Exon Skipping Skips Ahead by Margaret Wahl Stephen Wilton, an MDA grantee, and colleagues at the University of Western Australia in Perth, with scientists at AVI BioPharma in Corvallis, Ore., report significant progress in two sets of experiments that make use of the technique known as exon skipping. This strategy coaxes cells to produce some functional dystrophin — the protein missing or inactive in Duchenne muscular dystrophy (DMD) — despite the presence of a genetic mutation, by skipping over exons (regions of genetic information) in the part of the gene with the defect. The skipping is accomplished by delivering antisense oligonucleotides (AOs) to cells. A clinical trial is scheduled to begin in London by the end of the year. Success in Dog Cells On May 25, Graham McClorey and colleagues reported online in Gene Therapy that they’d achieved success in causing muscle cells taken from dogs with a disorder similar to human DMD to produce functional dystrophin.   Stephen Wilton They used AOs that carried a tag to enhance cellular delivery. These morpholino AOs, which appear to be more stable and taken up by cells more efficiently than other AO chemistries, block parts of the canine dystrophin gene transcript near the site of a serious genetic mutation. “This allows the treated canine cells to ‘spit out’ the bad part of the defective dystrophin gene transcript to restore the reading frame [the way the cell reads the instructions] and allow synthesis of a functional gene product,” Wilton said. The canine mutation mimics some that human patients have. The exon skipping treatment allowed production of nearly full-length dystrophin, with no apparent adverse effects on the cells, the researchers say. “The induction of dystrophin in the [dog] model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials,” the investigators write. More Targets Abbie Fall and colleagues, in a study published online May 24 in Genetic Vaccines and Therapy, applied the strategy to a wider range of genetic mutations than had previously been targeted. Using cocktails of morpholino AOs, these researchers induced cells to skip a large section of the dystrophin gene in mice carrying a “stop signal” mutation in exon 23. Previous successes in these mice only eliminated exon 23, splicing together exons 22 and 24, but this time, the researchers induced skipping of exons 19 to 25. They found detectable, apparently functional dystrophin eight weeks after a single intramuscular injection into 11-day-old and 16-week-old mice. In the younger mice, dystrophin appeared only in the area around the injection site but was strong and consistent. In the older mice, dystrophin was more widely distributed, but was patchy. Analysis of dystrophin gene defects that give rise to mild cases of Becker muscular dystrophy, a less severe disease than DMD, has shown that loss of up to 66 percent of the dystrophin gene can still produce a fairly functional dystrophin protein and lessen disease severity, the investigators note. The researchers say targeting multiple exons appears to be a “feasible strategy,” and that they could theoretically target more than 75 percent of dystrophin mutations that cause human DMD with a relatively small set of AO cocktails. Clinical Trial A clinical trial to test exon skipping with antisense in nine boys with DMD is scheduled to begin recruiting by the end of the year. The trial is under the auspices of the Imperial College of London and the Department of Health of the United Kingdom.   Exon Skipping: As a cell prepares the final version of instructions for making a protein, it removes excess material and leaves only the exons, the parts that will form the final protein recipe. Laboratory-designed antisense compounds can make a cell eliminate a specific exon along with the other unwanted material. Investigators plan to inject varied doses of antisense constructs into a foot muscle and evaluate the safety of the pro-cedure and the amount and longevity of any dystrophin produced. For updated information, see www.clinicaltrials.gov; enter “Duchenne muscular dystrophy” in the search box and scroll to the antisense trial.

Scientists Share Gene Therapy Advances

Researchers, including MDA grantees, working on gene therapy for muscle and nerve diseases presented findings and exchanged ideas at the ninth annual meeting of the American Society of Gene Therapy, held in Baltimore in June.

Improved Cell Therapy Has Promise, Drawbacks

Transplantation of cells committed to forming muscle (termed myogenic precursor cells in this study) from donors to patients, using updated techniques, may result in considerable production of a needed protein in Duchenne muscular dystrophy (DMD), said molecular biologist Jacques Tremblay, of the Human Genetics Unit of Laval University in Quebec, and colleagues, at the meeting.

Jacques Tremblay

Tremblay, who’s had intermittent MDA funding since 1996, has improved upon myoblast transfer, a technique tested in the 1990s that transplanted cells located around the periphery of muscle fibers and capable of repairing muscle. The trials failed to keep the transplanted cells alive for long and didn’t help boys with DMD get stronger.

