exon skipping

What is exon skipping and how does it work?

In order to explain the concept of exon skipping, it is first necessary to explain how genes work and how mutations in the dystrophin gene can cause both Duchenne and Becker muscular dystrophy.

Contents:

• What are genes?
• What are exons?
• What happens in Becker muscular dystrophy?
• What impact does a Becker mutation have on the dystrophin protein?
• What happens in Duchenne muscular dystrophy?
• How can exon skipping help?
• Does this really work?
• Is there a clinical trial?
• Will it work for everyone with Duchenne muscular dystrophy?
• Will the same 'molecular patch' work for everyone? 
• Are ‘molecular patches' a cure?

What are genes?

DNA is an extremely long molecule which contains the instructions to create and maintain our bodies. A gene is a section of DNA that contains the instructions for the production of one specific protein. Proteins are essential parts of cells and play a role in every process occurring within the cell, as well as having structural or mechanical functions which help maintain the cells' shape. It is estimated that we have about 30,000 different genes.

What are exons?

Genes are divided into sections called exons and introns. Exons are the sections of DNA that code for the protein and they are interspersed with introns which are also sometimes called 'junk DNA'. The introns are cut out and discarded in the process of protein production, to leave just the exons. The dystrophin gene is our largest gene- it has 79 exons which are joined together like the pieces of a puzzle.

Exon structure of Dystrophin

What happens in Becker muscular dystrophy?

Let's zoom in on exons 68 to 75 to look at this a bit more closely:

Dystrophin exons 68-75

In Becker muscular dystrophy an exon is deleted, for example exon number 74 in the diagram:

Dystrophin exon 68-75 missing exon 74

Although a part of the gene is missing, exon 73 can join up with exon 75, and the puzzle can be completed to the end of the gene:

What impact does a Becker mutation have on the dystrophin protein?

The dystrophin protein normally sits in the membrane that surrounds muscle fibres like a skin, and protects the membrane from damage during muscle contraction. Without dystrophin the muscle fibre membranes become damaged and eventually the muscle fibres die.

Dystrophin is a very large protein with a section in the middle consisting of lots of repeated segments (in green below) and it is known that the protein can still work to some extent if some of these repeated segments are missing. Individuals with Becker muscular dystrophy have some of these repeated segments missing and have relatively mild symptoms- often being able to still walk into their 40s and 50s.

Diagram of the dystrophin protein

A man has even been known to be still walking at 61 years of age, despite having a deletion of 46% of the dystrophin gene!

Illustration of the dystrophin protein in Becker muscular dystrophy

What happens in Duchenne muscular dystrophy?

In Duchenne muscular dystrophy an exon, or exons are deleted which interfere with the rest of the gene being pieced together. In our example (using exons 50-57), exon 52 illustrates this:

Exon 51 can not join up with exon 53, which prevents the rest of the exons being assembled. For the dystrophin protein to work it must have both ends of the protein. Therefore, this mutation results in a completely non-functional dystrophin protein and the severe symptoms of Duchenne muscular dystrophy.

How can exon skipping help?

As the name suggests, the principle of exon skipping is to encourage the cellular machinery to 'skip over' an exon. Small pieces of DNA called antisense oligonucleotides (AOs) or 'molecular patches' are used to mask the exon that you want to skip, so that it is ignored during protein production. In our example, if we use a 'molecular patch' designed to mask exon 53: 

Exon 51 can now join up to exon 54 and continue to make the rest of the protein, with exons 52 and 53 missing in the middle:

Therefore, exon skipping may be able to reduce the symptoms of Duchenne muscular dystrophy, to those more like Becker muscular dystrophy.

Does this really work?

So far scientists have shown this technique to be effective in a mouse model of Duchenne muscular dystrophy (the mdx mouse) and in human Duchenne muscular dystrophy muscle cells grown in the laboratory.

Several clinical trials have now been conducted that show that injecting a molecular patch into the blood stream or under the skin results in the production of dystrophin in the muscles. No serious side-effects were observed. There are three companies involved in conducting clinical trials of exon skipping. The principal of exon skipping is the same for all of the clinical trials but the molecular patch being tested has a slightly different chemical formulation.

• The Dutch company Prosensa has formed a partnership with GSK to test a molecular patch to skip exon 51 .
• AVI Biopharma conducted a clinical trial in the UK of a exon 51 molecular patch developed by the MDEX consortium with funding from the Muscular Dystrophy Campaign. 

