Research Updates
ENZYME TREATMENT BENEFITS BABIES WITH POMPE'S DISEASE
Babies with a metabolic muscle disorder known as Pompe's disease, or acid maltase deficiency, usually don't survive infancy because they lack a vital enzyme that normally breaks down glycogen in the heart and skeletal muscle cells.
Now, thanks to a research partnership involving MDA grantee Yuan-Tsong Chen, a pediatric geneticist at Duke University Medical Center in Durham, N.C., and the biotechnology company Genzyme of Cambridge, Mass., there's new hope for such children.
This fall, Chen presented the results of a pilot clinical trial in which three infants with Pompe's disease were treated with a genetically engineered acid maltase enzyme that Duke researchers developed with MDA funding.
The infants weren't expected to survive more than a year with currently available treatments, but, as of the October presentation, they had lived 15, 17 and 20 months, and were showing signs of improved cardiac muscle function. Skeletal muscle function may also have improved, but this effect was harder to detect, according to information from Genzyme.
In at least one infant, muscle biopsies confirmed that significant reductions in toxic glycogen accumulation had occurred with the enzyme replacement therapy. Glycogen is stored sugar.
without mannose
6-phosphate tags
Without mannose 6-phosphate tags, acid maltase enzyme molecules can't enter muscle cells from the bloodstream. |
The Pompe's disease treatment story goes back to the mid-1990s, says Chen, when he and a team that included
Jer-Yuarn Wu, then an MDA-supported investigator at Duke, began work to improve on earlier treatment strategies.
Two experiments in humans to replace the missing enzyme as early as the 1970s failed, Chen explains, because the researchers weren't using the right form of the enzyme.
In one instance, the researchers used a version of acid maltase (also known as acid alpha-glucosidase) isolated
from a fungus. "That was not successful," Chen says.
A second attempt, this time with acid maltase isolated from human placentas, had more promise, but also failed. Chen explains, "What happened is that the placenta-derived enzyme is a mature form of the enzyme."
A molecular tag called mannose 6-phosphate "had already been cleaved off," he says, when the enzyme was chemically changed from its immature (precursor) form to its mature form. At this point, it was no longer able to enter muscle cells when injected into the bloodstream. It's the mannose 6-phosphate tag that allows muscle cells to take up the enzyme and target it to the cellular compartment where it does its metabolic work.
with mannose
6-phosphate tags
When mannose 6-phosphate tags are added to acid maltase enzyme molecules, the molecules stick to receptors (docking sites) on the muscle cells and are carried deeper inside the cells, where they're needed. |
With advances in molecular technology, by the 1990s it was a straightforward matter to insert a human gene into
nonhuman cells and have them make a desired enzyme for use in clinical studies. Chen's team chose to have Chinese hamster ovary cells take up the gene for the precursor form of the human acid maltase gene and make acid maltase with the mannose 6-phosphate tag.
Studies confirmed that the enzyme worked in human cells in a dish and then, in 1998, in live quails with Pompe's disease.
Next, researchers scaled up production and tried the treatment on babies. The October announcement, made at a meeting of the American Society of Human Genetics in Philadelphia, was a clear go signal for the enzyme strategy in humans.
Genzyme and Chen want to expand the clinical trial in babies and later in children and adults with a less severe form of acid maltase deficiency. MDA has offered to work with them on an expanded clinical trial of Pompase, the enzyme Chen and colleagues developed and to which Genzyme now holds commercial rights.
MDA has funded research in both gene therapy and enzyme replacement therapy for acid maltase deficiency.
At the same time, a Dutch pharmaceutical company, Pharming, has had some success with an early trial in infants using an acid maltase enzyme the company is manufacturing in rabbits carrying the human acid maltase gene. And Novazyme, a company in Oklahoma City, has received Orphan Drug status from the Food and Drug Administration for the development of its version of human acid maltase. Orphan Drug Status is a federal program that gives companies financial incentives to develop drugs that normally wouldn't be profitable.
