MDA-supported investigators are actively pursuing several approaches to halt or reverse the muscle damage caused by Duchenne muscular dystrophy.
Some of the front-running strategies include: inserting new dystrophin genes; changing the way cells interpret genetic instructions; changing the mutated dystrophin gene itself; manipulating other proteins in the body to compensate for the lack of dystrophin; increasing blood flow to skeletal muscles and the heart; combating inflammation; and using stem cells to repair damaged muscles.
Some studies are focused on the dystrophin-deficient heart.
MDA's DMD Clinical Research Network is focused on advancing human clinical trials in this disease.
Researchers are pursuing a number of strategies to sustain or improve heart function in BMD and DMD. They're testing existing medications for their possible benefits in the BMD/DMD-affected heart, and conducting basic research to understand and find new approaches to treating the heart in these diseases.
Understanding and treating dystrophin-deficient cardiomyopathy (cardiac muscle abnormalities) is a priority for MDA. The MDA DMD Clinical Research Network has made studying the natural history and treatment of this condition a primary focus. In addition, MDA sponsored a meeting of more than 40 leading clinicians and researchers from the United States and Europe in January 2011 to discuss optimal clinical care of the DMD/BMD-affected heart.
In 2009, scientists found that dystrophin gene mutations that cause cardiomyopathy in BMD affect specific regions of the dystrophin protein, not necessarily the same regions associated with skeletal muscle loss. The study will allow better prediction of cardiomyopathy in BMD and earlier consideration of cardioprotective treatments in this disease, as well as giving researchers insight into which parts of the dystrophin protein are essential to preserve when shortened dystrophin molecules are being considered as therapeutic strategies.
The drug sildenafil (Viagra) has been found to impart cardioprotective effects in mice with both an early- and late-stage DMD-like disease. Sildenafil, which is used to treat erectile-dysfunction, belongs to a class of drugs called phosphodiesterase 5 (PDE5) inhibitors, which relax the smooth muscles lining blood vessels, increasing blood flow to muscles and the heart. The cardiac effects of sildenafil in teens and men with DMD are being studied.
Laboratory studies have found that an experimental compound designed to help seal cell membranes, p188, benefited heart function in dystrophin-deficient dogs.
In 2011, MDA-supported researchers found that inhibiting the action of a protein called NF-kappa B improved cardiac function in mice with a severe DMD-like disease.
Inflammation has been shown to be present in, and particularly harmful to, muscles affected by DMD. Scientists are working on understanding and interfering with inflammation in and around muscle fibers that may contribute to the DMD disease course.
Corticosteroids (such as prednisone) are commonly are used in DMD to help preserve muscle strength and function. It's thought that they work, at least in part, by reducing inflammation. However, corticosteroids also cause unwanted side effects such as increased appetite, weight gain, loss of bone mass and cataracts.
It had been hoped that benefits might be maximized and side effects minimized by giving prednisone to people with DMD on a compressed, weekend-only schedule instead of daily. However, an MDA-supported study for which results were reported in 2011 found that the two prednisone schedules were about the same with respect to benefit and side effects in this disease.
Some MDA-supported researchers are working on developing modified corticosteroids that have fewer or less severe side effects than those currently in use.
An MDA-supported team reported in 2011 that the naturally occurring protein interleukin 10 (IL10) may help reduce harmful inflammation and promote muscle regeneration in DMD and potentially in other forms of muscular dystrophy. These researchers conducted experiments in both cell culture and in a mouse model of DMD.
Other researchers have focused on the possible role of a protein called osteopontin in causing DMD-associated inflammation and scarring in muscles. In 2009, these researchers showed that eliminating osteopontin was beneficial to mice with a DMD-like disease and concluded that reducing osteopontin should be investigated as a possible therarapy for DMD.
Exon skipping is a strategy currently being developed for DMD (although it may have application to other genetic diseases down the line) in which sections of genetic code (exons) are “skipped,” allowing the creation of partially functional dystrophin, the muscle protein missing in DMD. Exon skipping is not a cure for DMD, but potentially could lessen the severe muscle weakness and atrophy that is the hallmark of this disease, making it more like Becker muscular dystrophy (BMD).
Laboratory development of exon skipping began in the 1990s and has received significant funding from MDA since then.
Exon skipping uses molecules called antisense oligonucleotides to coax muscle fibers to ignore certain parts of the genetic instructions for dystrophin, thereby restoring the genetic "reading frame."
To understand this better, think of the genetic code for a protein as a sentence. Cells have to read the genetic “sentence” in units of three “letters” each.
