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"Any strenuous exercise (running, lifting, even just stair-climbing) is enough to tear the cellular membrane, leading to my muscles breaking down and never being able to repair themselves. So coping with the disease is a combination of conserving energy, taking supplements and steroids that help reduce inflammation and retain muscle strength."
- Hawken Miller, DMD patient

Once diagnosed with Duchenne's, physical options for the paitent are very limited. Although there is some Dystrophin left in the body, there is nowhere near enough to keep muscle cells intact. Any exercise or excessive muscle movement may cause what little cell attachment remaining to break, causing damage that cannot be repaired.

As a result, living with DMD is very limiting. As patients grow older, slowly possible activities become more limited. Then, patients are limited to a wheelchair, and very few actions are even possible.

PSA video:

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Our Boy Christopher - Living with Duchenne Muscular Dystrophy

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You can donate to Christopher Callahan's Special Needs Trust to help with the family's exorbitant medical expenses at their GoFundMe Page: https://www.gofundme.com/2qwq8tut This is a movie about a little boy with Duchenne Muscular Dystrophy. DMD has no treatment and no cure. It is terminal and progressive.

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DMD: Full name for Duchenne Syndrome, also known as Duchenne Muscular Dystropy(DMD)

This X-linked genetic disorder causes progressive muscle degeneration and weakness. This condition is one of nine types of muscular dystrophy, and is caused by the absence of the protein dystrophin, which keeps muscle cells intact. Symptoms start as early as 3 - 5 years old, and most people with this condition don't make it past their early 20's. This disorder is primarily in males.

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Symptoms start very early, and begin with muscle weakness and developmental delays at about the age of 3.

Muscles affected-
Hips/Pelvic area
Thighs
Shoulders
Later the arms, legs, and trunk are affected
By the early teens, the heart and respiratory muscles are affected

Abnormality walking
Difficulty standing
Trouble walking/running
Scoliosis(the curving if the spine)

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Signs and Symptoms

Children with Duchenne muscular dystrophy (DMD) are often late walkers.

In toddlers, parents may notice enlarged calf muscles (see image at right). This enlargement is known as pseudohypertrophy, or "false enlargement," because the muscle tissue is abnormal and may contain scar tissue.

A preschooler with DMD may seem clumsy and fall often. Parents also may note that children have trouble climbing stairs, getting up from the floor or running.

By school age, children may walk on their toes or the balls of their feet with a slightly waddling gait, and fall frequently. To try to keep their balance, they may stick out their bellies and pull back their shoulders. Children also have difficulty raising their arms.

Many children with DMD begin using a wheelchair sometime between ages 7 and 12. Transition to a wheelchair usually is a gradual process; at first, the chair may be required only to conserve the child's energy when covering long distances. (Children often experience renewed independence once they fully transition to a power wheelchair.)

In the teen years, activities involving the arms, legs or trunk may require assistance or mechanical support.Pain and sensation

Pain and sensation

The muscle deterioration in Duchenne MD isn't usually painful in itself. Some people report muscle cramps at times; these usually can be treated with over-the-counter pain relievers.

Because muscular dystrophy doesn't affect nerves directly, touch and other senses are normal, as is control over the smooth, or involuntary, muscles of the bladder and bowel, and sexual functions.

The heart

Lack of dystrophin can weaken the muscle layer in the heart ( myocardium), resulting in a condition called cardiomyopathy. Over time, sometimes as early as the teen years, the damage done by DMD to the heart can become life-threatening. The heart should be monitored closely, usually by a pediatric cardiologist. See Medical Management for more on cardiomyopathy in DMD.

Respiratory function

Beginning at about 10 years of age, the diaphragm and other muscles that operate the lungs may weaken, making the lungs less effective at moving air in and out. Although the child may not complain of shortness of breath, problems that indicate poor respiratory function include headaches, mental dullness, difficulty concentrating or staying awake, and nightmares.

Weakened respiratory muscles make it difficult to cough, leading to increased risk of serious respiratory infection. A simple cold can quickly progress to pneumonia. It's important to get flu shots, and when infections occur, to get prompt treatment. See Medical Management for more on respiratory care in DMD.

Learning

About a third of boys with DMD have some degree of learning disability, although few have serious mental retardation. Doctors believe that dystrophin abnormalities in the brain may have subtle effects on cognition and behavior. Learning problems in DMD occur in three general areas: attention focusing, verbal learning and memory, and emotional interaction.

Children suspected of having a learning disability can be evaluated by a developmental or pediatric neuropsychologist through the school system's special education department or with a referral from the MDA clinic.

If a learning disability is diagnosed, educational and psychological interventions can begin right away. The specialist may prescribe exercises and techniques that can help improve these areas, and the school also can provide special help with learning. See Medical Management for more about learning disabilities in DMD.

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Duchenne Muscular Dystrophy (DMD) - Signs and Symptoms | Muscular Dystrophy Association

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Children with Duchenne muscular dystrophy (DMD) are often late walkers. In toddlers, parents may notice enlarged calf muscles (see image at right). This enlargement is known as pseudohypertrophy, or "false enlargement," because the muscle tissue is abnormal and may contain scar tissue.

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Duchenne is a X - linked condition. The gene DMD, which produces the protein dystrophin, is located on the X chromosome. If the offspring is male and the female is a carrier, or both parent's X chromosomes have the mutation, the offspring will have the condition.

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Duchenne Syndrome was first described by French neurologist Guillaume Benjamin Amand Duchenne in 1860's. But, because of little known information about the cause and the condition itself until the 1980's(little was known about the human genome was known at this time as well). In 1986,  researchers identified the DMD gene, which causes Duchenne Syndrome. Later in 1987 the protein Dystrophin was found, named, and deemed the cause of Duchenne Syndrome.

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Diagnosis may occur when young males (3 - 5 years old) are showing muscle weakness or developmental delays. Methods for DMD diagnosis include:

Muscle Biopsy(taking a sample of the muscle).

Genetic testing(looking at the body's genetic instructions.

These, and a combination of clinical findings, family history, and Dystrophin levels confirm the diagnosis.

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Treatment And Different Methods(Listed Below)

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Treatment And Different Methods(Listed Below)

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Today, scientists are researching many possible solutions to Duchenne Syndrome. In the past few years, drugs to lessen symptoms and prevent further muscle deterioration have been approved by the FDA, creating treatment options for DMD victims for the first time.
But, over 209 million dollars has been funded for DMD research, and recently, great steps have been made for treatment options such as Gene Therapy, Exon Skipping, Stop Codon Read-Through, and Gene Repair.

Because of this research, DMD patients are living till their 50's, whereas the past life expectancy was 20 - 25 years.

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Research

Scientists around the globe are conducting intense research to understand what causes muscle dysfunction in Duchenne muscular dystrophy (DMD) and to apply that understanding to the development of effective treatments. Since inception, MDA has dedicated over $209 million to DMD research, with over $45 million of that investment coming in the past five years alone. The research efforts of thousands of scientists over the course of decades has resulted in many promising leads for potential therapies, some of which are currently being evaluated by the FDA for approval.

In DMD, the muscle is missing a key structural protein called dystrophin, making it more susceptible to injury. Eventually the body can't keep up with repair, which leads to muscle loss and weakness. Scientists therefore are working hard on the development of a number of complementary therapies that could replace dystrophin, protect the muscle from injury and/or promote muscle repair and regrowth. Doctors hope that someday these potential therapies may be used in combination for maximum benefit.

Many examples of these potential therapies already have advanced into clinical trials where they are being evaluated for benefits in people with DMD. See the DMD Clinical Trial Feed for a list of clinical trials currently being conducted for DMD.

MDA also has created a dedicated DMD Clinical Research Network that aims to advance human clinical trials in this disease

Inserting new dystrophin genes

Gene therapy, or gene transfer, refers to the delivery of genes as therapeutic agents. It is being developed in a wide range of disease areas because it has the potential to deliver healthy replacement genes to anyone with a gene mutation, or to deliver adjunct genes (sometimes called "surrogate" gene therapy) that can alleviate disease symptoms.

For gene therapy in DMD, a primary goal is to deliver a replacement copy of the dystrophin gene. Scientists hope that by introducing a functional dystrophin gene, gene therapy may offer a more permanent benefit than other therapies, but they are quick to caution that gene therapy for DMD is unlikely to completely halt or reverse the disease.

To accomplish gene transfer in DMD, some researchers aim to utilize the action of viruses. A virus works by inserting its own genetic material into a host, which causes the host's cells to manufacture viral proteins and create more virus (and in doing so, elicits an immune response that makes the host "sick"). But researchers believe it may be possible to swap out the viral genes for a dystrophin gene, which will trick the virus into delivering the dystrophin gene to people with DMD where the person's muscle cells will use it to manufacture dystrophin protein. To accomplish this without making the patient sick, scientists are utilizing viruses that do not cause illness in humans.

Research into the development of gene therapy for DMD is robust, but some challenges remain. The key difficulties researchers are working to overcome include dealing with the large size of the dystrophin gene, delivering a sufficient quantity of the new genes to muscle (while avoiding other tissues), and avoiding an unwanted immune response to the proteins made from the new genes.