The next step is to find out if the increase in dystrophin in the muscles results in improved muscle function or at least slows the deterioration of the muscle. Clinical trials have been started to determine this and continue to monitor the safety of exon skipping.

Is there a clinical trial?

AVI Biopharma now plans to test higher doses of the molecular patch to try to gain a consistently strong response to the molecular patch. They are planning to conduct this trial in the US. 

GSK and Prosensa have started an international phase 3 clinical trial of exon 51 skipping. 

The challenge with all of these trials is to produce enough dystrophin in as many muscles as possible (including the heart) to prolong and improve the quality of life for boys with Duchenne muscular dystrophy.

Will it work for everyone with Duchenne muscular dystrophy?

It is thought that skipping one or two exons would be able to treat around 83% of the genetic errors causing Duchenne muscular dystrophy.

Will the same 'molecular patch' work for everyone?

No, the dystrophin gene is very large and the genetic errors associated with Duchenne muscular dystrophy occur in different places along this gene. There are however some common areas for mutations and initially 'molecular patches' will be made for these. The clinical trials are starting with exon 51 which would be applicable for around 13% of boys. Once the technology has been shown to be effective for a particular error it will be possible to design other 'patches'. A clinical trial has been started for a exon 44 molecular patch which is applicable to about 6 percent of boys. The companies have said that they have started preclinical research on patches for exon 50, 45, 52, 53 and 55.

Are 'molecular patches' a cure?

Scientists hope that this type of therapy will halt or even reverse the symptoms of Duchenne muscular dystrophy so that the symptoms are more like those of boys with Becker muscular dystrophy. It will not be a cure because if proven to be effective, this treatment would need to be repeated regularly- how often will become apparent during clinical trials.

Side effects reported in GSK exon skipping trials

Side effects reported in GSK exon skipping trials

GlaxoSmithKline (GSK) has announced that several boys taking part in clinical trials of drisapersen (a molecular patch) have received hospital treatment for side effects including a reduced number of a type of blood cell and protein in the urine. We would like to reassure our supporters that we are working closely with GSK and that we remain confident that the safety of boys in the trial is of paramount importance and that all boys are being carefully monitored for side effects.

In a presentation in Rome last week, GlaxoSmithKline (GSK) announced that several boys in the trials of drispersen (a potential exone skipping drug) have experienced serious side effects. The company reported that a small number of boys in the trial had required hospital treatment for thrombocytopenia (a reduction in the number of cells called platelets which can lead to problems with blood clotting) or proteinuria (too much protein in the urine which can be a sign of kidney damage).

Since the presentation, we have contacted GSK to ask for more information. They have assured us that the safety of the boys in the clinical trials is of "paramount importance" and confirmed that all the boys taking part in the trial are being carefully monitored for any signs of side effects. The vast majority (about 96%) of boys who started the trial are still taking part and GSK is confident that their monitoring program will make sure that all boys participating in the trial are safe. They also said that any boy who shows signs of these side effects will be admitted to a hospital for treatment and recommended that "anyone participating in a drisapersen study that has questions or concerns should discuss these with their study investigator."

 Dystrophin-Deficient Dogs Benefit From Gene Therapy

University of Missouri.

Jan 15 2013.

MDA-supported investigators found that intramuscular injections of microdystrophin genes

 improved muscle health in dystrophin-deficient dogs, a response not previously seen in large

 animals or humans

MDA research grantee Dongsheng Duan and colleagues have found that dogs with a DMD-like disease can be successfully treated with microdystrophin gene therapy

Article Highlight:

• Researchers supported in part by MDA used highly miniaturized dystrophin (microdystrophin) genes encased in AAV9 delivery vehicles to treat six dystrophin-deficient dogs that had a disease mimicking human Duchenne muscular dystrophy (DMD).
• The gene injections were made directly into the dogs’ front leg muscles.
• Muscle fibers that received the gene therapy showed good dystrophin protein production, improvements in muscle appearance, and partial protection from the weakness that occurs in dystrophin-deficient muscles after repeated contractions.
• No specific immune responses were detected against the newly made microdystrophin protein or the vehicle used to deliver the new genes, although immune system T cells were seen in the treated muscle fibers.

