WO2024160936A1

WO2024160936A1 - Rna formulation

Info

Publication number: WO2024160936A1
Application number: PCT/EP2024/052461
Authority: WO
Inventors: Daniela STRANGES; Emanuele TOMBA
Original assignee: Glaxosmithkline Biologicals Sa; CureVac SE
Priority date: 2023-02-03
Filing date: 2024-02-01
Publication date: 2024-08-08

Abstract

The present invention provides improved RNA-LNP formulations with lower amounts of RNA adduct, As well as methods and uses to reduce the amount of RNA adduct in RNA- LNP formulations, in particular mRNA-LNP formulations.

Description

RNA formulation

FIELD OF THE INVENTION

The present invention relates to RNA formulations in which RNA quality and stability are improved.

BACKGROUND OF THE INVENTION

Tremendous development has occurred in recent years with respect to the use of nucleic acids, in particular RNA molecules such as mRNA and siRNA, in therapy, and also in other fields e.g. crop protection. RNA based therapeutics include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs) and single-guide RNAs (sgRNAs)-mediated CRISPR-Cas system.

RNA molecules are considered to be significantly safer than DNA, as RNA molecules are cleared quickly out of the organism and cannot integrate into the genome and influence the cell's gene expression in an uncontrollable manner. It is also less likely for RNA therapeutics to cause severe side effects like the generation of an autoimmune disease or of anti-DNA antibodies (Bringmann et al., 2010). Transfection with RNA requires only insertion into the cell's cytoplasm, which is easier to achieve than into the nucleus.

RNA molecules are typically encapsulated in lipid nanoparticles (LNPs) that protect the RNA from degradation and help cellular uptake. LNPs that are used to encapsulate RNA, for example mRNA, generally comprise a cationic lipid which is an ionizable aminolipid. It has been shown that the ionizable amino-lipid might bring or generate impurities (N- oxide) upon oxidation of the tertiary amine group of the lipid (Packer et al., 2021 , https://doi . orq/10.1038/s41467-021 -26926-0) . N-oxide can indeed hydrolyze to aldehydes, which might form adducts through the covalent addition to the nucleobases of mRNA strands. Packer et al. (2021) have shown these adducts can be detected and quantified through Reversed Phase-Ion Pair High Performance Liquid Chromatography (RP-IP HPLC) and that they might be associated with a loss in in vitro protein expression. This occurrence when detected in RP-IP-HPLC is referred to as “late eluting peak” or “late eluting sPecies” (“LP” or “LEP”).

Given the relation demonstrated between the LP and protein expression, formation of the LP is thought to be associated with a loss of potency and/or stability of RNA therapeutics such as mRNA vaccines (Packer et al., 2021). One challenge upon formulating RNA into LNPs is preserving RNA quality, avoiding the formation of RNA adducts, and for mRNA, preserving the ability to translate into protein.

There is therefore a need in the field to reduce or prevent the formation of RNA adducts when RNA is formulated with an ionizable amino-lipid.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprising an RNA molecule, suitably mRNA, an ionizable amino-lipid and an antioxidant. In another aspect, there is provided the composition of the invention for use in therapy, suitably for use as a vaccine.

In another aspect, there is provided a method for treating or preventing a disease, comprising administering the composition of the invention to a patient in need thereof.

In another aspect, there is provided a method for treating or preventing an infectious disease, comprising administering the composition of the invention to a patient in need thereof.

In another aspect, there is provided the use of the composition of the invention for the manufacture of a medicament or vaccine.

In another aspect, there is provided the use of an antioxidant to stabilize a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid. In another aspect, there is provided a method for stabilizing a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid, comprising adding an antioxidant to said composition.

DESCRIPTION OF THE FIGURES

FIG. 1 : Analysis of pre-Lyo samples. Panel A: mRNA encapsulation % by Ribogreen. Panel B: LNP size by DLS.

FIG. 2: mRNA encapsulation % of Pre-Lyo vs Lyo samples at time 0. Panel A: Samples with Sucrose as cryoprotectant. Panel B: Samples with Threalose as cryoprotectant.

FIG. 3: LNP size of Pre Lyo vs Lyo samples at time 0. Panel A: Samples with Sucrose as cryoprotectant. Panel B: Samples with Threalose as cryoprotectant.