Tremblay and colleagues have changed the way in which the satellite cells are processed after being retrieved from donors’ muscles. They also administered the powerful immunosuppressant tacrolimus to the cell recipients, and spaced the cell injections every 1 to 2 millimeters in recipients’ muscles.

They say eight out of nine DMD-affected boys, all of whom received myogenic precursor cells from their parents, showed some benefit from the transplant, with dystrophin-producing fibers ranging from 4 percent to 26 percent four weeks after injec-tions into a very small area in a leg muscle.

Tremblay’s group plans to conduct a new trial of cell transplantation in 10 men with DMD or its milder variant, Becker MD, who are at least 18 years old. (The study is accepting only Canadians.)

Concerns remain about the safety and effectiveness of long-term immunosuppression and the practicality of administering intramuscular injections hundredths of an inch apart throughout the body.

However, Tremblay said, he thinks that “transplantation of cells derived from satellite cells will one day be a treatment for most skeletal muscles.”

Cells With SMA Coaxed to Make Needed Protein

Conference attendees saw some exciting data from MDA grantee Christian Lorson at the University of Missouri-Columbia, and colleagues, who presented a new molecular strategy to coax cells affected by spinal muscular atrophy (SMA) to make needed SMN protein molecules. (A paper from this group is in the July issue of Molecular Therapy.)

In people with SMA, cells usually make a shortened, nonfunctional version of the SMN protein when guided by SMN2 RNA instructions. University of Missouri investigators have created a bifunctional RNA that sticks to the SMN2 instructions (A) and attracts compounds (B) that lead to production of full-length SMN.

People with SMA have either mutated or no SMN1 genes, which normally produce the full-length SMN. But they have SMN2 genes, which produce some full-length SMN protein molecules but mostly short, nonfunctional SMN molecules.

Fortuitously, instructions for making full-length SMN are embedded in the SMN2 genetic recipe, at the RNA stage (made from the DNA genetic code), but these instructions are for the most part ignored by SMA-affected nerve cells. Now, Lorson’s group has figured out an elegant way to coax the cell into following them.

The Missouri investigators designed bifunctional RNA molecules that stick to the SMN2 RNA instructions at a specific place (one function). They then recruit so-called splicing factors, proteins that lead to the production of full-length SMN (a second function).

They delivered the molecules to SMA-affected cells in a lab dish, in some cases using altered adeno-associated viruses to continuously make the desired RNA pieces from DNA.

If the approach were used in people with SMA, Lorson said, “the idea would be that a single administration would last for many months or years. Therefore, it would not require frequent boosts, but it’s very likely that multiple muscle groups would need to be targeted at the time of initial treatment.”

Duan Receives Award

Dongsheng Duan

MDA research grantee Dongsheng Duan, at the University of Missouri-Columbia, received a prestigious Outstanding New Investigator Award from the society.

The $1,000 award is given to a scientist who’s held a full-time faculty position for no more than seven years. Duan is an as-sociate professor in the Department of Molecular Microbiology & Immunology.

Duan’s current MDA grant is for the development of gene therapy for heart disease in a mouse model of Duchenne muscular dystrophy (DMD).

 

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Stem Cells Restore Movement in Rats; May Apply in SMA, ALS

A research team led by MDA grantee Douglas Kerr at Johns Hopkins University in Baltimore has developed a multistep regimen that improves the functional effect of transplanted stem cells in paralyzed rats and could have implications for motor neuron diseases like spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS).

Deepa Deshpande at Hopkins, with colleagues at Upstate Medical University in Syracuse, N.Y., and Curis Inc. of Cambridge, Mass., describe in their June 26 online publication in Annals of Neurology how their strategy restored functional nerve-to-muscle connections and movement in the back legs of rats that had been paralyzed by a virus.

In a four-step process, the investigators transplanted mouse embryonic stem cells and injected the nerve growth factor GDNF into the sciatic nerve near the spinal cord. At the same time, they infused two compounds that counteract the effects of myelin, a normal substance that ensheathes nerve fibers but which has been found to interfere with the growth of new fibers.

