16 February 2016

Cure Duchenne myodystrophy

Competition of groups, unity of methods

Anna Petrenko, "Biomolecule" 

Duchenne muscular dystrophy is the most severe X–linked disease, for which there is still no effective treatment. In one of the latest issues of Science, as many as three articles were published about the successful testing of CRISPR/Cas9 technology for the treatment of this disease on mouse models. Maybe this approach has a chance to get to clinics as well?


The principle of gene therapy of Duchenne/Becker myodystrophy. Duchenne myodystrophy (DMD) is caused by mutations of the dystrophin gene (DMD), leading to a shift in the reading frame, and milder Becker myodystrophy (BMD) is caused by mutations without shifting the reading frame. There is no cure for this disease yet. Gene therapy will help improve or even restore muscle function. Figure from [17].

Duchenne muscular dystrophy, which affects one of 3600-5000 newborn boys, is caused by the absence of dystrophin, a protein that connects the cytoskeleton and extracellular matrix in the muscle fiber and ensures its stability during contraction (Fig. 1). Due to mutations of the DMD gene, the reading frame shifts during translation of its mRNA, and protein synthesis prematurely stops. The congenital disease progresses very quickly: it is diagnosed at the age of about four years, and by the age of 10 the child usually already needs a wheelchair. This is because without dystrophin, the fibers are damaged, and as soon as the regenerative capacity of muscle fibers is exhausted, they are replaced by fibrous and adipose tissues [1]. Studies show that cognitive functions in a child can also be impaired [2]. More than 30 years with such a disease, as a rule, do not live, and death occurs from cardiac and respiratory complications. A milder type of myodystrophy associated with the DMD gene is Becker muscular dystrophy, when mutations do not lead to a shift in the reading frame [3].

Dystrophin is located on the intracellular surface of the sarcolemma along the entire length of the muscle fibers and is part of the dystrophin-associated glycoprotein complex (DAGC, DGC). It binds at one end to F-actin of the cytoskeleton, and at the other end to β–dystroglycan, which stabilizes the fibers during contraction. The dystrophin gene is one of the longest in humans.


Figure 1. Mutations in dystrophin are the cause of the development of Duchenne myodystrophy. a–Dystrophin binds to actin filaments (part of the cytoskeleton) via the N-ABD and ABD2 domains) and to DAGC via the CR and CT domains.
b is the crystal structure of N-ABD dystrophin.
Actin binding zones are shown in yellow, and four well–studied mutations causing the disease are shown in red. Figure from [18].

Duchenne muscular dystrophy cannot be cured yet, and today's therapy is aimed at slowing the progression of the disease and treating complications [4, 5]. The "gold standard" is corticosteroids, which were proposed as a treatment several decades ago. However, their use causes many side effects.

It is not surprising that many groups of geneticists and molecular scientists are engaged in the development of pre- and postnatal treatment of Duchenne myodystrophy. The disease is mainly studied on various lines of mice. In one of the latest issues of Science, three independent papers on the treatment of Duchenne muscular dystrophy were published at once [6-8]. The research groups were led by Eric Olson from the University of Texas, Amy Wagers from Harvard University and Charles Gersbach from Duke University. All groups used the exon skipping technique to restore muscle function, in which one or more exons are removed from the mRNA (Fig. 2). In this case, the protein turns out to be shorter, but still can perform its supporting and anchoring functions in the muscle fiber, and the "annoying circumstance" – an extra stop codon - also turns out to be "missed."


Figure 2. Missing exons in the dystrophin gene in Duchenne myodystrophy. a – In patients with DMD, there are mutations in the DMD gene that violate the reading frame during protein synthesis.
For example, when exon 50 is deleted, an "out-of-cell" mRNA appears, which leads to the synthesis of truncated non-functional or unstable dystrophin (left). In one of the therapeutic approaches, an antisense oligonucleotide "masks" exon 51, and it is "skipped" during splicing, the reading frame is restored. The result is a shorter but partially functional dystrophin (right). In the new works, "extra" exons are simply cut out of the genome using CRISPR/Cas9. b is a multi–zone pass in MDD therapy.
If we skip exons 45-55, mutations of which occur in about 63% of patients, the resulting short dystrophin will lead to the transformation of the standard MDD phenotype into an asymptomatic or milder MDB phenotype. Figure from [19].

