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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 19 of 35

The ASO Inotersen (Tegsedi) is a 20-mer gapmer similar to that represented in Figure 5. It leads to the
degradation of TTR mRNA, with minimal off-target effect. It is fully PS-modified (phosphorothioate
backbone), with 5 ribose on both 5’ and 3’ends carrying 2’MOE (2’O-methoxyethyl) moieties [Figure 4C].
The 10 central nucleotides have a natural ribose. The nucleotidic sequence of Inotersen is
UCUUGGTTACATGAAAUCCC, with the nucleotides UCUUG and AUCCC carrying a 2’OMOE. As for
Nusinersen, all cytosines and uracil of Inotersen are 5’-methylated (uracil replaced by thymine and cytosine
by 5-methyl cytosine). The Inotersen IUPAC formula is detailed in Table 2.

After subcutaneous (SC) injection, Inotersen leads to a robust decreasing effect on the level of both variant
and wild-type transthyretin. It clearly inhibits TTR production and slows down the progression of the
[107]
[7]
disease but does not seem to induce any reverse effect on already-formed amyloid aggregates . Reported
Inotersen adverse events include thrombocytopenia. This could be linked to the presence of
phosphorothioate linkages, which have been shown to be a potent platelet activator [108,109] ,
[110]
glomerulonephritis, and hepatic toxicity .

siRNA patisiran for the treatment of transthyretin hereditary amyloidosis
Patisiran is a siRNA. The antisense guide strand formula is A-U-G-G-A-A-Um-A-C-U-C-U-U-G-G-U-
Um-A-C-dT-dT). It is complexed with the complementary passenger sense strand (G-Um-A-A-Cm-Cm-A-
A-G-A-G-Um-A-Um-Um-Cm-Cm-A-Um-dT-dT (A, adenosine; C, cytidine; G, guanosine; U, uridine;
Cm, 2’-O-methylcytidine; Um, 2’-O-methyluridine; dT, thymidine). Thus, Patisiran bears eleven 2’O-
[111]
methylated pyrimidines, which increases its lipophilicity. All inter-ribose linkages are phosphodiesters .
To protect this phosphodiester siRNA from rapid degradation and kidney filtration, the siRNA active
component of Patisiran is formulated into a lipid nanoparticle (LNP). In addition to this protecting effect,
LNP also facilitates Patisiran delivery to the liver. It has long been known that some colloidal delivery
systems, such as nanoparticles or liposomes, accumulate rapidly into the liver and spleen upon IV
administration, at a ratio superior to 90% [74,112-115] . Nanoparticles or liposomes containing cationic and/or
ionizable lipids thus represent a very favorable system for high-yield encapsulation of negatively charged
siRNA and for preferential liver uptake, as previously demonstrated for DNA.

The LNP formulation which has been selected for Patisiran includes buffer components (disodium
hydrogen phosphate, heptahydrate potassium dihydrogen phosphate, anhydrous sodium chloride), as well
as the lipid DLin-MC3-DMA [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31¬ tetraen-19-yl-4-(dimethylamino)
butanoate], an amine-containing ionizable lipid with a pKa of 6,4. In addition, the formulation comprises a
neutral lipid distearoylphosphatidylcholine, cholesterol, and the PEGylated lipid DMG-PEG 2,000 [
Figure 10A] . Each 1 mL Patisiran also contains 6.2 mg cholesterol USP, 13.0 mg DLin-MC3-DMA, 3.3
[111]
mg DSPC, and 1.6 mg α-(3'-{[1,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-ω-methoxy,
polyoxyethylene (PEG2000 C-DMG0). In such a typical LNP formulation, cholesterol is added to provide
rigidity to the 40-100 nm nanoparticles, and the neutral lipid distearoylphosphatidylcholine attenuates the
charge repulsion between the DLin-MC3-DMA cationic heads. In addition, the PEGylated lipid PEG2000
C-DMG0 ensures colloidal stability through the highly hydrated polyethyleneglycol chains forming a
protecting shield around the nanoparticle.

The mechanism by which the LNPs are targeted to the liver hepatocytes is schematized in Figures 10B and
10C. It is caused by the natural binding of the plasma protein ApoE to the PEGylated LNP. Since liver
hepatocytes express a high concentration of the ApoE receptor, LNPs decorated with ApoE bind to the
hepatocyte surface [Figure 10B]. The resulting ApoE clustering induces internalization by a clathrin-