key: cord-0783488-pnwuz2aa
authors: Suzuki, Yuta; Ishihara, Hiroshi
title: Difference in the lipid nanoparticle technology employed in three approved siRNA (Patisiran) and mRNA (COVID-19 vaccine) drugs
date: 2021-10-10
journal: Drug Metab Pharmacokinet
DOI: 10.1016/j.dmpk.2021.100424
sha: 5260c2682919dd91eb9fb213a94cae4702842ea3
doc_id: 783488
cord_uid: pnwuz2aa

Nucleic acid therapeutics are developing into precise medicines that can manipulate specific genes. However, the development of safe and effective delivery system for the target cells has remained a challenge. Lipid nanoparticles (LNPs) have provided a revolutionary delivery system that can ensure multiple clinical translation of RNA-based candidates. In 2018, Patisiran (Onpattro) was first approved as an LNP-based siRNA drug. In 2020, during the coronavirus disease 2019 (COVID-19) outbreak, LNPs have enabled the development of two SARS-CoV-2 mRNA vaccines, Tozinameran (Comirnaty or Pfizer-BioNTech COVID-19 vaccine) and Elasomeran (Spikevax or COVID-19 vaccine Moderna) for conditional approval. Here, we reviewed the state-of-the-art LNP technology employed in three approved drugs (one siRNA-based and two mRNA-based drugs) and discussed the differences in their mode of action, formulation design, and biodistribution.

In the 21 st century, nucleic acids are attracting attention as next generation modality beyond a 2 small molecule and antibody. Since the approval of the world's first nucleic acid drug 3 "fomivirsen" in 1998, a total of 16 nucleic acid medicines (9 antisense oligonucleotides 4 (ASO), 4 short interfering RNAs (siRNA), 1 aptamer, and 2 messenger RNAs (mRNA)) have 5 been approved in some areas of US, EU, and Japan, as of May 2021 (1). More than 80% of 6 these drugs (13 out of 16) have been approved since 2016. This rapid expansion of nucleic 7 acid therapeutics has been triggered by scientific breakthrough in drug-delivery systems. 8 Tremendous efforts toward the development of delivery carriers (2-9) have identified lipid 9 nanoparticles (LNPs) as a clinically validated platform technology that can deliver both long 10 (i.e. mRNA (10, 11)) and short nucleic acids (i.e. siRNA (12)) into target cells. Lipid 11 nanoparticle technology has played a central role in the development of the world's first drugs 12 associated with two completely independent phenomena, namely RNA interference by siRNA 13 and vaccination by mRNA. In this review, we have focused on three approved drugs using stored in a freezer (approximately -20 ºC) or in ultra-cold storage (-80 ºC to -60 ºC). 2 Intravenously administered LNP-formulated siRNA accumulates in the liver, the primary site 3 of transthyretin (TTR) production. After the uptake of LNPs by hepatocytes in the liver, 4 siRNA harnesses the endogenous RNA interference pathway, degrades TTR mRNA, and 5 reduces the production of TTR protein. 8 The EMA assessment report has detailed the process associated with mRNA vaccine at the 9 injection site as follows: "Intramuscular administration of LNP-formulated RNA vaccines 10 results in transient local inflammation that drives recruitment of neutrophils and antigen 11 presenting cells (APCs) to the site of delivery. Recruited APCs are capable of LNP uptake and 12 protein expression (i.e. spike of SARS-CoV-2) by translation and can subsequently migrate to 13 the local draining lymph nodes where T cell priming occurs (37)." 1 nuclease digestion. LNPs are neutral in physiological pH due to the ionizable lipid and 2 polyethylene glycol (PEG)-lipid, thereby reducing non-specific interactions with serum 3 proteins. Second, following dissociation of the PEG-lipid, cells take up LNPs via 4 apolipoprotein E (ApoE)-dependent and/or ApoE-independent pathways. Finally, protonated 5 LNPs, upon acidification in the endosome, induce hexagonal phase structures, disrupt the 6 membranes, and release RNA molecules into the cytoplasm. The released RNA molecules 7 lead to either down regulation (siRNA) or up regulation (mRNA) of target proteins. Details 8 regarding the uptake of LNPs into hepatocytes have already been reported, revealing that 9 LNPs are internalized via LDL receptors following the ApoE-dependent pathway (38). Details 10 regarding the uptake into antigen-presenting cells have not yet been clarified; however, 11 efficient localization of nucleic acids in the cytoplasm of antigen presenting cells has been 12 reported (42).