The large size of the dystrophin gene poses a challenge because gene therapy relies on engineered viruses, and there is a limit to the size of the load that these viruses can carry. To address this, MDA-supported scientists have created smaller, but still functional, versions of dystrophin to use in gene therapy. Mini-dystrophin (rAAV2.5-CMV-minidystrophin) is a miniaturized, working dystrophin gene that has been tested in boys with DMD. At the end of clinical testing, scientists determined that the treatment was safe, but unfortunately some of the boys experienced an unwanted immune response to the dystrophin protein. This immune response prevented the newly synthesized mini-dystrophin protein from being used properly by muscle cells, and thereby limited the effectiveness of this approach.

Several research groups are now working toward the development of an even smaller version of dystrophin, called micro-dystrophin. Micro-dystrophin contains the minimum amount of information from the dystrophin gene needed to produce a functional protein. The most advanced of these therapies, rAAVrh74.MCK.Micro-Dystrophin, is being developed by a team at Nationwide Children's Hospital and currently is undergoing phase 1 clinical testing in boys with DMD.

To address delivery of genes specifically to muscles (while avoiding other tissues), researchers have introduced a muscle specific promotor that functions like a control switch for the new dystrophin gene. Muscle specific promoters selectively promote activation of the gene in the muscle, but in other tissues the gene remains dormant so it cannot create unwanted side effects. Researchers led by Dr. Jerry Mendell are developing gene therapies that are activated by the muscle-specific promotor muscle creatine kinase (MCK). Potential therapies that utilize muscle specific promoters are currently in phase 1 clinical testing for DMD (rAAVrh74.MCK.micro-Dystrophin) and also phase 1-2 testing for LGMD (scAAVrh74.tMCK.hSGCA).

Controlling how cells read genetic instructions
Exon skipping
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 (AONs) 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.

For example:

Exon skipping is being tested in clinical trials in boys with DMD in the United States and in other countries.

On Sept. 19, 2016, the U.S. Food and Drug Administration granted accelerated approval to eteplirsen (brand name Exondys 51) as the first disease-modifying drug for DMD. Exondys 51 specifically targets a section of genetic code called exon 51 in the dystrophin gene. It's estimated that 13 percent of boys with DMD could benefit from skipping exon 51. For more, see Top 5 FAQs: Eteplirsen (Exondys 51) for DMD Treatment.

Additional exon skipping drugs that could impact other forms of DMD are moving through the drug development pipeline. At this time, researchers currently are working on development of exon skipping drugs to target exons 44, 45, 50, 52, 53 and 55, as well as strategies to target multiple exons.
Stop codon read-through
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. Between 5-10 percent of people with DMD have premature stop codon/nonsense mutations and may therefore someday benefit from read-through therapies.

A company called PTC Therapeutics, in conjunction with Genzyme Corp., and with 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 those in the high-dose or placebo groups (although all groups' walking distance declined over the course of the trial). Ataluren (Translarna), now owned wholly by PTC, will soon be evaluated by the FDA for approval in the US. It is currently available to DMD patients in the EU.

Researchers at a company called Nobelpharma have developed another stop codon read-through therapy called NPC14. This potential therapy may function similarly to Ataluren and is currently undergoing clinical testing in Japan.

Driving muscle growth
Myostatin Inhibitors
A prominent symptom of DMD is loss of muscle, so researchers aim to develop therapies that promote regrowth of muscle and, in turn, increase muscle strength.

One strategy that has received considerable MDA support involves inhibiting the actions of a naturally occurring protein called myostatin that limits muscle growth. In healthy muscle, myostatin performs an important role: it pushes back against growth signals to maintain muscle at a reasonable size. But in DMD, where muscle loss contributes to a decline in function, myostatin exacerbates the problem. Thus, researchers hope that blocking myostatin may allow DMD muscles to grow larger and stronger.

Inhibitors of myostatin have received much attention from the neuromuscular disease research community since it was found years ago that people and animals with a genetic deficiency of myostatin appear to have large muscles and good strength without apparent ill effects.

Initial efforts to develop therapies based on myostatin inhibition did not bear out. In 2008 a therapy called MYO-029 (developed by Wyeth) failed to meet its endpoint during a clinical trial of people with a variety of dystrophies, though not including DMD. Additionally in 2011, a DMD trial with ACE-031, which was developed by Acceleron Pharma, had to be discontinued due to safety concerns. Although these myostatin inhibitors were not successful, much was learned from these efforts.

Currently, Pfizer is developing a myostatin inhibitor called PF-06252616, which is in clinical trials to test its effectiveness in DMD boys. Bristol-Myers Squibb is also developing a myostatin inhibitor drug, BMS-986089. This potential therapy is currently in a phase 1/2 study to determine its safety in DMD boys. In addition to these two potential DMD medicines, Eli Lilly (LY2495655), Regeneron (REGN1033) and Novartis (BYM338) are also developing drugs that inhibit myostatin, but these are being tested in clinical studies for their effects in cancer wasting (cachexia), sarcopenia and inclusion-body myopathy (IBM), respectively.

Another unique strategy to block the action of myostatin uses gene therapy to introduce follistatin, a naturally occurring inhibitor of myostatin. 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. Furthermore, monkeys that received follistatin gene transfer had stronger, larger muscles. A gene therapy for delivering follistatin to people with DMD called rAAV1.CMV.huFollistatin344 is currently being developed by Milo Biotechnology. This potential therapy is now being tested in an early-stage (phase 1) clinical trial.

Researchers are also exploring the possibility of inducing muscle growth in DMD patients through activation of androgen receptors using drugs called Selective Androgen Receptor Modulators (SARMs). Androgen receptors bind to testosterone and other steroids to induce muscle growth, but anabolic steroids cause many undesirable side effects.

SARMs may offer a way to reap the benefits of muscle androgen mediated growth but with fewer side effects. The drug DT-200 is an oral SARM in development by Akashi Therapeutics, and has shown positive effects in early studies. Akashi next aims to study the safety of this compound in a clinical trial with healthy volunteers. Their long-term plan is to develop DT-200 for treatment of DMD.

A second company called GTx Inc. also is developing SARMs for a variety of muscle wasting conditions, including muscular dystrophy and cancer. Their DMD program is in the preclinical development phase.

Accelerating muscle repair
Stem cell transplantation
In DMD, muscle is more susceptible to injury because it is missing the dystrophin protein. The muscle will continually work to repair itself, but will eventually fall behind. So scientists hope to accelerate repair by transplanting stem cells into DMD muscle.

Stem cells are normally present in a variety of tissues, including muscle, where they play a crucial role in repair and maintenance. Stem cells can be thought of as cells that are in the very early stages of development, before they have become specialized ( differentiated) to perform specific roles in tissues. They may be precursors to 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 adults and children, stem cells reside in muscle tissue where they remain dormant until the tissue is damaged by injury or disease. Following tissue damage, stem cells become activated and then serve to replenish the damaged tissues. For treatment of DMD, then, scientists hope that stem cells could someday be transplanted (from healthy donors) to grow new muscle.

One type of stem cell that holds a lot of promise for transplant is called induced pluripotent stem cells (iPS cells). These cells are derived from adult cells in a process that was pioneered by Dr. Shinya Yamanaka 's lab in Japan. In 2012 he was awarded the Nobel Prize for this work. Building off of this breakthrough, MDA-supported scientists led by Dr. Radbod Darabi have demonstrated that human skin-derived iPS cells are able to differentiate into muscle and can be successfully transplanted into mice that model DMD. The mice are able to accept the new cells, and the cells produce the dystrophin protein.

A team of MDA-supported scientists led by Dr. Rita Perlingeiro have invented and continue to refine a method to efficiently generate immature skeletal muscle cells from iPS cells. In addition, they have shown that, once transplanted, the cells integrate with existing skeletal muscle cells. Next, the team is working to determine the efficiency with which genetically corrected iPS cells can increase muscle regeneration in DMD mice.

To ensure that stem cells grow to take on the proper architecture of a muscle, MDA-supported scientists led by Dr. Deok-Ho Kim are focused on developing materials that serve as scaffolds. Since muscle is a highly ordered tissue, with cells oriented in specific directions, these scaffolds may serve a critical purpose at the cellular scale to promote optimal growth of muscle in the most tissue-like patterns. In practice, the scaffolds are used for growing muscle from stem cells in the laboratory, which can then be transplanted into living organisms to replace damaged muscle. This work ultimately aims to generate a functional muscle patch capable of providing long-term muscle strength and regenerative capacity, and to improve symptoms in people with DMD.

Stem cell transplantation remains a very promising approach for treating the muscle loss that occurs with DMD. Stem cells continue to be a major area of investigation for MDA-supported researchers. At this time, there are no therapeutic stem cell clinical trials being conducted in the U.S. in DMD. However, five trials (phases 1 and 2) are being conducted by researchers outside of the U.S., in the Middle East and Asia.
Activating satellite cells
In addition to stem cell transplantation, scientists are exploring the potential to use a more traditional drug approach to activate a patient's own muscle stem cells. These muscle stem cells, called satellite cells because of their position next to muscle cells, naturally exist to perform muscle repair. Preliminary work by several MDA-supported labs has shown that satellite cells become activated following inhibition of enzymes called histone deacetylases (HDACs). In a mouse model of DMD, HDAC inhibition induces muscle growth, decreases inflammation and reduces fibrosis. Building off of this preclinical work, researchers at a company called Italfarmaco are developing an HDAC inhibitor called Givinostat. This potential therapy is currently being tested in a phase 2 clinical trial in Italy.