MDA GRANTEES MAKE GENETICS ADVANCES
The American Society of Human Genetics meeting, held in Philadelphia in October, showcased more than 100 neuromuscular research projects, many of which were funded by MDA.
In a dramatic step forward for treatment of a fatal neuromuscular disease, MDA grantee Y.T. Chen announced positive results of a treatment trial in infants with acid maltase deficiency. (See "Enzyme Treatment Benefits Babies".)
Other meeting highlights were:
Mouse Model for SBMA
J.R. Morrison and D.M. de Krester and colleagues at Monash University, Australia, have developed the first mouse model of spinal bulbar muscular atrophy (SBMA or Kennedy's disease). In this slowly progressive disease, muscle-controlling nerve cells are affected.
The new mouse model, which carries the human mutation, seems to faithfully reproduce the neurological problems seen in humans with SBMA and should prove a useful tool for investigating the causes of the disease.
Genes that Protect Against ALS Identified in Mice
MDA-funded scientist C.B. Kunst of the Eleanor Roosevelt Institute in Denver and colleagues have noted that some lines of mice don't develop symptoms of amyotrophic lateral sclerosis despite having the same genetic mutation that causes an inherited form of the disease in humans. Similarly, some humans don't develop ALS despite having an identified ALS-causing mutation.
The group bred the mice selectively in an attempt to identify a specific gene or genes that convey protection from ALS. They've localized protective effects to regions on three mouse chromosomes. One of the regions contains
a group of genes thought to be involved in another motor neuron
disease, spinal muscular atrophy. Understanding whether, and how, the SMA genes might be involved in ALS could significantly advance knowledge of ALS pathology.
DMD Research Progresses
Diagnosis: As many as 40 percent of boys with DMD lack the large "gaps" (deletions) in their dystrophin genes that can be detected with common genetic diagnostic tests. Instead, these boys have other kinds of gene flaws, known as point mutations, which are tiny "spelling errors" in the DNA code or tiny additions or subtractions of DNA that nevertheless render the gene nonfunctional. (See "Simply Stated," for an explanation of different kinds of gene flaws.)
Detecting such tiny changes in the very large dystrophin gene is a time-consuming process that isn't done routinely as a part of diagnosis. Now, as an offshoot of the MDA-funded trial to test the ability of the antibiotic gentamicin to overcome a specific point mutation in DMD, MDA grantee Jerry Mendell of Ohio State University in Columbus and colleagues have developed a long-needed, fast and accurate method for finding these changes in the dystrophin gene. Using a technique they call DOVAM-S or Detection of Virtually All Mutations-SSCP, the group was able to identify specific mutations in more than 90
percent of DMD patients who tested negative for the more common large dystrophin deletions.
Large-Scale Changes: Two MDA-supported groups headed by Louis Kunkel of Children's Hospital in Boston and Eric Hoffman of Children's National Medical Center in Washington, D.C., have used cutting-edge microarray (computer chip) technology to study the activity patterns of thousands of genes in the muscles of people with DMD, limb-girdle MD and congenital MD muscle and normal muscle. They've found that, in each of these
disorders, the protein production by several genes is altered and can be measured.
By studying muscle tissue under different circumstances, they hope to obtain new insights into the disease process and potentially discover new molecular targets for therapy.
Therapies: On the gene therapy front, MDA grantee Jeffrey Chamberlain of the University of Michigan has developed a series of "micro-dystrophins." These forms of the protein missing in DMD are small enough to fit into an adeno-associated virus designed to deliver correct genes to muscle cells, while retaining the ability to correct muscle pathology, at least to the level of the milder Becker MD, Chamberlain said.
The British group led by MDA grantee Kay Davies of Oxford University reports that increasing dystrophin's
sister molecule utrophin in all tissues isn't toxic to mice and it effectively
protects against the lack of dystrophin. The group is still screening for a small molecule capable of increasing utrophin production in muscle, an approach that may result in a drug therapy for DMD.