FIG. 4: LNP PDI of Pre Lyo vs Lyo samples at time 0. Panel A: Samples with Sucrose as cryoprotectant. Panel B: Samples with Threalose as cryoprotectant FIG. 5: mRNA integrity of Pre Lyo vs Lyo samples at time 0. Panel A: Samples with Sucrose as cryoprotectant. Panel B: Samples with Threalose as cryoprotectant

FIG. 6: LEP in Pre Lyo vs Lyo samples at time 0. Panel A: Samples with Sucrose as cryoprotectant. Panel B: Samples with Threalose as cryoprotectant

FIG. 7: mRNA encapsulation % after 2 weeks at 50°C.

FIG. 8: LNPs size. Applied thermal stress: 2 weeks at 50°C

FIG. 9: LNPs Pdl. Applied thermal stress: 2 weeks at 50°C.

FIG. 10: mRNA integrity. Applied thermal stress: 2 weeks at 50°C.

FIG. 11 : LEP. Applied thermal stress: 2 weeks at 50°C.

FIG. 12: mRNA encapsulation %. Applied thermal stress: up to 8 weeks at 40°C.

FIG. 13: LNPs size. Applied thermal stress: up to 8 weeks at 40°C.

FIG. 14: LNPs Pdl. Applied thermal stress: up to 8 weeks at 40°C.

FIG. 15: mRNA Integrity. Applied thermal stress: up to 8 weeks at 40°C.

FIG. 16: LEP. Applied thermal stress: up to 8 weeks at 40°C.

FIG. 17: mRNA encapsulation %. Applied thermal stress: up to 11 weeks at 25°C.

FIG. 18: LNPs size. Applied thermal stress: up to 11 weeks at 25°C.

FIG. 19: LNPs Pdl. Applied thermal stress: up to 11 weeks at 25°C.

FIG. 20: mRNA integrity. Applied thermal stress: up to 11 weeks at 25°C.

FIG. 21 : LEP. Applied thermal stress: up to 11 weeks at 25°C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding by the inventors that the addition of an antioxidant to RNA-lipid formulations can improve the stability of the RNA over time and help preserve RNA quality. In particular, in the context of mRNA, addition of an antioxidant to mRNA-lipid formulations can help preserve the mRNA’s ability to translate into protein.

In one aspect, the present invention provides a composition comprising an RNA molecule, an ionizable amino-lipid and an antioxidant. In another aspect, there is provided the composition of the invention for use in therapy, suitably for use as a vaccine. In another aspect, there is provided the use of an antioxidant to stabilize a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid. In another aspect, there is provided a method for stabilizing a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid, comprising adding an antioxidant to said composition. In another aspect, there is provided a method of formulating a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid, comprising adding an antioxidant to said composition.

As used herein, the term “antioxidant” is a moiety which has the ability to protect another compound from oxidation. Without wishing to be bound by theory, it is hypothesized that the antioxidant may act as a scavenger for aldehyde impurities, providing a competitor that could prevent their reaction with RNA, especially if used in large excess. In particular, methionine is also known to be a good antioxidant in therapeutic protein-based parenterals (see for example Dion et al. (2018), Pharm Res. 2018 Oct 2;35(11):222), therefore it is hypothesized that including it as an excipient may prevent lipid oxidation during manufacturing process steps or during storage. Suitable antioxidants include amino acids, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propylgallate and sodium ascorbate.

In one embodiment, the antioxidant is an amino acid. Suitably, the amino acid is selected from methionine, tryptophan, histidine, lysine, cysteine, arginine and tyrosine. In a preferred embodiment, the antioxidant is methionine.

Suitably, the antioxidant is present in the composition according to the invention in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose. In one embodiment, the antioxidant is present in the composition according to the invention in an amount of 0.45 mg per dose.

As used herein, a “dose” refers to the amount of the composition that is to be administered to a subject, suitably a human subject. Typically, when a composition is in liquid form, the volume of a dose is suitably selected from 0.3, 0.4 and 0.5 ml. In one embodiment, the volume of the dose is 0.3 ml. Typically, when a composition is in a dry from, such as a lyophilized, freeze-dried or spray-dried form, the dose is mixed with a liquid, suitably an aqueous liquid such as sterile water or saline, to form a “reconstituted liquid dose” prior to administration to a patient. Suitably, the volume of the reconstituted liquid dose is selected from 0.3, 0.4 and 0.5 ml. In one embodiment, the volume of the reconstituted liquid dose is 0.3 ml.

Suitably, the antioxidant is present in the composition according to the invention at a concentration between 0.1 and 5 mg per ml, more suitably at a concentration between 1 and 3 mg per ml, for example at a concentration selected from 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 mg per ml. In one embodiment, the antioxidant is present in the composition according to the invention at a concentration of 1 .5 mg per ml.