MDA also has provided funding to support development of a protein known as WNT7a, which has been demonstrated to drive an expansion of the satellite stem cell population and facilitate muscle regeneration, resulting in significant functional improvement in preclinical models of muscular dystrophy. Based on these findings, a company called Fate Therapeutics is developing a drug-like compound base on WNT7a. This program is currently in preclinical development.

Safeguarding muscle from damage
Fortifying DMD muscle
DMD muscle is more susceptible to damage because it is missing dystrophin, a protein that sits at the muscle cell membrane and is critical for maintaining the muscle's structural integrity. Thus, MDA supported scientists have devised a number of potential therapies that they hope will make DMD muscle more resistant to damage.

One potential strategy is to compensate for the lack of dystrophin with a similar muscle protein, such as utrophin. Utrophin is a naturally occurring structural protein that closely resembles dystrophin and performs a very similar function in the muscle. A key difference is that utrophin is produced during development or early stages of repair of muscle, and then is turned off and replaced by dystrophin (except for the area around where the nerve meets the muscle, where utrophin continues to be present in adult tissue). But scientists believe that if utrophin production were turned back on, it may be able to compensate for the absence of dystrophin.

MDA-supported scientists have identified a potential therapy that promotes production of utrophin protein in mice. This therapy, SMT C1100, increases strength and fatigue resistance in a mouse model of DMD. SMT C1100 is being developed by Summit Therapeutics and has completed phase 1 safety testing in DMD boys. Summit is planning additional clinical testing to determine the effectiveness of SMT C1100 in DMD. Summit also is developing a second generation utrophin upregulator therapy called SMT022357, which is currently in preclinical development.

Another potential strategy to protect muscles from damage is to fortify DMD muscles using another naturally occurring structural protein called biglycan. Scientists reported in 2011 that systemically injecting the human form of 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, importantly, recruited utrophin to the muscle-fiber membrane. MDA has supported research by a company called Tivorsan that has developed an engineered version of biglycan called TVN-102 that could be injected into patients. Tivorsan currently is preparing to take TVN-102 into early-stage clinical testing.

A third strategy to fortify DMD muscle is to introduce a different naturally occurring structural protein called Laminin 111. MDA has supported foundational research for this approach, where introduction of Laminin 111 was shown to improve exercise capacity and reduce muscle damage in a mouse model of DMD. Based on this work, a company called Prothelia Inc., in partnership with Alexion Pharmaceuticals, has developed an engineered version of laminin 111 called PRT-01. This potential therapy is currently in preclinical development for DMD as well as other dystrophies.

Researchers also are exploring whether muscle integrity could be strengthened by introduction of an enzyme called beta1, 4-N- acetylgalactosamine galactosyltransferase (GALGT2) using gene therapy. GALGT2 is involved in adding sugar molecules to a protein called alpha-dystroglycan, which is part of a cluster of proteins located at the muscle-fiber membrane. This cluster is abnormal in DMD muscle and previous work by MDA-supported scientists has shown that raising GALGT2 levels in mice can help to compensate for the loss of dystrophin and protect muscle from damage. Since these findings were reported, GALGT2 gene therapy has been effectively delivered to calf muscles in macaque monkeys. Now, researchers at Nationwide Children's Hospital aim to begin a phase 1 clinical study for GALGT2 gene therapy (rAAVrh74.MCK.GALGT2) for DMD in 2015.

In addition to identifying proteins such as utrophin, biglycan, laminin 111 and GALGT2 that may fortify against the loss of dystrophin, scientists are exploring other methods for protecting muscles from damage. For example, MDA-supported scientists have identified a compound called Carmaseal-MD (Polaxamer 188 NF), which acts as a membrane sealant in a mouse model of DMD. In mice, this compound acts by filling the rips in the membrane that result from damage. The company that is developing Carmaseal-MD, Phrixus Pharmaceuticals, hopes to begin clinical testing in people with DMD in 2016.
Suppressing the damage response
The repeated bouts of damage that occur in DMD muscle have been shown to eventually induce death ( necrosis) of the muscle cells. Scientists believe that therapies which block the signals that contribute to cell death may one day be used to slow muscle degeneration and, in turn, loss of function.

Researchers have reported that an enzyme called hematopoietic prostaglandin D ₂ synthase (HPGDS) may contribute to the necrosis of muscle cells in DMD. To prevent muscle cell death, an inhibitor of this HPGDS, TAS-205, is being developed by Taiho Pharmaceuticals. This compound was shown to reduce necrosis and improve running in a mouse model of DMD. TAS-205 is now in early-stage (phase 1) clinical testing in Japan.

Scientists also have identified regulation of calcium in muscle as a potential strategy for therapeutic intervention. This approach is based on the fact that accumulation of calcium inside the cell, which is thought to happen in DMD, causes degradation of muscle proteins and triggers cell death. There is evidence that calcium does in fact play an important role in disease progression in DMD muscle, so several groups are investigating ways to ramp down accumulation of calcium in DMD muscle cells.

For example, an MDA-supported company called ARMGO has identified a compound called ARM210 that restores the appropriate balance of calcium within muscle cells by correcting the activity of a type of channel called the ryanodine receptor calcium channel complex (RyR). RyR functions to keep calcium safely sequestered away inside the cell until it is needed, but in DMD this channel has been shown to be leaky. But in a mouse model of DMD, ARM210 corrects the calcium leak occurring through the RyR and improves daily activity, strength, muscle force, and muscle damage. ARMGO is continuing preclinical validation of ARM210 and aims to begin clinical testing soon.

A second strategy to correct calcium accumulation in the cell is being developed by Akashi Therapeutics. The compound, called AT-300 (formerly GsMTx4), inhibits one of the channels that allows calcium to enter the muscle cell from the outside, called a stretch-activated calcium channel. AT-300 is based on a natural tarantula venom peptide, and acts to block calcium entry into muscle cells when they are being stretched. This compound is currently in preclinical development, but Akashi plans to bring it into clinical testing in the near future.

Combating inflammation in muscle

During healthy muscle repair, inflammatory responses are activated and are known to aid in the cleanup and restoration of damaged muscle. In DMD, however, these inflammatory responses are chronically activated and therefore become detrimental to the repair process. MDA-supported scientists therefore are working to understand and interfere 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, to prevent scoliosis and to prolong the time that people with DMD can walk. 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.

The FDA on Feb. 9, 2017, approved deflazacort (brand name Emflaza) to treat DMD. For more, see FDA Approves Emflaza for Treatment of Duchenne Muscular Dystrophy.

MDA has invested heavily in researchers who are working on developing new anti-inflammatory therapies that have fewer or less severe side effects than the corticosteroids currently in use. Many of these potential therapies are being evaluated in clinical trials. These include VBP-15, HT-100, CAT-1004, and deflazacort. VBP-15, made by ReveraGen BioPharma, is currently undergoing phase 1 safety study in healthy volunteers. HT-100, made by Akashi Therapeutics, is undergoing a phase 1-2 clinical study in DMD boys to determine whether it is both safe and effective. CAT-1004, made by Catabasis, will soon undergo testing in a phase 1 clinical study to determine its safety.

Another anti-inflammatory therapy, called Idebenone (Raxone/Catena), is being developed by Santhera Pharmaceuticals. In clinical trials, Idebenone had a positive impact on a measurement of respiratory function (Peak Expiratory Flow, or PEF) in non-ambulatory DMD patients who were not taking steroids. Santhera has announced that it plans to apply for approval from the FDA for Idebenone in the near future.

Additionally, there are earlier-stage development efforts, such as IMO-8400 that are being developed by Idera Pharmaceuticals. IMO-8400 employs a unique strategy: inhibiting Toll-like Receptors (TLRs), proteins that are inappropriately upregulated in a mouse model of DMD. Idera is currently preparing to take IMO-8400 into phase 1 clinical trials with a goal of developing this drug for DMD.

Blocking muscle fibrosis

As muscles degenerate in a person with DMD, the muscle fibers are replaced by fat and connective tissue in a process called fibrosis. Fibrosis is similar to scarring, and the presence of fibrotic tissue in muscle impedes proper function and repair. In fact, scientists have observed that the extent of fibrosis in DMD muscle tracks with diminished function. Thus, MDA-supported researchers are investigating whether muscle function might be protected by medications, termed anti-fibrotics, that reduce this fibrosis. In addition, scientists hope that reducing fibrosis also may help increase the efficacy of other potential therapies. MDA has invested almost $4 million into investigating the potential of such therapies.

The most advanced drug in this class is currently being tested in a phase 2 trial - Akashi Therapeutics' HT-100. HT-100 is a delayed-release formulation of halofuginone, a compound that has been studied for years for its effects on autoimmune and fibrotic diseases. Halofuginone exerts its effects by inhibiting the formation of collagen 1, a major component of fibrotic tissue. MDA has strongly supported preclinical and clinical work with this compound that continues to show promise.

A second approach to reducing fibrosis is to introduce a peptide called angiotensin 1-7. In a mouse model of DMD, angiotensin 1-7 has been shown to reduce the formation of fibrotic tissue in muscle and also suppress some of the signals that have been implicated in the growth of fibrotic tissue. A company called Tarix Orphan LLC. is developing a therapy based on angiotensin 1-7, called TXA127. This therapy will undergo phase 2 clinical testing in 2016.

A third approach is to target connective tissue growth factor (CTGF), a common mediator of fibrotic disease. MDA has supported foundational work demonstrating a benefit of inhibiting CTGF in a mouse model of DMD. This work showed that CTGF reduces the ability of damaged muscle cells to repair themselves and promotes muscle fibrosis, and that inhibiting CTGF reduces muscle fibrosis and improves muscle function. Based on this preclinical work, a company called FibroGen is now developing a CTGF inhibitor, called FG-3019. FibroGen has recently received clearance from the FDA to begin phase 2 clinical testing of FG-3019 in DMD. In addition, FG-3019 also has been tested in phase 2 clinical studies for idiopathic pulmonary fibrosis, where it was shown to reverse fibrosis in a significant number of patients.

Maximizing blood flow to muscle
Phosphodiesterase Inhibitors
People with DMD experience inadequate blood flow to their muscles while exercising, which may contribute to fatigue and reduced performance. Thus, scientists hope that drugs that restore proper blood flow to muscles may benefit muscle strength and function.

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 and pulmonary arterial hypertension.

On the basis of these and other findings, researchers have started investigating the possibility that phosphodiesterase inhibitors can improve muscle function in people with DMD or BMD.

In 2010, an MDA-supported trial testing the effects of tadalafil on blood flow to muscles began in men with BMD. This study demonstrated that tadalafil could restore normal blood flow regulation after a single dose. Further study is ongoing to determine whether this effect will lead to improved muscle function in BMD.

A later study conducted in DMD found that treatment with either sildenafil or tadalafil restores blood flow to muscles during exercise or rest in DMD patients. Additional clinical studies are planned to determine whether this increased blood flow will translate to increased muscle function in DMD.

Of note, some research has focused on the effects of phosphodiesterase inhibitors on heart function in DMD and BMD. Unfortunately, a trial with sildenafil showed that it was unlikely to help heart function in these patient groups. A second study with sildenafil, which had enrolled a small number of participants, was in fact terminated due to concerns about the potential for negative impact on cardiac function in adults with Duchenne and Becker muscular dystrophy. Due to the small study size, questions remain about whether sildenafil is in fact unsafe for the BMD heart.
Nitric Oxide Donors
Scientists also are exploring the effect of modifying blood flow in DMD and BMD muscle with nitric oxide. Nitric oxide is the product of an enzyme called nNOS (which is absent in DMD and BMD muscle) and works in a similar manner to the phosphodiesterase inhibitors described above. Naproxinod is a potential therapy in preclinical development by a French pharmaceutical company called NicOx. Naproxinod is a version of naproxen (Aleve) that has been modified to release nitric oxide into the body. This therapy has been tested clinically for arthritis and other indications, and has demonstrated promising effects in a mouse model of DMD. The hope is that this therapy could increase blood flow to muscles and reduce inflammation, both of which may help to improve the function of DMD muscle.

Scientists in Italy are developing a combination therapy for DMD, using ibuprofen (a nonsteroidal anti-inflammatory drug) and isosorbide dinitrate (a nitric oxide donor). This combination therapy has undergone two clinical trials (named Isofen 1 and 2) to determine its safety and to establish the most effective dose of each drug. Additional clinical trials are planned to determine whether this combination has a beneficial effect in people with DMD.

Finally, researchers from Cedars-Sinai Medical Center currently are studying the effects of sodium nitrate, a nitric oxide donor, in people with BMD in two separate clinical trials. The first trial is a phase 2-3 study to determine whether sodium nitrate improves blood flow to muscle, as the phosphodiesterase inhibitors have been shown to do. The second is a phase 1 clinical study to determine whether there is a functional benefit to muscle that results from treatment with sodium nitrate.

Protecting the dystrophin-deficient heart

DMD heart muscle, like skeletal muscle, is more susceptible to damage because it is missing dystrophin, an important structural component of the cell membrane. As a result, some patients may develop cardiomyopathy or heart damage that weakens the heart's ability to contract (pump). More information about cardiomyopathy in DMD can be found here and here.

Understanding and specifically treating dystrophin-deficient cardiomyopathy is a priority for MDA. The MDA DMD Clinical Research Network has made studying the natural history and treatment of this condition as 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.

To address DMD-associated cardiomyopathy, researchers are aggressively pursuing a number of strategies to sustain or improve heart function. They're testing already existing medications for their possible benefits in the DMD affected heart, and also conducting research to find new approaches to specifically treat the dystrophin-deficient heart.

Since a number of cardiomyopathy drugs have been developed over the years to treat heart failure in older patients, doctors already have some tools at their disposal for treating the DMD heart. These therapies center on ways of reducing the burden on the pumping heart. To that end, doctors may prescribe angiotensin converting enzyme inhibitors (ACE inhibitors) and angiotensin receptor blockers (ARBs) that make blood vessels open up wide and thereby reduce the resistance to the heart's pumping action. Doctors may also prescribe diuretics to remove extra water from the blood, so that there is less volume for the heart to pump. Finally, doctors may prescribe beta-blockers to slow the heart rate, giving the DMD heart sufficient time to empty and refill with each beat so that it can pump blood more efficiently.

Researchers are continuing to study existing drugs to determine the best regimen to preserve heart function in DMD. Currently, there are several clinical studies aimed at determining the best combination and dose to prevent decline of heart function. These include studies of the relative efficacy of the aldosterone receptor antagonist, spironolactone and eplerinone, which are diurectics. This is a phase 3 clinical study led by Dr. Subha Raman at Ohio State University. Prior work by this group demonstrated that treatment with eplerinone (along with ACE inhibitors or ARBs) slowed the decline of cardiac function in boys with DMD over the course of one year. Additional studies investigating the optimal drug regimen for slowing cardiac decline in DMD include a phase 4 study in Italy comparing the effects of carvedilol (a beta blocker) with Ramipril (an ACE inhibitor), and a phase 3 study in France examining the effects of nebivolol (a beta blocker).

One promising and completely new therapy in development specifically for DMD is called CAP-1002 and is being developed by Capricor Therapeutics. CAP-1002 is a therapy based on cardiac stem cells (jump to stem cell transplantation) derived from donor heart tissue. Researchers aim to transplant these therapeutic stem cells into people with DMD with the hope that they will promote repair of the heart. Studies with CAP-1002 in a mouse model of DMD have demonstrated positive effects on the heart, and the company has since initiated a phase 1-2 clinical study to test the safety and effectiveness of CAP-1002 in boys and men with DMD.

Another new therapy, called PB1046, is in development by PhaseBio Pharmaceuticals. PB1046 is an engineered version of Vasoactive Intestinal Peptide (VIP), a neuropeptide which has been shown to be ionotropic (increases contraction of the heart) and lusitropic (speeds relaxation of the heart). VIP has also been shown to prevent fibrosis and inflammation in cardiac and skeletal muscle. PhaseBio has reported that their engineered version of VIP, PB1046, slowed cardiac functional deterioration in two mouse models of DMD and showed positive safety data in a clinical trial with volunteers who had essential hypertension. PhaseBio plans to begin phase 1 clinical testing to determine the safety of PB1046 in people with DMD in 2016.

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Duchenne Muscular Dystrophy (DMD) - Research | Muscular Dystrophy Association

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Scientists around the globe are conducting intense research to understand what causes muscle dysfunction in Duchenne muscular dystrophy (DMD) and to apply that understanding to the development of effective treatments. Since inception, MDA has dedicated over $209 million to DMD research, with over $45 million of that investment coming in the past five years alone.

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The transplantation of normal genes into cells in place of missing or defective ones in order to correct genetic disorders.

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"For gene therapy in DMD, a primary goal is to deliver a replacement copy of the dystrophin gene. Scientists hope that by introducing a functional dystrophin gene, gene therapy may offer a more permanent benefit than other therapies, but they are quick to caution that gene therapy for DMD is unlikely to completely halt or reverse the disease."
- Muscular Dystrophy Association on gene therapy

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Exon Skipping is the idea that bad genes, or exons, are skipped over through splicing. This creates a shorter but function DMD gene, hopefully lessening symptoms

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Prosensa's Video Animation on Exon Skipping

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Abstract

INTRODUCTION

In some organisms, in an mRNA-independent manner, UAG, UAA or UGA are reassigned from stop to specify an amino acid ( 1). In contrast, dynamic mRNA-specific redefinition of stop codons ( 2) is often influenced by mRNA context features that increase the chances of near-cognate tRNA successfully competing with release factor for ribosomal A-site acceptance. In the form of stop codon redefinition where the non-universal amino acids selenocysteine or pyrrolysine are specified, the identity of the amino acid encoded is of key functional significance. In other cases, commonly known as stop codon readthrough, the identity of the amino acid specified is unimportant and the key feature is the downstream encoded C-terminal extension. Until recently the repertoire of documented stop codon readthrough cases was limited mostly to viral genes where several cases have been described ( 3-7). Comparative sequence analysis and ribosome profiling studies are now, however, showing that readthrough is much more widespread in chromosomal gene expression than was previously appreciated.

Bioinformatic analysis of orthologous genes from 12 diverse Drosophila genomes revealed protein coding signatures in the zero frame downstream of the annotated stop codon for 283 protein coding genes ( 8, 9). Some of these were experimentally tested and verified as bona fide readthrough cases ( 9) and three had been previously characterized. One experimentally studied case of developmentally regulated readthrough in Drosophila is in decoding of the kelch gene which is essential for the organization of the female ring canal actin cytoskeleton ( 10). Readthrough of the UGA stop codon in kelch extends the standard 76 kDa protein product to 160 kDa. The proportion of extended product is commonly ∼5% but in imaginal discs during metamorphosis, it is at a higher level ( 11).