The exon removal strategy even has advantages over recreating the full length of the gene: it is easier to develop than to restore individual deletions of each patient [7].

To cut out "extra" nucleotide sequences, the researchers used the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas9 (CRISPR-associated protein 9)* genome editing technology [9], which, by the way, has just been allowed to be used in experiments on embryos by a London institute [10].

* – You can read more about this technique, borrowed from bacteria, in the articles: "CRISPR systems: immunization of prokaryotes", "Mutagenic chain reaction: genome editing on the verge of fiction" and "Should we take a swing at ... genome change?" [11-13]

Competing laboratories: who will be the first to translate technology into therapy for humans?

Scientists from three laboratories have successfully applied the technology of exon skipping in vivo on a standard object – mice – and have shown that their method helps to restore the reading frame and partially restore the synthesis of dystrophin. Since even its low level (3-15% of normal) brings therapeutic benefits, the results of the work can be called successful.

This is not the first time Eric Olson's group has used the CRISPR/Cas9 method in their work on Duchenne muscular dystrophy. In 2014, scientists corrected a mutation in the germ line of mice and prevented the development of the disease. However, since prenatal genome editing on human embryos (yet?) it is forbidden, the researchers had to come up with a way of postnatal application of the technology.

In their latest work, an adeno-associated virus-9 (AAV9, adeno-associated virus-9) was used to deliver the components necessary for editing into tissues [6]. The researchers tested several ways to administer AAV9 on different days after the birth of mice. In all cases, the expression of the dystrophin gene in the cardiac and skeletal muscles recovered, but to varying degrees. Moreover, protein production increased from 3 to 12 weeks after injection, and skeletal muscle function improved 4 weeks after injection. "Now the challenge for researchers from the Wellstone Center is to transfer the findings from the mouse model to patients with myodystrophy," says Pradeep Mammen, co–director of the Wellstone Center.

Amy Wadgers' group conducted a similar experiment in many ways [8]. After many preparatory stages of genome editing and exon skipping on cells and animals, their experience was also crowned with success: programmable CRISPR complexes composed of an adeno-associated virus (AAV) were delivered by local and systemic administration to differentiated skeletal fibers, cardiomyocytes and satellite muscle cells of newborn and adult mice. If the editing is aimed only at muscle fibers, then the effect may eventually come to naught. However, as Wadgers notes, gene editing in satellite cells can provide a much longer-lasting result. It can lead to the creation of a pool of regenerative cells carrying the edited dystrophin gene, and as a result of conventional muscle repair, the edited gene will also appear in muscle fibers.

Finally, as everyone has already guessed, scientists led by Charles Gersbach also discovered the therapeutic effect of using AAV-CRISPR/Cas9 in a mouse model [7]. Intraperitoneal administration of the viral vector to newborn mice led to the restoration of dystrophin synthesis in the abdominal muscles (abdominal muscles), diaphragm and heart seven weeks after injection. According to the authors, therapy of the heart and lung muscles is extremely important, since it is their failure that often leads to the death of patients with Duchenne disease. Intravenous administration of AAV vectors to six-week-old mice also led to a significant recovery of dystrophin production in the heart muscle. "There is still a lot of work to do to transform [the technology] into therapy for humans and confirm its safety," says Gersbach. "But the results of our first experiments are already very encouraging." The group is going to optimize the delivery system and evaluate the effectiveness and safety of the strategy on larger animals (Fig. 3). Which of the three laboratories will overtake the others and be the first to conduct tests on humans?

Duchenne myodystrophy therapy: old and new approaches

According to Olson, the main difference between the new strategy using a vector containing components for genome editing from other therapeutic methods is that it eliminates the cause of the disease. And what other approaches are scientists developing?


Figure 3. Animal models of Duchenne myodystrophy. a – Manifestations of Duchenne myodystrophy in mice and dogs.
Above: mdx mice show symptoms only in old age, and they are prone to the formation of rhabdomyosarcomas – tumors of muscular origin. The size of mice with atrophin/dystrophin and integrin/dystrophin gene knockouts is significantly smaller than their wild-type peers (BL10 and BL6). Below: manifestations of the disease in a five-month-old sick dog. Differences between healthy and sick two-year-old dogs. b – Comparison of the life expectancy of healthy and sick people, dogs and different lines of mice.
Figure from [17].