Suitably, methionine is present in the composition according to the invention in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose. In one embodiment, methionine is present in the composition according to the invention in an amount of 0.45 mg per dose.

Suitably, methionine is present in the composition according to the invention at a concentration between 0.1 and 5 mg per ml, more suitably at a concentration between 1 and 3 mg per ml, for example at a concentration selected from 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 mg per ml. In one embodiment, methionine is present in the composition according to the invention at a concentration of 1 .5 mg per ml.

Suitably, the antioxidant prevents or reduces the formation of RNA adduct. More suitably, the antioxidant prevents or reduces the formation of lipid-RN A adduct.

As used herein, the term “RNA adduct” refers to an RNA molecule which differs from the intended RNA molecule. In particular, an RNA adduct may be a dysfunctional RNA molecule, for example an mRNA molecule whose ability to translate into a protein is impaired or even entirely abolished. As used herein, a “lipid-RNA adduct” refers to an RNA molecule which is “lipidated” through covalent addition of lipid residues to nucleobases forming part of the RNA. In particular, a lipid-RNA adduct may be a dysfunctional lipidated RNA molecule, for example a lipidated mRNA molecule whose ability to translate into a protein is impaired or even entirely abolished, or a lipidated siRNA whose ability to silence translation of a target mRNA is impaired or even entirely abolished.

Chromatographic methods may be used for the detection and quantification of RNA- adduct, in particular of lipid-RNA adduct. Suitably, detection and/or quantification of lipid- RNA adduct in the context of the invention is performed using analytical high performance liquid phase chromatography (HPLC), suitably analytical reverse phase-ion pair HPLC (RP- IP-HPLC), for example as described in Packer 2021. In case chromatography is used, quantification of RNA-adduct, in particular of lipid-RNA adduct, may be based on comparing the peak area (or “area under the peak”) of the full length functional RNA and of the late peak (LP) in a chromatogram. The peak area may be determined by any suitable software which evaluates the signals of the detector system. The process of determining the peak area is also referred to as integration.

Typically, a test sample of the composition comprising the ionizable amino-lipid (and optionally other lipids to form an LNP) and the RNA (suitably mRNA), may be treated with a detergent to dissociate the LNP comprising the ionizable amino-lipid and to release the RNA. The released RNA may be captured using suitable binding compounds, e.g. Agencourt AM Pure XP beads (Beckman Coulter, Brea, CA, USA) essentially according to the manufacturer’s instructions. Following preparation of the RNA sample, analytical (RP)HPLC may be performed to determine the integrity of RNA. Typically, for determining RNA integrity, the RNA samples may be diluted to a concentration of 0.1 g/l using e.g. water for injection (WFI). About 10pl of the diluted RNA sample may be injected into an HPLC column (e.g. a monolithic poly(styrene-divinylbenzene) matrix). Analytical RP-HPLC may be performed using standard conditions, for example: Gradient 1 : Buffer A (0.1M TEAA (pH 7.0)); Buffer B (0.1M TEAA (pH 7.0) containing 25% acetonitrile). Starting at 30% buffer B the gradient extended to 32% buffer B in 2min, followed by an extension to 55% buffer B over 15 minutes at a flow rate of 1ml/min. HPLC chromatograms are typically recorded at a wavelength of 260nm. The obtained chromatograms may be evaluated using a software and the relative peak area may be determined in percent (%) as commonly known in the art. The relative peak area indicates the amount of RNA that is not affected by formation of lipid-RNA adduct. Late eluting sPecies (i.e. lipid-RNA adduct) is quantified as relative percentage of the total chromatographic peak area. To evaluate mRNA total amount in the analysis a standard curve is prepared using not encapsulated mRNA previously quantified with an orthogonal method and injected at different concentrations. Total recovery is extrapolated by linear regression calculation executed by the software.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 25°C for a period of 6 weeks is at least 10% lower, suitably at least 20%, at least 30%, at least 40%, at least 50%, lower than in a corresponding composition in which the antioxidant is not present.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 25°C for a period of 11 weeks is at least 10% lower, suitably at least 20%, at least 30%, at least 40%, at least 50%, lower than in a corresponding composition in which the antioxidant is not present.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 40°C for a period of 4 weeks is at least 10% lower, suitably at least 20%, at least 30%, at least 40%, at least 50%, lower than in a corresponding composition in which the antioxidant is not present. Suitably, the amount of I ipid-RN A adduct after storage of the composition according to the invention at 40°C for a period of 8 weeks is at least 10% lower, suitably at least 20%, at least 30%, at least 40%, at least 50%, lower than in a corresponding composition in which the antioxidant is not present.

