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Page 14 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

DMD belongs to the larger group of rare genetic progressive muscular dystrophies called
dystrophinopathies, which also include Becker muscular dystrophy (BMD) and a symptomatic form in
female carriers. Dystrophinopathies present a spectrum of severity ranging from progressive skeletal and
cardiac muscle wasting and weakness (DMD, BMD) to less severe muscle weakness or isolated
cardiomyopathy affecting carrier females. At the mildest end of the spectrum exercise-induced muscle
cramps and myoglobinuria may be the only feature, while at the severe end, there may be a complete loss of
muscle function, cardiomyopathy, and respiratory failure. BMD presents a mild phenotype and a broad
spectrum of clinical severity, with the onset of symptoms occurring from early childhood to as late as sixty.
A very severe, rapidly progressive, X-linked dilated cardiomyopathy (code Orphanet 262) may also be
[77]
caused by mutations in the dystrophin gene .

Muscle damage in DMD is caused by the complete absence of the cytoplasmic sarcolemmal protein
dystrophin, which participates in a complex connecting muscle fiber cytoskeleton to the surrounding
extracellular matrix through the cell membrane. Dystrophin protects myotubes’ integrity during muscle
contraction . Dystrophin possesses a central rod domain of twenty-four spectrin-like repeats. Its primary
[79]
muscular transcript measures about 2,100 kilobases. The mature mRNA, which is formed by the junction of
seventy-nine exons, measures 14.0 kilobases and encodes a protein of 3,685 amino acid residues. Hence,
[80]
intensive splicing is necessary for dystrophin biosynthesis .
Dystrophinopathies are allelic conditions caused by deletions, duplications, and mutations in the dystrophin
gene. While DMD genetic variants are frameshift, BMD variants are in-frame. While severe DMD results
from dystrophin complete absence, the moderate BMD form is only observed when one or several of the
spectrin-liked repeats is missing. Deletions identified in DMD patients are shifting the translational open
reading frame (ORF), thus resulting in a non-completed abnormal protein product of which the COOH
terminal fragment is either non-functional or lost. On the opposite, BMD patients’ deletions maintain the
in-frame translational ORF for amino acids and predict a shorter protein of lower molecular weight. This
indicates that the smaller protein product devoid of a certain number of internal spectrin-like domains
remains semi-functional, resulting in the milder Becker clinical phenotype [81,82] . From this, it was predicted
that, for some DMD patients, an in-frame skipping of the exon containing a nonsense mutation or deletion
could have a therapeutic value. Figure 7 illustrates an exon skipping obtained by the binding of a steric
blocker ASO targeting an intronic splice acceptor or donor site or an exonic splice enhancer site.

Given the fact that several thousands of variants have been reported for DMD patients, a search identified
which in-frame exon skipping was favorable to treat a high proportion of patients. As shown in Figure 8,
skipping exon 51 results in an in-frame skipping and leads to a partially functional protein in several
deletion scenarios. Such partially internally shortened proteins would contain the following junctional
exons, respectively: 50-53, 49-52, 49-52, 48-52, 47-52, 46-52, and 44-52. The deletions displayed in Figure 8
correspond to about 15 to 17% of the DMD population [83,84] .

An elegant in vivo exon skipping proof of concept leading to the expression of a partially functional
dystrophin molecule devoid of a certain number of spectrin-like domains was obtained by using an AAV
viral vector delivering an antisense moiety born by a U7 or U1 small nuclear RNA [85,86] . The muscle force
recovery was restored in these experiments. These very promising results prompted the development of
synthetic steric blockers ASOs. It has led to the drug Eteplirsen (AVI-4658; Exondys 51), which has been
approved by FDA but not by EMA, indicating that its efficacy is a matter of debate . Eteplirsen is a thirty
[87]
m o r p h o l i n o n u c l e o t i d e o l i g o m e r d e l i v e r e d i n t r a v e n o u s l y . I t s s e q u e n c e i s
CTCCAACATCAAGGAAGATGGCATTTCTAG. Other commercialized ASOs for DMD are skipping