CRISPR & Duchenne muscular dystrophy

The success of CRISPR in a model of Duchenne muscular dystrophy

Original story from Duke University

Researchers at Duke University have shown that by using genome editing technology, CRISPR can safely and stably correct a genetic condition such as Duchenne Muscular Dystrophy (DMD). The study appears in the journal Nature Medicine. Links here


CRISPR/Cas9 is, by far, the most effective technique to correct DNA defects. Described as a “molecular toolkit,” this intelligent and sophisticated technique enables researchers to perform cut and paste” operations on DNA.


How does CRISPR work?

This technology works as follows:

  • CRISPR is a type of gene sequence found naturally in specific organisms, including viruses. It can recognize a particular DNA sequence, i.e., find the place on a DNA strand where a disease-causing genetic defect (mutation) lies.
  • Cas9 acts like a pair of scissors. It “cuts” DNA at a specific location, i.e., where an action is needed to repair or eliminate a genetic defect.
  • With this technology, researchers can remove a genetic mutation or insert a fix for a faulty gene sequence.
  • Using standard DNA repair mechanisms, the cell will then naturally reattach the severed DNA strands.


About Gersbach’s latest research

In 2016, Charles Gersbach, a Rooney Family Associate Professor of Biomedical Engineering, was in the first published positive results concerning the CRISPR technique likely to be translated into human therapy. Since then, many other examples have been released, and several genome editing therapies are targeting human diseases that are currently undergoing clinical trials, and others are underway.

Gersbach’s latest research focuses on a mouse model of DMD, that is unable to produce dystrophin.

The genetic cause of DMD

Dystrophin is encoded by a gene containing 79 exons responsible for producing a protein, called dystrophin. If one or more exons are disrupted or removed by a genetic mutation, the chain is not built, causing the muscle to deteriorate slowly. Most patients use a wheelchair before the age of 10 and do not live in their twenties or early thirties.


Gersbach has been working on potential genetic treatments for Duchenne muscular dystrophy since 2009. His lab was one of the first to begin focusing on CRISPR/Cas9. Cas9. CRISPR/Cas9 is used to cut into the dystrophin gene around the exons responsible for the genetic mutation that causes DMD. Then, the body’s natural DNA repair system, picks up the previously cut gene, to create an abbreviated version of the dystrophin gene.


“As we continue to work to develop CRISPR-based genetic therapies, it is critical to test our assumptions and rigorously assess all aspects of this approach. A goal of our experiments was to test some ideas being discussed in the field, which will help us understand the potential of CRISPR to treat genetic diseases in general and Duchenne muscular dystrophy in particular. This includes monitoring the long-term durability of the response in the face of potential immune responses against the bacterial Cas9 protein.” – Charles Gersbach


“It is widely believed that gene editing leads to permanent gene correction. However, it is important to explore theoretical possibilities that could undermine the effects of gene editing, such as losing treated cells or an immune response.”  – Charles Gersbach


The purpose of this new study

The purpose of this new study is to explore the factors that may alter the long-term effects of CRISPR/Cas9-based gene editing.

A single dose of the CRISPR therapy was administered intravenously to adult mice and newborn mice carrying a defective dystrophin gene. The following year, the researchers measured how many muscle cells had been successfully edited and what types of genetic modifications had been made, as well as the possibility of an immune response against the bacterial CRISPR protein, Cas9, which acts as the “scissors” that cuts into the gene.

An immune reaction against CRISPR

Other studies have reported that the mouse immune system can mount a response to Cas9, which could potentially interfere with the benefit of CRISPR therapies. Several groups have also said that some people have preexisting immunity to Cas9 proteins.


“The good news is that even though we observed both antibody and T cell responses to Cas9, neither appeared to result in any toxicity in these mice. The response also did not prevent the therapy’s ability to edit the dystrophin gene and produce long-term protein expression successfully.” – Christopher Nelson, a post-doctoral fellow in Gersbach’s lab.


Some results suggest approaches to face potential challenges. When two-day-old mice without a fully developed immune system are treated intravenously, no immune response is detected. Genome editing by CRISPR has remained stable and, in some cases, even strengthened in one year. We could imagine that administering therapy to infants would be a method of circumventing an undesirable immune response.

