ARTICLE IN BRIEF
In animal models of congenital muscular dystrophy type 1A, researchers identified two proteins that, when working together, strengthened muscle and prolonged their lifespan — from weeks to more than two years. The proteins could be the “missing links” to correcting the defect in this form of muscular dystrophy, the research team said.
Researchers genetically engineered mice with a congenital form of muscular dystrophy to express two smaller proteins that, when combined, strengthened muscle and prolonged their lifespan — from weeks to more than two years, according to a June 28 study published in Science Translational Medicine.
The research team, led by Markus A. Rüegg, PhD, a professor at the Biozentrum of the University of Basel in Switzerland, said the proteins could be the missing links researchers have been looking for to correct the defect in congenital muscular dystrophy type 1A, a common and severe form of the disease that is caused by mutations in LAMA2, the gene encoding laminin-alpha 2, the heavy chain of the heterotrimeric basement membrane protein laminin-211.
The two smaller engineered proteins — mini-agrin (mag) and alpha-LNNd — seem to act as “linkers” in the mice to restore defective connections between muscle fibers and the supportive sheath, the basement membrane.
The findings provide a mechanistic understanding of LAMA2 muscular dystrophy and could pave the way for a novel treatment strategy for infants with the disorder, Dr. Rüegg said.
In LAMA2-related muscular dystrophy, there is a disconnect between the extracellular matrix of muscle cells and the intracellular scaffolding, Dr. Rüegg explained.
Muscle cells need a strong connection between the inside and outside of cell — like a sturdy bridge — to withstand the mechanical force generated during a contraction. The lack of laminin-211 causes structural instability to contracting muscle fibers; this leads to muscle degeneration and triggers secondary events that also accelerate the disease process.
“The bond between the muscle plasma membrane and the basement membrane is crucial for muscle fiber stability and signal transduction,” Dr. Rüegg said. Without laminin-211, infants are born floppy and usually do not develop enough strength to stand or walk on their own. They also have respiratory problems, which could be fatal.
At the same time, he said, there seems to be compensatory overexpression of a laminin isoform called laminin-411. The laminin-alpha 4 chain in laminin-411 can't bind to the muscle receptors, which are necessary in building a sturdy bridge. (Normally, laminin-alpha 4 disappears from the muscle postnatally but appears to remain in these muscular dystrophy patients and in the transgenic mouse models of the disease.)
Dr. Rüegg and his colleagues have been searching for small linker proteins that could fit onto both ends of laminin-411 and mimic the activity of laminin-211. A decade ago, they identified a linker protein they called mag that contains binding sites for laminins and its receptor on muscle, alpha-dystroglycan. It seemed to improve basement membrane stability, though that wasn't enough to fix the problem. The second linker protein, alphaLNNd, allowed stable networking or polymerization of the laminin in the basement membrane.
Study co-author Peter Yurchenco, MD, a professor in the department of pathology and laboratory medicine at the Robert Wood Johnson Medical School at Rutgers University, designed the small linker protein alphaLNNd. Together with mag, the two linkers bind to both ends of laminin-alpha 4. Then, they made double transgenic animals: The laminin-alpha 4 now linked up to basement membrane and the inside of the muscle cell, and formed a laminin network that was working.
The expression of both transgenes increased body mass by more than 90 percent, with an increased median life span of 81 weeks. Five of the 16 mice lived more than two years. They also had increased muscle size and strength. The linker proteins were helping the laminin-411 to bind to muscle cells and to polymerize. Both steps are necessary to get the muscle cells responding and surviving at near-normal levels.
The question for future study, independent experts told Neurology Today, is whether this strategy could fix problems postnatally, after the onset of the disease — a research problem the Swiss scientists said they are going to address in the future. For it to work in patients, scientists need to be sure that they could deliver these linker proteins (by way of gene therapy vectors) so that they link up to laminin-411 in muscle and do the work of laminin-211.
There are many reasons to hope that the strategy would work in patients, Dr. Rüegg and his colleagues wrote. The linker proteins are small enough to pack into gene vectors. (The laminin-alpha 2 is too large to use for gene therapy.) In other models, they wrote, adeno-associated virus (AAV)-mediated gene transfer “into skeletal muscle allows expression of the gene of interest for at least 10 years in human patients.” And the linkers are derived from proteins that the body already knows, and that cuts down the chance that the immune system wages an attack on the cells.
“Clearly, there is no sufficient linkage between the surface muscle and the basement membrane without laminin 211, but is also clear that once an effective linkage is re-established somehow, this severe disease can be expected to be ameliorated in a major way,” Carsten G. Bönnemann, MD, chief of the neuromuscular and neurogenetic disorders of childhood section of the National Institute of Neurological Disorders and Stroke, told Neurology Today.
“They have used smaller pieces of naturally-occurring proteins and linked them so that the present laminin-411 can now make a connection between the muscle surface and the basement membrane and crosslink them with each other. Now, it works like an artificial laminin-211.”
“It's a nice piece of ingenuity and a very rational approach to designing a treatment for this devastating disorder,” Dr. Bönnemann continued. “These linkers are small enough to package into AAVs for gene therapy and likely would be tolerated from an immunological point of view since they are based on proteins the patient's immune system would know.”
He added that there are good working animal models for this disease. “It will now be important to package the linker proteins in two different AAV vectors and deliver them at different times after birth to see if it still works to protect the muscle as this is disease is already established at birth,” he said. “Then, we can think about a clinical trial.”
“This is a very important study,” added Jeffrey Chamberlain, PhD, professor of neurology and the McCaw Endowed Chair in Muscular Dystrophy at the University of Washington School of Medicine, and director of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center of Seattle. “This team determined why the laminin-411 doesn't work very well; it needs binding partners to maintain the strong link between the inside and outside of the muscle. Dr. Rüegg and his colleagues came up with a way to add on an adaptor that strengthens this weak link. These two proteins strengthen the binding on both ends of the laminin-411. The study showed that it restores tight binding and protects the muscles.”
“It could be very therapeutic if you can get these adaptors into patients,” he said.
EXPERTS: ON THE ‘MISSING LINKS’ FOR CORRECTING LAMA-2 MD
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