For the first time, gene therapy using a highly miniaturized dystrophin gene resulted in significant improvement in muscle structure and function in dogs with a disorder mimicking human Duchenne muscular dystrophy.

The MDA-supported findings may help advance the development of microdystrophin gene therapy for DMD and the related disorder Becker muscular dystrophy (BMD), both of which result from a deficiency of the dystrophin protein.

MDA research grantee Dongsheng Duan, a professor in the Department of Molecular Microbiology and Immunology at the University of Missouri in Columbia, coordinated the research team, whose findings were published online Jan. 15, 2013, in the journal Molecular Therapy.

Microdystrophin gene therapy has been in development for more than a decade but so far has shown better results in dystrophin-deficient mice than in dystrophin-deficient dogs or humans with DMD.

This is the first time that significant muscle-related benefits have been seen with microdystrophin gene therapy in a large animal model of DMD.

Although the results were encouraging, the researchers note that the response of the dogs to microdystrophin gene therapy was not as robust as the response of dystrophin-deficient mice in previous experiments. They say further studies are needed to create an optimal microdystrophin gene, AAV delivery system and regimen of immunosuppressive drugs.

Immune response has been a challenge

MDA-supported investigators reported in late 2010 that four out of six boys with DMD who received microdystrophin gene therapy into a biceps muscle showed evidence that their immune systems rejected the newly synthesized dystrophin protein.

Previously reported studies of microdystrophin gene therapy in dystrophin-deficient dogs also have shown less than optimal results and some evidence of rejection of the therapy by the immune system.

It is widely believed that the dog model of DMD is more like the human disease than are mouse models. The canine immune system may also replicate the human immune system better than the mouse immune system does.

Improvements seen in muscle fiber structure and function

Six dystrophin-deficient dogs received injections into front leg muscles of microdystrophin genes, each encased in a delivery vehicle made from a modified type 9 adeno-associated virus (AAV9). Four of the dogs received a single gene therapy injection into one front leg muscle; two dogs received a single gene therapy injection into each front leg muscle.

All six dogs received temporary immunosuppression using two drugs, cyclosporine and myocophenolate mofetil (CellCept), intended to help them tolerate the gene transfer.

Two months after the gene injections, when treated muscle fibers were compared with untreated muscle fibers, the investigators saw:

• robust production of dystrophin from the microdystrophin genes (microdystrophin protein);
• restoration of a cluster of proteins at the muscle-fiber membrane that is disrupted when dystrophin is absent;
• much less calcification of muscle;
• substantially less scar tissue (fibrosis);
• less invasion of muscle tissue by inflammatory cells;
• more normal muscle fiber size; and
• significant protection of muscle fibers against the weakness that occurs with repeated contractions in dystrophin-deficient muscles.

The investigators saw no evidence of an immune response against either the newly made microdystrophin or the viral delivery vehicle. However, somewhat surprisingly, they saw an abundance of immune system T cells in the treated muscle fibers. The effects of the T cells and the reasons for their presence remain unclear.

What makes these experiments different

Commenting on the relative success of the current gene therapy experiments in dystrophin-deficient dogs compared to other DMD dog gene transfer experiments, the investigators note that

• the specific microdystrophin construct used in these experiments was different from that used in other experiments;
• the experimental design was different from that used in other studies;
• the gene delivery strategy was different; and
• the age of the dogs was different (older).

"In summary," the researchers say, "our results have cleared uncertainty on microdystrophin therapy arisen from other dog studies. However, compared with what was reported in the mouse model, the improvement we saw in dystrophic dogs remained suboptimal."

NEW RESEARCH SHOWS HOPE OF CURE FOR MUSCULAR DYSTROPHY

TOSHIFUMI YOKOTA A RESEARCHER IN   THE DEPARTMENT OF MEDICAL GENTICS AT THE UNIVERSITY OF ALBERTA, POSES FOR A PHOTO IN HIS LAB, THURSDAY AUG. 9, 2012.

           There is hope in sight for those suffering from muscular dystrophy after a breakthrough discovery at the University of Alberta that could someday cure the disease.

Dr. Toshifumi Yokota, a researcher at the university's faculty of medicine and dentistry, has been working on a gene treatment therapy for over five years that targets the gene responsible for causing Duchenne muscular dystrophy.

"This is one of the most common and devastating disorders worldwide," Yokota said.

