MDA-supported research at the University of Pittsburgh shows that a miniaturized version of the gene for agrin can markedly improve the health and longevity of mice with congenital muscular dystrophy (CMD) resulting from a lack of the crucial laminin alpha-2 chain of the laminin protein.

This type of CMD, usually called merosin-deficient CMD by physicians, generally begins at or near birth. The absence of merosin (laminin), a protein linking the muscle cell membrane to structures outside the cell, probably weakens a sheath around each muscle cell (the basal lamina), and destabilizes the membrane.

Chunping Qiao	Xiao Xiao

MDA grantee Chunping Qiao and colleagues, including senior investigator Xiao Xiao, who has received MDA funding for the development of viral gene transporters, published their results in the Aug. 23 issue of Proceedings of the National Academy of Sciences. Their hypothesis was that high levels of the muscle protein agrin would help compensate for the missing laminin.

In merosin-deficient congenital MD, laminin (merosin) is partially or completely missing, which breaks the connection between alpha-dystroglycan and the basal lamina. Adding agrin to merosin-deficient muscle cells seems to partially compensate for the lack of merosin.

The Pittsburgh researchers performed a set of experiments, delivering a miniaturized agrin (miniagrin) gene by two methods, each of which used a slightly different viral transporter.

In one experiment, they delivered miniagrin genes, each inserted into the shell of an AAV2 (adeno-associated virus 2) directly into the leg muscles of mice with laminin-deficient CMD. Two months after the injections, the treated leg muscles showed more agrin, less scarring and general signs of improvement in muscle health compared to the same muscles in untreated mice.

In another experiment, they delivered the same genes, this time inserted into AAV1 (adeno-associated virus 1) shells, injected into the abdominal cavity of the mice as a form of systemwide therapy.

The systemic delivery resulted in far-reaching therapeutic effects. About four months after treatment, the mice showed increased agrin levels and improvement in the appearance of several skeletal muscle groups, as well as the respiratory diaphragm, other breathing muscles and the heart.

The treated muscle fibers were larger and didn’t show the scarring that reflects dystrophy-related muscle damage. The hearts of the treated mice were nearly indistinguishable from those of normal mice, the investigators say.

Only 50 percent of untreated mice with laminin-deficient CMD were alive at the age of 4 weeks, while 50 percent of those systemically treated with the miniagrin genes were alive at more than 17 weeks.

Xiao said the results are encouraging and could bode well for future development of agrin-based therapy for patients with merosin-deficient CMD.

The University of Pittsburgh is one of three muscular dystrophy centers of excellence co-funded by MDA and the National Institutes of Health.

MDA grantee Elizabeth McNally, a cardiologist and molecular geneticist at the University of Chicago, recently led a research group that found that genes other than those directly responsible for two forms of limb-girdle muscular dystrophy (LGMD) can influence disease severity.

In some types of limb-girdle MD, one of the four sarcoglycan proteins is missing from the cell membrane, which destabilizes the rest of this group of proteins.

The investigators, who published their findings in the October issue of Neuromuscular Disorders, studied mice with a type of LGMD resulting from an absence of gamma-sarcoglycan, a protein in the muscle-fiber membrane, and other mice missing delta-sarcoglycan, another such protein. The mouse muscular dystrophies resulting from these missing proteins correspond to the human disorders known as LGMD2C and 2F, respectively.

They studied these two forms of sarcoglycan-related LGMD in mice from several different genetic families (strains) to see whether the various strains would respond differently to a sarcoglycan deficiency.

In LGMD2C-affected mice, the resulting dystrophy was least severe in mice from a strain called 129 and most severe in mice from the DBA strain. They measured the severity of the disease by whether the cells were permeable to a blue dye (an indication of muscle cell membrane fragility), and by how much scarring there was in the muscle tissue.

Elizabeth McNally

In the 129 strain, mice missing delta-sarcoglycan fared worse with respect to membrane fragility and scarring than did mice missing gamma-sarcoglycan.

But in another strain, called C57, there was no difference between the gamma- and delta-sarcoglycan-deficient animals.

“Uncovering these other gene modifiers will help us predict who will be more or less severely affected by their muscular dystrophy,” McNally said.

Simultaneously delivering genes for the muscle protein dystrophin and for the muscle-specific form of insulin-like growth factor 1 (IGF1) rescues muscle fibers of mice with a disease closely resembling Duchenne muscular dystrophy (DMD) more effectively than does either compound alone, say researchers at the University of Washington in Seattle.