Suitably, the amount of I ipid-RN A adduct after storage of the composition according to the invention at 50°C for a period of 2 weeks is at least 10% lower, suitably at least 20%, at least 30%, at least 40%, at least 50%, lower than in a corresponding composition in which the antioxidant is not present.

Suitably, the proportion of RNA that forms a lipid-RNA adduct after storage of the composition according to the invention at 25°C for a period of 6 weeks is lower than 10%, suitably lower than 9, 8 or 7%, for example is about 6%.

Suitably, the proportion of RNA that forms a lipid-RNA adduct after storage of the composition according to the invention at 25°C for a period of 11 weeks is lower than 10%, suitably lower than 9, 8 or 7%, for example is about 6%.

Suitably, the proportion of RNA that forms a lipid-RNA adduct after storage of the composition according to the invention at 40°C for a period of 4 weeks is lower than 10%, suitably lower than 9, 8 or 7%, for example is about 6%.

Suitably, the proportion of RNA that forms a lipid-RNA adduct after storage of the composition according to the invention at 40°C for a period of 8 weeks is lower than 10%, suitably lower than 9, 8 or 7%, for example is about 6%.

Suitably, the proportion of RNA that forms a lipid-RNA adduct after storage of the composition according to the invention at 50°C for a period of 2 weeks is lower than 10%, suitably lower than 9, 8 or 7%, for example is about 6%.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 25°C for a period of 6 weeks is not more than 3 times higher, 2,5 times higher or 2 times higher than prior to storage.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 25°C for a period of 11 weeks is not more than 3 times higher, 2,5 times higher or 2 times higher than prior to storage.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 40°C for a period of 4 weeks is not more than 3 times higher, 2,5 times higher or 2 times higher than prior to storage.

Suitably, the amount of lipid-RNA adduct after storage of the composition according to the invention at 40°C for a period of 8 weeks is not more than 3 times higher, 2,5 times higher or 2 times higher than prior to storage. Suitably, the amount of I ipid-RN A adduct after storage of the composition according to the invention at 50°C for a period of 2 weeks is not more than 3 times higher, 2,5 times higher or 2 times higher than prior to storage.

Suitably, the antioxidant is not linked to a PEG moiety. Suitably the antioxidant is not linked via a linker to a PEG moiety.

The composition according to the invention may be provided in liquid or dry (e.g. lyophilised) form.

In one embodiment, the composition is a liquid composition. Suitably, the liquid composition does not comprise a surfactant. Suitably, the liquid composition does not comprise a non-ionic surfactant. Suitably, the liquid composition does not comprise a nonionic surfactant selected from polysorbate 20 (PS20), polysorbate 60 (PS60), polysorbate 80 (PS80) and poloxamers.

In one embodiment, the composition is a dry composition.

The term “dry composition” as used herein may be a composition that has been lyophilized (e.g. according to WO2016165831 , WO2011069586, WO2022/232585, WG2022/101461 , WO2022/076562, WO2012/170889 or WG2022/036170), or spray-dried, or freeze-dried (e.g. according to WO2016184575, WO2016184576 or WO2021/216541) to obtain a dry composition, suitably a temperature stable composition, for example in the form of a powder.

In one embodiment, the composition according to the invention is a lyophilized, freeze-dried or spray-dried dry composition comprising one or more further excipients selected from cryoprotectants, plasticizers and polymers. Suitably, the lyophilized, freeze- dried or spray-dried dry composition is mixed with a liquid, suitably an aqueous liquid such as sterile water or saline, to form a “reconstituted liquid formulation” prior to administration to a patient.

As used herein, a “cryoprotectant” (or “lyoprotectant”) is an excipient which has the ability to preserve structural integrity, in particular LNP structural integrity, upon freezing. Suitably, the cryoprotectant is selected from sucrose, trehalose and hydroxypropyl-beta- cyclodextrin, more suitably from sucrose and trehalose. In a preferred embodiment, the cryoprotectant is sucrose.

