CMD: Aiming Simultaneously at Two Biological Targets

Targeting multiple pathways in mice with a disorder resembling merosin-deficient congenital muscular dystrophy shows more promise than aiming at one pathway at a time.

Researchers at Boston University, supported in part by MDA, say their experimental two-pronged strategy for merosin-deficient congenital muscular dystrophy (MDC1A) was highly successful in a mouse model of this disease and should be further investigated as a potential treatment approach for patients.

Better knowledge of the molecular underpinnings of congenital muscular dystrophies has prompted several experimental strategies in recent years, but available treatments remain largely aimed at relieving symptoms.

Now, MDA research grantee Mahasweta Girgenrath and colleagues have shown that simultaneously counteracting cell death and providing a growth-promoting protein improved muscle health and function in mice with an MDC1A-like disorder.

The investigators, who published their results online June 16, 2013, in Human Molecular Genetics, note that their study "is proof of concept that demonstrates simultaneous targeting of these two disease drivers leads to a near complete amelioration of disease pathology."

They also speculate that the effects of the combined therapy may reflect more than just the sum of two treatments.

Targeting the "noxious environment" found in MDC1A-affected muscle with one type of treatment, they say, may allow another treatment — growth promotion — to be effective.

Missing merosin leads to multiple muscle abnormalities

Merosin-deficient CMD is a form of congenital-onset muscular dystrophy in which a protein called merosin — also known as laminin 211 — is deficient. Laminin 211 normally forms a bridge between the muscle-fiber membrane and its surrounding matrix. This bridge is thought to be essential to both physical anchoring of the muscle fiber and biochemical signaling between the fiber and other cells.

Laminin-deficient muscle tissue in children and in mice shows evidence of at least three abnormalities: inflammation, fibrosis(scarring) and diminished ability for muscle regeneration.

The diminished muscle regeneration appears to arise in part from an excess of apoptosis, a normal cell death process. Apoptosis, derived from a Greek word that means "falling off," is a normal pruning process in which cells die and are replaced at intervals. However, too much of it can shift the balance in a tissue away from regeneration and toward degeneration.

Combining two gene-based therapies

Girgenrath and colleagues conducted experiments in laminin 211-deficient mice that show MDC1A-like abnormalities, including muscle degeneration with limited or no regenerative capacity, muscle inflammation, muscle fibrosis and a shortened life span.

Girgenrath’s earlier, postdoctoral work showed that laminin 211-deficient mice that are also deficient in the apoptosis-promoting protein BAX — because of genetic alteration before birth — lived longer than untreated laminin 211-deficient mice.

Also in earlier experiments, they found that laminin 211-deficient mice genetically engineered before birth to overproduce insulin-like growth factor 1 (IGF1), a growth-promoting protein, showed better overall growth as well as longer survival than untreated mice.

However, neither of these single treatments resulted in complete recovery, leading the investigators to conclude that "a single therapy may not be sufficient to treat MDC1A."

In their current project, Girgenrath and colleagues combined these two approaches to see if they could improve outcomes.

Two treatments better than one on most tests

First, the investigators developed laminin 211-deficient mice that were genetically deficient in BAX and simultaneously genetically engineered to overproduce IGF1.

Next, they compared these laminin 211-deficient, BAX-deficient, IGF1-overproducing ("combined treatment") mice to mice that were:

• laminin 211-deficient and not treated;
• laminin 211-deficient mice solely genetically engineered not to produce BAX ("single treatment"); and
• laminin 211-deficient mice solely genetically engineered to overproduce IGF1 ("single treatment").

When they compared "combined treatment" mice to "single treatment" or untreated mice with laminin 211 deficiency, the study investigators saw the following:

• The combined treatment mice had significantly higher body weights at 8 weeks of age than mice that were only BAX-deficient or mice that only overproduced IGF1. Untreated laminin 211-deficient mice had the lowest body weights.
• Leg muscles from all treated mice were significantly larger than those from untreated mice. Hind-limb muscle weights in the combined treatment group were significantly higher than they were for mice receiving only the IGF1 treatment. Some leg muscles in the combined treatment group weighed more than these same leg muscles in the mice that were only BAX-deficient.
• In a measure of muscle activity — how many times the mice spontaneously stood on their hind legs — the combined treatment mice showed significantly better activity than either singly treated group or untreated mice.
• In another measure of muscle activity — how long mice can hold their legs in an extended position when suspended by their tails — the combined treatment mice were able to maintain leg extension for a longer time than singly treated mice or untreated mice.
• Combined treatment mice showed an increase in regenerating muscle fibers compared with mice treated with BAX inhibition alone.
• The size (cross-sectional area) of muscles in the combined treatment group was significantly greater than that of the untreated mice or the IGF1-overproducing mice but not greater than that of the BAX-deficient mice.
• Serum creatine kinase (CK) levels — a measure of muscle damage — in the combined treatment group were significantly lower (a sign of less damage) than in the untreated group or the IGF1-treated group. They were slightly lower than in the BAX-deficient group.
• All treatments resulted in a significant reduction in the number of apoptotic (dying or dead) cells compared to no treatment, but the greatest decrease in apoptotic cells was seen with the combined treatment.
• The muscles of the combined treatment group showed the least number of inflammatory cells, indicating a less inflamed muscle environment. Single treatment groups showed only a slight decrease in inflammatory cells compared to untreated mice.
• BAX inhibition treatment resulted in drastic reduction in one marker of inflammation compared to no treatment, but in this case, adding IGF1 to BAX did not further reduce the inflammatory marker.
• Neither BAX inhibition alone nor IGF1 overproduction alone reduced the amount of scar tissue in muscles, but combined treatment resulted in a significant decrease.

In addition:

• The majority of the untreated mice died within 40 days of birth, but no deaths had occurred in the combined treatment group at the time the animals were last assessed at the age of 8 weeks.
• Muscle structure in the combined treatment group resembled that of normal mice, with very little space between the fibers and few inflammatory cells.

Drug-based IGF1 similar to gene-based IGF1

Since gene-based therapies require a lengthier and more complex development pathway than more conventional drug treatments, the researchers decided to substitute IGF1-based drug therapy — a drug called Iplex — for the IGF1-based gene treatment strategy in another set of experiments.

They found that Iplex treatment, given after the mice were born for seven weeks, had similar effects to IGF1 genetic treatment.

Specifically, they saw the following:

• Laminin 211-deficient, BAX-deficient mice treated with Iplex showed substantially higher body weights than BAX-deficient mice not treated with Iplex.
• On muscle function tests involving standing on their hind legs and extension of their legs during tail suspension, the Iplex-treated, BAX-deficient mice performed significantly better than mice that were BAX-deficient but not treated with Iplex.
• BAX-deficient, Iplex-treated mice had healthier-appearing muscles compared to mice that were BAX-deficient but not treated with Iplex.
• BAX-deficient, Iplex-treated mice showed less muscle fibrosis than BAX-deficient mice not treated with Iplex.

The researchers did not test anti-apoptotic medications in the mice, but they note that two of these — doxycycline and omigapil — should be tested in combination with Iplex in a future study. (These drugs have been tested in laminin 211-deficient mice but not in combination with Iplex.)