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Therapies for Muscular Dystrophy: Stem Cells and CRISPR-Cas9 [Part Two]

Editor: This article is the second of a two-part series. Check out Part One here.

Muscular dystrophy, as its name suggests, refers to a range of genetically inherited diseases which affect our muscles. It is characterised by the progressive weakening of muscles and muscle wasting (i.e. the reduction in muscle mass due to malnourishment and insufficient physical activity).

In this article, we are going to explore the different types of muscular dystrophies, placing a greater focus on discussing the life-threatening characteristics and therapeutic complications of Duchenne Muscular Dystrophy (DMD) specifically, for it is by far the most well-studied and understood form of muscular dystrophy. On top of that, we shall take a look at possible novel therapies using CRISPR-Cas9 gene editing techniques.

Muscular dystrophies - there are too many variations!

The age at which people start exhibiting symptoms varies depending on the disease type. In fact, there may be no noticeable age trend for certain diseases!

For example, individuals affected by Duchenne Muscular Dystrophy (DMD) could display symptoms from an early age (e.g. between two to six years old), whereas individuals affected by facioscapulohumeral muscular dystrophy (FSHD) may start to present symptoms by the time they enter the 20- to 30-year-old age group. What’s more, even the types of muscles affected can differ between the variants of muscular dystrophies (take a look at Table 1).

Considering there are approximately seven billion people in the world, muscular dystrophies are classified as rare diseases due to its relatively low disease prevalence. In the UK, about 70,000 of the citizens are living with some form of muscular dystrophy. Whilst in the US, it has been estimated that one in every 5,600 to 7,700 male children would be affected by the two most common types of muscular dystrophies, Duchenne and Becker.

As is the case with any other rare diseases, clinicians would not come across these medical conditions very often, and so they tend to have a difficult time with the diagnostic process. To further complicate matters, the severity of the disease and range of muscles affected vary from one individual to the next!

Since diagnosing muscular dystrophies is difficult as its symptoms are challenging to be characterised, it will be tough for us to decipher the root cause of the disease as part of developing therapies.

Let’s take a look at Table 1, where it summarises a few different types of muscular dystrophies and their characteristics.

Table 1: The features of common muscular dystrophies. The age of disease onset varies from one type to the next, and even within the same type of muscular dystrophy. The commonly affected muscles are also summarised. Usually at the beginning of the disease, only one set of muscles is affected. Taking FSHD as an example, the facial weakening of facial muscles is the first symptom, and an increasing number of muscles may be affected as the disease progresses. Meanwhile, the genes responsible for muscular dystrophies are usually different. Nonetheless, dystrophin is the only gene known so far that has the ability to cause two different types of muscular dystrophies. BMD and DMD are examples of this, though BMD is sometimes referred to as a less severe form of DMD since there are lower amounts of functional dystrophin protein expressed in BMD (leading to milder symptoms) compared to DMD. Besides that, multiple genes can contribute to pathology of a muscular dystrophy as is the case for EMD. *At the point of writing, the mutation in DUX4 is believed to be responsible for FSHD. However, other genes could potentially be involved too. This table is modified from Lovering et al (2005).

Introducing Duchenne muscular dystrophy (DMD)

In the previous article, we talked about skeletal muscle stem cells and the environment they reside in, and how each of the surrounding cells have a part to play to maintain the healthy function of skeletal muscles.

Since each type of muscular dystrophy has its own unique characteristics in terms of genetics, biological pathways that get disrupted, and even symptoms, this article will focus on the most common and extensively studied muscular dystrophy - Duchenne muscular dystrophy (DMD).

First question - what exactly is DMD?

DMD is a muscular dystrophy that leads to progressive wasting of muscular tissues – particularly skeletal and heart muscles. The age of the disease onset is quite early (ranging between childhood and early teenage years). Though in most cases, the symptoms DMD are found to manifest during a person’s early childhood.

The early signs of DMD that we ought to look out for include a child’s delayed ability to sit, stand (using what is known as the Gower’s sign), walk (look out for enlarged calf muscles), and impairment in speech. Essentially, these children are not able to carry out activities requiring balance, and by 12 years old, they would usually have become wheelchair-bound. Furthermore, spinal deformity or scoliosis may also develop, and patients could also display intellectual disabilities that are independent of the degree of muscle weakness.

As the disease progresses, serious life-threatening conditions may surface due to the weakening of heart (cardiomyopathy) and lung muscles, causing cardiovascular and breathing complications.

Delving into the genetics of DMD

DMD is an X-linked recessive disorder, which means males would typically be the ones inheriting this disease (whereas this disease tends to be very rare amongst females).

You may have come across X-linked disorders in your A Levels. But if that is not the case, here is a simple explanation.