Suitably, the cryoprotectant is present in the lyophilized, freeze-dried or spray-dried dry composition in an amount between 9 and 60 mg per dose, for example in an amount selected from 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, 50, 55 and 60 mg per dose. In one embodiment, the cryoprotectant is present in the lyophilized, freeze-dried or spray-dried dry composition in an amount of 15 mg per dose. Suitably, the cryoprotectant is selected from trehalose, sucrose or a combination of trehalose and sucrose, and is present in the lyophilized, freeze-dried or spray-dried dry composition in an amount between 9 and 60 mg per dose, for example in an amount selected from 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, 50, 55 and 60 mg per dose. In one embodiment, the cryoprotectant is selected from trehalose, sucrose or a combination of trehalose and sucrose, and is present in the lyophilized, freeze-dried or spray-dried dry composition in an amount of 15 mg per dose.

Suitably, prior to lyophilization, freeze-drying or spray-drying, the cryoprotectant is present at a concentration between 30 and 200 mg per ml, for example at a concentration selected from 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg per ml. In one embodiment, prior to lyophilization, freeze-drying or spray-drying, the cryoprotectant is present at a concentration of 50 mg per ml. Suitably, the cryoprotectant is selected from trehalose, sucrose or a combination of trehalose and sucrose, and prior to lyophilization, freeze-drying or spray-drying the cryoprotectant is present at a concentration between 50 and 150 mg per ml, for example at a concentration selected from 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg per ml. In one embodiment, the cryoprotectant is selected from trehalose, sucrose or a combination of trehalose and sucrose, and prior to lyophilization, freeze-drying or spray-drying, the cryoprotectant is present at a concentration of 50 mg per ml.

Suitably, the cryoprotectant is present in the reconstituted liquid formulation at a concentration between 30 and 200 mg per ml, for example at a concentration selected from 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg per ml. In one embodiment, the cryoprotectant is present in the reconstituted liquid formulation at a concentration of 50 mg per ml. Suitably, the cryoprotectant is selected from trehalose, sucrose or a combination of trehalose and sucrose, and is present in the reconstituted liquid formulation at a concentration between 30 and 200 mg per ml, for example at a concentration selected from 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg per ml. In one embodiment, the cryoprotectant is selected from trehalose, sucrose or a combination of trehalose and sucrose, and is present in the reconstituted liquid formulation at a concentration of 50 mg per ml.

As used herein, a “plasticizers” is an excipient which has the ability to protect lipid membrane structures, in particular LNP surface structures. Suitably, the plasticizer is selected from glycerol, sorbitol and PEG4000, more suitably from sorbitol and PEG4000.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises glycerol in an amount between 0.3 and 3 mg per dose, for example in an amount selected from 0.3, 0.5, 0.75, 1.0, 1 .25, 1.5, 1 .75, 2.0, 2.5, 2.5, 2.75 and 3.0 mg per dose. In one embodiment, glycerol is present in the composition according to the invention in an amount of 1 .5 mg per dose.

In one embodiment, prior to lyophilization, freeze-drying or spray-drying, glycerol is present at a concentration between 1 and 10 mg per ml, for example at a concentration selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg per ml. In one embodiment, glycerol is present in the composition according to the invention at a concentration of 5 mg per ml.

In one embodiment, glycerol is present in the reconstituted liquid formulation at a concentration between 1 and 10 mg per ml, for example at a concentration selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg per ml. In one embodiment, glycerol is present in the composition according to the invention at a concentration of 5 mg per ml.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises sorbitol in an amount between 0.3 and 9 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.25, 1.5, 1.75, 2, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 and 9 mg per dose. In one embodiment, sorbitol is present in the composition according to the invention in an amount of 3 mg per dose.

In one embodiment, prior to lyophilization, freeze-drying or spray-drying, sorbitol is present at a concentration between 1 and 30 mg per ml, for example at a concentration selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 mg per ml. In one embodiment, sorbitol is present in the composition according to the invention at a concentration of 10 mg per ml.

In one embodiment, sorbitol is present in the reconstituted liquid formulation at a concentration between 1 and 30 mg per ml, for example at a concentration selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 mg per ml. In one embodiment, sorbitol is present in the composition according to the invention at a concentration of 10 mg per ml.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises PEG4000 in an amount between 0.03 and 3 mg per dose, for example in an amount selected from 0.03, 0.05, 0.075, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 and 3 mg per dose. In one embodiment, PEG4000 is present in the composition according to the invention in an amount of 0.3 mg per dose.

In one embodiment, prior to lyophilization, freeze-drying or spray-drying, PEG4000 is present at a concentration between 0.1 and 10 mg per ml, for example at a concentration selected from 0.1 , 0.25, 0.5, 0.75, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 and 10 mg per ml. In one embodiment, PEG4000 is present in the composition according to the invention at a concentration of 1 mg per ml. In one embodiment, PEG4000 is present in the reconstituted liquid formulation at a concentration between 0.1 and 10 mg per ml, for example at a concentration selected from 0.1 , 0.25, 0.5, 0.75, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 and 10 mg per ml. In one embodiment, PEG4000 is present in the composition according to the invention at a concentration of 1 mg per ml.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition does not comprise a plasticizer.

