Category: Muscular Dystrophy

Rationale

A muscular dystrophy is a group of diseases that cause progressive weakness and loss of muscle mass. It is a genetic X-linked recessive inherited disorder which primally effects males. It is passed down through the mother who is a carrier of the gene. Becker Muscular Dystrophy (BMD) is one of the nine different types of Muscular Dystrophy. There is no cure.

Muscular Dystrophy is a mutation of one or more genes which interfere with the production of proteins called dystrophin. Dystrophin is required for normal muscle function (Becker MD, 2019), and it is mostly found in skeletal muscle used for movement. The symptoms of a patient with BMD are progressive muscle weakness of the leg and pelvis. Muscle weakness becomes apparent later in childhood or adolesce, and eventually loss of ambulation (Becker Muscular Dystrophy, 2011). Loss of ambulation means a person is no longer able to walking around, and often people with Becker Muscular Dystrophy will require a wheelchair.

Clinical trials using gene therapy are exploring ways to find a cure or reduce the symptoms of Muscular Dystrophy. Due to muscle weakness of the leg and pelvis being the main characteristics of BMD, a strategy for improving ambulation in BMD patients is to boost muscle mass growth in the legs. One possible this could be done using somatic gene therapy to introduce a protein call Follistatin directly into the muscle. Follistatin is a protein that can increase muscle mass by suppressing myostatin, the body’s muscle regulator. The Follistatin protein is injected into the weakened muscle using a virus so that it binds to the muscle. The hope is that the muscle will build and strengthen for the BMD patient. Unfortunately, because this approach is not germline gene treatment, meaning it does not target any of the reproductive genes, this type of therapy will only help the patient and not their bloodline descendants.

This report will investigate whether Follistatin gene therapy can prolong the ambulation of a male with Becker Muscular dystrophy.

Research Question: Can somatic gene therapy prolong ambulation of a male with Becker Muscular Dystrophy?

Sources

Source 1: Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model

• Rodino-Klapac, L. R., Janssen, P. M. L., & Shontz, K. M. et al (2013). Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model. Human Molecular Genetics, 22(24), 4929–4937. doi: 10.1093/hmg/ddt342

Source 2: Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates

• Kota, J., & Handy, C. R. (2009). Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates. Science Translational Medicine, 1(6). doi: 10.1126/scitranslmed.3000112

Source 3: A Phase 1/2a Follistatin Gene Therapy Trial for Becker Muscular Dystrophy

• Mendell, J. R. (2015). A Phase 1/2a Follistatin Gene Therapy Trial for Becker Muscular Dystrophy. Molecular Therapy, 23(1), 192–201. doi: 10.1038/mt.2014.200

Analysis and Interpretation of the evidence in the sources

Source 1

This study was conducted in dystrophic mice. This means that the mice are lacking in the protein dystorphin, the same as a patient with Muscular Dystrophy. Two approaches were examined to determine which somatic gene therapy method is the most effective:

• Micro-dystrophin, the introduction of a small amount of the dystrophin protein to inhibit muscle loss, or
• Follistatin, the introduction of the protein to build muscle.

Both approaches were delivered by a virus by administering an injection into the muscle at the front on the leg. When the mice reached 1 year of age, the muscle injected with the protein was isolated from each mouse. Muscle physiology measurements were the primary outcome.

• C57 = normal field mouse with no muscular dystrophy
• Mdx = mouse with Muscular Dystrophy gene
• µ-Dys = Mouse with micro-dystrophin gene therapy
• FS = Mdx mouse with Follistatin gene therapIn

The graphs below the mice that have been treated with either type of gene therapy show improvements when compared to mice with the muscular dystrophy gene. Not surprisingly, the muscle performance of a healthy mouse (C57) is still better. However, positive improvements in Mdx mice that had gene therapy include improvement in tetanic force which means the muscle can hold a contraction without relaxing. The trials also show the improvement in fast and slow twitch muscle which are important because it looks at how the muscle use the energy (i.e. Fast twitch muscle – sudden bursts of energy; Slow twitch muscles – support long durance activities)

Although these studies show the positive impacts of gene therapy improving muscular function for mice with muscular dystrophy, it is not known if these results could be replicated in larger muscles. It is also questioned if trials on mice have the same outcome in comparison to a human with a different DNA structure and chromosome orders.

This clinical trial is effective because it has used mice that are missing the dystrophin protein, the same as a person with Becker Muscular Dystrophy. This replicates muscle deterioration, and the trial provides evidence that Follistatin does build and strengthen muscles and is therefore an effective gene therapy solution.

Source 2

Building on the evidence from Source 1, the effects of Follistatin gene therapy are explored further in this source. The trials are performed on cynomolgus macaques – monkeys – and inject Follistatin using a virus directly into the quadricep muscle using a specific (MCK) and nonspecific (CMV) promotor. It is important to highlight that this research targets the quadricep muscle because sever weakness of this muscle is a defining feature of Becker Muscular Dystrophy. This research is also significant for future human trials because testing also included the overall health of the monkey, which after 15 months did not reveal any organ damage.

The results of this trial are shown below. All monkeys saw an increase in the size of their quadricep. The muscle grew quickly in the first 8 weeks, and then appeared to slow between weeks 8 to 12, with sustained or slowed growth after week 12.

Only half of the monkey were tested for increase to muscle strength. It is not evident from this small sample size (3 out of 6 monkeys) to draw conclusions regarding trends. However, what can be seen is that all monkey have improvements in twitch and tetanic force. This supports the claim there is a relationship between muscle size and muscle strength. Interestingly, the biggest improvement was observed in Monkey 2 that had the weakest muscle strength in the controlled leg.

Table 1: Strength measurements of treated quadriceps in macaques (measured in newtons N)

Twitch Force

Tetanic Force

Control Leg

Injected Leg

Difference

Control Leg

Injected Leg

Difference

Monkey 1

17.0

19.0

11.8%

65.0

73.0

12.3%

Monkey 2

4.2

5.7

35.7%

24.0

42.0

77.9%

Monkey 3

19.0

24.0

26.3%

64.0

72.0

12.5%

The limitation of this research is that although there was a control group used in this trial, the monkeys did not have a gene disorder where they were missing dystrophin which leads to degeneration of muscles like muscular dystrophy.

Overall, this study demonstrates that Follistatin gene therapy is successful in muscle growth and strengthening. The benefit of no organ damaged being found in any of the monkeys as a result of this research means that it Follistatin gene therapy could be tested in humans.

Source 3

This source specifically looks at Becker Muscular Dystrophy and the loss of movement that is seen in this condition. It is the first human trial of Follistatin gene therapy. There are two groups, shown in Table 1 – low dose and high dose group. Patients in the low dose group were observed for one year, and the patients in the high dose were observed for 6 months. All patients were ambulatory which allowed the six-minute walking test (6MWT) to measure the effectiveness of the observation period.

Table1: Characteristics of Becker Muscular Dystrophy patients enrolled in trial

Cohort

Patient ID

Age (years)

Gene Mutation

1 – Low dose

1

30

del exon 48 – 49

2

35

point mutation exon 8

3

37

del exon 45 – 48

2 – High does

4

34

del exon 45 – 48

5

24

del exon 45 – 47

6

30

del exon 13

The below charts show an uphill trend in most of the patients over the observation period, however the success did vary. Two patients – one from each group – experienced significant improvement (patient 02, 125m; and patient 05, 108m). One patient experienced a negative improvement (patient 04, -14m). Of the two patients that had significant improvement (patient 02 and 05) it is worth highlighting that age does not appear to have a relationship with improvement with the patients being 24 years and 35 years old respectively.

There is no evidence to suggest that the high dose was the reason for the better results in the 6MWT or why if a patient reached milestones quicker. It appears that improvements gained in the first six months do not continue at the same rate and are slowed (or even stopped) as the 12-month period approaches. It is encouraging to note that (other than in patient 4) improvements do not decline.

In the low-dose group (patients 01, 02 & 03) whose observation period was for 12 months the trend from 6 months to 12 months remains fairly flat.

Testing of patients throughout the trial did not show any health concerns to warrant not continuing this type of gene therapy.