The phylogenetic analyses of the 12 Drosophila genomes were by their nature designed to identify signatures of evolutionarily conserved readthrough. Therefore, they did not reveal instances of readthrough that either emerged recently or are not under strong evolutionary selection. This gap in our knowledge has recently been filled by ribosome profiling analysis. Deep sequencing of ribosome-protected mRNA fragments of Drosophila melanogaster early embryos, and S2 tissue culture cells, provided experimental evidence for over 300 readthrough events not predicted by phylogenetic approaches; and for some of these, functional domains in the extensions have already been demonstrated ( 12). A number of these instances of readthrough appear to be developmentally regulated; many more will doubtless be identified when other tissues are examined. The more modest number identified to date in [] Saccharomyces cerevisiae ( 12-14) likely does not reveal the full importance of readthrough in that organism since the balance of various factors, and most notably [], liberates evolutionarily important diversity ( 15-17). [The [] variant of termination factor eRF3 enhances readthrough (review, 18).]

Analysis of stop codon readthrough in mammalian gene expression is at an early stage. Phylogenetic analysis predicted four candidates, SACM1L, OPRK1, OPRL1 and BRI3BP ( 9, 19), and a readthrough isoform of myelin P0 was studied experimentally ( 20).

While the identity of the nucleotide 3′ adjacent to a stop codon alone has a substantial effect on termination stringency ( 21-23), the context effect is not limited to a single nucleotide. This is known to be exploited by some readthrough cases to elevate its efficiency. The 3′ hexanucleotide, CARYYA, is important for synthesizing the 57 kDa readthrough extension to a Tobacco Mosaic Virus protein ( 24). It also stimulates readthrough in many other cases, especially in plant viruses ( 25, 26). A similar sequence (CARNBA) can stimulate readthrough in yeast ( 27).

At an early stage a 3′ stem loop was identified for a case of Drosophila readthrough ( 28). Interestingly in Barley Yellow Dwarf Virus, there is a stimulatory structure that involves base pairing between long distant segments, >700 nucleotides 3′ of the UAG stop codon ( 4). Stimulation of readthrough by 3′ mRNA structures is much more commonly utilized than was appreciated until recently, for example in Drosophila kelch ( 6). Further, 3′ adjacent stem loops are involved in a subset of cases of eukaryotic selenocysteine specification ( 29, 30). RNA pseudoknot structures are also able to stimulate readthrough as in the case of Murine Leukemia Virus (MuLV) GagPol ( 31). The MuLV pseudoknot stimulation of readthrough involves a change in its conformation ( 32).

There is a highly conserved stem loop structure of high potential stability 3′ of the stop codon in the human SACM1L ( 7). However, studies in a heterologous expression system suggested very low levels of readthrough at SACM1L mRNA ( 33).

Sequences 5′ of stop codons are also relevant, in part because of effects at the nascent peptide level. A major effect of C-terminal proline on termination efficiency has been revealed by many studies (e.g. 34). Earlier systematic studies are also relevant for their insights into the effects that the identity of the last two amino acids of the nascent polypeptide have on termination efficiency in bacteria ( 35). However, as there are substantial differences in context effects in bacteria and eukaryotes ( 21), the 5′ context effect studies in both S. cerevisiae ( 36) and mammalian cells ( 37) are more relevant to the discovery of the new natural stimulatory motif reported here.

The present work experimentally tests readthrough of the published human candidates, SACM1L, OPRK1, OPRL1 and BRI3BP ( 9) as well as three new candidates identified here.

MATERIALS AND METHODS

Plasmids

Sequences flanking the stop codons of the seven predicted readthrough candidates were chemically synthesized (Integrated DNA Technologies: g blocks) and cloned with XhoI and BglII restriction enzymes into pDluc dual-luciferase vector. The SACM1L (NM_014016.3) insertion sequence range was 1950-2061 with a T to C mutation at 2032 to remove the second stop codon while still maintaining the predicted secondary structure. The sequence boundaries of the remaining six candidates were as follows: ACP2 (NM_001610.2) 1372-1419; MAPK10 (NM_138982.2) 2064-2120; BRI3BP (NM_080626.5) 876-989; AQP4 (NM_001650.4) 1017-1119; OPRK1 (NM_000912.3) 1348-1462; OPRL1 (NM_182647.2) 1511-1616. Mutation constructs were derived by two-step polymerase chain reaction with appropriately designed primers (Integrated DNA Technologies). Corresponding readthrough controls were generated for every reporter construct by mutating TGA stop codons to TGG or else TAA and TAG stop codons to TAC (Supplementary Figure S5C).

HA-AQP4-TGA and HA-AQP4-TGG were made by Gibson Assembly (NEB) using three overlapping synthetic DNA fragments (Integrated DNA Technologies: g blocks) based on the AQP4 CDS (NM_001650.4). Assembled gene fragments were cloned in-frame with the influenza hemagglutinin tag in pcDNA3-HA (Invitrogen). All constructs were verified by DNA sequencing.

Cell culture and transfections

HEK-293T cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1 mM L-glutamine and antibiotics. HEK-293T cells were transfected in quadruplicate with Lipofectamine 2000 reagent (Invitrogen), using the 1-day protocol in which suspended cells are added directly to the DNA complexes in half-area 96-well plates. For each transfection the following were added to each well: 25 ng of each plasmid plus 0.2 μl Lipofectamine 2000 in 25 μl Opti-Mem (Gibco). The transfecting DNA complexes in each well were incubated with 4 × 10 ⁴ cells suspended in 50 μl DMEM + 10% FBS. Transfected cells were incubated at 37°C in 5% CO ₂ for 24 h.

Dual-luciferase assay

Firefly and Renilla luciferase activities were determined using the Dual Luciferase Stop & Glo® Reporter Assay System (Promega). Relative light units were measured on a Veritas Microplate Luminometer with two injectors (Turner Biosystems). Transfected cells were washed once with 1 x PBS and then lysed in 12.6 μl of 1x passive lysis buffer (PLB) and light emission was measured following injection of 25 μl of either Renilla or firefly luciferase substrate. Readthrough efficiencies (% readthrough) were calculated by averaging relative luciferase activities (firefly/Renilla) from replicate wells of test constructs and dividing by average luciferase activities from replicate wells of control constructs (stop mutated to sense). Readthrough efficiencies of at least three independent experiments are graphed.

Western analysis

Cells were transfected in 6-well plates using Lipofectamine 2000 reagent, again using the 1-day protocol described above, with 3 μg of each indicated plasmid. The transfecting DNA complexes in each well were incubated with 2.4 × 10 ⁶ HEK-293T cells suspended in 3000 μl DMEM + 10% FBS and incubated overnight (3 days for HA-AQP4 transfections) at 37°C in 5% CO ₂. Transfected cells were lysed in 100 μl 1x PLB. For HA-AQP4 transfections cells were lysed in subcellular fractionation buffer (250 mM sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl ₂, 1 mM EDTA, 1 mM EGTA and 1 mM DTT). Proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Protran), which were incubated at 4°C overnight with primary antibodies. Immunoreactive bands were detected on membranes after incubation with appropriate fluorescently labeled secondary antibody using a LI-COR Odyssey® Infrared Imaging Scanner.

Antibodies

An affinity purified rabbit polyclonal antibody to a predicted antigen (DRTESRQDSLELSS) within ORF2 of AQP4 was prepared by GenScript. The following commercially available antibodies were also used. Mouse anti-Renilla (MBL), mouse anti-HA (Covance), mouse anti-β-actin (Sigma) and goat anti-AQP4 (Santa Cruz).

RESULTS

Phylogenetic analysis reveals evolutionary constraint 3′ of the stop codon in seven human genes

Previous searches for evolutionary conserved readthrough cases in humans identified four potential candidates: SACM1L, OPRL1, OPRK1 and BRI3BP ( 9, 19). Because all four of these candidates have highly conserved UGA stop codons, as do the vast majority of readthrough candidates predicted in other metazoa ( 9), we searched for additional candidates specifically among genes with highly conserved UGA stop codons using PhyloCSF ( 38) with a correspondingly relaxed multiple-hypothesis correction. PhyloCSF uses substitutions and codon frequencies among 29 mammals to distinguish coding from non-coding regions irrespective of nucleotide-level conservation. We found three additional candidates using this method, ACP2, AQP4 and MAPK10 (Supplementary Figure S1A-C). Each of the seven candidate readthrough extensions has a PhyloCSF score higher than 99.96% of similar-sized non-coding regions of 3′-UTRs (Supplementary Figure S2). We used RNAz ( 39) to predict that ACP2 has a 42-nucleotide conserved RNA stem loop starting six nucleotides 3′ of the predicted readthrough stop codon (Supplementary Figure S1D). RNAz did not predict conserved RNA secondary structures in either AQP4 or MAPK10. To help define sequences that could potentially stimulate readthrough we sought to identify non-mammalian orthologs of the seven readthrough candidates. For AQP4, MAPK10, OPRK1, OPRL1 and SACM1L, conserved C-terminal extensions were observed beyond mammals (Supplementary Figure S3A-E). In AQP4 and SACM1L, the conservation extends to at least the last common ancestor of mammals and cartilaginous fishes. For MAPK10 the extension is conserved to at least the last common ancestor of mammals and lampreys and in OPRK1 and OPRL1 it goes to at least the last common ancestor of mammals and bony fishes.