In one embodiment, a plasticizer is present in the composition according to the invention prior to lyophilization, freeze-drying or spray-drying.

In one embodiment, a plasticizer is not present in the reconstituted liquid formulation.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition does not comprise glycerol.

In one embodiment, glycerol is present in the composition according to the invention prior to lyophilization, freeze-drying or spray-drying.

In one embodiment, glycerol is not present in the reconstituted liquid formulation.

Polymers may improve physiochemical properties of lipid particles, and in particular of LNPs, through complexation of lipids and polymer. Suitably, the polymer is polyvinyl pyrrolidone (PVP).

In one embodiment, the composition according to the invention comprises PPV in an amount between 0.3 and 3 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.75, 1.0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg per dose. In one embodiment, PVP is present in the composition according to the invention in an amount of 1.5 mg per dose.

In one embodiment, the composition according to the invention comprises PVP at a concentration between 1 and 10 mg per ml, for example at a concentration selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg per ml. In one embodiment, PVP is present in the composition according to the invention at a concentration of 5 mg per ml.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises methionine and sucrose.

Suitably, the lyophilized, freeze-dried or spray-dried dry composition comprises RNA, suitably mRNA, an ionizable amino-lipid, methionine in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose, and sucrose in an amount between 9 and 60 mg per dose, for example in an amount selected from 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, 50, 55 and 60 mg per dose.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises

RNA, suitably mRNA, an ionizable amino-lipid, methionine in an amount of 0.45 mg per dose, and. sucrose in an amount of 15 mg per dose.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises methionine, sucrose and sorbitol.

Suitably, the lyophilized, freeze-dried or spray-dried dry composition comprises RNA, suitably mRNA, an ionizable amino-lipid, methionine in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose, sucrose in an amount between 9 and 60 mg per dose, for example in an amount selected from 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, 50, 55 and 60 mg per dose, and sorbitol in an amount between 0.3 and 9 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.25, 1.5, 1.75, 2, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 and 9 mg per dose.

RNA, suitably mRNA, an ionizable amino-lipid, methionine in an amount of 0.45 mg per dose,, sucrose in an amount of 15 mg per dose, and sorbitol in an amount of 3 mg per dose.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition comprises methionine, sucrose and PEG4000.

Suitably, the lyophilized, freeze-dried or spray-dried dry composition comprises RNA, suitably mRNA, an ionizable amino-lipid, methionine in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose, sucrose in an amount between 9 and 60 mg per dose, for example in an amount selected from 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, 50, 55 and 60 mg per dose, and

PEG4000 in an amount between 0.03 and 3 mg per dose, for example in an amount selected from 0.03, 0.05, 0.075, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1 .75, 2, 2.25, 2.5, 2.75 and 3 mg per dose.

RNA, suitably mRNA, an ionizable amino-lipid, methionine in an amount of 0.45 mg per dose,. sucrose in an amount of 15 mg per dose, and

PEG4000 in an amount of 0.3 mg per dose.

Suitably, the amount of lipid-RNA adduct in the composition according to the invention after mRNA lyophilization is not more than 3 times higher, 2 times higher, 1 ,5 times higher than prior to mRNA lyophilization.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after mRNA lyophilization is at least 50%, suitably at least 60%, at least 70%, at least 80% or at least 90% equal to mRNA translation efficiency prior to mRNA lyophilization.

Suitably, the lyophilized, freeze-dried or spray-dried dry composition has a water content of less than about 10%, suitably a water content of between about 0.5% and 5%, more suitably a water content of between about 0.5% and 3%.

Dry compositions may be reconstituted in a liquid form prior to administration to a subject. Suitably, liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. The requirement for buffer and/or tonicity modifying agents will depend on both the content of the container being reconstituted and the subsequent use of the reconstituted content. Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. The buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4. The composition of the invention comprises an ionizable amino-lipid.

Herein, an “ionizable amino-lipid” is an ionizable lipid that has an amino group. An “ionizable lipid” is a lipid which becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the ionizable amino-lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. Suitably, the ionizable amino-lipid is selected from a tertiary ionizable amino-lipid and a secondary ionizable amino-lipid. More suitably, the ionizable amino-lipid is a tertiary ionizable amino-lipid.

