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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 3 of 35

Figure 1. Non-RNase H-dependent ASO steric blocking effects. In yellow is indicated the complementary targeted sequence on the
mRNA. ISS: Intronic splice silencer. A: mRNA Translation inhibition; B: Antagomir; C: Inducing skipping of a pathologic exon carrying
stop or out-of-frame mutations; D: Splice modulator inhibiting a non-desired naturally occurring exon skipping.

antisense blocking effect on mRNA translation might be responsible for part of the observed decreased
protein expression, it was later found that the loss of protein expression was also due to mRNA degradation
induced by RNase H nuclease. This RNase specifically recognizes heteroduplexes and cleaves the associated
mRNA. This nucleolytic mechanism of action drives most of the actual ASOs’ clinical applications.

Antisense ASOs have the potential to induce numerous other steric blocking effects, which has led to major
therapeutic successes. These are schematized in Figure 1.

MicroRNA (miRNAs) are a group of one to two thousand small non-coding RNA molecules containing 21
to 23 nucleotides. They play important biological functions as post-transcriptional regulators of gene
expression. The miRNAs anneal to complementary sequences on mRNA molecules, leading to gene
silencing by several mechanisms: mRNA cleavage mediated by the RNA-induced silencing complex RISC
(see below the detailed section on RNA interference), mRNA destabilization by poly(A) tail shortening, or
blocking of mRNA translation. As shown in Figure 1B, ASO steric blocking can antagonize these cellular
“bandmasters”, and thus display an antagomir action, which presents interesting therapeutic applications in
neuronal and neuromuscular diseases .
[11]
An antisense blocker ASO can be used by sterically hindering a splice acceptor or splice enhancer site, thus
promoting exon-skipping [Figure 1C]. This mechanism of action is of great interest for treating genetic
diseases caused by stop or out-of-frame missense variants, and where the skipping of one or o several exons
leads to a still functional or partially functional truncated form of the protein product. This strategy has led
to clinically approved ASO RNA drugs for Duchenne Dystrophy. Exon skipping can also be of interest in
cases where a pathologic alternative splicing of variant genes occurs, which leads to the inclusion of an
additional exon, causing the expression of a non-functional protein. Finally, blocker ASOs can also treat
cases where an intronic pathogenic variant results in aberrant inclusion of an intron segment into the
mRNA transcripts, thus abolishing protein function.

Figure 1D displays the use of an ASO to inhibit a naturally occurring undesired splicing event leading to
exon N skipping and translation of a non-functional truncated protein. By blocking this undesired exon
skipping, therapeutic restoration of a complete functional protein is obtained. As will be described in Spinal
muscular atrophy, this occurs in the case of spinal muscular atrophy (SMA), where a non-functional