The three LNP-based drugs share multiple similarities in their formulation, and hence, behave 2 similarly as nanoparticles in vivo. Importantly, all LNPs are composed of four types of lipids; 3 ionizable lipid, phospholipid, cholesterol, and PEG-lipid ( 4 Figure 3) . All 3 ionizable lipids have tertiary amine group with pKa 6.0-6.7. These lipids switch 5 its charge from neutral to cationic based on the neutral pH in the blood and the acidic pH in 6 endosomes. The 3 PEG-lipids have dialkyl chains 14-carbon long, which are important for the 7 rapid dissociation from the surface of LNPs once inside the body (43). The biodegradable 8 design of ALC-0315 (44) and SM-102 (11) is described later.

Lipid composition of the three drug products is summarized in Table 3 . All three LNPs were 10 composed of four lipids in similar molar ratios. The major differences are in the storage 11 condition of drug products. A recent review estimated that mRNAs, not LNPs, are the main 12 factors for low storage stability of drug products, and thus mRNA vaccines need to be 13 supplied at freezing (about -20 °C) or ultra-freezing (-80 to -60 °C) conditions to slow down 14 mRNA hydrolysis (45) . Sucrose is used as a cryoprotectant to maintain the physical properties 15 of LNPs during freeze-thaw cycles (46). In contrast to the two mRNA vaccines, Patisiran 16 needs to avoid freezing temperatures, probably because lipid nanoparticles dissolved in PBS,

The major difference in biodistribution of the three LNP-based drugs arises from the 2 administration route (Table 4) . Regarding Patisiran, a study on rats using 14 C-labeled 3 ionizable lipid ( 14 C-MC3) had revealed that with a single intravenous dose of siRNA-LNP, 4 approximately 90% of the administered radioactivity was detected in the liver 4 h after 5 administration (21). Regarding Tozinameran, a study on rats using 3 H-labeled cholesteryl Besides that in the administration site, the total radioactivity recovery rate for the dose was 10 highest in the liver (up to 18%) 8-48 h after administration (22). Subcutaneously administered 11 liposomes (approximately < 0.1 µm) did not enter the blood capillary owing to their size; they 12 remained at the injection site and were drained through lymphatic capillaries to nearby lymph 13 nodes (50). The innate biodistribution of nanoparticles is favorable for mRNA vaccines, since 14 1) adaptive immune response occurs at lymph nodes, and 2) unwanted systemic exposure is 15 reduced. A detailed study had been conducted with intramuscularly administered mRNA-16 LNPs in non-human primates (51).

Ideal drug carriers are rapidly eliminated from the body once their purpose is served. Various Biologics, modified with PEG, produce anti-drug antibodies (ADA), more specifically anti-5 PEG antibodies, in vivo (54). siRNA-LNP (55) and mRNA-LNP (56) have been shown to 6 generate anti-PEG antibodies in mice, leading to accelerated blood clearance (ABC) upon 7 repeated administration. In the phase 3 APOLLO trial, ADAs to Patisiran were evaluated by 8 measuring antibodies specific to PEG2000-C-DMG that is exposed on the surface of LNPs 9 (57). The overall incidence of treatment-emergent ADAs was 3.4% (5 of 145 patients) in the 10 Patisiran group and 1.3% (1 of 77 patients) in the placebo group. Treatment-emergent ADAs 11 to PEG2000-C-DMG occurred at a low frequency, were transient and appeared to have no 12 effect on PK, PD, safety or efficacy (57). To the best of our knowledge, no study has been 13 reported on anti-PEG antibody yet, which causes accelerated blood clearance due to 14 intramuscularly administered LNPs.

In relation to anti-PEG antibody, a severe allergic reaction, named anaphylaxis, has been 16 reported to occur (though rarely; a couple of cases per million doses) due to the 17 administration of two mRNA vaccines (58). The trigger of anaphylaxis is considered to be due 18 to anti-PEG IgE against PEG-lipid (59, 60).

1 This review focused on the differences in LNP technology across three approved drugs. With 2 the initial success of siRNA therapeutics (14), the technology platform has become well-3 established and its scope has expanded from siRNA to mRNA (15). In early 2020, two 

Drug Metabolism & siRNA Delivery

siRNA-lipid nanoparticles with long-term storage 17

The Importance of Apparent pKa in the Development of 30

Suppression of RNA recognition by Toll-like 44. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids

mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability

Achieving long-term stability of lipid nanoparticles: examining Advanced drug delivery reviews

Efficient Targeting and Activation Vaccines in the US

mRNA Vaccines to The journal of allergy and clinical immunology In practice

Anti-PEG IgE in 6 anaphylaxis associated with polyethylene glycol. The journal of allergy and clinical immunology In 7 practice

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

A Single Immunization 12 with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses 13 against SARS-CoV-2 in Mice

mRNA-based SARS-CoV-15 2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection 16 in rodents

CureVac COVID vaccine let-down spotlights mRNA design challenges

A single dose of self-transcribing 20 and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice