Advances in BMD: Analyzing 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.

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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