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A Novel Gene Therapy Shows Promise for Limb Girdle Muscular Dystrophy Type 2B

Article In Brief

Researchers reported that a single dose of human acid sphingomyelinase delivered to the liver by an adeno-associated virus holds the potential to allow extended histopathological and functional muscle improvement in limb girdle muscular dystrophy type 2B. Our experts say the novel delivery system shows great promise for targeted therapy.

A single dose of a gene, delivered by adeno-associated virus (AAV), provides long-lasting muscle repair in a mouse model of limb girdle muscular dystrophy type 2B (LGMD2B), according to the study published online January 4 in the Journal of Clinical Investigation.

LGMD2B is caused by mutations resulting in the loss of a muscle repair protein, dysferlin. The dysferlin gene is too large to be packaged in an AAV and requires delivery to the muscle. The new single dose gene therapy, which uses the gene for acid sphingomyelinase (ASM)—a downstream target of dysferlin—delivers the ASM gene payload to the liver, so the liver can produce and provide it to the muscles.

“This is an ingenious approach,” commented Carsten Bonnemann, MD, chief of the neuromuscular and neurogenetic disorders of childhood section and senior investigator and acting chief of the neurogenetics branch of the National Institute of Neurological Disorders and Stroke, who was not involved in the study.

Dysferlin's role in repair is to promote the release of ASM, which then circulates systemically, Dr. Bonnemann explained. “This approach takes advantage of the mechanistic link between dysferlin and ASM. By letting the liver produce and release it, it is essentially using an enzyme replacement therapy to treat a muscle disease.”

More About LGMD2B

LGMD2B is an autosomal recessive, adult-onset muscular dystrophy that causes progressive muscle weakness and wasting. Dysferlin is a membrane protein that facilitates lysosomal vesicle fusion with the sarcolemma; the absence of dysferlin is associated with the inability to repair muscle membranes after exercise-induced injury.

An early step in normal muscle repair is the fusion of lysosomal vesicles with the sarcolemma, allowing the release of ASM. ASM then converts membrane sphingomyelin to ceramide, which helps damaged sections of the membrane to be internalized and replaced—a process that enables membrane repair.

Recent work, including by the laboratory of Jyoti Jaiswal, PhD, the senior author of the current study, has shown that dysferlin's key role is to promote the fusion of the lysosome and sarcolemma, a function facilitated by several so-called C2 domains.

“Proteins with C2 domains are involved in vesicle fusion,” said Dr. Jaiswal, professor of genomics and precision medicine at George Washington University School of Medicine and Health Sciences, and associate director of academics at the Center for Genetic Medicine Research at the Children's National Research Institute in Washington, DC. Such proteins include the synaptotagmins, which promote the release of neurotransmitters from synaptic vesicles.

While the absence of dysferlin causes LGMD2B, replacing it therapeutically has been a challenge. The gene is too large for a single AAV vector, leading researchers to develop dual-vector strategies, delivering part of the gene in each followed by rejoining them in the target cell. But the approach requires very large amounts of vector, raising safety concerns, and for the moment this strategy remains untested in humans.

In 2014, Dr. Jaiswal and colleagues showed that dysferlin's role in promoting ASM release was critical for membrane repair, which prompted him to consider ASM as a therapy for LGMD2B.

“In dysferlin patients, there is no lack of acid sphingomyelinase,” he said. “Instead, there is a lack of its release upon injury to the muscle fiber.” Once released, ASM circulates freely; therefore, he reasoned, supplying the enzyme systemically should be therapeutic, obviating the need to transfect muscles. That led him to ask whether the liver might be the best target for transfection, since it is a large organ designed to manufacture and release multiple products, for which many types of AAV have a natural tropism.

Study Details

In the new paper, Dr. Jaiswal and colleagues showed the promise of this approach. After demonstrating that human LGMD2B muscle cells treated with human ASM protein improved their ability to self-repair through an increase in ASM-driven membrane endocytosis, they turned to the mouse model of the disease.

Treated with a single dose of the gene for human ASM under control of a liver-specific promoter and delivered with a hepatotropic AAV, mice expressed high levels of ASM in the liver and increased their serum ASM concentration significantly compared with controls, without any signs of liver injury. Treated mice had a two- to threefold reduction in exercise-induced injury in the major muscles, and a pattern of loss of force due to injury that matched healthy controls.

At the cellular level, treated mice showed reduced inflammation, indicating less muscle damage, and the number of centrally nucleated myofibers was reduced by half, indicating less need for regeneration. Fibrosis, a late consequence of exhaustion of regeneration capacity, was also dramatically reduced.

“Our findings indicate that a single dose of human acid sphingomyelinase delivered to the liver by AAV holds the potential to allow extended histopathological and functional muscle improvement in limb girdle muscular dystrophy type 2B,” Dr. Jaiswal said.

“What surprised us was that the extent of improvement we found by addressing the downstream consequence of the dysferlinopathy compared very well to benefits seen in mice using the dual vector approach for gene replacement. Until dual vector therapy becomes available, we feel this may be a treatment strategy that patients can benefit from.”

While downstream targeting is a relatively new concept in neuromuscular disease, Dr. Jaiswal added, “it is the standard of care in cancer,” where the function of the housekeeping gene target is often too critical to knock out. “But it should be remembered that LGMD2B is a chronic disease, and that may present special challenges not faced in similar approaches in oncology.”


“Our findings indicate that a single dose of human acid sphingomyelinase delivered to the liver by AAV holds the potential to allow extended histopathological and functional muscle improvement in limb girdle muscular dystrophy type 2B.”—DR. JYOTI K. JAISWAL

Experimental treatment of acid sphingomyelinase deficiency type A/B (formerly known as Niemann-Pick disease) with recombinant ASM protein is moving forward. The European Medical Agency recently accepted for accelerated review a marketing authorization application for olipudase alfa, Sanofi's version of ASM, which is delivered by infusion every three weeks. For LGMD2B treatment, Dr. Jaiswal said, this type of bolus therapy poses challenges. “What we need is to have increased ASM circulating at all times, which can be achieved with gene therapy.”

Expert Commentary

“The approach used in this study is very interesting,” commented Isabelle Richard, PhD, research director at the Centre National de la Recherche Scientifique at Genethon in Evry, France, whose research includes development of two-vector therapy for LGMD2B.

“Because the gene product circulates in the bloodstream, it can come into contact with muscles throughout the body, without having to target them all directly. This is what makes it an interesting strategy.”

“The ASM gene is small enough to fit easily into AAV,” noted Dr. Bonnemann.

“Further, you only have to effectively transduce the liver, and targeting the liver is relatively easy with AAV; in fact, in muscle gene therapy we try to avoid the liver, because so much of the vector typically ends up there.” An additional advantage, he said, is that the ASM protein is already produced widely in the body, and so the immunogenic risk is smaller than for dysferlin, which is not produced in abundance in LGMD2B patients.

Other advantages include that ASM replacement is already in clinical development for ASM deficiency disease, “which means there is considerable clinical experience with it already. And liver production of therapeutic proteins has also been tried and found practical, including for Pompe disease. So this strategy is not an outlandish one and may be more likely to succeed than not.”

Finally, Dr. Bonnemann said, “This is a great opportunity for combinatorial therapy. Even under the best circumstances, gene replacement therapy is not going to be 100 percent effective, so having another approach that can be added to that is promising. That is the future of gene therapy in neuromuscular disease, and LGMD2B is a candidate for that.”


Dr. Jaiswal had no conflicts to disclose.