What these graphs do not show is the type of gene mutation that each patient has (Table 1). If you consider the minimal success of patients 03 and 04 in this trial, they share a common gene mutation. This could be a relevant factor impacting the success of the Follistatin gene therapy trial.

There are also no patients that were already in a wheelchair, or not ambulant, in the trial. It would be interesting to see if Follistatin gene therapy could take a BMD patient from a wheelchair to walking a small distance.

Considering this is the first human clinical trial of Follistatin gene therapy, and two of the three patients in each group improved in their 6MWT, this is inspiring research and provides evidence that injection of the quadriceps muscles does improve muscle strength.

Conclusion

In conclusion, the results of the three clinical trials suggest that somatic gene therapy using the Follistatin protein injected directly to the quadriceps muscle can improve the ambulation of a male with Becker Muscular Dystrophy. Effects of the therapy may vary from patient to patient, and take several months to be realized, however based on the evidence presented here it is realistic to expect that this muscle therapy can improve leg strength and therefore ambulation. There is no evidence to suggest that a patient with no ambulation will benefit from this type of therapy.

Evaluation of Quality of Evidence

These sources are very reliable as they are printed by the US National Library of Medicine National Institutes of Health, a very well-known publishing press. The represent actual clinical trials that were conducted in America and have been reviewed by many different scientists. These sources were written to provide insight and detail into how Follistatin gene therapy can help enhance muscle growth. The source shows that the statement ‘Follistatin gene delivery enhances muscle growth and strength in nonhuman primates’ is true as it provides evidence and test results to give reasoning which is why this source is reliable.

These sources are all trials that have been conducted in the last 20 years, and build on the learnings from the previous clinical trial. The clinical trials relate directly to the research question.

Extrapolation of findings

This research demonstrates that gene therapy using Follistin can improve muscle size and strength, which leads to improved ambulation in the majority of patients. Because this research looked at rebuilding muscles it could be extended beyond just Becker’s Muscular Dystrophy to all types of Muscular Dystrophy, or perhaps anyone with muscle degeneration.

It could also be expected, that females would have a similar response to the gene therapy. The trial with the monkeys included some female monkeys and there were no health impacts so it is likely to be safe in clinical trials were to include women.

The research question only includes the Becker’s Muscular Dystrophy in males when it could also be extended to females as well

Improvements and extensions

Some improvements that could be made to this investigation would be to explore how much further a patient that received Follistatin protein in both left and right quadricep could walk. It would also be interesting to test how the muscles that have stopped working respond to this type of therapy. Is it possible for the twitch and tetanic force to return to muscles that no longer work.

It would also be beneficial to know how the patients progressed after the clinical trial. Did they require any further injections or did the one injection last them a lifetime? This would provide detail towards the side effects of the therapy.

Reference List

1. Amthor, H., Macharia, R., Navarrete, R., Schuelke, M., Brown, S. C., Otto, A., … Patel, K. (2007). Lack of myostatin results in excessive muscle growth but impaired force generation. Proceedings of the National Academy of Sciences, 104(6), 1835-1840. doi:10.1073/pnas.0604893104
2. Becker MD. (2019, September 1). Retrieved from https://www.mda.org.au/disorders/others/bmd/
3. Becker muscular dystrophy: Medlineplus medical encyclopedia. (n.d.). Retrieved from https://medlineplus.gov/ency/article/000706.htm
4. Becker muscular dystrophy. (2011). Retrieved from https://ssl.adam.com/content.aspx?productId=117&pid=1&gid=000706&site=ssfhs-mychart.adam.com&login=SSFH6566
5. Follistatin gene transfer to patients with becker muscular dystrophy and sporadic inclusion body myositis – full text view – clinicaltrials.gov. (n.d.). Retrieved from https://www.clinicaltrials.gov/ct2/show/NCT0151934 9
6. Kota, J., & Handy, C. R. (2009). Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates. Science Translational Medicine, 1(6). doi: 10.1126/scitranslmed.3000112
7. Mendell, J. R. (2015). A Phase 1/2a Follistatin Gene Therapy Trial for Becker Muscular Dystrophy. Molecular Therapy, 23(1), 192–201. doi: 10.1038/mt.2014.200
8. Rodino-Klapac, L. R., Janssen, P. M. L., & Shontz, K. M. et al (2013). Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model. Human Molecular Genetics, 22(24), 4929–4937. doi: 10.1093/hmg/ddt342
9. T., Ross. (2019, March 7). Myostatin Inhibitors – Do They Work? – Is There Another Way to Do It? Retrieved from https://www.researchedsupplements.com/myostatin-inhibitors

Introduction:

An ongoing investigation has indicated that CRISPR can be utilized as a generative method that can treat Duchenne muscular dystrophy. Because of an examination in mice, it could be created as a remedial choice for humans Duchenne muscular dystrophy is caused by a defective gene for dystrophin. Duchenne muscular dystrophy occurs in about 1 out of every 3,600 males commonly between 3 to 6 years. As this is an inherited disorder, risks include a family history of Duchenne muscular dystrophy.

In 2016, Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke, incorporated one of the first successful uses of CRISPR to treat an animal model of the genetic disease with a technique that can potentially be used for human treatment. Gersbach effectively showed in a mouse model that CRISPR can recover muscle suffering the Duchenne muscular dystrophy (DMD). His methodology utilizes CRISPR to cut out dystrophin exons around the genetic mutation, leaving the body’s normal DNA fix framework to repair the remaining gene back together to make a shortened, however useful adaptation of the dystrophin gene.

Other studies have reported that the immune system within mice can stimulate a response to CRISPR, which could possibly intervene with the benefit of CRISPR therapies within the human body. Multiple groups have also reported that some people have developed immunity to CRISPR proteins, likely due to previous exposure to the bacterial host, however, where this would occur is unknown.

Recent years have witnessed the development of tools which actively assist researchers in performing CRISPR-Cas9 experiment optimally. These tools specifically aim to maximize on-target while also minimizing potential off-target effects by analysing the features of the target site, within the Dystrophin gene, causing the inherent disorder.

About the biotechnology:

CRISPR (clusters of regularly interspaced short palindromic repeats) is a simple however powerful tool which is able to edit genomes. It has recently allowed researchers to alter DNA sequences and modify gene function. CRISPRs are specialized stretches of DNA with two major characteristics: the presence of nucleotide repeats and spacers.

The CRISPR model allows for targeted genome editing of DNA sequences and strands. The biotechnology is focused to the DNA through contact with a guide RNA (gRNA) molecule, which then binds to the targeted DNA block through base complementarity and then allows precise DNA cleavage. This cleavage is then repaired through multiple pathways, which can be modified to the required outcomes.

Forcing a gene to be inoperative (knockout) can then be attained via error-prone repair through the Non-homologous end Joining pathway, which has the ability to introduce mutations and disrupt gene function. Targeted incorporated of a sequence can be achieved via the Homology Directed Repair pathway, which uses a provided DNA template to repair the cleavage of the Dystrophin gene.

Activation or repression of a gene can be accomplished through focusing catalytically inert Cas9 combined to a transcription activator or repressor to the promoter. All of these methods require the precise and effective focusing of the CRISR framework to the ideal location. The achievement of an analysis utilizing the CRISPR framework consequently depends on the right recognizable proof of the ideal objective site and ensuing structure of the complimentary gRNA).

Contributions of the use of biotechnology (how could it be applied in the context of your choice and comparison with current methods and the effect of biodiversity)

Steroid medications can slow the loss muscle strength, however, there is yet no cure of Duchenne muscular dystrophy. Treatment intends to control symptoms to improve quality of life. Other treatments include medications which include Corticosteroids, such as prednisone and deflazacort, which can help muscle strength and delay the progression of certain types of muscular dystrophy.

Many forms of therapy and assistive devices can improve the quality and in some cases the lifetime of individuals diagnosed with muscular dystrophy, examples include, range-of-motion and stretching exercises. Muscular dystrophy can restrict the flexibility and mobility of joints, these Limbs often draw inward and become fixed in that position. Range-of-motion exercises can help to keep joints. However, unlike a CRISPR used approach for treatment there is no guaranteed results and many methods have side effect symptoms.

