Research Updates Fall 2010

Article Highlights:

The latest research as of September 2010

by Quest Staff on October 1, 2010 - 12:15pm

QUEST Vol. 17, No. 4

A new generation of molecules can help cells permanently repair errors in the dystrophin gene, fixing the underlying cause of Duchenne muscular dystrophy (DMD) — and potentially fixing the underlying genetic causes of other neuromuscular diseases as well, researchers report.

The research group, headed by MDA grantee Carmen Bertoni at the University of California, Los Angeles, published its findings online June 23, 2010, in the journal Human Molecular Genetics.

Although Bertoni’s team and others have been developing gene repair technology for several years, the process showed limited success until development of “peptide nucleic acid single-stranded oligodeoxynucleotide,” or PNA-ssODN, molecules, which yielded a 10-fold increase in gene repair efficiency.

The technology — although greatly improved — still has a ways to go before it can be tested in human clinical trials. But these new results demonstrate “proof of concept” for gene repair, as well as representing a dramatic increase in efficiency.

PNA-ssODNs cause more DNA repair

The researchers found that the new PNA-ssODN molecules, which are structurally similar to DNA, are more effective than previous molecules at targeting the region of the dystrophin gene containing the genetic defect.

In experiments on cultured cells and in mice with a flawed dystrophin gene, PNA-ssODNs stimulated DNA repair levels more than 10 times greater than those achieved by the previous class of targeting molecules. The muscle cells containing new, unflawed dystrophin genes successfully produced normal dystrophin protein at levels consistently higher than muscle cells treated with the older-generation molecules.

How PNA-ssODNs mend broken genes

Gene repair with the designer molecules follows a simple principle, Bertoni explained.

First, the targeting molecule is injected into muscle, where it seeks out the DNA in cell nuclei and locates the defective region of the dystrophin gene. The molecule then aligns itself with the part of the dystrophin gene containing the error, flagging the site and activating the cell’s own DNA repair system, leading to correction of the flaw.

PNA-ssODN targeting molecules repair broken genes

This approach is particularly attractive because it avoids technological challenges inherent in gene replacement and other strategies.

For now, gene repair is limited to correcting single-letter errors (point mutations) in a gene, which account for approximately 20 percent of cases of DMD. In these cases, DNA correction would result in restoration of full-length (normal) dystrophin protein production.

Gene repair vs. exon skipping

Gene repair has the potential to treat deletions or other large-scale gene mutations in a way that’s similar to another very promising strategy under development for DMD, exon skipping. In exon skipping, regions of genetic code (called exons) are “skipped over” by the cell’s protein-building machinery, resulting in production of a shorter but still functional dystrophin protein.

One significant difference between DNA repair and exon skipping, Bertoni noted, is that gene repair restores normal (full-length) dystrophin, while exon-skipping strategies result in a shorter, but still partially functional, dystrophin protein. The main advantage of using ssODNs is that correction at the DNA level is permanent — “in other words, no need to continuously deliver the ssODNs.”

Bertoni said gene repair offers the possibility of “nipping DMD in the bud” by directly reversing the genetic mutation leading to the disease.

Further refinement required

“Although the results we have obtained are very encouraging,” Bertoni said, “the frequencies of gene repair are still in the 1 percent to 5 percent range — too low to be considered therapeutically relevant.”

Work must be done before this technique can progress to human clinical trials, including refinement of the targeting molecules, studies to determine the most effective delivery methods, and testing in different animal models.

Potentially, the gene repair strategy could be applied to essentially any genetic disease caused by an identified point mutation, which includes many of the more than 40 neuromuscular diseases covered by MDA.

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MDA awards more than $14 million in research funding

In July, MDA approved 38 new research grants for neuromuscular disease research, at a cost of more than $14 million.

Eleven new grants totaling more than $3.5 million have relevance for Duchenne (DMD) and Becker (BMD) muscular dystrophies, including projects designed to further development of exon skipping; DNA repair; the effects of improved blood flow on damaged muscles and the use of magnetic resonance imaging (MRI) as a noninvasive method for measuring muscle changes; protection of skeletal muscle in DMD via control of skeletal muscle metabolism; and stem cell mediated gene therapy.

Ten new projects totaling nearly $3.5 million are aimed at better understanding and developing therapies for ALS (amyotrophic lateral sclerosis, or Lou Gehrig’s disease).