“I do see these findings as a preliminary step that could ultimately apply to either or both ALS or SMA,” Kerr said, adding that he was particularly optimistic about the implications for infantile SMA. He noted that “the environment is less hostile [than in ALS], because the disease is [limited] to motor neurons; infants have a short distance to bridge between spinal cord and muscle; the peripheral nerve components remain primed to receive new axonal input because of the rapid nature of motor neuron death in infants; and there is no or little myelin to inhibit growth in infants.”

Merg1a Protein Could Be New Target in Fighting Atrophy

Better understanding of muscle atrophy — the wasting of muscle tissue that occurs in various disease states or when weight bearing doesn’t occur for some time — may reveal new targets for muscular dystrophy treatment, say researchers at Purdue University in West Lafayette, Ind., and the University of Pittsburgh.

Xun Wang and colleagues, who published their results online May 24 in the FASEB Journal, found that activation of the protein Merg1a may set off an atrophy program in muscle cells.

When mice that had been prevented from bearing weight on their back legs for seven days were given the drug astemizole, which blocks Merg1a function, they experienced significantly less atrophy than mice that didn’t get the drug.

Amber Pond and Kevin Hannon at Purdue’s School of Veterinary Medicine, who led the study, said their group has since experimented with mice affected by a disorder resembling Duchenne muscular dystrophy (DMD). “We have tested mdx [DMD-affected] mice for the Merg1 protein, and cursorily, it appears to be more abundant in the mdx mice than in control mice,” Pond said.

Pond emphasized that astemizole can’t be safely used in humans because of its dangerous effects on cardiac muscle. She noted, however, that in the heart, it’s believed that there are two different Merg1 proteins — Merg1a and Merg1b — whereas in skeletal muscle, there’s only Merg1a. That might make it possible, she said, to target the skeletal muscle form without af-fecting the heart protein.

Androgen Receptors, Protein Blocking Explored in SBMA

Spinal-bulbar muscular atrophy (SBMA) is caused by an extra-long androgen receptor protein and a loss of normal androgen receptor protein function, say MDA grantee Albert La Spada at the University of Washington-Seattle and colleagues.

The extra-long androgen (male hormone) receptor (docking site) occurs because of an extra-long stretch of DNA on the X chromosome and has been known for years to cause neurodegeneration in SBMA. The question of whether the loss of androgen receptor activity is a cause of SBMA symptoms has until now been controversial.

In fact, therapies to reduce androgens have been proposed and even tested, on the theory that if receptors aren’t functioning normally, it might be better to have less androgen for them to receive.

Albert La Spada

In a series of experiments, mice bred without normal androgen receptor function and only with SBMA-affected androgen receptors had more severe symptoms of nerve cell degeneration and hormonal abnormalities than did mice with some normal receptor function and some SBMA-affected receptors.

Patrick Thomas Jr. and colleagues (in La Spada’s group), who published their results online June 13 in Human Molecular Genetics, caution that treatments based on knocking out abnormal genetic information might actually make SBMA worse, since remaining androgen receptor function might be lost in the process. La Spada, who was on the team that identified the SBMA-causing genetic mutation in 1991, later commented that blocking androgen activity with medications also might be detrimental.

In another paper, published online June 4 in Nature Neuroscience, Gerardo Morfini at the University of Illinois-Chicago and the Marine Biological Laboratory at Woods Hole, Mass., with colleagues at these institutions and the University of Texas Southwestern Medical Center in Dallas, identified activation of a protein known as JNK as an important downstream effect of the underlying genetic mutation that leads to SBMA.

Activation of JNK, they say, may turn on a cell death program in the nervous system. The investigators say blocking JNK is a promising SBMA therapeutic target.

FARA Launches Friedreich's Registry

The Friedreich's Ataxia Research Alliance (FARA) has launched a Web-based patient registry to identify and recruit potential participants with Friedreich's ataxia for future clinical trials.

To participate, see www.CureFA.org/registry, where you’ll be asked to agree to an online consent and enter some basic demographic and clinical data. For more information, go to www.CureFA.org, or call FARA in Arlington, Va., at (703) 426-1576.

 

MORE MDA RESEARCH NEWS

For up-to-the-minute news on MDA research developments, visit MDA’s Web site at www.mda.org. Click on "Research" for information on current research developments and active clinical trials, and links to major medical/research sites. Look at the Web site’s "News" section for news bulletins about breaking research announcements. For research news about amyotrophic lateral sclerosis, see The MDA/ALS Newsmagazine or go to www.als-mda.org.