There have also been past gene-editing technologies, for example, gene replacement. Gene replacement therapy is the technique of recognizing a faulty gene, then applying a piece of DNA in its correct form though a viral vector to the gene, in turn being able to override the identified faulty gene with the now correct copy. Gene replacement seeks to alter genes to correct genetic defects and thus prevent or cure genetic diseases. Three various gene therapy methods can be utilized to restore dystrophin expression. These are gene repair, exon skipping, and dystrophin gene replacement.

What was previously attempted with gene editing was to manipulate genetic information in blocks, basically in big pieces. The power of CRISPR technology. Thus, the precision of CRISPR is greater than other biotechnologies. This allows scientist to make changes in DNA of actual genes where they can “Turn off” harmful genes or they can potentially repair genes that have got mutations within them where the code is written incorrectly.

A successful gRNA must maximize on-target activity while also minimizing potential off-target effects. Adjusting both requirements can be a combinatorial task and thus, a significant effort in the ongoing years have been aimed at creating computational tools to aid in the design of gRNAs. These tools are aimed to help researchers in the selection of desired target sites by helping them exclude unwanted targets based on anticipated low productivity or a high potential for off-target impacts.

Researchers have recently successfully demonstrated in a mouse model, how CRISPR can regenerate muscle suffering from Duchene muscular dystrophy. To treat muscular dystrophy, a viral delivery system could provide patient cells with the instructions to make the Cas9 protein, as well as the RNAs that target specific regions of DNA. This has led to the possibility of CRISPR to treat the large population with the condition.

Risks and benefits (consider bioethics, and social impacts)

CRISPR can be an extremely effective tool in editing genes and to potentially treat Duchene muscular dystrophy. However, it still must be confirmed as an effective technique. This has caused various researchers to strive for improvements in this area, to make the process increasingly precise and effective, for future potential benefits.

CRISPR technology is also simple and cost-efficient unlike other gene-editing techniques. This technology can also be used to analyse the interaction of genes and the relationship between genetic differences and expression (phenotype). If CRISPR is successful in treating Duchene muscular dystrophy, it will inevitably lead to a significant decrease in the disease, as it is inherited. This will in turn lead to a significant decrease in investment put into research to find a cure for the disease, thus providing more funding into other illnesses.

Within previous months, Increased concerns have been raised regarding CRISPR. Various studies have suggested that CRISPR technology being used on humans could possibly cause cells within human bodies to lose their cancer-fighting ability. Thus, doing more damage to genes than originally thought. This raises many concerns about whether CRISPR should be used in humans as though it may cure Muscular Duchenne Dystrophy for children affected at young ages it may cause further health issues throughout their life which may be more life-threatening than Duchenne Muscular Dystrophy.

Future directions of the biotechnology

The currently available tools of CRISPR cannot just simply predict the success of an experiment as prediction accuracy depends on various factors including the accuracy of the approximations of tested models and how similar the experimental setup matches the data the model was based off of. Some patients were reported to have pre-existing immunity against Cas9, as they were once exposed to Cas9-carrying bacteria.

Fusing environmental data in future predictive models will help improve the precision of data collected through conducted experiments and this is essential if the technology is confirmed to be successful where it can be applied within the clinics and hospitals for children. Such modeling will also allow for the selective targeting of individual muscles, which contain the defective gene, causing illnesses in children.

Incorporation of chromatin environments would further likely improve off-target predictions of ways to treat the illness, as it will be more suitable to assisting accessibility of Chromatins. Other than chromatin data, potential off-target pipelines should also concentrate on including variant data. A recent study demonstrated that the differences between individuals have dramatically affected the off-target ability, with point mutations creating and destroying potential off-target sites.

Future models may also not only simply predict the success of CRISPR editing within the Dystrophin gene, but additionally the increase the success of potential results. By targeting sites with microhomology and exploiting the microhomology-mediated repair pathway, researchers may be able to delete specific DNA segments and subsequently control the results of CRISPR-Cas9 editing within these children, thus being a potential remedy to the illness.

Conclusion

As researchers’ understanding improves regarding CRISPR within Duchene Muscular Dystrophy, there will be larger incorporation into new features into predictive models to increase their accuracy. Gaining an informed understanding through various research and experiments conducted are essential for applying CRISPR-Cas9 in clinical applications, where individuals are left vulnerable. Until CRISPR technology is capable of being used to treat Duchenne Muscular Dystrophy, scientists must sustain this understanding to ensure this cure is effective for human beings.

Inquiry Question:

Should funding be allocated towards further research into genome editing as a clinical treatment for Duchenne Muscular Dystrophy?

Introduction:

Duchenne Muscular Dystrophy is a form of inherited neuromuscular disease found in children, causing progressive degeneration of muscle stability. It is as a result of a mutation in the DMD gene coding for the protein called dystrophin. A mutation is defined as the change in the base sequence of an organism. This mutation is inherited as it is passed on through germline cells to then be inherited in offspring. Approximately 1 in 3500 male births worldwide are affected by this recessive trait, making it the most prominent form of childhood muscular dystrophy. The prevalence of this condition sparked research in the scientific community into a cure for those affected. With the evolution of technology, genome editing has become an innovative tool that has the potential to cure a currently permanent disease. The groundbreaking technology called Crispr was created in 2015, that allows for the deletion, insertion or modification of DNA sequences. The discovery into the capability of gene editing through Crispr to act as a potential cure for the disease led to the development of this investigation. This report will research the potential of Crispr to be used as a treatment, taking into account the benefits and risks of the technology with the aim of evaluating the inquiry of ‘Should funding be allocated towards further research into genome editing as a clinical treatment for Duchenne Muscular Dystrophy?’

Duchenne Muscular Dystrophy:

Duchenne Muscular Dystrophy, or Pseudohypertrophic Muscular Dystrophy, is an inheritable muscular disease that begins to affect individuals from early childhood. Symptoms can appear as early as infancy, typically presenting themselves between the ages of 1 and 6. A delayed development for milestones such as sitting, standing and walking independently are all considered initial symptoms of the disease. Between the ages of 6 -11 there is a continual progressive weakness of muscles, with most affected individuals being wheelchair restricted by adolescence. The condition can result in breathing difficulty, and its presence on cardiac muscles induces Cardiomyopathy, a condition where the heart becomes enlarged, by the age of 18. In most cases, ventilators become a 24 hours necessity to ensure breathing. The disease is life-threatening and it is rare to see survival past the age of 30. Duchenne Muscular Dystrophy originated from a mutation in the DMD gene. There are multiple possible mutations of the DMD gene that can result in the disease, with the most prominent cause being a large scale deletion which removes one or more exons from the gene. This would remove numerous amino acids from the polypeptide chain drastically changing the composition, structure and function of the protein. The other notable type of mutation is a large duplication which produces extra copies of the nucleotide sequence in the gene that distorts the resulting protein by interfering with the correct and ordered sequence of amino acids that should in the polypeptide chain. The other possible types of mutations occur less frequently but include deletions or substitutions to bases that then change the protein that eventuates. As a result of any of these mutations, the subsequent effect is a defective DMD gene which consequentially impairs the dystrophin protein that was originally intended. The dystrophin protein is found in skeletal and cardiac muscle and its role is to strengthen, stabilise and protect muscle fibres. A mutation in the DMD gene has severe consequences on dystrophin and hence, the muscles in the body. A dysfunctional or incompetent protein deprives muscles of required protection. when coupled with the repeated contraction and relaxation of muscles through use over time, this damages the muscle, leading to the death of these fibres. The DMD gene which codes for dystrophin is found on the X chromosome, therefore making Duchenne Muscular Dystrophy an X-linked trait. The mutated gene is also recessive with the original being dominant over it. This means that if there is one mutated gene in either paternal or maternal X chromosomes and an unmutated gene in the other, the original DMD gene will be expressed and the individual’s phenotype will show no characteristics of muscular dystrophy., they will just be a carrier. Both alleles are required to be altered in order for the disease to be expressed. This requirement for expression is true for women as they possess two X chromosomes, but as males only have one X chromosome they are at an increased risk of the disease. If a male’s mother has or is a carrier of the disease and they inherit the mutated gene, they will express phenotype of muscular dystrophy as there is no alternative allele to fall back on. As a result of this, the prevalence of Duchenne muscular Dystrophy is significantly larger in males than females. Currently, there are no approved clinical treatments for curing the disease but diagnostic testing is available. The most common method of diagnosis is a genetic test, generally performed through a blood test that is able to detect the presence and type of mutation there is in the dystrophin gene. Muscle biopsies are also conducted to examine the amount and location of dystrophin in muscle tissue. Once confirmed, assistive therapy is provided to affected individuals through medication, physiotherapy and electric muscle stimulation to prolong muscle use. [LINK]

