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Page 10 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

phosphodiester backbone has been replaced by the peptide linkage analog N-(2-aminoethyl)-glycine units,
[45]
which allows using convenient peptide synthesis technology . However, it has been reported that PNAs
[46]
had the disadvantage of rapid kidney elimination .
Finally, base modifications have been introduced, such as the base analog 5-methylcytosine, because this
increases nuclease resistance while reducing innate immune response [Figure 4D]. However, the risk of
genomic incorporation of these non-natural bases has hampered up to now their clinical use, except for 5-
methyl cytosine, while pseudouridine (Psi) is not used in RNA drugs but in mRNA vaccines to alleviate the
innate immune response against double-stranded RNA stretches.

More chemical refinements have been proposed to improve the pharmacokinetics and blocking properties
of ASOs. For instance, it was found that a mixture of LNA with 2’O-methyl and 2’F nucleotide together with
[47]
a PS backbone was most efficient in inhibiting miRNAs . Such mixed oligomers are called “mixmers” and
are schematized in Figure 5.

Other means to improve ASOs bioavailability to the desired tissue and cells imply nanoparticle
encapsulation (see section Type 1 myotonic dystrophy: different ASO modes of action), or covalent coupling
to a penetration enhancer or to a targeting moiety. Coupling morpholino nucleic acids to a peptide rich in
alanine and the cationic amino-acid arginine has been reported to increase tissue delivery and the efficacy of
exon skipping or exon restoration in models of Duchenne dystrophy and spinal muscular atrophy [48,49] .
Linking ASO to a fatty acid chain showed promising results in a spinal muscular atrophy model . The use
[50]
of triantennary N-acetyl-galactosamine (GalNac) for targeting and high-performance delivery to liver
hepatocytes via the asialoglycoprotein receptor (ASGPR) represents one of the most successful strategies,
both for ASOs and siRNAs. It has led to impressive therapeutic achievements [51-54] (see below Tri-GalNac
siRNA Vutrisiran for transthyretin hereditary amyloidosis treatment). Attempts to deliver ASOs through the
intestine and blood-brain barrier have been reported [55-56] .

Enhanced bioavailability and nuclease resistance are sufficient conditions for achieving the distinct ASOs
therapeutic mechanisms of cation illustrated in Figure 1: steric block of mRNA translation, microRNA
inhibition (antagomir effect), exon skipping, and exon restoration. Several FDA-approved drugs
demonstrate the success of these chemical modifications for treating rare diseases, which will be further
detailed below for muscular and neuromuscular diseases.

ASO chemical optimization for RNase H - induced mRNA cleavage
The necessity to maintain RNase H activity represents a major constraint that limits the use of many of the
chemical modifications presented above. Phosphorothioate linkages are compatible with RNase H activity.
Inversely, methylphosphonate substitution must be finely optimized. In a typical model study, duplexes
formed with deoxy oligonucleotides or phosphorothioate analogs were allowing mRNA cleavage by RNase
H, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides
alternating with normal deoxynucleotides was not permissive to RNase H attack. The mRNA susceptibility
to cleavage by RNase H increased in parallel to a reduction in the number of methylphosphonate
[38]
linkages .
Sugar modifications such as morpholino or LNA are not tolerated by RNase H. Uniformly modified 2'-
deoxy-2'-fluoro phosphorothioate oligonucleotides led to antisense molecules with strong binding affinity,
high selectivity for the RNA target, and stability towards nucleases, but they did not support RNase H
activity on mRNA. However, the incorporation of a mixture of these modifications into "chimeric"