Research Updates Summer 2010

Article Highlights:

Updates on the latest research as of June 2010

by Quest Staff on July 1, 2010 - 4:03pm

QUEST Vol. 17, No. 3

Recent research in spinal muscular atrophy (SMA) has concentrated on three different ways to raise levels of the protein known as SMN that’s required for the health of nerve cells that control muscle function.

SMN (“survival of motor neuron”) protein is lacking in SMA. A similar but shorter protein is produced by people with SMA, but unfortunately, the shorter protein can’t do the job of full-length SMN, possibly because it’s broken down too quickly in cells. Full-length SMN protein molecules are made from a gene called SMN1, while the shorter SMN protein molecules are made from a gene called SMN2.

The three approaches below focus on either increasing producing of full-length SMN or improving the longevity or functionality of short SMN.

Transferring SMN genes

A multicenter U.S. research team has reported dramatic improvement of motor function and brain-to-muscle signals, as well as longer survival, in newborn mice with an SMA-like disease that received an intravenous injection of SMN genes. These results were published online Feb. 28, 2010, in Nature Biotechnology.

The U.S. research group, which included Brian Kaspar and Arthur Burghes at Ohio State University in Columbus (who have both received MDA funding for related work), found that a “window of opportunity” exists during early life in which SMA gene therapy can have optimum benefit.

The gene delivery method used in the mice involved encasing SMN genes in type 9 adeno-associated viral delivery vehicles called “AAV9 vectors.” The same technique was also used successfully in a macaque monkey and investigators are moving the treatment toward human testing.

A British research group based at the University of Sheffield saw similar results with SMN gene transfer in mice with an SMA-like disease, using intravenous gene transfer and AAV9 vectors. This group announced its findings in April 2010 at the annual meeting of the American Academy of Neurology, held in Toronto.

Christian Lorson (left) and colleagues
Christian Lorson at the University of Missouri-Columbia, left, and colleagues, are working to increase SMN protein levels as a potential SMA treatment.

Trans-splicing to increase SMN

A research team at the University of Missouri-Columbia that included Christian Lorson, a member of MDA’s Scientific Advisory Committee, has found that a strategy called “trans-splicing” has increased the production of full-length, functional SMN protein molecules and lengthened life span in mice with an SMA-like disease. The team published its findings Jan. 6, 2010, in the Journal of Neuroscience.

The trans-splicing technique allows full-length SMN protein to be produced from the SMN2 gene, which usually produces mostly shortened, nonfunctional SMN protein molecules.

Saving short SMN

The short SMN protein molecules made from SMN2 genes may not be as nonfunctional as previously thought, a research team at the University of Pennsylvania has found. This group, which published its findings in the March 2010 issue of Genes & Development, found that if the short SMN protein molecules stick around longer in the cell, they do provide benefit.

The investigators showed that the shorter SMN protein made from the SMN2 gene contains a molecular “degradation” signal, which causes its rapid destruction. When Gideon Dreyfuss at the University of Pennsylvania, and colleagues, manipulated SMN2 genetic instructions to force the addition of five amino acids (protein building blocks) to the end of the short SMN protein, the resulting molecule was stable and able to serve its critical function of providing support to cells.

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Several therapeutic strategies for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are based on increasing levels of functional dystrophin, the muscle protein that’s missing in DMD and diminished in BMD.

Updates on three such strategies were presented in April 2010 at the annual meeting of the American Academy of Neurology (AAN).

Immune response seen in dystrophin gene therapy

Andrew Kilbarger, a child with DMD, participated in the dystrophin gene therapy trial. He's shown here with his mother, Julie Kilbarger, and neurologist Jerry Mendell, in 2006.

Unwanted responses by the immune system to dystrophin were seen in a small, MDA-supported clinical trial of gene therapy for DMD. Rather than a setback, the finding is “the beginning of a new way of thinking” about gene therapy, said Jerry Mendell, director of the Center for Gene Therapy at Nationwide Children’s Hospital in Columbus, Ohio, and a longtime MDA research grantee and MDA-associated clinician. Mendell was the neurologist on this trial.

“I don’t think this is the end of gene therapy for Duchenne dystrophy,” Mendell said. “Now that we know that the immune response can be a problem, we can start dealing with it.”

Gene therapy for DMD involves injecting genes for a functional version of the muscle protein dystrophin. Extensive analyses of trial data revealed unwanted immune responses to the newly synthesized dystrophin protein in four out of the six trial participants.

