn’t receive treatment for their hearts.

In the May issue of Molecular Therapy, MDA grantee DeWayne Townsend at the University of Michigan in Ann Arbor and colleagues describe experiments conducted to answer this question that reveal the possible hazard of treating skeletal muscles alone in this disease.

Townsend, with MDA grantee Soichiro Yasuda, also at the University of Michigan, and others, bred mice that produced dystrophin only in their skeletal muscles but not in their hearts. In comparison to fully dystrophin-deficient mice, the mice with dystrophin in their voluntary muscles spent more time running in their exercise wheels, but they also sustained five times the amount of cardiac damage and had significant cardiac muscle dysfunction.

“The finding suggests that, in the context of patients with DMD, caution needs to be exercised when considering potential therapeutic approaches that may have efficacy in skeletal and respiratory muscles but very little activity in the heart,” the researchers note.

Other researchers are addressing this issue and looking closely at gene delivery to the heart.

Gene therapy improved cardiac health of dystrophin-deficient mice

Gene therapy using a microdystrophin gene inside an AAV9 vector markedly improved cardiac function when it was given to newborn mice missing the dystrophin protein, a new study shows.

MDA grantee Dongsheng Duan at the University of Missouri, and colleagues, who published their findings online June 17 in Human Gene Therapy, found this combination led to production of a shortened dystrophin protein throughout the hearts of DMD mice four months after a single intravenous injection.

It also led to complete correction of abnormalities seen in these mice on an electrocardiogram and partial correction of cardiac muscle function.

The investigators say microdystrophin gene transfer using AAV9 can “significantly improve electrophysiological defects in [dystrophin-deficient] mice.” They say its inability to completely normalize cardiac muscle function may reflect a limitation of the highly miniaturized microdystrophin.

Injecting full-length genes improved health of mice with severe MD

Scientists in Japan have used modified AVs, which are larger than AAVs, to transport full-length dystrophin genes into the muscles of mice missing the dystrophin protein and a similar protein, utrophin. These mice, which have a DMD-like disease but are more severely affected than mice missing only dystrophin, lived longer and had better motor function than their untreated counterparts.

Most gene therapists targeting muscle tissue are using AAVs with miniaturized dystrophin genes. But Yasushi Maeda of Kumamoto (Japan) University Graduate School of Medical Science, and colleagues, who published their findingsin the May issue of Molecular Therapy, say the muscle fibers of the mice that received full-length dystrophin genes showed a more complete restoration of a cluster of proteins at the muscle-fiber membrane than can be achieved with AAV6-microdystrophin gene compounds.

They paid particular attention to the restoration of a protein called nitric oxide synthase, which isn’t restored with microdystrophin gene transfer and which, they say, may be important for DMD treatment.

They say AVs caused a slight immune response but are unlikely to activate the immune system more than it’s already activated in dystrophic muscles.

Gene therapists confront the immune system

At the ASGT meeting this spring, many sessions focused on the problem of overcoming potentially harmful immune-system responses to gene therapy.

Jude Samulski, a molecular biologist at the University of North Carolina at Chapel Hill and Asklepios BioPharma in Chapel Hill, and neurologist Jerry Mendell at Nationwide Children’s Hospital in Columbus, Ohio, both of whom have received substantial MDA support to develop gene therapy for DMD, addressed this issue. Both are investigators in a phase 1 clinical trial of dystrophin gene transfer in DMD using an AAV vector.

Mendell said he thought the presence of so-called revertant fibers — isolated muscle fibers that produce dystrophin despite a dystrophin gene mutation — would likely protect many boys with DMD from rejecting full-length dystrophin that would be synthesized from a transferred gene. However, he said, analysis of the final results of the current DMD gene therapy trial will reveal whether that’s the case.

Samulski concentrated his presentation on concerns about the potential immune-system rejection of the surface proteins found on the AAV vectors used in gene therapy.

He emphasized that, so far, they have seen no serious adverse events and no immune-system cells known as cytotoxic lymphocytes attacking these proteins in the DMD gene therapy clinical trial. However, he said, it’s still “early in our understanding” and we “can’t infer too much from one study.”

Jude Samulski
Jude Samulski says that, so far, there have been no serious adverse events in the clinical trial of gene therapy for DMD.

The vector being used in the DMD trial is called AAV2.5. It’s a laboratory-engineered viral shell designed to maximize muscle-cell entry and minimize unwanted immune responses. Samulski said the field is moving toward such designer vectors and away from settling for what nature has provided.

Other investigators are concentrating on transplanting muscle precursor cells, allowing genes to be delivered to ailing muscle tissue without the use of viral vectors.

Jacques Tremblay, from Laval University in Quebec City, discussed his experiences with intramuscular injections of muscle precursor cells from healthy parents into their DMD-affected children (see "Scientists Share Gene Therapy Advances," September-October 2006).

In his phase 1A trial, Tremblay gave each of 10 boys 25 or 100 injections into a very small area of a leg muscle. The boys also received the immunosuppressant drug tacrolimus. Several of the patients showed good dystrophin production in the injected area.

Tremblay has now proceeded to a phase 1B trial, designed to test the effect of 300 to 600 injections into an arm muscle. (He is not seeking trial participants at this time.) So far, one patient has received 300 injections and then 600 injections a month later. Surprisingly, only 1 percent of this patient’s muscle fibers in the injected area were producing dystrophin six months after the second set of 600 injections.

Tremblay speculated that the separation of more than a year between the participation of this patient in the phase 1A clinical trial and the phase 1B clinical trial may have allowed the patient’s immune system to become “primed” against the donated cells and to reject the cells injected in phase 1B.

Also experimenting with cell transplantation is Maruilio Sampaolesi from the University of Pavia (Italy), who’s transplanting blood-vesselassociated cells called mesoangioblasts into dystrophin-deficient dogs. Mesoangioblasts are able to become the type of cell that surrounds them, including muscle.

Dogs that received a leg-artery injection of donated mesoangioblasts from healthy dogs and also received the immunosuppressant cyclosporine produced dystrophin and showed increased muscle force.

Sampaolesi and colleagues are now testing the safety of intramuscular injections of mesoangioblasts in children in Italy.

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