OPRL1 and OPRK1 are paralogs encoding G protein-coupled opioid receptors. The amino acid sequences of their respective extensions, except for six of the last eight residues, show little similarity between the paralogs (See Weblogo ( 40) in Figure 1A and B, Supplementary Figure S4). However, the extensions in OPRK1 homologs are almost always exactly 28 residues long and conservation at the amino acid level is strongest near the C-terminus (Supplementary Figures S3C and S4). The extensions in OPRL1 also show high amino acid conservation near the C-terminus but their lengths vary from 28 to 34 residues with the deletions and insertions concentrated in the central region (Supplementary Figure S3D). The nucleotide sequences surrounding the stop codon of the main open reading frames (ORFs) in OPRK1 and OPRL1 exhibit a striking level of conservation (Supplementary Figure S3C and D). The sequence 5′ of the stop codon shows conservation primarily at the amino acid level although the last sense codon is only moderately conserved-predominately GUA in OPRK1 and GCA in OPRL1. The stop codon is always UGA. The four nucleotides immediately 3′ of the UGA stop codon-CUAG-are universally conserved within the two paralogous groups and their respective orthologs. In OPRL1, but not OPRK1, the next two nucleotides, GC, are also absolutely conserved and the following 12 nucleotides are moderately conserved, the first six of them clearly primarily at the nucleotide level. No other sequences 3′ appear conserved strictly at the nucleotide level alone in either OPRK1 or OPRL1. The features of the OPRL1 and OPRK1 extension described above suggest that their readthrough capability predates their divergence.

MAPK10 is a member of the mitogen activated protein kinase family. Its readthrough extension is always 13 residues long (Supplementary Figure S1C). The penultimate and the last sense codons of the first ORF encoding this protein are always the same-UGC-AGG. The three nucleotides following the stop codon are always CUA and in more than 90% (68 of 73 cases) the next nucleotide is G, forming the same tetranucleotide sequence, CUAG that is universally conserved at the same position in OPRK1 and OPRL1 (Figure 1 and Supplementary Figures S1 and S3). The next eight nucleotides are also highly conserved, primarily at the nucleotide level and not the amino acid level. Seven of the nucleotides in this region have the potential to base pair with conserved nucleotides of the last two sense codons and the stop codon of ORF1.

AQP4 belongs to the aquaporin family of membrane proteins and is involved in conducting water through the cell membrane in kidneys and cells of neuronal origin. The readthrough extension of AQP4 is typically 28 residues (Supplementary Figure S1B). Like OPRK1 and OPRL1, conservation of the extension at the amino acid level is concentrated near the C-terminus. Unlike in OPRK1 and OPRL1 the last sense codon of the first ORF is always the same-GUA (Figure 1D and Supplementary Figure S3A). Remarkably, the four nucleotides following the stop codon, CUAG, are highly conserved, with the first three universally conserved, and are identical to the universally conserved nucleotides in the same position in OPRK1, OPRL1 and MAPK10 genes. Of the other nucleotides 3′ of notable conservation are those present at positions 13-24 after the stop codon. However, only two of them are absolutely conserved.

Suppressor of actin 1 (SACM1L) is a membrane associated protein. Its readthrough extension appears highly variable in size, from 19 to 38 residues (Supplementary Figure S3E). The four C-terminal amino acids are highly conserved. Like AQP4 and MAPK10 the last sense codon of the first ORF is always the same-GAC. We previously proposed that the sequence 3′ of the stop codon of SACM1L, at least in mammals, can be folded in an energetically stable RNA secondary structure that potentially can be involved in stimulating the readthrough ( 9, 19). The present analysis including sequences from non-mammalian vertebrates indicates that the proposed RNA secondary structure, as previously proposed, is only conserved in mammals. A homologous putative structure, supported by co-variant nucleotide substitutions, lacking part of one stem, is present throughout vertebrates, including cartilaginous fishes and coelacanth, but is completely absent from ray-finned fishes.

Experimental evidence of readthrough for OPRK1, OPRL1, MAPK10 and AQP4

To verify the functionality of the seven predicted readthrough candidates, local sequences for each (see Figure 2A for boundaries) were cloned in-frame between Renilla and firefly luciferase genes. Lysates from HEK-293T cells transfected with each construct were assayed for luciferase activities and expression levels. The 3′ firefly luciferase gene lacks an initiation codon and its expression is dependent on readthrough of the candidate's stop codon. Readthrough efficiencies were determined by comparing relative luciferase activity (firefly/Renilla) of test constructs with controls for each construct in which the UGA stop codon is changed to UGG (TRP). Efficient stop codon readthrough was observed for the candidates OPRL1, OPRK1, MAPK10 and AQP4 by western blotting with anti-Renilla luciferase antibodies (Figure 2B). Readthrough efficiencies were determined by dual-luciferase assay and were as follows: ∼31% ( OPRL1), ∼13% ( OPRK1), ∼14% ( MAPK10) and ∼7% (AQP4) (Figure 2C). SACM1L, ACP2 and BRI3BP stop codon cassettes failed to promote levels of readthrough greater than 1% in these cells.

Identification of a conserved 3′ readthrough motif

Though context dependent, several studies have shown UGA to be the leakiest of the three stop codons. The annotated stop codon of all seven readthrough candidates is UGA, however, strikingly, only those candidates confirmed here experimentally ( OPRK1, OPRL1, MAPK10 and AQP4) contain the highly conserved CUAG tetranucleotide sequence immediately 3′ of their annotated UGA stop codons as described above (boxed in Figure 1 and underlined in Figure 2A). To assess the possible role of nucleotides 3′ of the stop codon on readthrough we made several sets of 3′ nested deletions for OPRK1, OPRL1, MAPK10 and AQP4 readthrough. For all four candidates, when the CUAG motif was completely deleted, readthrough levels became almost undetectable (Figure 3A). While there are several instances of readthrough stimulation by RNA secondary structures 3′ of the stop codon, here, only 12 nucleotides 3′ of the stop codon (+15 in Figure 3A) appears to be sufficient for maximal readthrough of OPRK1, OPRL1, MAPK10 and AQP4. Though deletion of the CUAG motif results in the most dramatic decrease in readthrough efficiency for each of the readthrough genes, deletion of nucleotides +9 to +15 in OPRK1, OPRL1 and MAPK10 results in ∼2-fold decrease in readthrough efficiency.

Analysis of OPRL1 5′ sequences

Because of the relatively high level of readthrough observed for OPRL1 (∼31%), we decided to focus on this candidate for systematic mutational analysis to delineate local sequence requirements effecting readthrough levels. The only difference between the 12 nucleotides immediately 3′ of both OPRK1 and OPRL1 annotated coding regions are two nucleotides at positions +8 and +15 (where the first nucleotide of the UGA stop codon is assigned +1), yet the readthrough efficiency for OPRL1 is almost 2.5 times greater than that for OPRK1 (∼31% versus ∼13%). Swapping the 12 nucleotides immediately 3′ of OPRL1 with those of OPRK1 did not reduce readthrough levels of OPRL1 to those of OPRK1 levels (Figure 3B). Similarly, swapping the 12 nucleotides 3′ of OPRK1 with those of OPRL1 did not increase the efficiency of OPRK1 readthrough. This suggests that OPRL1 sequences 5′ of the main ORF stop codon are responsible for the difference in readthrough levels between OPRL1 and OPRK1.

Complete deletion of the OPRL1 5′ sequences reduced readthrough levels to ∼5% when 87 3′ nucleotides are present (Supplementary Figure S5A). Together these results indicate that while the CUAG motif is important for readthrough, an additional stimulation is exerted by sequences 5′ of the UGA stop codon. In the case of OPRL1, this additional stimulation is ∼5-fold (since readthrough decreases from ∼31% to ∼5% when OPRL1 5′ sequences are deleted).

The sequential restoration of OPRL1 codons 5′ of UGA had dramatic effects on readthrough efficiency with only six codons required to regain maximum OPRL1 readthrough (Figure 3C). The addition of just one codon 5′ of the OPRL1 stop codon resulted in ∼50% restoration in readthrough efficiency (cf >10% readthrough in Figure 3C to ∼5% readthrough in Supplementary Figure S5A when the last codon is not GCA). The nucleotide immediately 5′ of readthrough stop codons has been shown previously by others ( 41) to have an influence on readthrough efficiency and in agreement with this we see that a purine in this position is preferable (Supplementary Figure S5B).

Analysis of OPRL1 3′ sequences

To better understand the importance of each nucleotide 3′ of the UGA stop codon, we systematically mutated each of the thirteen 3′ nucleotides to each of the other three possible nucleotides in turn and then assessed readthrough efficiency by dual-luciferase assay (Figure 4A). Substitutions furthest 3′ of the UGA stop codon had the least effect on readthrough whereas those immediately 3′-adjacent had the most dramatic outcome. As reported previously for UGA readthrough ( 22, 23), a cytosine immediately 3′ is especially important, however, in the OPRL1 context the identity of the following three nucleotides 3′ of the cytosine are also critical. Most other mutations in this series had only modest effects on OPRL1 readthrough.