The DMD gene therapy trial, supported by MDA through grants to Asklepios Biopharmaceutical (AskBio) of Chapel Hill, N.C., opened in March 2006. Conducted at Nationwide Children’s, the trial included six boys with DMD, ages 5-12. Each received a placebo injection into one biceps muscle and an injection into the other biceps of Biostrophin, a patented product developed by AskBio consisting of miniaturized dystrophin genes encased in adeno-associated viral (AAV) delivery vehicles. Three boys received a low dose of Biostrophin, and another three received a higher dose.

In January 2008, the investigators announced that the gene transfer procedure was safe and said they were considering proceeding to a third and higher dosage level of Biostrophin.

That didn’t happen, however.

The unwanted immune responses to dystrophin seen in four of the six trial participants were detectable only at the microscopic level, through complex analyses of blood samples and examination of biopsy slides.

Additional analyses showed that, in some cases, the production of a small amount of dystrophin protein from some muscle fibers in boys with DMD actually “primed” the immune system to react to the new dystrophin created by gene therapy. These few dystrophin-producing muscle fibers (seen in some boys with DMD) are called “revertant” fibers.

Investigators had previously believed that revertant fibers, with their pre-existing, low-level dystrophin production, might make patients tolerant of new dystrophin from gene therapy, because the dystrophin would be familiar to the immune system and so wouldn’t be attacked as an invader. This was not the case.

On the other hand, there was a partial “disconnect” between what was seen of the immune response in blood tests and what was actually seen in some of the muscle biopsy samples, said Scott McPhee, director of research and development at AskBio.

McPhee cautioned that a blood-cell immune response does not necessarily mean that the immune system will actually attack muscle tissue. “There are a lot of steps that must occur for a blood immune cell that recognizes a specific protein to actually infiltrate the tissue to target the cell expressing the [protein],” McPhee said.

He called the study outcomes “exciting and informative,” adding, “Using gene delivery, we have uncovered a clinically relevant biological process in a subpopulation of DMD patients, related to the unexpected ability of the immune system to recognize naturally occurring revertant muscle fibers. It remains to be determined what exact role this immune recognition plays. There were no clinical adverse events related to the gene delivery in any of the patients, but it will be another marker for clinical researchers to measure and evaluate when carrying out any form of dystrophin gene or protein modification.”

McPhee also noted that no trial participant showed an immune response to the AAV delivery vehicle.

Based on these results, McPhee said, AskBio plans to advance to the next phase of DMD clinical studies, involving whole-limb delivery of Biostrophin, within the next 1.5 to two years. Investigators will prescreen trial participants to assess their immune status before enrollment.

An immune response to dystrophin also could occur with other experimental treatments that seek to raise levels of this protein, and the immune response needs to be considered and monitored as the field moves forward with dystrophin-enhancing therapies, experts say.

Meanwhile, AskBio emphasized that it plans to continue to develop gene therapy for DMD, albeit with additional prescreening for an immune response to dystrophin. “This study was the first to carefully assess dystrophin immunity in a clinical trial,” said McPhee, “and it will help us design the ongoing and future trials of different classes of DMD therapies to be sure that we continue to learn more about the nature and clinical implications of any immune response. It will also help us identify and enroll patients most likely to benefit and most unlikely to have an adverse clinical response.”

Exon skipping safe and increases dystrophin

The Dutch biotechnology company Prosensa, in conjunction with the multinational pharmaceutical company GlaxoSmithKline, announced promising results from their 12-person trial of an experimental “exon-skipping” therapeutic for DMD. Exon skipping is a strategy in which cells are coaxed to ignore (skip) certain targeted regions (exons) of a gene in which an error occurs and use the surrounding genetic instructions to produce a functional protein.

The molecule developed by Prosensa and GSK, known as PRO051/GSK240968, was well-tolerated, and almost all the analyzed muscle fibers from the treated trial participants contained dystrophin. In general, the higher the dose, the more dystrophin was produced. Some participants began excreting protein in their urine, a concerning effect that the investigators said requires follow-up.

A lab at PTC Therapeutics, where ataluren was developed.

Development of PRO051/GSK240968 to skip exon 51 is likely to continue, and the companies will now tackle skipping of exon 44 of the dystrophin gene with a molecule called PRO044. Testing of this molecule, at least initially, will be in Europe.