The immune system of the mouse works differently from the human immune system. Screening for DMD in newborns is not yet widespread; most Duchenne diagnoses occur when children are three to five years old. The suppression of the immune system during treatment might be an approach.


“We were pleased to observe that all the mice were doing well a year after treatment, but our results show that there needs to be more focus on the immune response as we move toward larger animal models,” – Christopher Nelson


Some studies have shown that CRISPR can cut out genetic sections much more significant than intended or that pieces of DNA can embed at the site of the cut.

To exhaustively map all the changes in the dystrophin gene, Nelson used a DNA sequencing approach. Surprisingly, many types of modifications have been made in addition to the intended removal of the targeted exon, including a high level of DNA sequence insertion.


“None of these edits would necessarily be a cause for concern in this case because the dystrophin gene is already defective. That being said, any unintended results could potentially take away from the efficiency of the gene editing you are trying to achieve, which supports the importance of designing ways to identify and mitigate alternative edits in future studies objectively. Moving forward, this phenomenon needs to be monitored carefully and better understood. Methods that avoid these alternative edits and increase the frequency of the intended edit will be important to maximizing the potential of genome editing to treat disease.”- Christopher Nelson


An encouraging step in the fight against DMD

Duchenne muscular dystrophy (DMD) results from an error in the “writing” of the dystrophin gene. In children with DMD, the dystrophin gene is corrupt—it contains a genetic defect (mutation). The gene is so badly “written” that the cellular machinery cannot accurately “read” the genetic instructions to produce dystrophin—a protein that’s essential for proper neuromuscular function.

Researchers are using CRISPR/Cas9 to remove the corrupt part of the genes (mutations). “Erasing” problem areas on a DNA strand may restore accurate protein decoding. With this technology, DMD researchers have been able to restore the production of dystrophin.

Researchers have found a way to transport this molecular toolkit aboard viruses with a particular attraction to muscle cells. They injected this mixture into mice models that could not synthesize dystrophin. After a few weeks, the rodents’ muscles began to produce dystrophin.

A brilliant idea!





(Also known as study 041)

The clinical trial of ataluren in nonsense mutation Duchenne muscular dystrophy (NMDMD)

Study 041 is a new clinical trial for boys and young men who are living with nonsense mutation Duchenne Muscular Dystrophy (nmDMD).

Who Can Enter Study 041?

To ensure that participants who enroll in Study 041 get the most benefit, and to ensure their safety, there are strict criteria around who is able to enter the study. Your doctor will help to decide whether you/your child can take part.

Main Inclusion Criteria

  • Documentation proving that you have undertaken a genetic test which shows you/your child has nmDMD.
  • You/your child is male and five years of age or older.
  • You/your child is able to walk at least 150 meters (165 yards) without help during a six-minute walking test.
  • You/your child is able to undertake certain timed tests.
  • You/your child has been on corticosteroid treatment (i.e. prednisone, prednisolone, or deflazacort) for at least 12 months immediately before the start of study treatment, with no significant change in dose within three months before the start of the study (other than for body weight change).


Trial Design

Stage One

Stage One of Study 041 is looking at how the investigational treatment, ataluren, affects the ability to walk and endurance in males aged five years and older with nmDMD, compared to placebo (a substance that looks and tastes the same as ataluren but does not actually contain the drug). The study will also look at the ability of participants to climb up and down stairs, walk/run certain distances, use their upper limbs and measure lung function. The safety of ataluren will also be assessed.

Stage Two

Stage Two of Study 041 will compare results in participants who started on ataluren from the beginning of the trial with those who started taking ataluren from the start of stage two. All participants will be involved in both stages of the trial.

If you/your child decides to enroll in Study 041, you may help drive a better understanding of nmDMD, as well as current and future treatments, which may benefit nmDMD patients in the future.


Frequently asked questions

What is ataluren?