A number of other grants will go toward elucidating the molecular causes of, and development of therapies for, a host of other diseases in MDA’s program: central core disease (CCD), Charcot-Marie-Tooth disease (CMT), congenital myasthenic syndrome (CMS), Emery-Dreifuss muscular dystrophy (EDMD), inclusion-body myositis (IBM), limb-girdle muscular dystrophy (LGMD), mitochondrial myopathy, type 1 myotonic muscular dystrophy (MMD1, or DM1), periodic paralysis (PP), spinal-bulbar muscular atrophy (SBMA) and spinal muscular atrophy (SMA).

For more information about these new grants, visit MDA’s website and view Grants at a Glance, a new online feature showcasing MDA’s grants with photos and information.

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Two genetic changes needed to cause FSHD

Facioscapulohumeral muscular dystrophy (FSHD) requires the presence of not one but two genetic changes, both on chromosome 4, MDA-supported scientists have found.

MDA grantee Silvère van der Maarel at Leiden (Netherlands) University Medical Center coordinated the multinational study team, which announced its findings online Aug. 19, 2010, in the journal Science.

Two genetic changes — a contracted segment of DNA on chromosome 4, and a "permissive" signal near it on the same chromosome — are necessary to complete the FSHD puzzle.

The investigators found that two genetic requirements, located near each other on the tip of chromosome 4, must be combined for FSHD symptoms to appear. One requirement is a deletion of some of the DNA in a region of chromosome 4 called D4Z4. Its contribution to FSHD has been recognized for many years. The second requirement, newly recognized, is a particular variant of DNA further toward the tip of chromosome 4 than the D4Z4 region.

The variant contains a “polyadenylation” signal, which stabilizes otherwise fragile genetic instructions called RNA transcripts, after they’re synthesized from DNA (genes).

The presence of a polyadenylation signal makes it more likely that genetic instructions will stick around long enough to be translated into proteins, the final product of DNA and RNA instructions. In this case, the signal appears to make it possible for one or more potentially toxic proteins to be produced.

More than 300 people with FSHD and more than 2,000 people without the disease were studied. All the people with FSHD had a contracted D4Z4 region on chromosome 4 and at least one of three “permissive” DNA sequences further out toward the tip of the same chromosome.

Among the more than 2,000 people without any FSHD symptoms that the investigators studied, some had contracted D4Z4 regions on chromosome 4. However, they all had “nonpermissive” signals further out on the chromosome.

Without a “permissive” polyadenylation signal, the researchers believe, genetic instructions (RNA) from the D4Z4 region don’t last long enough to cause muscle-cell damage.

The new findings will make it easier to diagnose FSHD in someone with symptoms and predict who will develop the disease among those without symptoms.

Once the identities of the toxic proteins or RNA instructions are established, therapeutic strategies to block them could potentially be developed.

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Nonwalkers with SMA face high risk for weight gain

Children and adults with types 2 and 3 spinal muscular atrophy (SMA) who are no longer walking have an increased risk of being overnourished and overweight if they otherwise have relatively good motor function, a multicenter study shows. The results emphasize the importance of a “dedicated and experienced nutritionist” in SMA medical management, the researchers say.

The findings were reported in the July 2010 issue of Neuromuscular Disorders by Douglas Sproule at Columbia University Medical Center in New York, with colleagues there, as well as at St. Luke’s Roosevelt Hospital in New York; Strong Memorial Hospital in Rochester, N.Y.; Children’s Hospital of Philadelphia (CHOP); Children’s Hospital Boston; and Catholic University in Rome.

MDA did not fund the study, but physicians who direct MDA neuromuscular disease clinics at Columbia, CHOP and Children’s Hospital Boston were among the authors of the study.

The authors noted that a previous study, conducted in Italy, found that SMA patients who have difficulty swallowing can be at risk for being undernourished and underweight.

Investigators analyzed data from 53 people with types 2 or 3 SMA, divided into three groups.

Group one consisted of 19 people with SMA2 whose average age was 11, who were unable to walk, and who had generally low motor function (defined as a score of less than 12 on the expanded Hammersmith functional motor scale).

Group two, also with an average age of 11, contained 17 people with SMA2 or SMA3 who were unable to walk, but whose Hammersmith motor scores put them in a “high-functioning” category.

Group three, consisting of 17 people with SMA3 and an average age of 15, was for walkers with SMA.

The investigators found that the group 2 study subjects — those with relatively high motor function scores but no walking ability — had more body fat than those with lower motor scores who were not walking (group one) and those with higher motor scores who were walking (group three).