Exon skipping is being tested in clinical trials in boys with DMD in the United States and in other countries.
In August 2011, AVI BioPharma, with supplemental support from MDA, began testing its exon-skipping drug eteplirsen in boys with DMD still able to walk at Nationwide Children's Hospital in Columbus, Ohio. The pharmaceutical company GlaxoSmithKline began testing its exon-skipping drug GSK2402968 in nonwalking boys with DMD at the same hospital in 2010.
Although still in very early stages of development, repair of the dystrophin gene at the DNA level shows promise. This strategy is aimed at helping cells permanently repair errors in the dystrophin gene, fixing the underlying cause of DMD.
In experiments on cultured cells and in mice with flawed dystrophin genes, "designer" gene-repair molecules stimulated DNA repair levels more than 10 times greater than those achieved by a previous class of targeting molecules. The muscle cells containing rejuvenated dystrophin genes successfully produced normal dystrophin protein at levels consistently higher than muscle cells treated with the older-generation molecules.
Although the results so far have been encouraging, the frequencies of gene repair are in the 1 to 5 percent range — too low to be considered therapeutically relevant. And, for now, the gene repair strategy is limited to correcting "single-letter" errors (point mutations) in a gene.
Work must be done before this technique can progress to human trials, including refinement of the targeting molecules, studies to determine the most effective delivery methods and testing in different animal models.
Gene therapy, or gene transfer, refers to the delivery of genes as therapeutic agents. Since genes carry the instructions for protein synthesis, they can lead to production of proteins that are directly or indirectly therapeutic in neuromuscular diseases. Because transferred genes potentially can continue to produce protein for some time, gene therapy may offer a more permanent fix than other therapies. But gene therapy faces many technical challenges, as well as a high bar set by regulatory agencies like the U.S. Food and Drug Administration (FDA).
The key challenges are delivering the genes to the targeted tissue while avoiding off-target tissues, and avoiding unwanted immune response to the proteins made from the new genes, or to the delivery vehicles in which the new genes are delivered.
MDA-supported scientists have created a miniaturized, working dystrophin gene that has been tested in boys with DMD. Although the treatment appeared to be safe, some of the boys experienced an unwanted immune response to the dystrophin protein that limited the effectiveness of the gene transfer. This immune response is undergoing further investigation.
Blocking the myostatin protein via a protein called follistatin is a strategy that has potential for treating DMD and likely many other neuromuscular diseases. Mice with a DMD-like disease that received genes for the follistatin protein showed an overall increase in body mass and weight of individual muscles. Monkeys that received follistatin gene transfer had stronger, larger muscles.
Experiments have shown that, when dystrophin is missing from the muscle-fiber membrane, it causes another protein, known as nNOS, to be missing as well, and that this results in an inability of blood vessels supplying muscles to adequately dilate during exercise. When nNOS-deficient mice were treated with a phosphodiesterase inhibitor, which dilates blood vessels, their exaggerated fatigue response to exercise was eliminated. Phosphodiesterase inhibitors are a class of drugs that include sildenafil (Viagra) and tadalafil (Cialis), both used to treat erectile dysfunction.
Other investigators found that treatment with sildenafil significantly improved heart function in mice missing the dystrophin protein.
On the basis of these and other findings, researchers have started investigating the possibility that phosphodiesterase inhibitors can improve skeletal-muscle or heart-muscle function in people with BMD or DMD.
In 2010, an MDA-supported trial testing the effects of tadalafil on blood flow to muscles began in men with BMD.
Other trials of phosphodiesterase inhibitors also are under way to test their effects on skeletal-muscle and heart-muscle function in DMD and BMD.
A strategy that has received considerable MDA support involves inhibiting the actions of a naturally occurring protein called myostatin, which limits muscle growth. Researchers hope that blocking myostatin may allow muscles to grow larger and stronger.
Inhibitors of myostatin have received much attention from the neuromuscular disease research community ever since it was found several years ago that people and animals with a genetic deficiency of myostatin appear to have large muscles and good strength without apparent ill effects. In 2010, a study showed that mice lacking dystrophin and showing a DMD-like disease benefited from treatment with a "decoy" that lured myostatin away from their muscles.
The biotechnology company Acceleron Pharma then developed a drug based on this decoy and began testing it, with MDA support, in boys with DMD. Unfortunately, unexpected safety issues arose during that trial, causing Acceleron to terminate it in 2011.
The company hopes to resolve these safety issues and resume testing ACE-031, or a modified version of ACE-031.
