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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 11 of 35

Figure 5. Different types of ASOs developed at the preclinical and clinical stages. Squares, circles, and assorted colors represent the
diverse types of nucleotides displayed in Figure 4. The linkage between each nucleotide might also vary, for instance, the
phosphorothioate Sp and Rp diastereoisomers. In a gapmer, the yellow circles represent non-modified nucleotides allowing cleavage by
RNase H of the annealed complementary mRNA.

oligonucleotides has been shown to activate mammalian RNase H-mediated degradation . Consequently, a
[57]
sizable proportion of RNase H-dependent ASOs are “gapmers,” in which a gap of 10 unmodified
deoxyribose is flanked by 3’ and 5’ “wings” whose partial composition in chemically modified nucleotides
leads to metabolic stability, enhanced binding to target, and cellular availability[Figure 5].

A large variety of gapmer geometries can be envisioned. A reduced gap size confers more precision in the
cleavage zone and potentially contributes to allele specificity. Different flanking wings have been proposed
to increase affinity and selectivity to the target RNA. However, care should be taken that the cleaved mRNA
must dissociate to initiate another mRNA degradation. Thus, ASOs’ affinity for the mRNA target must not
be too high. For instance, with LNA-containing wings, an increased binding affinity has been reported,
[58]
which leads to an optimal size of 12 to 15 nucleotides . On the opposite, the clinically approved gapmer
Inotersen for treating transthyretin amyloidosis is a fully phosphorothioate-modified ASO with five 2’-O-
methoxyethyl ribonucleotides on each side, thus consisting of a 5-10-5 structure) . In addition, a too strong
[7]
affinity might induce off-target binding and RNase H cleavage of mismatched mRNAs (see section
Challenges faced by the synthetic ASO and siRNA technology). Due to a high ASO concentration observed in
the liver, off-target hepatic toxicity has been reported for 2’Fluoro or LNAs [59-61] .

Chemical and delivery optimization of siRNAs
Contrary to ASOs which might be efficient as steric blockers with no concomitant nuclease activity, siRNAs
efficacy strictly depends on argonaute 2 nucleolytic activity. As for RNase H-dependent ASOs, the
modifications introduced for increasing metabolic stability are limited and must be finely optimized for the
guide siRNA strand. More freedom for modifications is allowed on the passenger sense strand, which is not
involved in the RISC-guided nuclease activity. An additional constraint to be considered is to limit the
innate immune response induced by natural RNA duplexes, which has initially represented a major obstacle
to the development of the technology.

A useful mapping of the functional domains of a typical siRNA gives guidance on where chemical diversity
can be introduced [Figure 6A]. While most siRNA designs are based on a non-covalent two strands double
helix [Figure 6A and 6B], corresponding to the presently clinically approved RNA drugs (see Chemical and
delivery optimization of siRNAs), other concepts have been proposed and shown to possess promising
properties at least at the preclinical level, such as di-siRNA composed of two double-helical siRNAs made of
one double-sized passenger strand and two guide strands Figure 6C . The geometry developed by the
[62]
DIcerna company is that of a nicked hairpin [Figure 6D]. While the guide antisense strand is of 22 bases
canonical length, the sense passenger strand is longer (36 bases), and it auto-hybridizes through a GC-rich
sequence (GCAGCC hybridized to GGCUGC) to form a GAAA loop at its extremity. Hence, a nick of the