The investigators, who published their results in the September issue of Molecular Therapy, found that miniaturized genes for dystrophin (microdystrophin) helped muscle fibers resist mechanical injury from muscle contraction, while genes for the muscle form of IGF1 made the muscles larger. Together, the dystrophin and IGF1 made from the two genes increased muscle mass and strength and increased resistance to contraction-related injury.

The researchers inserted the genes into an AAV6 (adeno-associated virus type 6) and injected the viral particles into the leg muscles of the mice.

In Duchenne MD, muscle cells lack dystrophin, which affects the stability of the rest of the proteins in the membrane cluster. Utrophin can stand in for dystrophin, but there isn't much of it in mature cells.

Team members Paul Gregorevic and Jeffrey Chamberlain have MDA funding for closely related work. Chamberlain directs the University’s Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, which is co-funded by MDA and the National Institutes of Health.

“Microdystrophin delivery is showing great promise as a potential treatment for muscular dystrophy,” Chamberlain said. “However, the smaller dystrophins are not quite as effective as the normal-sized gene. This new study shows that delivering muscle-building agents such as IGF1 with them leads to a further improvement in muscle strength.”

MDA grantee Ching Wang, a pediatric neurologist at Stanford (Calif.) University, was the senior investigator on a research team that recently demonstrated that adding the drug hydroxyurea to white blood cells taken from people with spinal muscular atrophy (SMA) increased the ability of these cells to produce SMN1, a protein needed but deficient in SMA.

Wang’s team added the drug to blood cells from five people with type 1 SMA (the most severe), five with type 2 (medium severity), five with type 3 (least severe) and five without SMA. It was effective at increasing SMN1 in all groups, although the increase was most significant in the SMA-affected cells.

The investigators, who published their results in the August issue of Annals of Neurology, say they believe the increase in SMN1 protein resulted from a change in the output from SMN2 genes, which carry almost identical instructions for making the SMN protein but can only produce a small amount of it. SMN2 genes mostly produce a closely related, but shorter, SMN protein.

Ching Wang

The hydroxyurea apparently caused the SMN2 genes, which people with SMA have, to produce more of the longer, SMN1 protein, which those with SMA normally lack because they don’t have functional SMN1 genes.

“We are very encouraged by our findings that hydroxyurea is able to increase SMN protein production in white blood cells isolated from SMA patients,” Wang said. “We hope that we can see the same effects when we use hydroxyurea to treat SMA patients.”

Wang and colleagues are testing hydroxyurea in people younger than 10 years with all types of SMA. For more information, contact Tony Trela at (650) 498-7658, or sma@stanfordmed.org; or visit http://sma.stanford.edu.

Investigators at the University of Michigan in Ann Arbor have found that a synthetic compound known as poloxamer 188 (p188) can protect heart muscle cells in mice lacking the protein dystrophin, which have a muscle disease resembling Duchenne muscular dystrophy (DMD).

Soichiro Yasuda and colleagues, who published their results online July 17 in Nature, found that when they added p188 to heart muscle cells from dystrophin-deficient mice, the cells’ resistance to stress matched that of cells from healthy mice. They believe the drug may shore up the fragile cell membranes seen in DMD.

Next, they gave some of the mice an intravenous infusion of dobutamine, a drug that increases heart rate and blood pressure, and another group an infusion of dobutamine preceded by intravenous p188.

Several of the 10 DMD-affected mice in the first group experienced acute heart failure, which didn’t occur in the mice that received p188.

John Quinlan

“If issues of dosing and long-term safety can be addressed, our results indicate that membrane-sealing poloxamers could represent a new class of therapeutic agents” for heart muscle damage associated with DMD and possibly other types of MD involving defects in the muscle-cell membrane, the authors say.

John Quinlan, an MDA grantee at the University of Cincinnati who is studying cardiac problems in DMD-affected mice and is also interested in p188, said, “This work is exciting and cause for hope. The Michigan team has provided us with a better understanding of how DMD attacks cardiac function on a cellular level. Most importantly, they showed how p188 has both beneficial action on reversing cellular damage and improving heart function under stressed conditions.”

Flaws (mutations) in a chromosome 1 gene that instructs for the mitofusin 2 protein account for

approximately 20 percent of type 2 Charcot-Marie-Tooth disease (CMT) cases, says a study in the July 26 issue of Neurology.

The mitofusin 2, or MFN2, gene was first linked to CMT2 last year (see “Research Updates,” July-August 2004). It affects the behavior of mitochondria, the energy-producing units in cells.

Now, researchers in the laboratory of Kevin Flanigan at the University of Utah School of Medicine in Salt Lake City have identified mutations in the MFN2 gene in three large families with CMT2.