They concluded that individuals with relatively high motor function skills but without walking ability may be “at particular risk” of excess weight gain because they can take in more calories than their relatively low energy expenditure levels utilize.

Group one, the group with low motor function, may have less access to calories, researchers speculate (i.e., they’re unable to independently take food out of the refrigerator). It’s possible that swallowing dysfunction also plays a role for some. The walking group probably has more opportunity to burn off calories.

The researchers recommended that individuals with SMA receive expert nutritional guidance as part of their care. Those with SMA who have difficulty eating may need special nutritional supplements, while those who have no difficulty eating but can’t exercise may need to practice calorie restriction to avoid obesity.

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'Antisense' treatement restores full-length SMN in SMA mice

Scientists have found that mice with a disease resembling a mild form of spinal muscular atrophy (SMA) known as SMA type 3 showed more production of a needed protein in their spinal cords and more normal-looking ears and tails after treatment with a gene-modifying molecule that researchers hope could become a treatment for human SMA.

Adrian Krainer is exploring antisense in SMA.

MDA-supported Adrian Krainer, at Cold Spring Harbor (N.Y.) Laboratory, with colleagues at Isis Pharmaceuticals in Carlsbad, Calif., and Genzyme Corp., in Framingham, Mass., announced the new findings online July 12, 2010, in the journal Genes & Development.

They treated the SMA type 3 mice with a molecule called an antisense oligonucleotide, one of a class of experimental therapeutic molecules designed to target genetic instructions at the RNA stage. The RNA stage is an intermediate step between DNA (the original genetic instructions) and protein synthesis inside cells.

At the RNA stage, genetic instructions are “spliced” (some sections are left in and some are removed) prior to the creation of the final protein “recipe.” The use of antisense to change RNA splicing — and, by extension, the final protein recipe — is a promising experimental technique for treatment of genetic diseases.

In SMA, very little full-length SMN protein is made; most SMN protein is produced in a short, unstable form. Full-length SMN is needed by muscle-controlling nerve cells (motor neurons) in the spinal cord. Without it, loss of motor neurons from the spinal cord occurs, with resulting weakness and atrophy of the voluntary muscles.

Adult mice missing functional genes for the full-length form of SMN, but carrying four human genes for the short form of SMN showed a “robust and long-lasting increase” in full-length SMN protein in their spinal cords and in the motor neurons themselves after the experimental antisense treatment, the investigators said. (Because the mice don’t exhibit muscle weakness, it wasn’t possible to assess any functional improvements.)

When a single injection of the antisense molecule was given into the brains of newborn or embryonic mice, their ears and tails looked much more normal than those of such mice that didn’t receive the treatment. Loss of tissue in the ears and tail is believed to be a blood-flow problem, perhaps related to the motor neuron loss or other nervous-system defects. The investigators consider the normalizing of these tissues in the mice a sign that the antisense was beneficial when delivered directly to the nervous system.

There are many strategies in development for SMA. Some of these involve direct injection of genes for full-length SMN, while others involve changing the splicing of the short SMN genes so that long SMN can be made from them. Still others involve stabilizing the short SMN protein molecules.

This antisense molecule appears particularly beneficial, allowing mice with short-form SMN genes to make much more of the full-length protein than they would otherwise and improving the outward signs of SMA that these mice display.

The molecule, like all other experimental therapies, will need to undergo extensive laboratory testing before it can be tested in humans.

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New molecular strategy helps myotubularin get into muscle fibers in MTM

MDA-supported researchers at 4s3 Bioscience, a biotechnology company in Medford, Mass., are using a new molecular strategy to transport a potentially therapeutic protein into muscles, as an experimental treatment for X-linked myotubular myopathy (MTM), a genetic muscle disease that results from a deficiency of the enzyme myotubularin.

The treatment will be tested in a MTM research mouse that doesn’t make myotubularin, and displays signs and symptoms similar to those in humans with X-linked MTM.

“This is a really exciting strategy, because it has applicability to a number of diseases,” said Jane Larkindale, portfolio manager for MDA Venture Philanthropy, a division of MDA’s research program that specializes in funding small biotechnology companies with innovative ideas. “We’re especially thrilled that 4s3 has decided to pilot this technology in myotubular myopathy.”

Dustin Armstrong, vice president of research at 4s3 Bioscience and the company’s co-founder, is the primary investigator on the project. He’s been awarded a grant totaling $260,291 from MDA to develop this experimental MTM treatment.