Another molecule designed to coax skipping of exon 51 has been developed by AVI BioPharma of Bothell, Wash., and encouraging results for this molecule also were presented at the April AAN meeting. This agent, AVI4658, appears safe and resulted in increased dystrophin production in some of the 19 participants, who received it intravenously. In May, the company announced that one participant’s muscle samples showed 3 percent dystrophin-positive fibers before treatment, compared to 55 percent after treatment. AVI intends to continue testing AVI4658, and there are plans to open a trial site at Nationwide Children’s Hospital in Columbus, Ohio, as soon as possible.

Stop codon read-through: Less may be more with ataluren

Another strategy for increasing dystrophin production in DMD/BMD involves encouraging cells to ignore, or “read through,” a type of mutation known as a premature stop codon in the dystrophin gene. These genetic errors, also called “nonsense” mutations, cause cells to stop synthesizing dystrophin protein molecules before the process has been completed.

PTC Therapeutics, of South Plainfield, N.J., in conjunction with Genzyme, of Cambridge, Mass., has developed a stop codon read-through drug called ataluren, designed to cause cells to read through premature stop codons in the dystrophin gene. (MDA has provided significant funding for the development of ataluren.)

In March 2010, the companies reported disappointing results from their phase 2b trial of 174 participants with DMD or BMD due to stop codon mutations. Initial reports indicated that, at the end of a 48-week trial, there was no difference in walking ability in the ataluren-treated and the placebo-treated trial participants. The primary measure of effectiveness in this trial was the distance a participant could walk in six minutes before and after treatment.

However, at the April AAN meeting, study investigator Brenda Wong, who directs the MDA clinic at Cincinnati Children’s Hospital, presented a more detailed analysis of the trial results, showing that the low-dose ataluren group actually did better on the six-minute walk test after almost a year of treatment than either the high-dose or the placebo group. The high-dose and placebo groups’ walking distances each declined by an average of 42 meters (138 feet) after 48 weeks, while the low-dose group’s distance declined by an average of only 13 meters (43 feet).

These and other outcomes, including measurement of dystrophin levels in muscle biopsy samples, are undergoing more thorough analysis to determine whether ataluren could be effective if given at the right dosage.

At the same meeting, researchers reported results of an MDA-supported trial of intravenous gentamicin, an antibiotic in the aminoglycoside family that’s been found to induce stop codon read-through in the dystrophin gene. Details of this study also were reported online March 15, 2010, in Annals of Neurology.

In this study, six of the 12 participants who received gentamicin for the longest period of time (six months) showed increased dystrophin production.

No significant improvements in strength or function were seen in any of the participants, although hints of effectiveness raised the question about whether a higher dose would be beneficial. An immune response appears to have destroyed newly synthesized dystrophin in one trial participant.

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Magnetic resonance imaging machines, like this one, could one day become routine in assessing muscle health in DMD/BMD.

The use of magnetic resonance imaging (MRI) as an assessment tool in boys with Duchenne muscular dystrophy (DMD) is being studied by former MDA grantee Krista Vandenborne at the University of Florida in Gainesville, through a $7.5 million grant from the National Institutes of Health (NIH).

The goal of the study is to assess whether MRI technology can be used as a precise, noninvasive measure of muscle tissue, judging both disease progression and the effectiveness of therapies tested in children with DMD.

The NIH grant was based on evidence from an earlier, MDA-supported study that suggested MRI boasts a number of advantages over traditional muscle biopsies, which are invasive and provide researchers with an incomplete view of the muscle tissue.

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Charcot-Marie-Tooth disease (CMT) is a disorder of the sensory and motor nerves that run between the brain and spinal cord. About 30 different forms of CMT exist, all of which result from various mutations in any of several genes that affect the nerve fibers themselves, the myelin insulation that surrounds them, or both. Several reports of progress in CMT research were presented at the April 2010 American Academy of Neurology (AAN) meeting.

NT3 gene therapy improves strength, function in mice

Mice with a CMT-like disease treated with NT3 genes performed better on a rotating rod than did untreated mice.

Zarife Sahenk, a professor of pediatrics, neurology and pathology at Ohio State University in Columbus, presented her findings in mice with a CMT-like disease that received therapy with genes for the protein neurotrophin 3 (NT3).

The mice in these experiments had a mutation in the PMP22 gene, the same gene affected in humans with type 1A CMT. They received NT3 genes, which code for a protein that supports nerve-fiber health in general, encased in type 1 adeno-associated viral delivery vehicles, via direct injection into a leg muscle.