Type 2 CMT results from abnormalities in the nerve fibers (axons), while type 1 results from defects in the insulating sheath surrounding the axon and containing proteins and carbohydrates (myelin sheath). These are distinguished by nerve conduction testing.

The investigators suggest that genetic testing of CMT2 patients begin with a screen of the MFN2 gene. (Such screening is commercially available.)

Neurotrophin 3 Improved Sensation in CMT1A

A pilot study of eight people with type 1A Charcot-Marie-Tooth (CMT) disease, a disorder in which signals in the peripheral nervous system are impaired, has found that treatment with neurotrophin 3 (NT3) improved sensory function and nerve regeneration.

Zarife Sahenk

Neurologist Zarife Sahenk at the Columbus Children’s Research Institute Neuromuscular Program at Ohio State University led the MDA-funded study team. Jerry Mendell, a neurologist and MDA clinic co-director at OSU Hospitals, was also an investigator.

NT3 is a natural neurotrophic (nerve-nourishing) factor.

The investigators, who published their findings online July 6 in Neurology, studied people with a form of CMT that results from an abnormally duplicated PMP22 gene on chromosome 17.

In CMT1A, the function of the PMP22 protein, which normally contributes to an insulating sheath that covers nerves running between muscles and the spinal cord (peripheral nerves), is disrupted, impairing sensory and motor signals.

After establishing that NT3 was apparently effective in mice with PMP22 defects, the research team gave four adults with CMT1A injections of NT3 three times a week for six months. The other subjects received a placebo.

At the end of the study, the placebo group’s scores on a standardized scale of neuropathy-related impairment had worsened, while scores in the treated group had improved.

There were no significant changes in specific sensory or motor tests in the placebo group at six months, but the NT3 group showed improved vibratory sensation assessed by a tuning fork test. Their reflexes also improved. Motor function didn’t improve in either group.

Biopsies of the sural nerve, located in the calf, showed some regeneration of the nerve tissue in the NT3-treated participants.

“We hope that this approach with neurotrophic agents can be applied to peripheral neuropathies, where there are few treatment options,” Sahenk said.

Pregnancy and Delivery to Be Studied in FSH MD

Investigators at the University of Rochester (N.Y.) Medical Center are seeking 200 women with facioscapulohumeral muscular dystrophy (FSHD) who are at least 18 years old to participate in a questionnaire-based study to better understand pregnancy and delivery in this disorder.

The questionnaire, which takes about 30 minutes to complete, asks about pregnancy, genetic counseling, fertility, FSHD and other medical issues. Participants are also asked to release obstetrical/gynecological and neurological medical records to the investigators.

URMC is the site of a Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, co-funded by MDA and the National Institutes of Health.

For information, contact Alexis Smirnow in Rochester at (585) 275-4715 or alexis_smirnow@urmc.rochester.edu.

CellCept Being Tested in Myasthenia Gravis

Two multicenter clinical trials are under way to test mycophenolate mofetil (brand name CellCept) in adults with myasthenia gravis (MG). CellCept was developed in the 1990s and is marketed by Roche Pharmaceuticals to reduce the immune response to transplanted organs. This unwanted immune response bears some similarity to the immunologic attack on muscle cells in MG.

For details, see www.clinicaltrials.gov. Enter “CellCept” and “myasthenia gravis” into the search box.

SMA Researchers Need Pre-Trial Patient Data

Three forms of spinal muscular atrophy can show symptoms in infancy. Researchers who are part of the Pediatric Neuromuscular Clinical Research Network (see www.urmc.edu/sma) are seeking 270 people with types 1, 2 or 3 spinal muscular atrophy (SMA) whose disorder was diagnosed before age 19, for a study to gather data necessary for a future clinical trial. Participating centers are in Boston, New York and Philadelphia, and several visits to these centers are necessary. Participants will undergo physical exams, lab tests and biopsies. Contact Jessica Rascoll at (212) 342-5767 or jr2024@columbia.edu for more information.

Life Satisfaction With Disability Probed

Graduate student Roy Chen at Michigan State University in East Lansing is investigating the relationship between self-acceptance of a disability and life satisfaction. Chen, who has muscular dystrophy, is seeking people who are at least 18 years old, have a progressive neuromuscular disorder, and are willing to fill out a questionnaire online or in hard copy format.

The online survey can be found at www.surveymonkey.com/s.asp?u=344351246794. For more information or a hard copy of the survey, contact Chen at (517) 355-8091 or chenroy@msu.edu.