Update for Duchenne Muscular Dystrophy -
Hope for Causal Treatment of a Disorder
Presenting in Early Childhood?
Gerhard Franz Walter*
International Neuroscience Institute Hannover, Germany
Submission: March 31, 2022; Published: May 11, 2022
*Corresponding author: Gerhard Franz Walter, International Neuroscience Institute Hannover, Germany
How to cite this article:Walter GF. Update for Duchenne Muscular Dystrophy - Hope for Causal Treatment of a Disorder Presenting in Early
002 Childhood?. Acad J Ped Neonatol 2022; 11(4): 555872. 10.19080/AJPN.2022.11.555872
Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease that affects 1 in 3,600 to 6,000 males. DMD is not curable until today. In this mini-review, myoblast transplantation and stem cell therapy, read-through therapy, antisense oligonucleotide-mediated therapy, vector-mediated gene therapy, and CRISPR/Cas9-mediated gene editing are shortly addressed, approaches which alone or combined have the potential for causal treatment of DMD.
The dystrophin gene (DMD gene) is the largest human gene. It is located on chromosome Xp21.2-p21.1 and comprises more than two million base pairs with 89 exons. Mutations in the dystrophin gene lead to a lack of dystrophin protein. “Big mutations” of the structural cytoskeletal dystrophin gene comprise deletions in which parts of the dystrophin gene are lost, or duplications in which parts of the dystrophin gene are doubled. Rarer “small mutations” are nonsense mutations with substitution of a single base-pair, thereby leading to the appearance of a stop codon where previously there was a codon specifying an amino acid; this premature stop signal terminates the production before the entire dystrophin protein is formed.
The dystrophin protein, in short dystrophin, is a subsarcolemmal rod-shaped protein with four functional domains, the actin-binding N-terminal domain, the central rod domain, the cysteine-rich domain, and the C-terminal domain [1-3]. Dystrophin is localized at the sarcolemma via the dystrophin-associated protein complex . Dystrophin serves as a sort of shock absorber or stabilizer of the muscle fibers by linking the cytoskeleton to the extracellular matrix to allow the transmission of force from the contractile elements inside the cell to extracellular matrix structures, while at the same time maintaining the integrity of the muscle fibers. Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. Its absence leads to dystrophinopathies, which were recently comprehensively reviewed .
Dystrophinopathies, dependent from site and extent of mutation, cover a spectrum of mild to severe dystrophies of the skeletal muscle. The effects of gene mutations are primarily determined by whether the variants disrupt the reading frame downstream of those mutations. In case of out-of-frame mutations, the open reading frame of the dystrophin gene is disrupted, leading to dystrophin deficiency and a severe phenotype presenting as Duchenne muscular dystrophy (DMD). Meanwhile, in-frame mutations preserve the reading frame, generating partially functional dystrophin protein and leading to less severe Becker muscular dystrophy (BMD). When the heart is primarily affected, an associated dilated cardiomyopathy (DCM) may occur . Mild forms present with asymptomatic increase in serum concentration of creatine phosphokinase (CK) and muscle cramps with myoglobinuria.
Dystrophinopathies are inherited in an X-linked manner. The risk depends on the genetic status of the mother. Heterozygous females (carriers) have a 50% chance of transmitting DMD in each pregnancy. Sons who inherit the pathogenic variant will be affected; daughters who inherit the pathogenic variant are heterozygous and may have a range of clinical manifestations.
Males with DMD usually do not reproduce. Carrier testing for atrisk
females, prenatal testing, and preimplantation genetic testing
are possible, if DMD in the family is known. DMD presents in early
childhood and occurs in one of 3,600 to 6,000 newborn boys.
Around 10% of female carriers can have symptoms of the disease.
In 30% of cases, there is no known familiar predisposition.
DMD causes delays in walking independently and standing
up from a supine position. Proximal weakness causes a waddling
gait and difficulty climbing stairs, running, jumping, and standing
up from a squatting position. DMD is rapidly progressive, with
affected children being wheelchair dependent about by age 12
years. Cardiomyopathy occurs in almost all individuals with
DMD after age 18 years. Few survive beyond the third decade,
with respiratory complications and progressive cardiomyopathy
being common causes of death due to left ventricular dilation and
congestive heart failure.
The diagnosis of a dystrophinopathy should be suspected in
any patient with a highly elevated creatine kinase level beyond
the context of rhabdomyolysis secondary to toxic or metabolic
myopathy and is established with the characteristic clinical
findings and elevated CK concentration and/or, on molecular
genetic testing, by identification of a hemizygous pathogenic
variant in the dystrophin gene in a male and of a heterozygous
pathogenic variant in a female. Females may – rarely – present with a classic dystrophinopathy or may be asymptomatic carriers.
Genetic testing for dystrophinopathy is highly sensitive and
specific, and identifying a proband will often lead to implications
for several relatives at risk for cardiomyopathy, weakness, or
anesthetic reactions . The genetic testing involves a deletions/
duplications analysis performed by quantitative technique such
as microarray-based comparative genomic hybridization (array-
CGH), multiple ligation probe assay (MLPA). Since traditional
methods for detection of point mutations and other sequence
variants require high cost and are time consuming, especially for a
large gene like dystrophin, the use of next-generation sequencing
(NGS) has become a useful tool available for clinical diagnosis
. However, genetic testing of DMD identifies a certain number
of variants of uncertain clinical significance (VUS). Recently,
as first systematic characterization of DMD splicing variants,
computational tools to prioritize VUS and a cell-based minigene
assay to confirm aberrant splicing were developed, whereby rare
and hitherto undetermined variants in exon and intron 10 of the
DMD gene were evaluated .