One promising approach is cell therapy. Although experiments with intramuscular injection of myoblasts from healthy donors have failed, technologies using stem cells and induced pluripotent stem cells (iPSCs) have so far been successfully tested on models of not only Duchenne myodystrophy, but also Alzheimer's, Parkinson's, Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, autism and schizophrenia [14-16]. For example, in 2013, researchers from Boston Children's Hospital's Stem Cell Program reprogrammed iPSCs from the skin of patients with Duchenne myodystrophy into muscle cells using a mixture of three small molecules (forskolin, the main fibroblast growth factor bFGF and a glycogen synthase kinase-3 inhibitor), which then successfully took root in mice. Cardiomyoblasts and neurons have now been obtained from IPSC [2].

Other studies show that restoring the normal level of nitric oxide (NO) synthesis, which decreases in patients due to impaired NO synthase activity (nNOS), weakens inflammation, increases the activity of their own stem cells and reconstructs the morphology and functions of skeletal muscles [3].

Givinostat, a histone deacetylase inhibitor that slows down the progression of the disease in a mouse model, is already in phase II clinical trials.

Such a massive experimental blow to Duchenne's myodystrophy is encouraging. Will the CRISPR/Cas9 technology become the leading one in the development of therapy that clinicians can adopt? Perhaps the publication of similar works on other diseases, where you need to get rid of mutations in a single gene, is not far off? We will learn this from the upcoming issues of Science (as well as other honorary journals).


  1. van Putten M., Hulsker M., Nadarajah V.D., van Heiningen S.H., van Huizen E., van Iterson M. et al. (2012). The effects of low levels of dystrophin on mouse muscle function and pathology. PLoS One. 7, e31937;

  2. Russo F.B., Cugola F.R., Fernandes I.R., Pignatari G.C., Beltrão-Braga P.C. (2015). Induced pluripotent stem cells for modeling neurological disorders. World J. Transplant. 5, 209–221;

  3. Falzarano M.S., Scotton C., Passarelli C., Ferlini A. (2015). Duchenne muscular dystrophy: from diagnosis to therapy. Molecules. 20, 18168–18184;

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  5. Bushby K., Finkel R., Birnkrant D.J., Case L.E., Clemens P.R., Cripe L. et al. (2010). Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care. Lancet Neurol. 9, 177–189;

  6. Long C., Amoasii L., Mireault A.A., McAnally J.R., Li H., Sanchez-Ortiz E. et al. (2016). Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 351, 400–403;

  7. Nelson C.E., Hakim C.H., Ousterout D.G., Thakore P.I., Moreb E.A., Castellanos Rivera R.M. et al. (2016). In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 351, 403–407;

  8. Tabebordbar M., Zhu K., Cheng J.K., Chew W.L., Widrick J.J., Yan W.X. et al. (2016). In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 351, 407–411;

  9. Elements: "The prokaryotic immune system will help edit the genome";

  10. Gallagher J. (2016). Scientists get ’gene editing’ go-ahead. BBC News;

  11. Biomolecule: "CRISPR systems: immunization of prokaryotes";

  12. Biomolecule: "Mutagenic chain reaction: genome editing on the verge of fiction";

  13. Biomolecule: "And whether we should take a swing at ... genome change?";

  14. Biomolecule: "Nobel Prize in Physiology or Medicine (2012): induced stem cells";

  15. biomolecule: "Alzheimer's disease: the gene I'm crazy about";

  16. Biomolecule: "How to save the Thirteenth? (Prospects for the treatment of Huntington's disease)";

  17. McGreevy J.W., Hakim C.H., McIntosh M.A., Duan D. (2015). Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Dis. Model. Mech. 8, 195–213;

  18. Singh S.M., Kongari N., Cabello-Villegas J., Mallela K.M. (2010). Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-β aggregates. Proc. Natl. Acad. Sci. USA. 107, 15069–15074;

  19. Goyenvalle A., Seto J.T., Davies K.E., Chamberlain J. (2011). Therapeutic approaches to muscular dystrophy. Hum. Mol. Genet. 20, R69–R78.

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