Genome Editing:

Genome editing is an emerging, innovative and contemporary technology recently developed that is showing significant potential to cure multiple diseases, in particular, Duchenne Muscular Dystrophy. Genome editing is defined as the insertion, deletion, modification or replacement of DNA in the genome of an organism. Recent studies have utilised the relatively new biotechnology called Crispr-Cas9 which enables permanent gene therapy, otherwise known as genome editing. Crispr has been depicted as a cut and paste tool for editing the human genome that is able to identify and modify DNA through the use of proteins and RNA molecules. A recent experiment conducted by the Technical University of Munich highlighted the potential of Crispr gene scissor to act as a treatment for Duchenne Muscular Dystrophy by correcting the mutated dystrophin gene in pigs. This is the most promising experiment conducted to date as pigs are biologically similar to humans in many ways which leaves the potential for clinical trials to begin eventually to cure the disease. Researchers modified the Crispr-Cas9 gene scissors to remove the mutated section of the gene sequence creating a shorter dystrophin protein. In Figure 1, the gene therapy process is displayed on a mouse. It shows an adeno-associated virus being used to deliver the Cas9 protein that then excised exons 21 to 23 of the DMD which was the mutated section. The Cas9 protein removes the exons after being guided by a molecule known as guide RNA which can identify the DNA sequence being targeted. The result of this process is a shorter, deficient but functional dystrophin protein that can restore partial muscle function. The result of this process is the mutations that were previously missense or nonsense become viable, thus creating a partially accurate sequence that can be used in protein synthesis to create functional dystrophin that can restore limited muscle function. The original experiment highlighted that a permanent therapy for muscular dystrophy could be achieved due to the fact muscle and cardiac cell life is prolonged. According to Professor Christian Kupatt, “One half of myocardial cells remain functional from birth throughout the entire lifecycle of a human being.” Therefore, through editing the genome of cells such as myocardial cells, longer, permanent correction becomes a reality, as it was for the pig. This technology is promising for those affected with the condition as clinical trials look to be a logical step in the near future. Trials using Crispr have already commenced for cancer and cystic fibrosis patients further enforcing the exciting prospect that it could be used for muscular dystrophy to permanently treat more than 80% of Duchenne patients. As a result of Crispr-Cas9 and recent scientific studies, the introduction of genome editing into medical care as clinical treatment is a viable and tangible thought.

Evaluation:

The research and development of Crispr-Cas9 has sparked curiosity in the scientific community for its potential application towards Duchenne Muscular Dystrophy, but with every new technology there are essential questions that need to be asked to evaluate the benefits, risks and ethical implications. Genome editing has an undeniable benefit for those affected with Duchenne’s as it holds the potential to treat what was previously untreatable. Through correcting the mutated DMD gene individuals will be relieved of symptoms, possess basic muscle function and have a better quality of life that will last longer. Although this technology does have numerous benefits, it does have limitations that have been discovered during testing. Crispr-Cas9 is able to edit the genome correcting mutations but within experiments conducted for Duchenne Muscular Dystrophy, only certain sections of the gene were removed. Consequently, full muscle function would be impossible to restore with this level of current technology, which is used as a valid argument against the proposals for further funding. Taking into account the profound potential this technology possesses to save and improve lives, its value and need to be apart of clinical application is irrefutable. This statement holds especially true after considering the lack of alternative approved clinical treatments available for individuals. Currently, there is only the ability to diagnose the disease and the type of underlying mutation, with assistive therapy being the only pathway after a diagnosis has been made. Although there are studies using drugs being experimented with, this type of treatment is not permanent, potentially dangerous to the chemistry of the body and expensive to sustain the purchase of medication. Treatment using drugs is not as beneficial to patients as Crispr is, due to the fact that it doesn’t personalise the treatment to each type of mutation, only catering for those with a specific mutation that the drug is applicable to. This is opposed to treatment through Crispr which takes into account each individual’s mutation by specifically coding the Guide RNA to their DNA. Despite the clinical benefits of the technology, the use of Crispr to treat disease distorts and divides society as to whether it should be done. From a legal aspect, there is contention on how laws should be formulated to determine when Crispr is deemed a valid medical response and when it becomes a way to edit the human race. Also taken into consideration is the regulation and construction of patents from corporations who may be able to control and heavily profit from development of the technology. Others completely disapprove of the use of Crispr because of possible personal or religious reasons with common arguments that humanity and the body is a product of nature and/or God, and therefore it should not be tampered with. Many are against the use of animal testing, protesting that it is unethical to make the decision and tamper with an innocent organism who is unable to express their willingness to be experimented on. As well as this, the issues of economic factors and accessibility have been called to attention with use of a new, experimental technology. Fortunately, Crispr is an accessible and cost effective treatment especially when compared to other technologies. Previously only 13% of Duchenne Muscular Dystrophy patients had access to experimental treatments due to cost. This is dramatically different with Crispr and with the ongoing development of the technology, the cost will likely decrease. Furthermore, genome editing is a permanent therapy reducing costs further by making it a single payment as opposed to alternative treatments. In an article by Stanford Medicine, it was also highlighted that there could be serious health risks associated with gene editing because the human immune system can be unpredictable in its reaction towards substances injection into the body. This is a major concern for Duchenne patients as the studied form of treatment relies on the injection of a virus into their system. Despite portraying the danger, they are aware and express that Crispr should be employed provided it is done with caution. This evaluation that Crispr has an overall benefit to society as a whole, in particular, patients with muscular dystrophy holds true regardless of the ethical and social considerations as it can have a significant positive impact on countless lives. Additionally, a study headed by the David Heart and Lung Research Institute of treatment displayed date of treatment in mice encapsulating the value and clinical utility of genome editing. As can be seen in Figure 2, four mice were treated and all four showed levels of dystrophin in their muscular cells. This is true testament to the potential of Crispr and act as effective reasoning to outweigh all social and ethical concerns.

Conclusion:

From this study, it can be clearly determined that genome editing is an innovative and powerful tool capable of making a significant difference in the lives of individuals affected by Duchenne Muscular Dystrophy. Thus, funding should be allocated towards further research into genome editing as a clinical treatment for Duchenne Muscular Dystrophy. As detailed, its impact on patients far outweighs the social and ethical implications with those concerns a cause for further funding. The funds would allow for the technology to be developed in a level of detail and caution that increases benefit through eliminating many of the physical concerns. The future of gene therapy through Crispr is unpredictable with the only guarantee being that it will become a successful technology capable of being an influential factor in modern medicine. In regards, to Duchenne Muscular Dystrophy, animal testing has yielded positive results and the next step looks to be human clinical trials where the effectiveness of the technology will be tested as a treatment. There is a genuine possibility that with further funding and research, Duchenne Muscular Dystrophy will be cured, becoming a treatable disease.