In muscle biopsy, the affected muscle tissue undergoes necrosis
of single muscle fibers, which were invaded by macrophages and
cleared away. The muscle fibers are replaced by connective tissue.
A lack of subsarcolemmal dystrophin can be documented (Figure
1). In the end stage of DMD, most muscle cells have been removed
and, during the time, the muscle tissue is almost entirely replaced
by functional fat and connective tissue (Figure 2).
DMD is not curable until today, but it is possible to slow
down the course of the disease and improve the quality of life.
Management is focused primarily on supportive care, and steroid
therapy is now standard-of-care. Oral corticosteroids improving
muscle strength and function have changed the natural history
of DMD, prolonging ambulation in affected boys between ages 5
and 15 years and reducing the risk of developing scoliosis and
subsequent surgical correction, improving cardiac health, and
increasing long-term survival [5,10].
Approaches to a causal treatment
Intensive research is focused on the potential for altering gene
expression and gives hope for a future causal treatment of DMD.
The dystrophinopathies represent model diseases to understand
the personalization of genetic treatment. The many details
regarding current approaches would exceed the given frame of
a mini-review, therefore, two recent extensive overviews are
recommended, also documenting details on current clinical trials
Myoblast transplantation and stem cell therapy
Almost 40 years ago this still interesting cell transplantation
approach was proposed. In mice, transplanted myoblasts fuse
with host myofibers and express donor genes. Subsequent
experiments verified the presence of dystrophin in treated animals
[15,16]. However, Western blot of patient biopsies indicated a
low percentage (0–5%) of dystrophin expression, which can
be partially explained by immune rejection or low survival of transplanted myoblasts . Human induced pluripotent stem
cell (iPSC) is another tempting cell source for reconstructing
dystrophic muscles. iPSCs cultured with cytokines and growth
factors were committed to myogenic linage . Recently, human
Wharton jelly mesenchymal stem cells with high expression
of Aurora kinase A were explored and might have therapeutic
efficacy against DMD .
To treat nonsense mutations, read-through therapies utilize
small molecules to interact with ribosome, which leads to
insertion of an alternative amino acid at the point of premature
termination codon to allow translational read-through, so that a
relatively functional dystrophin protein could be generated .
Antisense oligonucleotide-mediated therapy
Another approach for the treatment of DMD is antisense
therapy. Antisense oligonucleotides (AONs) alter pre-messenger
RNA splicing to promote exon exclusion, as in eteplirsen in DMD
which targets specific mutations and can be used to treat about
14% of DMD cases . AON-mediated exon skipping therapy
can be applied for the treatment of about 55% of DMD patients
by targeting affected exons with predesigned AONs to produce
a shorter but working version of dystrophin [22,23]. A new
approach is the conjugation of AONs with lipophilic compounds
to explore the influence of the lipophilic moiety on exon skipping
efficiency in DMD .
Vector-mediated gene therapy
Conceptually, replacing the mutated gene with a normal one
would cure the disease, but this task has encountered significant
challenges due to the enormous size of the dystrophin gene and
the distribution of muscle throughout the body. The former creates
a hurdle for viral vector packaging and the latter begs for wholebody
therapy such that all muscles are rescued. To address these
obstacles, a promising approach for treating DMD is gene transfer
of the highly abbreviated micro-dystrophin to restore dystrophin
expression, therefore using body-wide systemic gene transfer
with non-pathogenic adeno-associated viral vector (AAV). Studies
in transgenic mice had revealed that highly functional microdystrophin
cassettes could be generated with a size less than 4 kb
. Improved vectors could be generated from newly discovered
AAV serotypes (such as AAV6, 8 and 9) which, when injected into
the vasculature at high dose (in the range of 1014 vector genomes
per kilogram) could transduce all the striated muscles in adult
mice . Whilst micro-dystrophin gene transfer using AAV
vectors shows therapeutic success in large animal models of DMD,
translating this promising therapy from bench to bedside still
offers scope for many optimization steps [27,28].
CRISPR/Cas9-mediated gene editing
To obtain permanent dystrophin expression in DMD patients,
gene editing, particularly CRISPR/Cas9-based gene editing, has
been adopted to correct genomic deficits. The CRISPR/Cas9 system
consists of the endonuclease Cas9 and a single-guide RNA (sgRNA).
The Cas9 endonuclease produces DNA double-strand break at the
editing sequence targeted by sgRNA. Subsequent nonhomologous
end-joining leads to exon-skipping while homology-directed
repair could replace dystrophin gene mutations with correct
sequences and generates normal dystrophin protein .
Thus, adeno-associated virus (AAV)-mediated CRISPR
editing holds promise to restore missing dystrophin in Duchenne
muscular dystrophy (DMD). Intramuscular co-injection of Cas9
and sgRNA vectors resulted in robust dystrophin restoration in
short-term studies in the mdx mouse model of DMD. Intriguingly,
this strategy failed to yield efficient dystrophin rescue in muscle
in a long-term (18-month) systemic injection study which was
obviously caused by a selective loss of the sgRNA vector. It could
be shown that the sgRNA vector dose determines the outcome of
systemic AAV CRISPR therapy for DMD .
A multimodal approach might be hopeful. Sun et al. (2020)
emphasize that gene therapy combined with cell transplantation
and tissue engineering has the potential to lead to life-changing
therapy for DMD, which possibly is also applicable for other types
of muscular dystrophies including Becker muscular dystrophy
and distal muscular dystrophies. In summary, there are promising
novel genetically mediated therapies on the horizon, which point
towards a hopeful future for individuals with DMD.