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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 13 of 35

phosphorothioate linkages are introduced in the 5’ and 3’ ends of both strands, which ensures sufficient
metabolic stability against exonucleases. Another backbone modification has been recently introduced in
the form of a divalent siRNA, in which two passenger moieties are covalently linked. These di-siRNAs
display a favorable distribution in the central nervous system and promising efficacy in neurodegenerative
disease models in rodent and non-human primates .
[62]
While the maximum number of 2’fluoro must be controlled because of potential toxicity, other
modifications have proven their utility, such as 5’ carbon pyrimidines. Moreover, using a systematic
iterative screening technology, it has been shown that optimizing the positioning of 2'-deoxy-2'-fluoro and
2'-O-methyl ribose across both strands enhanced metabolic stability. This could be obtained with a low 2'-
[68]
deoxy-2'-fluoro content .

Numerous formulations have been proposed to improve siRNA pharmacokinetics, such as lipid
nanoparticles (LNP), a detailed reviewing of which is out of the scope of the present review. Lipid
nanoparticles have proven their efficacy in targeting the liver in vivo, leading to the clinically approved
Patisiran siRNA drug in transthyretin amyloidosis [69,70] . While liver targeting is presently well mastered using
either LNP or the GalNac technologies (seeExon-skipping ASO for Duchenne muscular dystrophy),
challenges remain for other organs, particularly the brain. Nonetheless, progress is being made in terms of
oral and ocular delivery [71-73] and intravenous (IV) delivery to inflammatory sites [74-76] .

The following sections illustrate the above concepts by a selection of typical examples of ASOs and siRNAs
approaches for treating muscular and neuromuscular disorders, and for which either marketing approval or
very promising results have been obtained. Since any genetic disease caused by a dominant negative variant
might benefit from an RNase H-dependent ASO or a siRNA approach, and since many others might benefit
from anti-miRNA or splice modulation properties, this review can by no means be fully exhaustive
concerning the ongoing preclinical studies.

Exon-skipping ASO for duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is caused by anomalies in the dystrophin gene located on the X
chromosome (Xp21.2). Because the dystrophin gene is the largest known gene in the human genome,
genetic variants and deletions occur at a higher frequency than in other genes, and DMD has one of the
highest prevalence rates among rare diseases (about 6/100,000). Diagnosis is suspected based on the clinical
picture, family history, and laboratory findings (serum creatine kinase being 100-200 times the normal
[77]
level). Genetic testing is a critical tool for accurate DMD diagnosis (Orpha 98,896) .
DMD onset occurs in early childhood, and affected boys may show a delay in walking accompanied by
speech and/or global developmental retardation. Autism and behavioral problems, such as ADHD
(attention deficit hyperactivity disorder), anxiety, and obsessive-compulsive disorder, are common.
Untreated DMD children rarely achieve the ability to run or jump. The condition progresses rapidly, and
the child develops a waddling gait and a positive Gowers sign. Proximal muscles are affected first, then
distal limb muscle. Climbing stairs becomes difficult and the child falls frequently. Loss of independent
ambulation occurs between the ages of 6 and 13 years, the average being 9.5 years in non-steroid treated
patients. Once ambulation is lost, joint contractures and scoliosis develop rapidly. Until recently, untreated
patients might not survive over late teens to early twenties because of respiratory failure and/or
[78]
cardiomyopathy, but life expectancy is increasing with adapted cardiac care and assisted ventilation .