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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 21 of 35

only eleven 2’O-Me nucleotides and only phosphodiester linkages, Vutrisiran is a heavily modified siRNA
with 6 phosphorothioate linkages at the end of each strand, 35 nucleotides carrying a 2’OMe ribose and 9
nucleotides modified with a 2’F ribose. Thus, the two Vutrisiran strands (one of 21 nt and one of 23 nt) are
fully modified [116,117] . Optimization of the number and respective positions of these modifications has been
performed in view of reducing toxicity, particularly hepatotoxicity [19,46,54,116,117] .

The most innovative feature of Vutrisiran is the functionalization of the sense passenger strand with a very
efficient hepatocyte targeting moiety, the tri-N-acetyl-galactosamine (tri GalNac). The triGalNac head binds
with high avidity to the ASGPR, a lectin that is present at a remarkably high density on the hepatocyte
surface (500,000 ASGPR per cell). Binding to the ASGPR actively promotes the cellular uptake of the ligand
upon clustering and aggregation of several receptors in coated pits. The ASGPR turnover is very fast since
only about 5%-10% of ASGPR are permanently accessible at the cell plasma membrane. After endocytosis,
ASGPR recycling occurs in ~15 min concomitantly with the release of the bound ligand in the acidified
endosomal compartment, in a process analog to that of the transferrin receptor cycle. Hence, high ASGPR
density on hepatocyte surface and fast turnover points Tri GalNac as an ideal targeting head candidate for
[118]
any liver-targeted RNA drug . The fact that more than 80% of an IV injected compound expressing
galactose or galactosamine is taken up by the liver has been evidenced by SPECT or luminescent imaging
techniques using lactosylated albumin [119,120] .

As shown in Figure 11, the tri-GalNac is covalently linked to the 3’ end of the siRNA sense strand, so as not
to interfere with the binding of the complementary antisense to RISC. The improved metabolic stability of
Vutrisiran and the very potent targeting efficacy of its tri-Gal-Nac lead to an exceptional intrinsic efficacy of
Vutrisiran. This is demonstrated by the therapeutic efficacy of very low doses and the exceptionally long
duration of action because a quarterly regimen is sufficient to achieve a similar therapeutic effect as
Patisiran [121-125] . Both polyneuropathy, cardiomyopathy, and wt-TTR are envisioned as Vutrisiran
indications.

As compared to the LNP formulation used for Patisiran, GalNac-siRNA conjugate allows simpler GMP
preparation and storage conditions, and a more convenient administration protocol (SC versus IV). In
addition, slow diffusion to capillaries through the extracellular conjunctive tissue creates a “depot” effect
which, together with increased metabolic stability, allows less frequent administration. Remarkably, the last
generation of GalNac siRNAs carrying a phosphate triglycan-end group allows a regimen as distant as twice
a year for multiple indications requiring silencing of a liver-expressed protein. A one-year duration after a
single dose has been reported for Cemdisiran, which has strong potential in the treatment of complement-
mediated diseases such as paroxysmal nocturnal hemoglobinuria (PNH). This is a historically never
achieved performance. Thus, the GalNac targeting technology represents actually the most promising
approach for siRNAs, as illustrated by the fast development of Givosiran (FDA approved in 2019 for adults
with acute hepatic porphyria), Lumasiran (FDA approved in 2020 for hyperoxaluria type 1), and Nedosiran
(in phase III for primary hyperoxaluria [116,123] . Similarly, the GalNac targeting technology is also being
pursued for ASO liver targeting [126,127] .

Type 1 myotonic dystrophy: different ASO modes of action
Myotonic dystrophy (also called dystrophic myotonia) is a multisystemic disease with a frequency of 1/8000
worldwide. The most severe form (DM1) includes symptoms such as myotonia, muscle weakness, cardiac
arrhythmias, cognitive dysfunction, and cataract. The genetic cause of DM1 comes from the repeated
expansion of a CTG triplet motif in the DM Protein Kinase (DMPK) gene. Normal DMPK gene contains 5
to 37 repeats, while variant DM1 DMPK gene might contain up to thousands of CTG repeats, and the