"We still have a long way to go, but our research has worked quite well, and now we are working on human cells."
This form of muscular dystrophy affects one in every 3,500 live male births, and works by mutating the gene responsible for muscle membrane support, causing the muscle to break down.

The effect that this has on those with the disease is crippling.

"It's quite a devastating disorder, the average life expectancy is 25 years," Yokota said.

But there is light on the horizon.

By creating DNA molecules that can remove the mutated part of genes, U of A researchers have seen positive results.

After testing on mouse cells and live mice, researchers found a 10 to 15% rise in protein levels in the mice that have been treated.

And while a 15% rise may not sound like a lot, the effect it will have will be monumental, Yokota said.

"A 10 to 15% leads to strong effects," he said.

"Patients have zero proteins so if you can rescue 15% there is a great scientific effect."

Quite simply, 15% is the difference between a wheelchair and walking. It also raises life expectancy from 25 years to up to 70 years.

Yokota's findings have made a splash in the muscular dystrophy community, and while his research solely targets Duchenne muscular dystrophy, it has given hope to all who suffer from the disease.

"His findings are very important because they continue to give hope for other treatments," said Marla Spiegel, the national director of research for Muscular Dystrophy Canada.

"It will allow patients with muscular dystrophy to lead healthier, better lives."

Yokota said human trials are still five to 10 years away but trials on human cells have begun.

Yokota left the Children's National Medical Center in Washington, DC, in October 2011 to become the lead researcher on this subject at the U of A.

New drug to be tested for use in the muscular dystrophies

thursday 22 November 2012
Muscular Dystrophy Campaign

Landon.

New drugs to be tested for use in muscular dystrophies

   Prof Dominic Wells, and Dr Susan Brown, at the Royal Veterinary College in London recently received a grant from the Medical Research Council (MRC) as part of an innovative scheme that saw the MRC partnering with pharmaceutical company AstraZeneca.   AstraZeneca made 22 of its chemical compounds available free-of-charge to scientists, who were encouraged to apply for MRC funding to use them in medical research with the ultimate aim of benefiting patients. AstraZeneca had conducted early trials of these compounds and validated their use for future research, but had put them on hold for further development. The aim of this partnership was to extend the possible application of these compounds for use in new areas.

       Prof Wells and Dr Brown will be testing a drug called AZD1236 for its ability to prevent scar tissue formation in muscular dystrophy.  AZD1236 was designed to block the activity of an enzyme called matrix metallopeptidase-9 (MMP-9) which is known to be involved in the formation of scar tissue. In the muscular dystrophies, there is a gradual breakdown of the muscle which leads to muscle weakness.  As the muscle breaks down it is replaced by fat and scar tissue further reducing the ability of the muscles to work properly. It is this process that the researchers hope to slow using AZD1236.

        The researchers will use the drug, originally developed by AstraZeneca to treat a type of lung disease known as chronic obstructive pulmonary disease, in two different mouse model of muscular dystrophy - Duchenne muscular dystrophy and limb girdle muscular dystrophy to see if they can slow progression of the disease. If successful, the study will provide the evidence they need to plan human trials of the drug to test whether it has a benefit for individuals with muscular dystrophy.

Research news (phase 1 exon skipping trial )

GlaxoSmithKline releases results of phase 1 exon skipping trial 

Tuesday 6 November 2012

The drug company GlaxoSmithKline has today announced results of a phase 1 clinical trial of a potential exon skipping drug -now called drisapersen - in boys with Duchenne muscular dystrophy who are unable to walk. This drug, a molecular patch for exon 51 of the dystrophin gene, has previously been tested in boys with Duchenne muscular dystrophy who are still able to walk and has generated encouraging results. The main aim of this trial was to make sure there are no additional safety issues for wheelchair users and to test whether different doses of the potential exon skipping drug were safe and well tolerated.

    The company recruited 20 boys who were given one of three doses of drisapersen or a placebo (an inactive substance). The participants received a single injection of the drug and were monitored for up to 6 months. All the boys who received the potential drug experienced some mild side effects such as redness or swelling of the injection site and all the boys given the highest dose of the potential drug experienced fever. However, the researchers say no serious side effects were observed.

hydrotherapy for muscular dystrophy (mayopathy)

aquatic therapy for DMD 
(MAYOPATHY-TAMILNADU-INDIA)

                            WATER SURVIVAL SESSION

                          POSTURE TRAINING SESSION

                                  DIVE START SESSION