New Tool for Developing SMA Treatments
A recent MDA-funded study paves the way for drug treatments that could compensate for the genetic flaw underlying spinal muscular atrophy (SMA), a disease that leads to paralysis through the destruction of motor neurons (nerve cells that control muscle).
Nearly all cases of SMA are caused by deletion of the SMN1 gene, which normally produces the essential survival motor neuron (SMN) protein. Everyone has a backup SMN gene called SMN2, but it produces very low levels of active SMN protein -- usually not enough to fully substitute for the missing SMN1 product.
But it's possible to boost production of SMN protein from SMN2, according to the new study, an international collaboration that included MDA grantees Christian Lorson of Arizona State University in Tempe and Elliot Androphy from the New England Medical Center and Tufts University School of Medicine in Boston. In theory, a high enough boost to SMN2 could be used to treat SMA.
Previous research showed that protein production from SMN2 is extremely sensitive to a molecular editing process that cuts and pastes RNA -- the chemical intermediate between DNA (the material that makes up genes) and protein. Because of a tiny difference between the SMN1 and SMN2 genes, that process often clips off a piece of SMN2 RNA that's critical to making fully functional SMN protein.
The new study, published in August in the proceedings of the National Academy of Science, shows that a protein called Htra-ß1 can attach to SMN2 RNA and protect it from this overzealous editing. When injected into isolated human cells, Htra-&sxlig;1 caused the cells to increase their levels of "full-length" SMN2 RNA by about threefold.
Although that effect is encouraging, it's probably not large enough to consider using Htra-&sxlig;1 as a treatment for SMA, Lorson said. Instead, he plans to use Htra-&sxlig;1 "in developing screens for small molecules and small compounds that can do the same thing" -- only better. Htra-&sxlig;1, he explained, will serve as a yardstick for measuring the effects of hundreds of thousands of potential drugs that might boost full-length SMN2 RNA to clinically beneficial amounts.
In another development, the drug gabapentin, marketed for seizure disorders as Neurontin by Pfizer, failed to show any benefit for people with SMA, according to study team member Robert Miller, an MDA-supported neurologist and clinical investigator at California Pacific Medical Center in San Francisco.
Gabapentin partially blocks glutamate, a natural central nervous system chemical that laboratory evidence suggested might be toxic to fragile nerve cells in SMA.
A separate trial of gabapentin in ALS recently failed to show any benefit in that disease.
Results of a one-year, MDA-supported study of 64 people with SMA types 2 and 3 ( less severe than type 1) showed no differences in results between those given gabapentin and those receiving a placebo.
The findings were presented Oct. 17 during a meeting of the American Neurological Association in Boston.
Based on these findings, the use of gabapentin in SMA cannot be recommended, say MDA advisers.
New Insights into Mitochondrial Diseases
Three recent studies have brought scientists closer to developing treatments for diseases caused by defects in mitochondria, the vital energy-producing factories found in most cells.
Like many diseases, mitochondrial diseases are caused by mutations (changes) in DNA (the chemical that makes up our genes). But only some mitochondrial diseases are caused by mutations in the best understood, most abundant type of DNA -- the kind packed into the cell control center known as the nucleus. Other mitochondrial diseases are caused by mutations in DNA housed in the mitochondria themselves -- DNA that's used to make essential mitochondrial proteins.
In two independent studies, scientists have produced the first mice that have mitochondrial diseases caused by mutations of mitochondrial DNA (mtDNA). Although neither mouse strain has the exact features of a particular human mitochondrial disease, they'll be useful for designing treatments that counteract the damaging effects of mtDNA mutations.
One mouse strain was created by scientists at Emory University in Atlanta, who presented their work at the American Society for Human Genetics meeting in October. The other mouse strain was created by a group of Japanese scientists, who published their work in the October issue of Nature Genetics. This strain has the same type of mtDNA mutation that causes the human mitochondrial diseases Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO).
KSS and PEO are caused by mtDNA mutations that eliminate a special type of mitochondrial RNA (the chemical intermediate between DNA and protein). This RNA -- called tRNA -- enables mitochondria to produce their own proteins; when it's missing, all of those essential proteins are missing, too.