Figure 2 (https://www.sciencedirect.com/science/article/pii/S1525001616309741)

Reference:

1. https://www.technologynetworks.com/biopharma/news/gene-therapy-for-duchenne-muscular-dystrophy-successful-in-pigs-329888
2. https://newatlas.com/medical/crispr-gene-editing-muscular-dystrophy-pigs/
3. https://www.biospace.com/article/experimental-gene-therapy-for-duchenne-muscular-dystrophy-shows-promise/
4. https://www.tum.de/nc/en/about-tum/news/press-releases/details/35877/
5. https://stanmed.stanford.edu/2018winter/CRISPR-for-gene-editing-is-revolutionary-but-it-comes-with-risks.html
6. https://www.youtube.com/watch?v=8xNh1qr43oo
7. https://www.youtube.com/watch?v=UKbrwPL3wXE
8. https://ghr.nlm.nih.gov/condition/duchenne-and-becker-muscular-dystrophy#genes
9. https://www.genome.gov/
10. https://www.sciencedirect.com/science/article/pii/S1525001616309741
11. Eldra Pearl Solomon. P.William Davis – Human Anatomy and Physiology (Holt-Saunders International Editions)

Duchenne’s Muscular Dystrophy is a genetic disorder linked to the X chromosome that is caused by a deficiency in the protein dystrophin (Mendell et al., 1995). This disease weakens skeletal and cardiac muscles, and may pose obstacles when sitting, standing, walking, and speaking. Treatment by myoblast transfer showed promising results in animal trials. This method strives to replace dystrophin through the injection of donor myoblast cells into skeletal muscles. The donor cells are able to replace dystrophin by fusing with muscle fibers and supplying the absent gene that causes the production of dystrophin. Clinical trials of this method were used to treat murine dystrophies, and they showed favorable results. One account showed dystrophin produced from the donor cells one month after the myoblast administration. However, other reports were not able to find any donor produced dystrophin or messenger RNA after the injection. From all of the controlled trials, there was no report of increased strength after the myoblast application. On the contrary, uncontrolled trials showed increases in strength. To avoid rejection of the myoblast cells, immunosuppressors were used in some of the trials. Furthermore, incorporation of anti-inflammatory factors affects the analysis of the myoblast administration; prednisone and cyclosporine have demonstrated the ability to increase strength in Duchenne’s muscular dystrophy patients. As a result of the promising conclusions of these studies, the researchers in this study hypothesized that myoblast transfer will be a viable treatment for replacing the lacking gene that causes the production of dystrophin in patients with Duchenne’s muscular dystrophy. Also, the researchers hypothesized that administering cyclosporine, in conjunction with the myoblast transfer, will help to increase muscle strength for these patients.

This study differs from previous studies in a few ways. In contrast to previous studies, this study conducted myoblast injections once a month for a six-month period (Mendell et al., 1995). Previous studies only administered myoblasts on a single occasion. Also, the measurement of dystrophin expression in this study was done using peptide antibodies that are distinct to the missing exons in the dystrophin gene. This allows examiners to differentiate between revertant fibers, or fibers from the host that return to normal, and donor cell-produced dystrophin-positive fibers. Lastly, the administration of cyclosporine or a placebo was randomly assigned. This allowed for the evaluation of the effect of cyclosporine on increasing muscle strength, and the determination of the need for immunosuppressors.

Results

Twelve boys with Duchenne’s muscular dystrophy took part in a double-blind trial of myoblast transfer. Normal human myoblasts were isolated and mixed with muscle samples from the biceps brachii of all donors. The samples were maintained until being dissociated in 0.01 percent trypsin, collagenases I and II, and 0.02 percent EDTA. The patient’s arms were then randomly assigned to receive a myoblast injection or a sham injection, both patients and researchers were unaware of which arm received myoblasts and which received sham injections. The maximal voluntary isometric strength of elbow flexion was the primary variable tested, the average of three consecutive trials was recorded. One year after the trial started (6 months after the final myoblast transfer) biopsies of biceps brachii were performed.

The results showed that myoblast transfer had no effect on muscle strength. Six myoblast transfers were completed, at month 0 and months 1, 2, 3, 4, and 5. Maximal voluntary isometric strength was tested the day before each myoblast transfer and at twelve months. The difference in the degree of change from the baseline test (month 0) between arms injected with myoblasts and sham injected arms. Additionally, there was no difference in the mean number of dystrophin-positive fibers between the myoblast injected and sham injected arms. Dystrophin mutations are what causes Duchenne muscular dystrophy. Peptide-specific antibodies raised against deleted exons was used to determine whether the dystrophin-positive fibers in the arms that received myoblast infusions were expressing donor-derived dystrophin or native mutated fibers. Only four of the twelve patients muscle fibers expressed donor-derived dystrophin. Of those patients, only one fiber was in the entire cross-section of Patients 3 and 6 and two positive fibers were found in the entire cross-section of Patient 12. These positive cross-sections made up less than 1 percent of the revertant fibers that express dystrophin in patients with Duchenne’s muscular dystrophy. However, patient 5 expressed that 10.3 percent of dystrophin fibers were donor-derived, which was 420 dystrophin-positive fibers from a total of 4080 fibers. Six boys were treated with 5 mg of cyclosporine per kilogram each day and the other sic were given a placebo. There was no significant difference in the average muscle scores of the boys treated with cyclosporine and those given a placebo. The loss of muscle strength was similar to the natural rate of decline. There was no significant difference between boys treated with cyclosporine and those given a placebo. There was no significant difference in muscular strength after six rounds of myoblast transfer.

Discussion

The study assessed the effects of myoblast transfer as a way to treat Duchenne’s muscular dystrophy. The patients each received a total of 660 million myoblasts to the biceps muscle over the course of 6 months, which differs from a previous study in which the patients received half the amount. None of the patients had any muscular strength increase following the conclusion of the treatment. It is suspected that treatment for muscular dystrophy depends on the expression of the dystrophin gene. Of the twelve patients that participated in this study, four had donor-produced dystrophin. One patient, in particular, had significant amounts of dystrophin-positive fibers, with a total of 10.3%. The positive fibers were found to be equally distributed throughout the muscle, in both the superficial and deep muscles. In a previous study, it was concluded that 1% of host muscle fibers expressed donor-produced dystrophin following a method using reverse transcriptase-polymerase chain reaction. The researchers also found no difference in muscular strength and dystrophin-positive fibers between myoblast-injected and sham-injected patients. It is suspected that the dystrophin fibers found in the sham-injected sites were originally mutant fibers that have reverted back to their original forms having emerged from a second mutation. In another study, a patient was observed to have 5% of dystrophin-positive fibers with no donor-produced DNA, further showing that detecting the source of dystrophin is very difficult.

Future studies would be beneficial in understanding the origin of dystrophin-positive fibers and discovering other ways to detect their source of them. It would also be helpful to use a bigger sample size than this study used, which was only 12 patients. By doing so, researchers would have a higher confidence level and would be able to differentiate between the source of dystrophin-positive fibers. Since this study had a secondary outcome regarding the use of cyclosporine and its effects, it would be interesting to note how that treatment coincides with myoblast transfer. A previous study observing the effects of cyclosporine found positive results in increased muscle strength after 12 weeks of treatment. This would be interesting to follow up using myoblast transfer to see if there would be further positive results or if the myoblast transfer has no effects at all.

In conclusion, this study showed no improvements of muscle strength in patients with Duchenne’s muscular dystrophy using myoblast transfer. This is despite the fact that the patients received high numbers of myoblasts to the muscles. It was demonstrated that myoblast transfer can result in cells with the capability of fusing with host fibers and expressing dystrophin-positive fibers. Future studies would help to further this one and assess the efficiency of myoblast transfer to attain positive results.

Introduction:

Muscular dystrophies are defined as a group of heterogeneous group of diseases characterized by muscle weakness (Rahimov et al, 2013). There are different kinds of muscular dystrophy like Duchenne, Becker, Myotonic, Congenital, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, Distal, and Oculopharyngeal (Table 1). The most common form is Duchenne muscular dystrophy (DMD). 1 in every 3500 to 5000 newborn males worldwide are affected by it. Symptoms of muscle weakness are seen typically between 3 to 7 years of age. Boys suffering from DMD show delayed development of motor skills like talking, sitting and walking and around puberty lose ambulation (Gao et al, 2015). DMD results from the loss of function due to a mutation in dystrophin present in the muscle cell. Dystrophin provides a structural link between muscle cytoskeleton and the extracellular matrix to maintain muscle integrity. It is rich in myofibers and concentrated into rib-like structures called costameres (structural components linking myofibrils to sarcolemma). Disruption of this protein causes the plasma membrane to be fragile and making it less stiff with an increase in leakiness of the muscle enzymes (Goldstein et al, 2010). Current research suggests the loss of dystrophin as being the primary reason for muscle weakness. However, a potential secondary reason could be the abnormal Ca2+ levels which play a role in muscle necrosis.

