Page 21 - Read Online

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

Figure 6. A: The siRNA functional regions. More modifications are tolerated on the passenger strand (red squares) which is not
involved in argonaute 2-mediated mRNA cleavage. A targeting moiety can be covalently linked to the 3’ strand (orange arrow). A stable
phosphonate resistant to dephosphorylation enzymes can be included instead of phosphate (blue rod) at the 5’end of the guide siRNA
(green square). Both are necessary and compatible for binding the guide strand to the RISC complex. Fewer modifications are allowed
on the seed region of the guide siRNA strand (nucleotides 2 to 8). An optimized guide strand must contain a flexible 5′ end (which can
be obtained by lowering base pairing and facilitates capture by the RISC complex), a high affinity ‘seed’ region, which drives the initial
base pairing between the guide strand and mRNA target, and a lower affinity 3′-region required for cleaved mRNA release. B: Frequently
used siRNAs are made resistant to exonucleases by 2 to 3 terminal phosphorothioate linkages on both 3’ and 5’ terminals of each
strand. Modified sugars 2’-OMe and 2’-fluoro have led to siRNAs highly resistant to nuclease, of improved bioavailability, and of high
binding affinity to the RISC complex. This leads to an unprecedented duration of action which can reach up to 6 months or more after a
single administration, due to the recycling mechanism displayed in Figures 4 and 5 inspired from [18,29] . C: Scheme of a di-siRNA [62,145] . D:
In this presented geometry, the antisense strand is of 22 bases canonical length, while the sense passenger strand is much longer (36
bases), and it auto-hybridizes through a GC-rich sequence (GCAGCC hybridized to GGCUGC) to form a GAAA loop at its extremity.

hairpin structure is located between the sense and antisense strands [63,64] . Targeting moieties such as GalNac
sugars have been linked to the GAAA loop, thus providing a high tetravalent sugar moiety for targeting liver
hepatocytes.

The presence of a 5’ phosphate on the siRNA guide strand is an essential factor for entry into the RISC
complex and loading to the Ago2 nuclease. Stable phosphate analogs, such as phosphonate, have been
introduced with a strong enhancing effect [Figure 6A] . A non-cleavable targeting moiety can be linked to
[65]
the passenger strand. With a tri-antennary GalNac targeting head that binds to the hepatocyte
asialoglycoprotein receptor, a dramatic increase in liver uptake and silencing efficiency has been
observed [66-67] , leading to several months silencing effect after a single dose. This represents one of the most
exciting perspectives of siRNA therapeutics for liver-associated diseases (see Exon-skipping ASO for
[53]
Duchenne muscular dystrophy).

While 2’fluoro ribose modification is well tolerated by the RISC machinery, phosphodiester linkage
substitution by phosphorothioate can only be introduced in a limited number, and this is outside the seed
region and cleavage site. Similarly, sugar modifications such as 2’OMe or LNA are more tolerated on the
passenger strand, and they are favored at the 5’end because they block passenger strand entry into the RISC
complex and consequently favor RISC exclusive loading with the guide strand. Two typical popular siRNA
geometries are displayed in Figure 6B. In the upper siRNA, 2’fluoro and 2’OMe are intercalated and face
each other to obtain a canonical alpha helix geometry. In the second more widespread example, stretches of
3 consecutive 2’fluoro and 2’OMe nucleotides are present and frequently facing each other. Two to three