When checked at 20 and 40 weeks after treatment, the mice showed improvements in strength, function and nerve-signal transmission compared to the untreated group. This positive result could mean that NT3 gene therapy has potential for the treatment of human CMT.

Curcumin-treated mice did a little better on rod test

Mice with a CMT-like disease treated with oral curcumin, an ingredient in the spice known as turmeric, and with a derivative of curcumin, did a little better on a functional test than did untreated mice, reported Agnes Patzko, a research associate at Wayne State University in Detroit. The mice performed slightly better than untreated littermates on a test of their ability to stay on a rotating rod, although they did not improve on other tests of motor function. Patzko was part of a team coordinated by MDA grantee Michael Shy, a professor in the Center for Molecular Medicine and Genetics at Wayne State.

The mice in these experiments had a mutation in the gene for myelin protein zero, the same gene involved in human type 1B CMT.

It’s believed that curcumin and related substances may break up cellular traffic jams, such as occur in CMT1B. Tests will continue in mice and, if they’re promising, the investigators will consider moving to human trials.

Streamlining CMT genetic testing

MDA grantee Michael Shy from Wayne State University in Detroit presented results of genetic testing of more than 1,000 people suspected of having CMT. The results could lead to streamlining of such testing.

Of the 1,019 participants who underwent CMT testing at Wayne State, 782 (77 percent) ultimately received confirmation of a CMT diagnosis. A specific genetic diagnosis was made for 519 of the 782 (66 percent), while no mutation was identified in 263 of participants (34 percent).

The most common CMT subtypes were CMT1A (PMP22 gene duplications or point mutations, chromosome 17); CMT1X (connexin 32 gene, X chromosome); HNPP (PMP22 gene deletions or point mutations, chromosome 17); CMT1B (myelin protein 0 gene, chromosome 1); and CMT2A (mitofusin 2 gene, chromosome 1). Less than 1 percent of participants in whom a genetic diagnosis was made had other subtypes of CMT.

The investigators proposed that these results could be used to develop a flow chart for more efficient, cost-effective genetic testing for CMT subtypes.

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Facioscapulohumeral muscular dystrophy (FSHD) is a form of MD that predominantly affects the facial, scapular (shoulder) and humeral (upper arm) muscles. Since the early 1990s, it’s been known that the underlying cause is a shorter-than-normal stretch of DNA on chromosome 4 in a region that’s been called D4Z4. However, the specific mechanisms by which the deleted DNA lead to FSHD are not clear. Research to uncover these mechanisms and exploit them for treatment strategies is a major part of MDA’s program. Two recent grants exemplify this effort.

RNA interference, antisense strategies to be studied

People with FSHD often have difficulty smiling because of weakness of the facial muscles.

Joel Chamberlain, an assistant professor of medical genetics at the University of Washington-Seattle, has received joint support from MDA and Friends of FSH Research to develop a strategy called “RNA interference” both as an investigative tool to help probe the molecular underpinnings of the disease and also as a basis for therapy.

RNA interference is a natural cellular mechanism that cells use to regulate levels of protein synthesis from RNA (a close relative of DNA).

Silvere van der Maarel, a professor of medical genetics at Leiden (Netherlands) University Medical Center, has received MDA support to explore FSHD using “antisense oligonucleotides.” These molecules also interfere with genetic instructions, but in a different way from RNA interference.

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Scientists at five U.S. institutions have successfully used gene therapy to improve muscle function in a single human subject with a hereditary form of inclusion-body myositis (IBM) caused by mutations of the GNE gene. Study results were published online March 30, 2010, in the Journal of Gene Medicine.

The results demonstrate positive “proof of principle” that the investigators’ strategy for intramuscular injection of a normal GNE gene is safe and that the resulting GNE gene activity can increase strength and muscle function in a human with the GNE-related form of hereditary IBM.

To deliver the GNE gene to the muscle, the researchers created a GNE gene “lipoplex” — a combination of lipid (fat) molecules and the genetic instructions for the GNE gene. The gene contains the genetic instructions for the GNE protein, an enzyme responsible for determining production levels of sialic acid, a crucial multifunctional molecule that resides on cell-surface membranes.

The researchers injected the treatment into muscles in the right and left wrists and into the left biceps muscle of a 40-year-old woman with GNE-related IBM. The wrist muscles showed a temporary increase in strength with the experimental treatment, but the biceps strength did not improve, an outcome the researchers attributed to its pretreatment deterioration and extensive scarring.

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