In some embodiments, ionizable amino-lipids as defined herein have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, suitably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention. In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11 , e.g., a pKa of about 5 to about 7.

The ionizable amino-lipid may be cationic. Unless a different meaning is clear from the specific context, the term “cationic” means that the respective structure bears a positive charge, either permanently or not permanently, but in response to certain conditions such as pH. Thus, the term cationic covers both “permanently cationic” and “cationizable”. The term “permanently cationic” means, e.g., that the respective compound, or group, or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge results from the presence of a quaternary nitrogen atom.

The term “cationizable” as used herein means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH of its environment. Also in non-aqueous environments where no pH value can be determined, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationizable or polycationizable compound, in particular the pKa of the respective cationizable group, at which pH or hydrogen ion concentration it is charged or uncharged. In diluted aqueous environments, the fraction of cationizable compounds, groups or atoms bearing a positive charge may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art. In some embodiments, it is suitable that the cationizable compound or moiety is predominantly neutral at physiological pH values, e.g. about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, the range of pKa for the cationizable compound or moiety is about 5 to about 7.

Suitable ionizable amino-lipids include, but are not limited to N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2- Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N- dimethylaminopropane (y-DLenDMA), 98N12-5, 1 ,2-Dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1 ,2-Dilinoleoyl-3- dimethylaminopropane (DLinDAP), 1 ,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S- DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1 ,2- Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), HGT5000, HGT5001 , DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K- DMA, DLin-K-XTC2-DMA, XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane) HGT4003, 1 ,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2- Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1 ,2- propanediol (DLinAP), 3-(N,N-Dioleylamino)-1 ,2-propanedio (DOAP), 1 ,2-Dilinoleyloxo-3- (2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-Dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N- dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1 ,3]dioxol-5- amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-

(dimethylamino)butanoate (MC3), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1 ,3]dioxol-5-amine)), 1 , 1’-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]- dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K- DMA), NC98-5 (4,7, 13-tris(3-oxo-3-(undecylamino)propyl)-N ,N 16-diundecyl-4,7, 10,13- tetraazahexadecane-l,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19- yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 Ether), 4- ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 Ether), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1 ,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable ionizable amino-lipids for use in the compositions and methods of the invention include those described in international patent publications WO2010053572 (and particularly, Cl 2-200 described at paragraph [00225]) and WO2012170930, both of which are incorporated herein by reference, HGT4003, HGT5000, HGTS001 , HGT5001 , HGT5002 (see US20150140070A1).

In some embodiments, the ionizable amino-lipid is an ionizable aminoalcohol lipidoid.

Ionizable aminoalcohol lipidoids may be prepared by the methods described in U.S. Patent No. 8,450,298, herein incorporated by reference in its entirety.

Suitable ionizable amino-lipids can also be the compounds as disclosed in Tables 1 , 2 and 3 and as defined in claims 1-24 of WO2017075531 A1 , hereby incorporated by reference.

In another embodiment, suitable ionizable amino-lipids can also be the compounds as disclosed in W02015074085A1 (/.e. ATX-001 to ATX-032 or the compounds as specified in claims 1-26), U.S. Appl. Nos. 61/905,724 and 15/614,499 or U.S. Patent Nos. 9,593,077 and 9,567,296 hereby incorporated by reference in their entirety.

In other embodiments, suitable ionizable amino-lipids can also be the compounds as disclosed in WG2017117530A1 (/.e. lipids 13, 14, 15, 16, 17, 18, 19, 20, or the compounds as specified in the claims), hereby incorporated by reference in its entirety.

In some embodiments, ionizable amino-lipids may also be selected from the lipids disclosed in W02018078053A1 (/.e. lipids derived from formula I, II, and III of W02018078053A1 , or lipids as specified in Claims 1 to 12 of W02018078053A1), the disclosure of W02018078053A1 hereby incorporated by reference in its entirety. In that context, lipids disclosed in Table 7 of W02018078053A1 (e.g. lipids derived from formula I- 1 to 1-41) and lipids disclosed in Table 8 of W02018078053A1 (e.g. lipids derived from formula 11-1 to II-36) may be suitably used in the context of the invention. Accordingly, formula 1-1 to formula 1-41 and formula 11-1 to formula II-36 of W02018078053A1 , and the specific disclosure relating thereto, are herewith incorporated by reference.

