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Page 20 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

Figure 10. A: RNAi encapsulated in LNPs are protected from the actions of nucleases and targeted to the liver. LNPs are specifically
targeted to the liver because they bind apolipoproteins E in the blood circulation and then bind to the ApoE-LDL receptor on the surface
of the hepatocytes. B: LNP decorated with Apo E bind to Apo E receptor on the hepatocyte plasma membrane. C: DLin-MC3-DMA
lipids at the LNP surface are neutral at pH 7. Inside the endosome, DLin-MC3-DMA becomes cationic, which favors either fusion with
the endosome membrane and siRNA release or endosome breakage caused by the “proton pump” osmotic effect.

dependent endocytosis mechanism. Inside the endosome, it is postulated that the ionizable DLin-MC3-
DMA lipid becomes cationic because of endosomal/lysosomal acidification. This induces endosome
breakage, for which two different mechanisms have been proposed.

1) In the first one, the ionizable lipid captures and titrates the H+ ions taken up into the endosomes by the
endolysosomal proton pump ATPase. This induces the sustained uptake of a large number of protons and
chloride anions which diffuse into the endosomes to equilibrate the proton cationic charges. This “proton
sponge” effect induces endosome swelling and breakage.

2) According to the alternative proposed mechanism, the ionizable DLin-MC3-DMA lipid at the LNP
surface becomes cationic in the acidic endosomal compartment, leading to the fusion of the positively
charged LNP surface with the negatively charged endosome membrane.

Both postulated mechanisms result in siRNA release into the cytosol and further interaction with the RISC
complex.

Patisiran was granted orphan drug status, fast track designation, priority review, and breakthrough therapy
designation due to its novel mechanism and the rarity of the condition it is treating. It was approved for
medical use in USA ad EU in August 2018. However, with the development of other transthyretin
amyloidosis treatments, such as Inotersen and Vutrisiran which is accumulated into the liver through a
triGalNac N-acetylglycosamine targeting moiety, there is now intense competition between these different
RNA drugs.

Tri-GalNac siRNA Vutrisiran for transthyretin hereditary amyloidosis treatment
Vutrisiran (AMVUTTRA™) is a subcutaneously administered TTR-specific siRNA developed for hATTR
and wtATTR treatment. Vutrisiran was FDA approved in 2022 for the treatment of hATTR amyloidosis
with polyneuropathy in adults, following fast-track designation in 2020 given the very promising efficacy of
this RNA drug.

The Vutrisiran sequence was designed to bind to a conserved sequence on all TTR mRNA variants. Because
Vutrisiran is administered as a naked siRNA, i.e., not associated with a lipid nanoparticle delivery system,
the drug must be metabolically protected and targeted to hepatocytes. Nuclease resistance has been achieved
by introducing much more modifications in Vutrisiran than in Patisiran. Indeed, while Patisiran contains