Armstrong and colleagues at 4s3 and Children’s Hospital Boston will fuse a fragment of an antibody (immune system protein) to the myotubularin protein molecule. The specific antibody fragment they’ll use has been shown to be capable of entering muscle fibers from the bloodstream. Preliminary tests also suggest that the antibody fragment is nontoxic and does not interfere with the action of the protein to which it’s attached.

The researchers will inject the construct intravenously into mice with an MTM-like disease and then determine whether the construct penetrates muscle fibers; whether myotubularin assumes its normal functions in the fibers; whether strength or function improve in the mice; and whether any toxic effects occur, including rejection by the immune system.

“Our goal is to develop a method that will ‘shuttle’ missing proteins into muscle more efficiently, and we’re excited about this opportunity to further validate our approach and test a novel therapeutic candidate for myotubular myopathy,” Armstrong said.

If the strategy proves safe and effective in mice, it may be tested in humans with myotubular myopathy. In fact, success with the myotubularin-deficient mice would bode well for broadening development of this strategy for use in other diseases, such as some muscular dystrophies and metabolic myopathies.

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Cystamine strengthens muscles in mice with an OPMD-like disease

Scientists in the United Kingdom have found that mice carrying a genetic mutation that causes symptoms similar to human oculopharyngeal muscular dystrophy (OPMD) benefited from treatment with a chemical called cystamine, provided in their drinking water.

David Rubinsztein and colleagues at the University of Cambridge, United Kingdom, announced their findings June 2, 2010, in Science Translational Medicine.

They found that mice with an OPMD-like disease treated with cystamine showed better muscle function and strength compared to untreated OPMD mice; and that their muscle fibers showed fewer abnormal clumps and markers of cell death than those of their untreated counterparts.

The investigators hypothesize that cystamine’s benefits result from its interference with an enzyme called transglutaminase 2 (TG2), which shows enhanced activity in OPMD mice, and suggest that targeting drugs to interfere with TG2 may be worthy of further consideration in the development of treatments for this disease.

The underlying cause of OPMD is a mutation in the gene for a protein called PABPN1 that causes extra alanine molecules to be inserted into the protein. The mice in these experiments had a mutant PABPN1 gene inserted that caused them to have extra alanines in each PABPN1 protein molecule, as do humans with OPMD.

In humans and mice, PABPN1 mutations result in the presence of abnormal clumps (“aggregates”) inside muscle fibers and in a cell-death process called apoptosis. It isn’t clear if the large aggregates themselves are the most toxic disease-related phenomena, although the aggregation process is likely to cause damage. (For more about OPMD, see Quest, October-December 2009, In Focus: Oculopharyngeal Muscular Dystrophy.)

Cystamine isn’t routinely used in humans, but cysteamine, which is converted to cystamine in the body, may be suitable for OPMD treatment, Rubinsztein said. He noted that cysteamine is used to treat cystinosis, a disease involving an abnormal accumulation of the amino acid cysteine.

“Trials of cysteamine could be considered in OPMD, and it may be possible to identify safer molecules that inhibit TG2 that could be suitable for long-term use in OPMD,” Rubinsztein said.

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Lumizyme now commercially available for late-onset Pompe disease

Lumizyme, the first treatment approved by the U.S. Food and Drug Administration (FDA) for late-onset acid maltase deficiency (AMD, or Pompe disease), now is commercially available in the United States.

Monique Griffin of Orlando, Fla., was among the first in the nation to receive FDA-approved Lumizyme for Pompe disease.

The FDA approved commercial sales of Lumizyme on May 25, 2010, making it available to be prescribed by physicians and covered by insurance.

Lumizyme, administered every two weeks via an approximately four-hour infusion into the arm or hand, is a laboratory-developed enzyme manufactured by Genzyme Corp., of Cambridge, Mass. It replaces the acid maltase enzyme deficient in people with Pompe disease, and is the first treatment approved in the United States specifically for people ages 8 and older with the late-onset form of the disease.

MDA-supported basic research played a role in the development of both Lumizyme and Myozyme, Genzyme’s enzyme replacement drug for infants and very young children.

Individuals ages 19 and older with severe Pompe disease have had access to enzyme infusions through Genzyme’s Alglucosidase Alfa Temporary Access Program (ATAP), which the company set up during the time it was waiting for FDA approval of Lumizyme, as a means of providing treatment for adults who are severely affected by the disease.

Those who have been receiving alglucosidase alfa through ATAP will continue to receive therapy as they transition to the commercial supply. Individuals who have been waiting for treatment should contact their physician about getting access to the new medication.

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