Ataluren is an investigational therapy being studied for the treatment of people with nmDMD who are still able to walk and aged five years or older. It is administered three times per day, mixed with a liquid or semi-solid food and taken orally. People with nmDMD are unable to produce a protein, dystrophin, which helps keep muscles healthy. Without dystrophin, muscles become weaker over time. PTC Therapeutics is studying ataluren to determine if it helps the body produce dystrophin, which may help slow down muscle weakening.

Is ataluren safe?

Ataluren has been studied in over 1,000 patients in clinical trials. During these clinical trials, ataluren was found to be well-tolerated in patients with nmDMD and has demonstrated a favourable safety profile for this condition.

Will I need to pay anything to be involved in the trial?

No, all costs involved in the trial, including physical examinations, screening, laboratory and other tests, as well as the cost of the drug, will be covered by PTC Therapeutics, who is funding Study 041.

You may be reimbursed for all reasonable*costs for travel, meals and accommodation necessary for your visits to the study site the clinic, which will be approximately every 12 weeks for the first stage of the trial, and every 24 weeks for the second stage of the trial.

How will ataluren affect other treatments that I am taking?

If you/your child are/is taking any medications, then the doctor will advise you/your child of potential problems and discuss treatment options with you/your child. However, to participate in the trial you/your child must be taking treatments known as corticosteroids, and ataluren can be taken alongside these medications.

What happens if/when ataluren is approved in my country? Can I leave the trial?

If you/your child enroll in Study 041, we would like a commitment that you/your child will follow through with the study obligations, unless a doctor determines you/your child should discontinue for health or safety reasons. However, participation in this study is entirely voluntary, and if you wish to do so, you/your child are able to withdraw from the study at any point for any reason, or no reason.

What do I have to do to be involved in Study 041?

If you/your child would like to participate in the trial, before being enrolled, you/your child will need to discuss the trial with your doctor, and agree (consent) to being involved. You/your child will be asked to sign a consent form(s). The consent form(s) provides detailed information, which will outline the risks and benefits of participating in the trial. If you/your child have/has any questions about this information, talk to your doctor as it is important that you are fully informed. The consent form(s) also explains the basic facts about the trial in age-appropriate language. When you/your child signs these forms you will be given copies to keep.

The next step involves some checks to ensure that you/your child is eligible to participate in the trial. These checks, known as screening procedures, will include tests that are timed to assess your/your child’s ability to run/walk 10 meters, climb four stairs, descend four stairs, and stand up from lying down on your back within 30 seconds. These checks will take place in the two weeks before your/your child’s trial starts. A genotyping test, to ensure that you/your child has a nonsense mutation of the dystrophin gene, will be administered at baseline, or the start of the trial.

What do I have to do during Study 041?

If you/your child are/is accepted into the trial, you will be randomly assigned to either the ataluren group or the placebo group.

Ataluren or placebo is taken through the mouth. Every day for the duration of the trial, you/your child will need to prepare and take the medication or placebo three times a day: in the morning, at midday and in the evening.

Ideally, there should be about six hours between morning and midday doses, about six hours between midday and evening doses, and about 12 hours between evening doses and the morning dose on the next day.


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New drug for DMD being reviewed by the FDA

Sarepta Announces FDA Acceptance of Golodirsen (SRP-4053) New Drug Application for Patients with Duchenne Muscular Dystrophy Amenable to Skipping Exon 53

Press release here:


Sarepta Therapeutics, Inc. announced the Food and Drug Administration, Division of Neurology had accepted its New Drug Application (NDA) seeking accelerated approval for golodirsen (SRP-4053) and provided a regulatory action date of August 19, 2019. Golodirsen is a phosphordiamidate morpholino oligomer* engineered to treat those individuals with Duchenne muscular dystrophy (DMD) who have genetic mutations subject to skipping exon 53 of the dystrophin gene.

The company completed its NDA at the end of 2018 as part of a rolling submission and requested priority review, which was granted. The company previously received orphan drug designation for golodirsen.

The study demonstrated statistically significant results in favour of golodirsen on all biological endpoints.

*a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. 


Doug Ingram, president and chief executive officer, Sarepta: “If approved, golodirsen will serve up to another 8 percent of the Duchenne community, bringing us closer to helping as many Duchenne patients as possible.