Table 1: Characteristics of the Types of MD

NOTE: Verhaert, D., Richards, K., Rafael-Fortney, J. A., & Raman, S. V. (2011). Cardiac Involvement in Patients With Muscular Dystrophies. Circulation: Cardiovascular Imaging, 4(1), 67-76. doi:10.1161/circimaging.110.960740

Skeletal Muscle Structure and Function:

In order to understand the complexities of muscular dystrophies, first we need to understand the basics of the structure of skeletal muscle and how muscle contraction works.

Structure of skeletal muscle

Muscle in our body is surrounded by a connective tissue called epimysium. The muscle is further divided into numerous bundles called fascicles of individual muscle cells by another connective tissue called perimysium. Hundreds of muscle fibers are contained in these elongated fascicles. Fusion of many cells results in the muscle fibers being multinucleated. These nuclei are found in a region below the plasma membrane of the muscle fiber called as sarcolemma. A semi-fluid cytoplasm called the sarcoplasm is present in each muscle fiber. Rod like elements, myofibrils along with the mitochondria surround the sarcoplasm. Each myofibril is composed of thick filaments called myosin and thin filaments called actin (Stanfield, 2013). The sarcoplasmic reticulum, a saclike membranous network surrounds each of the myofibrils, which is closely associated with other structures called transverse tubules (T tubules). The lateral sacs, enlargements of the sarcoplasmic reticulum are present near the T tubules and serve as a place to store calcium. Each T tubule along with two lateral sacs forms a triad. The sarcoplasmic reticulum and T tubules together help in activating muscle contraction. They send signals to the myofibrils from the sarcolemma further enabling a muscle cell to respond to neural input (Stanfield, 2013).

Figure 1: Structure of a skeletal muscle fiber showing its different components

Muscle contraction:

Skeletal muscle are referred to as striated muscle because of their striped like appearance. The protein fibers (thick and thin filaments) present in the myofibrils are arranged in an orderly way giving them the striations. Thick and thin filaments are present in a 2:1 ratio. These myofibrils are made up of repeating units called the sarcomere that is bordered by the Z lines on each of its sides. These Z lines are perpendicular to the long axis and support one of the ends of the thin filaments. M lines connecting the thick filaments together are also perpendicular to the long axis. On the sides of the A band, dark striations are visible due to the overlapping of the thick and thin filaments. However, the center of the A band is lighter because only thick filaments are present without any overlapping between the thick and thin filaments. This is the H zone. The region where only thin filaments are present without any with thick filaments the I band (Stanfield, 2013). In the center of the I band, there is a Z line connecting the thin filaments together. The thin filaments are made up of actin and the thick filaments are made up of myosin. They are arranged in a repeating fashion.

Each of the thin filaments contain the actin monomer call the G (globular) actin, which have a myosin-binding site. G actin are linked end to end forming the F (fibrous) actin strand. Two F actin strands are arranged in a double helix. The thin filaments also contain two regulatory proteins called the tropomyosin and troponin, which help in activating and deactivating contraction. The long fibrous tropomysin extends over the actin monomers blocking the myosin to bind preventing contraction. Troponin is made up of three proteins. One binds to the actin strand. The second protein binds to tropomyosin. Binding of troponin with tropomysion results in exposing of the myosin binding sites on the actin thin filament. The third protein contains calcium-binding sites (Stanfield, 2013).

Figure 2: (a) A myosin molecule (b) Two actin molecules forming the double-helical structure

Figure 3: Sarcomere structure showing the arrangement of protein filaments within the sarcomere

Note: Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.

The Mechanism of force generation in a muscle:

Muscle contraction occurs by a repeating cycle where the thick and thin filaments slide past each other. This cycle is called the cross-bridge cycle (Stanfield, 2013).

Each cross-bridge cycle involves the five steps:

1. Binding of myosin to actin: Myosin is in its energized form of ADP and Pi with high affinity for actin. It is first bound to the ATPase site of myosin head, which further binds to the actin monomer of the thin filament. This step requires the presence of calcium to take place.
2. Power stroke: In this step, the myosin and actin are bound together marking the release of Pi from the ATPase site. This step is called the power stroke because force is generated. In this step, the myosin head swivels towards the center of the sarcomere resulting in shortening of the sarcomere and muscle contracting.
3. Rigor: In this step, myosin goes to a lower energy state and releases the ADP from the myosin head. This stage is called as rigor because the actin and myosin are bound tightly together. An excess of calcium due to damaged cell membranes or less ATP production due to no energy production could result in the crossbridge cycle getting stuck at this stage. This could also be called as the rigor mortis, which is characterized by the stiffening of body after death.
4. Unbinding of myosin and actin: In this step, a new ATP is introduced to the ATPase site of the myosin head resulting in its conformational change. This conformational change decreases the affinity of myosin for actin. Hence, myosin unbinds from actin.
5. Cocking of the myosin head: In this step, energy is released. ATP bound to the ATPase site of myosin head is cleaved into ADP and Pi by ATP hydrolysis. Now myosin is in its high-energy form and is bound to the ATPase site of the myosin head. In presence of calcium, the cycle will repeat again starting from step 1.

This cross-bridge cycle continues endless if there is enough ATP available. Troponin and tropomyosin help in regulating this cycle by interacting with calcium and decreasing the available myosin-binding sites (Stanfield, 2013).

Figure 4: The cross-bridge cycle

Note: Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.

Excitation-Contraction coupling:

The axon terminal of a motor neuron or the presynaptic cell releases Acetylcholine (ACh). On diffusing into the postsynaptic cell or the muscle cell, the ACh binds to receptors in the motor end plate, which has a high density of acetylcholine receptors. This results in the activation of the many ACh receptors creating an end plate potential (depolarization). This end plate potential is higher in the presynaptic cell than a regular postsynaptic potential. This triggers an action potential in the muscle cell. This action potential generates throughout the sarcolemma and the T tubules. As the action potential propagates through the T tubules, calcium stored in the lateral sacs of the sarcoplasmic reticulum is released. Calcium then binds to the troponin resulting in a conformational change causing tropomyosin to move from its position exposing the myosin-binding sites. This calcium-binding to troponin marks the start of muscle cell contraction. When tropomyosin is not bound to troponin, it blocks the myosin-binding sites resulting in relaxation of the muscle (Stanfield, 2013).

Figure 5: Steps that take place during excitation-contraction coupling

Note: Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.

What is the role of Dystrophin in Muscular Dystrophy?

We have understood how the muscle works- how does it contract and relax as well as how force generation occurs in a muscle. In order to understand how the absence of dystrophin affects the weakening of muscle, we need to understand the function of dystrophin.

Dystrophin and its Associated Proteins:

DMD is known to be the largest known gene that encodes for the dystrophin (Goldstein et al, 2010). This rod-shaped protein is located primarily on the subsarcolemmal of the skeletal muscle plasma membrane. It is used for movement (skeletal muscles) and in heart (cardiac) muscle. However, small amounts of dystrophin are also found in the nerve cells in the brain. The dystrophin binds to the actin through actin binding domains (Culligan et al, 2002). In skeletal and cardiac muscles, dystrophin is part of a group of proteins, a protein complex. They work together to strengthen muscle fibers and protect them from injury during muscle contraction and relaxation. The dystrophin complex provides a structural link between each muscle cell’s cytoskeleton with the framework of proteins and other molecules outside the cell (extracellular matrix). The dystrophin complex may also play a role in cell signaling by interacting with proteins that send and receive chemical signals.

There is very little information about the function of dystrophin in nerve cells. Current research suggests that this protein is important for the normal structure and function of synapses that are specialized connections between nerve cells where cell-to-cell communication occurs (Culligan et al, 2002).