There are two key facts that you need to first understand. Number one, recessive genetic disorders require two copies of the mutated alleles (i.e. a variant of a gene) in order to cause a disease. This means that if one allele is mutated and the other copy is not, a functional protein will still be produced and the person will not have the genetic disease.

Number two, DMD is a gene that can only be found on the X chromosome. Naturally, females would carry two copies of the dmd gene since they have two X chromosomes. While in contrast, males carry only one X chromosome, so if the dmd gene that they inherit turns out to be mutated, they will display the disease phenotype.

As a result, due to the lack of a second DMD allele, X-linked disorders tend to affect males in a much higher frequency than females.

What does the dmd gene do anyway?

Here’s a fun fact - the dmd gene is 2.4 million base pairs in size!

It’s job is to basically code for a protein known as dystrophin, which is a cytoskeletal protein. This means that it is a protein that forms part of the cytoskeleton of a cell’s structure. And in the dystrophin’s case, this protein is found in the intracellular region of the muscle cell membrane (called the sarcolemma), where it associates with a number of other proteins to form the dystrophin-glycoprotein complex (DGC) (Figure 1).

DGC is important for providing both structural integrity and the stabilization of muscle fibres during muscular contractions. The loss of the dystrophin gene thereby causes the sarcolemma integrity to be compromised. As a result, muscular contraction actually ends up damaging the muscle fibres, leading to the release of intracellular substances.

And to this date, around 7,000 different mutations have been identified within the dmd gene.

Figure 1: A schematic of location of dystrophin protein. The extremely large protein is associated with other cytoskeletal proteins, such as actin, at one end. Actin, the most abundant protein in eukaryotic cells, is a cytoskeletal protein which is responsible for cellular shape and mobility. Meanwhile, dystrophin interacts with other proteins that are present at the sarcolemma (i.e. the muscle cell membrane) to form the dystrophin-glycoprotein complex (DGC). Moreover, the basal lamina is the extracellular matrix surrounding the muscle fibre, with the DGC essentially connecting the myofiber to the basal lamina. This figure is modified from Davies and Nowak (2006)’s Molecular mechanisms of muscular dystrophies: old and new players.

DMD therapy - which one will prevail?

Since DMD is caused by the loss of functional dystrophin in the cell, the whole idea of all the available therapies is to orchestrate a way to reintroduce functional form of dystrophin. Currently, there are two different methods that seem promising.

Stem cell therapy

Stem cell therapy refers to delivery of satellite cells (muscle stem cells, in other words) without causing any genetic mutations to the skeletal muscles, so that they would differentiate and give rise to non-dystrophic (i.e. healthy) myofibers and functional skeletal muscles.

The principle behind this is that muscular dystrophies are genetic disorders, so the satellite cell population present will also have genetic abnormalities.

So, it is essentially a cell transplantation method. Successful transplantation of satellite cells have been done in mdx mice. Mdx mice are model organisms for studying DMD. In particular, they have a mutation in the dystrophin gene, and this means that they are unable to express the dystrophin protein. In the transplantation experiments mentioned above, the researchers managed to present results showing that the “healthy” satellite cells introduced into mice subjects having successfully produced non-dystrophic muscle fibres.

This is good news, but there are problems associated with the process of transplanting satellite cells into human subjects.

Firstly, this is an invasive process as skeletal muscle sections will have to be extracted from the patient via a process known as muscle biopsy. Meanwhile, it is difficult to isolate a large number of satellite cells. In fact, for every 50 to 100 mg of skeletal muscle that is extracted, only 500 to 1000 cells are found per cubic millimetre. This might look like a lot at first glance. However, these cells are not homogenous, meaning that not all extracted cells will be satellite cells. Thus, at the end of the day, there might not be much satellite cells available for transplantation.

To further complicate things, recent experiments demonstrated that there are actually many different types of satellite cell populations, whereby each population expresses a different marker, so these different satellite cell populations may have different roles in our body. Not to mention, researchers have yet to identify which particular type of satellite cell population is the most appropriate variant for transplantation.

To tackle this particular issue, induced pluripotent stem cells (iPSCs) can be used, whereby iPSCs are usually somatic (i.e. body cells) which can be reprogrammed using transcription factors to create “reprogrammed” cells. These “reprogrammed” cells would then have the ability to give rise to most cell types within the body. Transcription factors are proteins which recognise specific DNA sequences and can regulate the expression of genes by altering the rate of transcription. And in the case of iPSCs, certain transcription factors can be used to alter the gene expression within a cell, so that the cell can be switched from one type into another. For example, the mouse fibroblast cells can be reprogrammed into muscle progenitor cells.

To shift gears slightly, another major hurdle that we face is that satellite cells transplanted from other individuals will be recognised as “foreign” objects in the patient. This therefore triggers an immune response. So, one way to overcome this problem would be to use the patient’s own cells. But, as mentioned previously, the patient would be carrying the dmd mutation in all of their body cells. For that reason, CRISPR-Cas9 was brought into the picture, in which the whole idea is to extract satellite cells or somatic cells from the patient, edit the mutated dmd gene using CRISPR-Cas9, and then transplant the edited cells back into the patient.