We look forward to working with the FDA toward advancing this important therapy and rapidly bringing it to individuals with Duchenne who are amenable to exon 53 skipping.”


What is Golodirsen?

Golodirsen uses exon-skipping technology and works by binding to exon 53 of the dystrophin sequence to exclude, or skip, this part of the sequence. Exon skipping is intended to allow for the production of an internally truncated but functional dystrophin protein.

Positive results

Golodirsen showed potential to treat Duchenne muscular dystrophy (DMD) in a first clinical trial of DMD patients. Press release

Why do we need to skip an exon?

DMD is caused by specific errors (mutations) in the gene that codes for dystrophin. Dystrophin is a protein that plays a crucial role in the function of muscle cells and protects them from damage as muscles contract and relaxes. These mutations in the dystrophin gene lead to a lack of dystrophin protein in muscles. Without enough dystrophin, muscles gradually grow weaker until they can’t move at all, and eventually breathing and heart function are lost.

The condition is universally fatal. Death usually occurs before the age of 30 generally due to respiratory or cardiac failure.



The zebra-striped ribbon as a symbol for rare diseases

The zebra is used as a symbol for rare diseases since about 1940. 

This comes from a quote by Dr. Theodore Woodward: “When you hear hoofbeats, think horses, not zebras.” and “When you hear hoofbeats behind you, don’t expect to see a zebra.”

This is the metaphor Dr. Woodward used to teach students basic concepts about the diagnosis of disease: when examining a patient’s symptoms, it’s better to think of a horse rather than a zebra. It’s a fact that horses are hoofed animals more commonly encountered than zebras, so you should automatically assume that if you hear the sound of hooves, it should be a horse, not a zebra, right?

The national awareness day is on February 29, a date that’s only on the calendar every four years. (It’s moved to February 28 on non-leap years.)

The day was started by the European Organisation for Rare Diseases and is now recognized globally. The symbol for rare disease awareness is a zebra-striped ribbon.

Statistics and facts

Here are a few statistics and facts to illustrate the breadth of the rare disease problem worldwide.

  • There are approximately 7,000 rare diseases and disorders, with more being discovered each day.
  • It is estimated that 350 million people worldwide suffer from rare diseases
  • If all of the people with rare diseases lived in one country, it would be the world’s 3rd most populous country. In Quebec, it is estimating that close to one in 20 people will be affected or have a rare disease, which means nearly 500,000 Quebeckers.
  • 80% of rare diseases are genetic in origin, and thus are present throughout a person’s life, even if symptoms do not immediately appear.
    • It can take several years to diagnose a rare disease. Many rare diseases have nonspecific symptoms such as pain, weakness, and dizziness, which can make them hard to diagnose.
    • Rare diseases can also be hard to diagnose because they’re unusual. Your doctor may never have seen a similar case and may not even realize a specific disease exists.
    • Besides, it could take weeks or months for you to get an appointment with a specialist. Then, if that specialist was not the right one, you might wait months before seeing the next one. Patients with rare diseases visit more than seven doctors on average before receiving an accurate diagnosis, according to a 2013 study published in the Journal of Rare Disorders.
  • Approximately 50% of the people affected by rare diseases are children.
  • 30% of children with rare disease will not live to see their 5th birthday
  • Rare diseases are responsible for 35% of deaths in the first year of life.
    • Newborn screening for rare diseases is recommended. Screening requirements for newborns vary by country, but they’re increasingly becoming routine.
    • Even without a cure for a particular condition, early diagnosis is essential to prevent death or disability and to help children reach their full potential.
    • Genetic testing can help diagnose many rare diseases, but not all. Genetic testing identifies a genetic cause in an estimated 25 percent to 30 percent of cases.
  • Only 5 percent of rare diseases have treatments. Drug research that helps a limited number of people can be cost-prohibitive for pharmaceutical companies. 



Finding a support group is important

A rare disease can be isolating for the patient as well as for the caregiver, especially when it’s your child who has the condition.

Connecting with others can be essential, not only for support but also to share information and resources.


Sources and interesting links