Another readthrough motif, CARYYA, also immediately 3′ of the stop codon, has been reported previously to stimulate readthrough ( 24, 26). We investigated whether the CARYYA motif could replace the hexanucleotide sequence of OPRL1 when placed immediately 3′ of the stop codon. The readthrough efficiency of OPRL1-CARYYA is ∼15% (Figure 4B) indicating that, at least in the OPRL1 context, CARYYA is a less efficient readthrough stimulator than the CUAG motif. Systematic mutational analysis of the OPRL1-CARYYA confirmed that all six nucleotides of the CARYYA appear to be important for readthrough (Figure 4B).

Stop codon identity is important for the CUA_G readthrough motif

To ascertain the importance, if any, of stop codon identity 5′ of the CUAG and CARYYA motifs we generated readthrough constructs with wild-type OPRL1 and OPRLI-CARYYA with either of the three stop codons. Changing the OPRL1 stop codon from UGA to either UAA or UAG reduced readthrough efficiency by ∼6-fold and 3-fold, respectively, indicating that UGA is the most efficient readthrough stop codon with this new motif (Supplementary Figure S5C). In contrast, we find that for the CARYYA motif, readthrough efficiency is very similar with either UGA or UAG, both of which are much more efficient than UAA.

Aminoglycoside effect on readthrough

Aminoglycoside antibiotics such as gentamycin are known to enhance readthrough of stop codons ( 42-47) and have been suggested as possible therapeutic interventions to suppress nonsense mutations (e.g. subsets of Duchene's Muscular Dystrophy and Cystic Fibrosis patients) ( 48, 49). Early studies on aminoglycoside-induced readthrough indicate that readthrough efficiency may be influenced by the sequence context surrounding the stop codon ( 50, 51) which could have implications for the treatment of patients with nonsense mutations in their DNA. We tested the effect of the aminoglycosides gentamycin, paromomycin and amikacin on readthrough of both wild-type OPRL1 and OPRL1-CARYYA constructs with each of the three stop codons (Figure 5). For wild-type OPRL1 constructs with either of the three stop codons, all aminoglycosides enhanced readthrough in a dose responsive manner. UGA readthrough was the most and UAA readthrough was the least responsive to aminoglycoside treatments. For OPRL1-CARYYA both gentamycin and amikacin had little effect on readthrough at UAG or UAA but did induce a dose-dependent increase at UGA stop codons. Paromomycin enhanced OPRL1-CARYYA readthrough on both UGA and UAG while having little effect on UAA stop codons. Together these results indicate that varying stop codons and 3′ contexts can respond dramatically to different aminoglycosides.

Immunoblotting demonstrates readthrough in natural context

In an attempt to identify readthrough for any of the four candidates in the context of the full coding sequence, we cloned each in-frame with sequences encoding an epitope tag (HA) so that expressed proteins have HA fused to their N-terminus. For each construct, sequences extending to the end of ORF2 were included. Constructs in which TGA was changed to TGG were also constructed so that the coding sequence and readthrough extension are expressed as a single ORF (readthrough control). Each construct was transfected into HEK-293T cells and lysates analysed by western blotting with antibodies against HA. HA-MAPK10 constructs were not well expressed and both HA-OPRL1 and HA-OPRK1 were expressed as multiple diffuse bands, making it difficult to identify potential readthrough products (not shown). However, HA-AQP4-TGA and HA-AQP4-TGG were well expressed and migrated as distinct proteins of ∼32 kDa (36.2 kDa predicted) and ∼35 kDa (39.6 kDa predicted), respectively (Figure 6, left-hand panel). For HA-AQP4-TGA a much fainter protein of ∼35 kDa is also evident. This protein co-migrates with the protein expressed from the HA-AQP4-TGG readthrough control. Furthermore, western analysis of the same lysates with a commercially available antibody against an AQP4 C-terminal epitope detected the same sized proteins as detected by the HA antibody (Figure 6, middle panels). Similar sized proteins are also evident in mock infected cells, suggesting that anti-AQP4 can detect endogenous expression of AQP4 and its readthrough product in these cells. However, AQP4 isoforms generated by leaky scanning are well documented ( 52) (leaky scanning does not explain the presence of putative readthrough proteins in cells expressing N-terminally HA-tagged AQP4-TGA since leaky scanning produces proteins with truncated N-termini).

To establish whether the putative AQP4 readthrough proteins are genuine, we raised antisera against an epitope within the predicted AQP4 readthrough extension. Immunoblotting of lysates transfected with constructs expressing either HA-AQP4-TGA or HA-AQP4-TGG detected only the ∼35 kDa protein, to varying levels, in both lysates (Figure 6, right-hand panels). Longer exposures revealed a protein of ∼35 kDa in mock infected cells that we assume is the endogenous AQP4 readthrough protein.

DISCUSSION

Before the advent of ribosome profiling the majority of published stop codon readthrough examples were found in viral decoding ( 3-7). Until very recently only two examples of mammalian readthrough had been confirmed experimentally ( 20, 53) although four more had been predicted by comparative genomic studies ( 9, 19). A study published in 2013 using ribosome profiling identified 42 genes displaying stop codon readthrough in human foreskin fibroblasts ( 12), although the proportion that is productively utilized is unknown. None of the seven candidates studied here was expressed to sufficient levels to have been properly assessed by this ribosome profiling study. This may not be so surprising since OPRL1, OPRK1 and MAPK10 expression is known to be confined to neuronal cells whereas AQP4 expression is predominantly in kidney and astrocyte cells. Here the prior comparative genomics analysis was extended leading to the identification of a further three mammalian readthrough candidates. We experimentally tested all seven and confirm readthrough in four.

TargetP analysis of the four readthrough examples did not reveal any cellular targeting motifs although AQP4, OPRL1 and OPRK1 all possess well-conserved serine and threonine residues (Supplementary Figure S4). AQP4, OPRL1 and OPRK1 are multiple pass transmembrane proteins where the readthrough extension would be predicted to reside in the cytoplasm. Serine and threonine residues could be sites of phosphorylation that may be involved in regulating desensitisation. Interestingly, readthrough of 3-phosphoglycerate in Ustilago maydis, a basidiomycete plant pathogen, reveals a functional peroxisomal targeting motif ( 54). The degree of importance of readthrough for generating dual targeted protein isoforms merits further investigation.

Factors affecting readthrough efficiency include tRNA abundance, RNA secondary structure, release factor levels, stop codon context and more recently posttranslational modifications of either the ribosome ( 55) or release factor ( 56). Since tRNA abundance and release factor levels are presumably invariant in our study and none of the confirmed readthrough genes have conserved RNA secondary structures it is most likely that context effects are the key determinants of readthrough efficiency for our candidates. Many groups have reported that readthrough is affected by the nucleotide sequence surrounding the stop codon ( 3, 27, 57-61). Mechanisms explaining the influence of downstream nucleotides and RNA secondary structures on the competition between productive near-cognate tRNA and release factor recognition of stop codons relevant to readthrough efficiency are still lacking. Several features have been considered including tRNA selection through stabilization of the A-site tRNA:mRNA interaction by stacking effects, interaction between the stop codon and the rRNA and interaction between the stop codon and the polypeptide chain release factor ( 62). Identity of the base 3′ adjacent to a stop codon was shown by binding studies to be important for release factor action ( 63). While the preferred termination signal differs in prokaryotic and eukaryotic species, a common feature appears to be a strong bias against a cytidine residue following a termination codon in all organisms ( 22, 23), implying that any termination codon followed by a C is a weak stop codon. Dramatic recent advances in the resolution of ribosome structure using cryoEM will likely reveal information about the ribosome conformational changes that influence A-site selection in response to recoding signals.

AQP4, MAPK10, OPRK1 and OPRL1 all possess CUAG immediately 3′ of their termination codons and our mutational analyses of the OPRLI context clearly demonstrate the importance of these nucleotides. A search of all human genes for CUAG immediately following a UGA stop codon indicated that there were 23 instances (∼39 expected assuming equiprobable nucleotide occurrence). Four had positive PhyloCSF scores and these are the four candidates confirmed in this study. Efficient readthrough cannot be ruled out for those genes with UGA_CUAG and negative PhyloCSF scores since readthrough may be highly efficient but unimportant for organism fitness. However, one gene (Malate dehydrogenase; MDH1) had a borderline negative PhyloCSF score. We tested MDH1 for readthrough using a dual-luciferase assay and confirmed ∼3.5% readthrough efficiency (not shown). This is the lowest level of readthrough that we observed for those candidates with CUAG 3′ of UGA stop codons and emphasises the importance of sequences extending beyond this motif. Interestingly, only one of the readthrough genes identified in the human foreskin fibroblast profiling study ( 12) possesses the UGA_CUAG stop codon context and this gene is MDH1.

Several groups have classified readthrough stop codons based on both nucleotide context and the presence of a 3′ RNA secondary structure ( 25, 26). One type is generally made up of diverse plant viruses containing UAG_CARYYA or UAG_CARNBA in yeast ( 27). Another class generally has a pseudoknot structure 3′ of UGA_G. Another set made up mostly of animal viruses has UGA followed by either CGG or CUA. Only a handful of viruses (alphaviruses) have UGA_CUA and these include Sinbis, Middleburg (UGA_CUAG), Ross river and Getah (UGA_CUAGGC that is the same sequence as OPRL1) and also Chikungunya (UGA_CUAG) that is dangerously spreading. UGA_CUA readthrough has also been identified in Mimivirus and Megavirus which are the best characterized representatives of an expanding new family of giant viruses infecting Acanthamoeba ( 64).