Figure 6: Muscle fiber showing the presence of dystrophin

Note: Duchenne Muscular Dystrophy (DMD) – Causes/Inheritance. (2018, January 31). Retrieved from https://www.mda.org/disease/duchenne-muscular-dystrophy/causes-inheritance

Membrane fragility caused by Dystrophin deficiency:

Dystrophin along with its associated proteins, are found at the sarcolemma along with the sarcomeric Z disc in structures known as costameres. Costameres are present over the Z disc within the plasma membrane and is perpendicular to the long axis of the myofiber. This intracellular position, dystrophin’s capacity to bind actin, argues for a role in transmitting force from the sarcomeres to the extracellular matrix and neighboring cells. A research paper by Rybakova et al. demonstrated the presence of cytoskeletal actin on the inner cytoplasmic face in a peeled membrane, indicating the tight linkage of dystrophin and actin (Goldstein et al, 2010).

Goldstein et al (2010) used mice as the model organism to study DMD. A peeled membrane from the myofibers of the mdx mouse which has a point mutation in the DMD gene, that lacks dystrophin was not found to retain actin. On the other hand, a peeled membrane from the laminin alpha 2-deficient dy mice retained both dystrophin and actin, hence, a specific role for dystrophin in the actin-binding interface. Recently, it was shown that dystrophin directly binds to tubulin and organizes the microtubule network. Because microtubules not only provide cell structure, but also scaffold the Golgi network and direct intracellular traffic, loss of this dystrophin– microtubule interaction is a plausible mechanism for the altered localization of many proteins in dystrophin- mutant animals.

Figure 7: (A) Normal myofiber (B) Dystrophin deficient mdx myofiber

Note: Goldstein, J. A., & Mcnally, E. M. (2010). Mechanisms of muscle weakness in muscular dystrophy: Figure 1. The Journal of General Physiology, 136(1), 29-34. doi:10.1085/jgp.201010436

Membrane leak and rupture caused by Dystrophin deficiency:

DMD releases muscle enzymes, particularly creatine kinase, into the circulation. Some other proteins like lactate dehydrogenase and aldolase also leak into the serum, and these serum proteins are effective as biomarkers of disease. It is unclear whether the leak of these myoenzymes contributes to muscle dysfunction in muscular dystrophy. The disrupted membrane allows for the entry of small molecules into the myofiber syncytium. Ca2+-sensitive dyes has been used effectively to trace calcium entry into muscle cells. The vital tracer Evans blue dye is a small molecule that can enter into muscle lacking dystrophin whereas normal fibers are typically impermeable (Goldstein et al, 2010).

Impaired calcium handling in muscular dystrophy:

The primary defect in DMD is the loss of the membrane cytoskeletal protein dystrophin resulting from specific mutations in the human DMD gene. However, the secondary molecular mechanisms leading ultimately to muscle degeneration is not clear. Abnormal Ca2+ homeostasis has been known to make the skeletal muscle cells more susceptible to necrosis, cell death. Recently, a study shows how Ca2+ levels are higher in individuals with DMD. A Ca2+ deposit-sensitive histochemical red stain called alizarin was used and an increase in positive-reacting cells in the DMD muscles was seen. When repeated on mdx skeletal muscle, a similar result was observed. Fura-2, a fluorescent Ca2+ indicator has also indicated an increase in Ca2+ levels in the dystrophic muscles. This was further confirmed by studying mdx fibres which showed increase in cytosolic Ca2+ levels particularly in dystrophin deficient muscles. This study was not confirmed universally since many other scientists reported no difference in the calcium levels between dystrophin deficient muscles and normal muscles. (Culligan et al, 2002).

Muscular dystrophy affects other body systems:

A patient suffering from muscular dystrophy has a high chance of experiencing respiratory failures. This results from weakening of breathing muscles which further result in a limited lifespan unless mechanically supported (Gao et al, 2015). In patients suffering from muscular dystrophy, the lung muscles lose their elasticity and become less distensible. The weakened muscle is marked by its inability to generate adequate levels of pressure and volume whereas the fatigued muscle is marked by their inability to produce the pressure in response to the constant load. The cause of this distensibility is unknown but one cause could be breathing at a low lung volume which is common in a MD patients. Respiratory muscle weakness varies from person to person depending on the intensity of the disease. The diagnosis can can be delayed since the symptoms are very subtle in the beginning. However, there is a tension time index (force developed by the inspiratory muscles to the time they are being used) which helps in understanding the respiratory muscle fatigue. Children suffering from MD show elevated values of the tension time index. This is indicative of the fact that inspiratory muscles are prone to fatigue beginning from childhood (Lo Mauro A. et al, 2016). In some forms of muscular dystrophy, heart is also affected further resulting in heart failure and irregular heart rhythms (Gao et al, 2015).

Treatments for muscular dystrophy:

There are not a lot of therapies for Muscular Dystrophy that have been discovered. One of the common successful one is the use of corticosteroids. They have proved to benefit the boys suffering from DMD by stabilizing their muscle strength and function and delaying the advancement of scoliosis and cardiomyopathy. Treatment continued even after the loss of independent ambulation showed some improvement in the patient and hence proved beneficial. Available literature and some clinical experience suggest that corticosteroid treatment should be considered for all patients suffering from DMD starting at an early stage of the disease (Mah, 2016). In addition, supportive care for the muscular dystrophy patients has also helped improve the life expectancy of many young adults suffering from DMD (Mah, 2016 and Leung et al 2013). This involves a lot of nonpharmacologic interventions, like physical therapy, occupational therapy, orthopedic surgery, and genetic counseling (Leung et al 2013).

Works cited:

1. Gao, Q. Q., & Mcnally, E. M. (2015). The Dystrophin Complex: Structure, Function, and Implications for Therapy. Comprehensive Physiology, 1223-1239. doi:10.1002/cphy.c140048
2. Goldstein, J. A., & Mcnally, E. M. (2010). Mechanisms of muscle weakness in muscular dystrophy: Figure 1. The Journal of General Physiology, 136(1), 29-34. doi:10.1085/jgp.201010436
3. Leung, D. G., & Wagner, K. R. (2013). Therapeutic advances in muscular dystrophy. Annals of Neurology, 74(3), 404-411. doi:10.1002/ana.23989
4. 4. Mah, J. (2016). Current and emerging treatment strategies for Duchenne muscular dystrophy. Neuropsychiatric Disease and Treatment, Volume 12, 1795-1807. doi:10.2147/ndt.s93873
5. Rahimov, F., & Kunkel, L. M. (2013). Cellular and molecular mechanisms underlying muscular dystrophy. The Journal of Cell Biology, 201(4), 499-510. doi:10.1083/jcb.201212142
6. Stanfield, C. L. (2016). Principles of human physiology. Upper Saddle River: Pearson.
7. Duchenne Muscular Dystrophy (DMD) – Causes/Inheritance. (2018, January 31). Retrieved from https://www.mda.org/disease/duchenne-muscular-dystrophy/causes-inheritance
8. Culligan, K. G., & Ohlendieck, K. (2002). Abnormal Calcium Handling in Muscular Dystrophy. Retrieved from http://www.bio.unipd.it/bam/PDF/12-4/02491Culligan.pdf
9. Verhaert, D., Richards, K., Rafael-Fortney, J. A., & Raman, S. V. (2011). Cardiac Involvement in Patients With Muscular Dystrophies. Circulation: Cardiovascular Imaging, 4(1), 67-76. doi:10.1161/circimaging.110.960740

Muscular dystrophy is a genetic disease that currently has no cure. There are medical treatments and medications that can help ease symptoms and manage the disease. We will discuss further later what these treatments and medications are and how they can help the patient. Let’s first briefly discuss the history of muscular dystrophy and how the disease propagates.

Muscular dystrophy comes in various types, one being Duchenne Muscular Dystrophy (DMD), and will be the focus of this discussion. According to an article in Medical News Today (2017) written by Tim Newman, the “symptoms [of Duchenne muscular dystrophy] normally start before a child’s third birthday; they are generally wheelchair-bound by 12 years and die of respiratory failure by their early-to-mid-twenties’ (Newman, 2017). So, what is muscular dystrophy?

