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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 7 of 35

Figure 2B and Figure 3, left side]. Since the guide sense is designed for a perfect match with its
complementary sequence on the targeted mRNA, the Ago-mediated mRNA cleavage is complete.

Once this has occurred, the RISC /guide siRNA complex can be used again to target and cleave another
pathologic mRNA. This recycling process associated with optimized chemistry enabling high siRNA
metabolic stability ensures a long duration of action . Indeed, in vivo effects have been reported for the
[22]
most recently developed siRNA drugs with a spectacular duration of action superior to 6 months after a
single administration .
[23]

Comparison between ASO and RNAi properties
The differences between natural phosphodiester ASOs and siRNAs are summarized in Table 1.

The ASO and siRNA characteristics and specificities listed in Table I are responsible for marked differences
in physicochemical and pharmacokinetic properties which dictate different required chemical optimization,
depending on the nature of the drug and the targeted tissue. A single-stranded ASO is more flexible and
accessible to an endonuclease and thus necessitates complete protection of each phosphodiester linkage
against nucleases. By contrast, a double-helix siRNA shows more resistance in its internal phosphodiester
linkages. A single-stranded ASO also displays hydrophobic moieties from its nucleic bases, which is not the
case for double-helical siRNA. Hence, ASOs will bind to plasma proteins such as albumin, which leads to
longer circulation time and enhanced biodistribution to tissues. On the contrary, natural siRNAs are rapidly
eliminated by kidney filtration and need a delivery vector such as a lipid nanoparticle (LNP) formulation or
a functional targeting moiety towards an extracellular receptor.

Other differences originate from the fact that only binding to the target mRNA is required to achieve a
blocker ASO function, as for mRNA degradation, the catalytic activity of RNase H (for ASOs) and of
Argonaute 2 (for siRNAs) must be maintained. Finally, it is generally considered that ASOs can block or
cleave both pre-mRNA and mRNA either in the nucleus or cytosol, while siRNA ensures the degradation of
the mature mRNA in the cytosol only. This represents an important point for diseases caused by nuclear
aggregation of variant mRNA, such as Myotonic Dystrophy Type 1 (MD1) (see section Type 1 myotonic
dystrophy: different ASO modes of action). This is, however, a disputed point, because several siRNAs and
shRNAs have been shown to lead to nuclear foci degradation in MD1 cellular and animal models [24-26] thus
suggesting that the RISC-induced cleavage and subsequent degradation could also occur in the nucleus.

Chemical optimization of steric blocker ASOs
After their initial discovery, the first enthusiastic attempts towards the clinical use of both ASOs and siRNAs
were very disappointing, leading to a drastic decrease in the investment into these genetic pharmacology
drugs. These initial approaches used natural oligonucleotides, which have poor pharmacokinetics because
they undergo fast degradation by endo- and exonucleases. They are degraded in less than 20 minutes after
intravenous administration. In addition, these first-generation ASOs and siRNAs were rapidly eliminated by
kidney filtration. This decreases tissue biodistribution and might induce undesirable glomerular toxicity
resulting from temporary elevated local concentrations of these biologically active agents. Moreover, natural
oligonucleotides are small hydrophilic polyanionic compounds due to the phosphodiester linkages, which
hinder cell penetration through the lipophilic plasma membrane. Finally, innate immune response to
double-stranded RNA, especially through the TLR3 receptor [27-30] , was found to represent a strong bottleneck
for the development of siRNA drugs. All these considerations led to the conclusion that chemical
modifications were required for the ultimate success of an ASO or siRNA drug.