Interestingly, in our mutagenesis studies, when the CARYYA motif is mutated by a single nucleotide from CAAUUA to CUAUUA in an otherwise OPRL1 context thus partially creating the CUAG motif, then wild-type levels of readthrough are restored (Figure 4B). This was not observed in work in a plant system ( 24) when CAAUUA was similarly mutated to CUAUUA 3′ of a UAG terminator rather than the UGA stop codon used here. Whether this difference reflects a plant versus mammalian difference or different stop codons has not been addressed.

It is interesting that little readthrough was detected in the two candidates with RNA structures ( SACM1L and ACP). Perhaps these structures are binding sites for RNA binding proteins that influence readthrough only in certain cell types, different from the one tested. This could also explain why studies in a heterologous yeast system ( 33) detected little readthrough for SACM1L and C18B2.6, the latter being a worm readthrough candidate with a stem loop.

Our analysis of the effects of aminoglycosides on readthrough sequences with either the CARYYA motif or the CUAG motif indicates that each motif responds differently and suggests that the context effects of patients with premature stop codons could respond differently to individual drugs. A simple test of a specific stop codon and their contexts could be performed before treatment to ascertain the most effective treatment.

We are very grateful to Dr Mike Howard (University of Utah) for providing dual-luciferase plasmid pDLuc.

FUNDING

Science Foundation Ireland [08/IN.I/B1889,12/IP/1492 to J.F.A.]; National Science Council of Taiwan [NSC 101-2917-I-564-049 to M.-Y.C.]; National Institutes of Health [NIH-1-R01-HG004037-07, NSF-DBI-0644282 and NIH-U41-HG007234 to M.K.]. Wellcome Trust Funding [094423 to P.V.B.]. Funding for open access charge: Wellcome Trust [094423].

Conflict of interest statement. None declared.

REFERENCES

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Evidence of efficient stop codon readthrough in four mammalian genes | Nucleic Acids Research | Oxford Academic

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Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes.

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Gene Repair is a technique used to correct a mutation at a specific site in an episome or chromosome. It uses synthetic oligonucleotides together with the cell's inherent DNA repair system to direct single base pair changes.

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Even though there are some drugs preserving dystrophin levels and muscle formation, Duchenne's is a terminal condition, and eventually the body nor medication will be able to stop the muscle degeneration.

However, not using muscles as much and preserving the little dystrophin in the muscle helps to keep muscles intact for longer periods of time.

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I was diagnosed with Duchenne Muscular Dystrophy when I was six years old.

I didn't have the same stamina as other kids on my soccer team, and my parents knew something was wrong. We finally realized that I had a rare genetic disease that affects my muscles after a trip to the doctor's office.

At that point I really didn't know what that meant, only that I couldn't keep up with my classmates anymore. I wouldn't grow up to be the football player I had always dreamed of.

Duchenne Muscular Dystrophy is a degenerative, genetic muscle disease that leaves sufferers with an average life expectancy of 25. It is a sex-linked genetic disease only affecting boys, hindering the production of a protein in your muscle cells that holds together your cellular membrane. In many cases, boys are left wheelchair bound after losing the ability to create that protein entirely.

My body still produces a small amount and I've stuck closely to a regime of stretching and resting. As a result, I am fortunately not yet at that stage.

Any strenuous exercise (running, lifting, even just stair-climbing) is enough to tear the cellular membrane, leading to my muscles breaking down and never being able to repair themselves. So coping with the disease is a combination of conserving energy, taking supplements and steroids that help reduce inflammation and retain muscle strength.

When I was diagnosed it came as a shock to us all, and at the time there were no real treatment options. It was scary, but my parents tell me I was more peaceful now I finally understood why I couldn't keep up with the other kids. W henever someone asked, I told them that I had "leg problems," without knowing the full extent of the disease I had.

My little blue scooter drew everyone towards me throughout elementary school and middle school. Everyone wanted a ride because they thought it was the coolest thing they'd ever laid their eyes on. I remember being so frustrated when asked why I needed my scooter - being able to walk didn't qual not needing a chair, and I wanted to be treated like a normal person.

At the time, there was a lack of work being done on the disease and no one really knew about it. My parents started their foundation, CureDuchenne, to try to jump start research for a cure.

There are numerous examples of parents helped me through my childhood when physical activity was too much. On the final eighth grade trip to Yosemite, my dad fashioned a backpack to carry me in up part of Half Dome. My mom was as equally as committed to helping me through my disease because she helped grow my faith both in God and myself. Even though Boy Scouts is a thought of a outdoors physical activity, I took to my dad's shoulders for long hikes and during my Eagle Scout project, I organized the physical labor without having to do any of it.

As I transitioned from lower school into high school, I became more involved in my parents foundation, speaking at fundraising events and accompanying my parents to network with the community.

High school was both easier and harder. For example, I was really into football at that time, so I became part of the team as a statistician and a "motivator". I loved being able to travel with the team - to games and got to know some of the players.

That said, I still felt left out. Some friends thought I wouldn't be able to participate in activities that they'd planned. At times I felt like being around them made extra work for them, helping me up and down stairs and the small physical things.

This lead to me being afraid of asking for help - I wanted to be able to do everything else on my own.

My dad was there again to help me through the physical challenges that came with international trips. He carried me through the Ho Chi Minh Mausoleum in Vietnam, they thought my scooter was a safety risk. In Argentina he carried me down the side of a cliff for a waterfall boat ride.

Since arriving at USC, I've had to learn to manage on my own.

When I was living at home my parents were always there. They made sure I took my medicine, took on anything around the house I wasn't able to do and they stretched me every night, to name a few things. It's not just one little pill everyday either.

I take around 10-15 pills in the morning, in the afternoon and night. Those range from a steroid that reduces inflammation, which also hampers my growth and maturity (I had to take human growth hormone for a couple of years), multivitamins, supplements that help strengthen muscle cells (some of the same ones you see bodybuilders downing), and general vitamins like D-3.

I'm now in charge of everything. I was able to coordinate with the USC Physical Therapy school to get stretched every day and I've been able to find alternatives with day to day exertions, butI have had to constantly remind myself to take my medicine.

USC has done it's best to accommodate my needs: giving me extra time, the use of a word processor on exams, a room which I can fit my power chair, stretching table and scooter and accessible facilities, but there's still a lot they can do to retrofit their old buildings to be wheelchair accessible. For example, m any of the doors on campus are not accessible via a push plate and some buildings don't have elevators to every floor.

Balancing all of my work and health has been an additional struggle for me. I need rest and cannot pull all nighters so I am constantly figuring out ways to manage my time better and unfortunately cut social activities that would interfere with the amount of sleep that I need. Even with that, I am doing as much as possible to take advantage of the countless opportunities there are at USC.

I am a part of the Navigators at USC, a Christian club that has a weekly service on campus and has semester retreats. Recently I drove up to the San Bernadino mountains with my friends to spend time in worship and prayer and have fun together. I am writing for the Daily Trojan and I was a Deputy News Editor last year, which I was still able to do with all of the other things I have to worry about. I am also writing for the Tab and working at the Annenberg Media center on Monday and Wednesday. Even though I am completely busy at USC, I wouldn't have it any other way.

Years ago, the future for boys with Duchenne like me was grim. The life expectancy was very low and many were wheelchair bound from an early age. Now, with the approval of a drug called Eteplirsen which treats 13 percent of the population, the odds are stacked in the favor of kids with the disease. 13 percent may not seem like a lot put its approval by the FDA has laid the groundwork for the development of other drugs that can treat different mutations.

Living with Duchenne has given me the chance to participate in groundbreaking biological research. I was poked and prodded and my cells were grown in a petri dish to further research the disease. I made two trips to the Netherlands, the original country of my ancestors, to give my body to science with the hopes that it would accelerate the research in Duchenne. I was almost selected to participate in a research trial in Italy, which unfortunately ended up falling through. Obviously there were countless other boys and families who sacrificed parts of their lives to jumpstart the research as well and I am also forever grateful for them.

This drug isn't an outright cure per se either; it won't reverse the effects of the disease and the only hope is that it will stop the progression. The drug works by skipping over an exon that produces the protein dystrophin so that instead of producing nothing, the cell produces a protein that acts the same way as dystrophin would. That's one part of the cure because the disease affects all parts of the body.

In order to stop the progression of the disease, scientists must employ medicine that targets each part of the body that the exon skipping drug doesn't affect - sort of like a drug cocktail. There's already been development in stem cell therapy with the heart and there's bound to be so much more.

With the way science is progressing today nothing is impossible, even curing a disease like mine.

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Living with Duchenne Muscular Dystrophy is frustrating and humbling

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I was diagnosed with Duchenne Muscular Dystrophy when I was six years old. I didn't have the same stamina as other kids on my soccer team, and my parents knew something was wrong. We finally realized that I had a rare genetic disease that affects my muscles after a trip to the doctor's office.

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Below is the listed contact information for a DMD family support group:

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Family Support - Parent Project Muscular Dystrophy

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© 2017 Parent Project Muscular Dystrophy | 800-714-5437 | info@parentprojectmd.org | Media | Site Map | Privacy Policy | Designed by Big Duck | EIN 31-1405490

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https://thetab.com/us/usc/2016/10/14/duchenne-mucsular-dystrophy-2436
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