The various different ways of utilising stem cell therapy are summarised in Figure 2. Additionally, you can read more about the methods and challenges associated with stem cell therapy to treat DMD here.

Figure 2: Diagram showing the two possible ways of using stem cell therapy to treat patients suffering from DMD. Firstly, satellite cells can be taken from unaffected individuals. However, since the presence of foreign cells can trigger an immune response (rejection) in the patient, another treatment method would be to obtain satellite or somatic cells from the patients themselves. As the patient’s dmd gene is mutated, the CRISPR-Cas9 technique can be employed to correct the mutation before reintroducing the edited cells back into the patient.


Since the discovery of CRISPR-Cas9 in the bacterial genome, this revolutionary system has been largely adapted as a gene editing tool.

Essentially, CRISPR-Cas9 gene editing requires two things to be delivered to the nucleus (Figure 3):

  1. The Cas9-nuclease, which cuts the DNA

  2. The single-strand guide RNA (sgRNA), which as its name suggests, guides the nuclease to the region of DNA where it should be cut (for example, a gene of our interest)

Once the DNA is cut, bacterial and mammalian cells have an endogenous system in place to repair the DNA. But, this repair can be error-prone and so the original sequence of our gene of interest may be altered. As a result, our gene of interest can no longer code for a functional protein.

Researchers have generally been very creative with the use of CRISPR-Cas9 - when we bring DMD into the picture, CRISPR-Cas9 has been used to modify portions of genes, as demonstrated in various DMD mice models.

Figure 3: Schematic of CRISPR-Cas9 system for gene editing. The single-stranded guide RNA (sgRNA) contains a sequence (dark green) that is complementary to a region within the target gene (light green). Upon interaction, the Cas9 nuclease cleaves the DNA within this complementary region. To better understand the process see this insightful video. This image has been modified from Wikimedia Commons.

Both of these techniques, stem cell therapy and CRISPR-Cas9, appear to be super promising therapeutic options for DMD. But, we have to remember that they have yet to be tested in humans!

So, the research process is still ongoing.

What about treating other types of muscular dystrophies?

The research progress for other types of muscular dystrophies is generally lagging behind DMD’s. In general, the causes behind their pathologies are not entirely clear, though it is more likely that the disruption of numerous biochemical pathways are contributing to the onset of these diseases.

Nevertheless, there is the possibility of developing a unique gene therapy for these variants of muscular dystrophy. For instance, the use of stem cell therapy (as mentioned above) can be used to treat some (if not all) other forms of muscular dystrophies.

Furthermore, CRISPR-Cas9 can be used to target muscular dystrophies that are caused by mutation in a single gene. An example of this would be FSHD, which occurs due to the abnormal expression of the dux4 gene. Essentially, since the dux4 gene is not expressed in healthy skeletal muscle cells, we can technically use the CRISPR-Cas9 system to edit the dux4 gene out of the patients.

However, in other cases of muscular dystrophy, there is still a possibility of other genes contributing to the disease pathology. And in those cases, it would be difficult for CRISPR-Cas9 to effectively edit numerous genes at once.

Based on the type of muscular dystrophy, it is entirely possible to find multiple biological pathways impaired in just a single disease. Thus, further research is required for us to be able to design drugs targeting multiple molecular pathways. Though, as is the problem with any other disease, we cannot cure it until we finally determine the underlying cause.

The therapeutic prospects for DMD

Currently, there is no set “cure” for DMD. Instead, treatments are focused on managing its clinical symptoms. Because of the wide range of clinical complications that DMD presents, managing symptoms are neither easy or cheap.

Medical specialists in different fields have to be involved. These include cardiologists, physiotherapists, neurologists, physiotherapists, and even geneticists.

On the other hand, patients have to be under constant supervision as the disease progresses. In severe cases, the use of specialised equipment such as ventilators may be required to assist breathing. The problem is - not all healthcare services across the world are equipped to offer such specialised treatment. Besides that, the psychological toll this illness exerts on both the patient and their families cannot be overlooked.

To end things on a positive note

There is clearly a desperate need for effective therapies to treat DMD. Fortunately, there has been an increased interest in this field.

As a matter of fact, there are numerous funding bodies and organisations established with the aim of encouraging muscular dystrophy research. On the other side, pharmaceutical companies are also looking to invest in the research and development (R&D) of drugs holding the potential to tackle muscular dystrophies. These efforts, in turn, help to generate awareness within our society regarding the impact of muscular dystrophies and why we need to take action today.

If you want to look further into some of the organisations and how they are planning to help, do take a look at Muscular Dystrophy UK and MDA (based in the US).

Author: Israt Jahan, BSc Biotechnology


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