There is a protein found in the skeletal muscle called dystrophin. Dystrophin is a” protein [that] helps anchor various components within muscle cells together and links them all to the sarcolemma” (Newman, 2017). This protein is vital for skeletal muscles to function appropriately including movement and repair. When this protein is missing, the muscle will weaken. In addition to progressively decreasing gross motor skills, there is even more happening on the cellular level. “Pathological changes include the absence of dystrophin at the membrane of the muscle fibers, increased adipose and connective tissue between muscle fibers, increased variability in muscle fiber size, infiltration of inflammatory cells, and centrally located nuclei, which are indicative of degenerating and regenerating muscle fibers” (Lovering, 2005). The absence of dystrophin is linked to genetic mutations to the X chromosome (2017). The “recessive X-linked disorder occurring 1 in every 3500 live male births [is] named after a French neurologist Guillaume Benjamin Amand Duchenne in 1860” (Sinha et al., 2017).

An article in the Journal of Family Medicine and Primary Care (Sinha, 2017) followed a case of a 12-year-old male who’s only complaint at the time was a decayed tooth. The article mentioned the parents of the child told the physician they noticed he was having difficulty climbing stairs, fatigue and falling. “On general physical examination, the child had an obese appearance and presented with difficulty in standing, walking, getting up from sitting position and climbing stairs, proximal weakness, calf hypertrophy, hamstring muscle contracture, and positive Gower’s sign” (Sinha, 2017). Gower’s sign was invented in 1879 by a neurologist by the name of Sir William Richard Gowers. This test is used to screen patients for muscle weakness commonly seen in DMD (Chang, 2012).

When a patient is being examined by his or her physician for possible signs of DMD, there are several factors that play into the official diagnosis. Since DMD is a severe disease and can lead to death, physicians use a variety of tools and test like the Gower’s sign, to determine if a patient has DMD or not. Newman (2017) lists several signs and symptoms that are commonly associated with DMD. Some of the early signs of DMD include: “a waddling gait, pain and stiffness in the muscles, difficulty with running and jumping, walking on toes, difficulty sitting up or standing, learning disabilities, such as developing speech later than usual, frequent falls” (2017). These symptoms will progress into more serious problems as time goes on. For example, patients will develop a full inability to walk, limited movement due to shortening of muscles and tendons, difficulty breathing and even cardiac problems because of weakened heart muscles (2017).

In addition to physical examinations of a patient suspected of having DMD, further tests will be ordered to confirm the diagnosis of DMD. These tests may include blood draws, X-Ray and/or CT scan, and even an EMG scan. In the case of the 12-year-old male patient mentioned previously, his physician ordered these tests to be completed. The results were as follows; an analysis of his serum found creatine kinase (CK) levels were elevated in addition to other chemical components. “On electromyographic examination, interference pattern analysis revealed myopathic pattern in the right vastus lateralis suggestive of primary muscle disease” (Sinha, 2017). Based on these finding, the patient’s physician determined a diagnosis of DMD was warranted and was referred for further evaluation and rehabilitation.

Since there is no cure currently for DMD, there are medication treatments and therapies available for patients with DMD. Patients may be prescribed corticosteroids to help strengthen muscle weakness, however, according to Newman (2017), “long-term use can weaken bones and increase weight gain” (2017). If the patient is experiencing cardiac problems, beta blockers and/or ACE inhibitors may be prescribed to help with symptoms.

In addition to drug therapy, physical therapy can help keep the patient’s muscles flexible for as long as possible. Unfortunately, the muscles will eventually fatigue to a point where there is no longer motion and the limbs will become “fixed in position” (Newman, 2017). To help the patient maintain some mobility, they may be required to use specialized devices like leg braces, canes, walkers and wheelchairs. Since it is inevitable that the patient’s limbs will become immobile, the goal of physical therapy is to prolong the mobility for as long as possible. Newman (2017) suggests, “Standard low-impact aerobic exercises such as walking and swimming can also help slow the disease’s progression” (2017).

DMD not only affects the patient’s limbs but can also affect other vital muscles such as the diaphragm, which is necessary to breath. “As the muscles used for breathing become weaker, it may be necessary to use devices to help improve oxygen delivery through the night. In the most severe cases, a patient may need to use a ventilator to breathe on their behalf” (Newman, 2017).

Researchers are currently working to develop long term treatments that may help replace the missing dystrophin in muscle tissue and may even help repair some of the damage DMD causes. An article in Muscular Dystrophy News (2018) written by Marta Figueiredo, announced a promising form of treatment for DMD. Researchers are currently running test trials of microdystrophin gene therapy that “robustly induces the production of a shorter, but functional, version of the dystrophin protein and reduces muscle damage in Duchenne muscular dystrophy (DMD) patients, according to preliminary results of a Phase 1/2 clinical trial” (Figueiredo, 2018). According to Figueiredo (2018), “Microdystrophin is a shorter version of the dystrophin gene, that contains the minimum amount of information needed to produce a functional dystrophin protein. It also directs the delivery of the microdystrophin gene specifically to muscle tissue (while avoiding other tissues), in particular the heart muscle, which is crucial for DMD patients, who frequently die from heart disease” (2018). This clinical research trial has found that microdystrophin gene therapy has increased the dystrophin levels in trial patients while also decreasing the levels of creatine kinase. Figueiredo (2018) stated, “Two months after treatment, all patients showed a significant decrease, more than 87%, in the levels of creatine kinase (CK), an enzyme used as a biomarker for muscle damage. This result suggests that the amount of microdystrophin being produced is effectively protecting the muscle” (2018).

In a more recent research study, a new medication is being investigated through clinical trials. According to IOS Press of Science News (2019), “The investigational drug edasalonexent, an oral NF-κB inhibitor, has the potential to slow the progression of the disease for all patients with DMD” (IOS Press, 2019). Dr. Joanne Donovan, who is following this clinical study suggests, “edasalonexent has the potential to limit disease progression for all patients affected by DMD, regardless of their underlying mutation, and can potentially reduce muscle inflammation and degeneration and enhance muscle regeneration” (IOS Press, 2019).

Even though DMD is rare, based on statistics mentioned previously, it is still very important to gain as much knowledge about the disease and find new ways to treat it. Since there is no cure, researchers are working hard to find new ways to help patients cope with the disease and prolong the use of their muscles. According to the Muscular Dystrophy Association (MDA) (2019), the “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” (MDA.Org, 2019). There is hope that one day a cure will be found.

Resources

1. Chang, R. F., & Mubarak, S. J. (2012). Pathomechanics of Gowers’ sign: A Video Analysis of a Spectrum of Gowers’ Maneuvers. Clinical Orthopedics and Related Research, 470(7), 1987–1991. doi:10.1007/s11999-011-2210-6. Retrieved online from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369091/
2. Figueiredo, M. (2018, June 22). Microdystrophin Gene Therapy Shows Promising Interim Results in Phase 1/2 Trial. Muscular Dystrophy News Today. Retrieved online from https://musculardystrophynews.com/2018/06/22/microdystrophin-gene-therapy-shows-promise-early-trial-results/
3. IOS Press. (2019, February 21). New Drug for Duchenne Muscular Dystrophy Clears Phase 1 Clinical Trial Testing in Boys. ScienceDaily. Retrieved online from www.sciencedaily.com/releases/2019/02/190221130242.htm
4. Lovering, R. M., Porter, N. C., & Bloch, R. J. (2005). The Muscular Dystrophies: From Genes to Therapies. Physical Therapy, 85(12), 1372–1388. Retrieved online from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496952
5. MDA.Org. (2019). Duchenne Muscular Dystrophy. Research and Special Programs. Retrieved online from https://www.mda.org/disease/duchenne-muscular-dystrophy/research
6. Newman, T. (2017, December 18). All About Muscular Dystrophy. Medical News Today. Retrieved online from https://www.medicalnewstoday.com/articles/187618.php.
7. Sinha, R., Sarkar, S., Khaitan, T., & Dutta, S. (2017). Duchenne Muscular Dystrophy: Case Report and Review. Journal of Family Medicine and Primary care, 6(3), 654–656. doi:10.4103/2249-4863.222015. Retrieved online from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787973/