Other strategies to inhibit myostatin, such as injecting genes for the myostatin-blocking follistatin, also are under consideration.
MDA scientists are using stem cells (cells from which specialized cells "stem") isolated from muscle, blood vessels or bone marrow to regenerate muscles in dystrophin-deficient laboratory animals.
Stem cells are cells in the very early stages of development. They may be destined to turn into a specific cell type (such as muscle or nerve cells), or they may still retain pluripotency — the ability to develop into any of a number of different cell types.
In 2006, MDA-supported researchers restored mobility to two dogs and stabilized function in a third using stem cells taken from muscle blood vessels.
In a study reported in 2007, a European research group successfully used a combination of genetic correction and stem cells to treat DMD research mice. The researchers in this study extracted muscle-generating stem cells from muscle tissue and blood in people with DMD, corrected the genetic error in the cells' dystrophin genes, and then injected the cells into dystrophin-deficient mice. The muscle-derived cells gave rise to better muscle regeneration than did the blood-derived cells.
In 2010, MDA-supported scientists in France reported they had identified a previously unknown type of muscle stem cell located in the spaces between muscle fibers in mice. Although still in the early stages of research, it's hoped the new cells, dubbed PICs, may play an important a role in muscle regeneration and repair.
Scientists reported in 2010 that formation of new muscle tissue first requires a controlled type of DNA damage. The new finding increases scientists' understanding of how immature muscle cells become muscle and could help them manipulate this process to treat several forms of muscular dystrophy.
Stem cells continue to be a major area of investigation for MDA-supported researchers. Some are continuing to study muscle satellite cells, a type of stem cell present in muscle tissue. Others are studying different cell types that are capable of surviving transplantation into muscle and producing the desired proteins. Still, others are studying the similarities and differences in the development of skeletal muscle and fat tissue.
In stop codon read-through, drugs target mutations known as premature stop codons (also called nonsense mutations), which tell the cell to stop making a protein — such as dystrophin — before it has been completely assembled. The drugs coax cells to ignore, or "read through," a premature stop codon in a gene.
A company called PTC Therapeutics, in conjunction with Genzyme Corp., and with some initial funding from MDA, developed an experimental stop codon read-through drug called ataluren to treat DMD or Becker muscular dystrophy due to a premature stop codon. In October 2010, PTC announced that a lower dose of ataluren appeared to work better than a higher dose. In a clinical trial, those on the lower dose walked an average of 29.7 meters (about 97 feet) more in six minutes than the high-dose or placebo groups (although all groups' walking distance declined over the course of the trial).
Another experimental drug designed to cause stop codon read-through in DMD is known as RTC13. In 2011, MDA gave a three-year grant to Carmen Bertoni at the University of California, Los Angeles (UCLA) to develop RTC13 so it can be taken orally. By the end of the trial, RTC13 is expected to cause a lot of muscle cells to ignore premature stop codon (nonsense) mutations and produce dystrophin, and improve symptoms in dystrophin-deficient mice with a DMD-like disease.
Laboratory evidence shows that raising levels of the muscle protein utrophin can, to some extent, compensate for a deficiency of dystrophin.
Utrophin closely resembles dystrophin but, unlike dystrophin, is normally produced and entirely functional in BMD. Therefore, raising utrophin levels is unlikely to provoke an unwanted immune response, while raising levels of dystrophin may do so. Increasing utrophin production has the potential to help compensate for dystrophin deficiency regardless of the specific dystrophin gene mutation.
Although utrophin is close to dystrophin in both structure and function, there’s at least one key difference between the two proteins. During fetal development and perhaps a little beyond, utrophin is present all around the muscle fiber, interacting with clusters of proteins stuck in its surrounding membrane. As the animal or person matures, utrophin is replaced almost entirely by dystrophin, with one exception. At the neuromuscular junction, utrophin remains throughout life.
Several strategies are being tried to increase utrophin. One is to identify and suppress whatever is inhibiting utrophin production — find the brake and release it, so to speak.
Another strategy is to inject a modified version of the utrophin protein itself into the body. A 2009 study found that modified utrophin protein conferred significant benefits when injected into mice lacking the dystrophin protein and showing a disease resembling DMD.
Scientists reported in 2011 that systemically injecting the human form of a protein called biglycan into mice with a disease resembling human DMD improved the resistance of mouse muscles to contraction-related damage; restored several proteins to their normal location at the muscle-fiber membrane; and recruited utrophin to the muscle-fiber membrane.