A study by an international team of scientists suggests mitochondrial diseases like KSS and PEO could be treated by replacing the missing mitochondrial tRNA with tRNA produced in the nucleus (which normally functions in parts of the cell outside mitochondria). In the study, published in the September issue of Science, the researchers identified signals that promote the import of nuclear tRNAs into mitochondria, and showed that those nuclear tRNAs could manufacture mitochondrial proteins.
Physical and Mental Fatigue Trouble Those With MG
Twenty-eight people with myasthenia gravis and 34 without any neuromuscular disease (a control group) were
studied to probe the role of fatigue in MG. Jonathan Goldstein, a neurologist who directs the MDA clinic at Yale University in New Haven, Conn.,
and James Gilchrist, a neurologist directing the MDA clinic at Rhode Island Hospital in Providence, were on the study team, which published its findings in the September issue of Muscle & Nerve.
In the most common form of MG, the immune system attacks the acetylcholine receptors, structures on muscle cells that receive signals from nerves. The immunologic attack causes fluctuating weakness, often described by patients as fatigue. Physical fatigue as a result of muscle weakness is expected in the disorder, the researchers say, but they were surprised to learn that cognitive (thinking-related) fatigue also occurs often in MG.
The investigators found that those with MG reported significantly more physical and cognitive fatigue than those in the control group and that both kinds of fatigue affected their mental, physical and social functioning.
The researchers say they can see no clear reason for the cognitive fatigue in MG, since the brain isn't thought to be involved in the disease. However, they note that physical fatigue may influence a person's perception of his level of
mental tiredness; and, they note, sleep apnea (periodic cessation of breathing during sleep) because of muscle
weakness could cause cognitive fatigue during the day.
Prednisone Side Effects Topic of Long-Term Study
The corticosteroid drug prednisone has been found to slow muscle deterioration in boys with Duchenne muscular dystrophy (DMD), but with many side effects.
The study was conducted by physicians at four MDA clinics: the University of Rochester (N.Y.) Medical Center; Ohio State University Hospitals in Columbus; Vanderbilt University Medical Center in Nashville, Tenn.; and Washington University Medical Center in St. Louis. Results were combined with those of investigators seeing patients through clinics associated with the University of Alberta in Edmonton, Canada.
The investigators documented prednisone-associated side effects in boys with DMD who started on the drug between 1986 and 1989. Data were collected in 1991, 1995 and 1999. Between 1991 and 1999, the number of patients being followed declined from 226 to 100, with 51 still on prednisone.
Their average age in 1999 was 18 years, and the average time on prednisone was seven to 12 years.
Of those patients or parents who chose to discontinue prednisone or reduce the dosage, 85 percent cited unacceptable weight gain as the reason for doing so. Some 13 percent gave mood swings or other behavioral problems as the reason.
Other complications that occurred during the long study period included cataracts in 19 boys; fractured vertebrae in four boys; fractures of long bones in conjunction with falls in 21 boys; high blood pressure in three boys;
diabetes in one boy; and short stature with delayed sexual maturation in seven boys.
The researchers suggest that improved management of excessive weight gain would permit a great number of patients to benefit from long-term treatment with prednisone.
The results were presented at a meeting of the American Neurological Association in Boston in October.
Yet Another Protein, Gene Implicated in LGMD
The gene for myotilin, a protein located in the part of the muscle cell that allows it to contract and relax, has been confirmed to play a role in causing limb-girdle muscular dystrophy type 1A. MDA grantee Marcy Speer at Duke University Medical Center in Durham, N.C., was on the team that pinpointed the myotilin gene as being responsible for LGMD in a large North American family of German descent. The finding is published in the Sept. 1 issue of Human Molecular Genetics.
At least a dozen different genes can each, when defective, lead to LGMD, now recognized as a group of disorders affecting mostly the muscles of the shoulder and pelvic girdles, which stabilize the upper arms and legs.  |