In some embodiments, ionizable amino-lipids may be derived from formula III of published PCT patent application W02018078053A1. Accordingly, formula III of W02018078053A1 , and the specific disclosure relating thereto, are herewith incorporated by reference. In some embodiments, the ionizable amino-lipid is selected from structures 111-1 to HI-36 of Table 9 of published PCT patent application W02018078053A1. Accordingly, formula 111-1 to III-36 of W02018078053A1 , and the specific disclosure relating thereto, are herewith incorporated by reference.

In some embodiments, the ionizable (cationic) lipid has the formula III:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

L¹ or L² is each independently -O(C=O)- or -(C=O)O-;

G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

G³ is CI-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene;

R¹ and R² are each independently, branched or linear, C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl;

R⁵ is H or Ci-Ce alkyl. ionizable (cationic) lipid has the formula III:

L¹ or L² is each independently -O(C=O)- or -(C=O)O-;

G¹ and G² are each independently unsubstituted C1-C12 alkylene;

G³ is C1-C24 alkylene;

R¹ and R² are each independently, branched or linear, C6-C24 alkyl;

R³ is OR⁵; and

R⁵ is H.

In some embodiments, the ionizable (cationic) lipid has the formula III and wherein R¹, R² or both R¹ and R² have one of the following structures:

In some embodiments, R² has the structure:

In some embodiments, the ionizable (cationic) lipid has the formula:

In some embodiments, the ionizable (cationic) lipid has the formula:

, , , , , WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601, WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625,

US20130178541, US20130225836 and US20140039032 and WO2017112865 specifically relating to ionizable (cationic) lipids suitable for LNPs are incorporated herewith by reference.

In other embodiments, the ionizable amino-lipid is a tertiary ionizable amino-lipid, suitably selected from

The amount of the ionizable amino-lipid may be selected taking the amount of RNA into account (N/P ratio). In this context, the “N/P ratio” is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the ionizable amino-lipid to the phosphate groups (“P”) of the RNA. The N/P ratio may be calculated on the basis that, for example, 1 pg RNA typically contains about 3 nmol phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The “N”-value of the ionizable aminolipid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and/or cationizable groups. If more than one ionizable amino-lipid is present, the N-value should be calculated on the basis of all ionizable amino-lipids comprised in the lipid nanoparticles.

In one embodiment, the composition has an N/P ratio from about 0.1 to about 20. In one embodiment, the composition has an N/P ratio from about 1 to about 20, suitably from about 2 to about 15, more suitably from about 3 to about 10, even more suitably from about 4 to about 9, most suitably about 6. In one embodiment, the composition has an N/P ratio from about 5 to about 20, more suitably from about 10 to about 18, even more suitably from about 12 to about 16, most suitably about 14.

Suitably, the composition according to the invention comprises one or more further lipids which together with the ionizable amino-lipid form a delivery system for the RNA. A range of delivery systems have been described which can be used to encapsulate (or complex) RNA in order to protect it and facilitate its delivery to target cells. In a preferred embodiment, the delivery system is a lipid nanoparticle (LNP).

The term “lipid nanoparticle” (or “LNP”) refers to a non-virion particle in which nucleic acid molecules, such as RNA, can be encapsulated. LNPs are not restricted to any particular morphology, and include any morphology generated when an ionizable (or cationic) lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and/or in the presence of a nucleic acid, e.g. an RNA. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle (LNP). LNP delivery systems and methods for their preparation are known in the art.

Suitably, at least about 80%, 85%, 90%, 95% of lipid-based carriers, suitably the LNPs, have a spherical morphology.

LNPs are typically suitable for intramuscular, intradermal and/or intravenous administration.

In some embodiments, the ionizable amino-lipid is present in the LNP in an amount from about 20 mol% to about 70 mol% (based upon 100% total moles of lipid in the LNP). Suitably, the ionizable amino-lipid is present in the LNP in an amount from about 30 mol% to about 65 mol%. In one embodiment, the ionizable amino-lipid is present in the LNP in an amount from about 40 mol% to about 60 mol%, such as about 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol%, respectively. In one embodiment, the ionizable amino-lipid is present in the LNP in an amount from about 47 mol% to about 48 mol%, such as about 47.0, 47.1 , 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0 mol%, respectively, wherein 47.4 mol% is particularly suitable.

The composition according to the invention can comprise two or more (different) ionizable amino-lipids as defined herein. Ionizable amino-lipids may be selected to contribute to different advantageous properties. For example, ionizable amino-lipids that differ in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the LNP (or liposomes, nanoliposomes, lipoplexes). In particular, the ionizable amino-lipids can be chosen so that the properties of the mixed-LNP are more desirable than the properties of a single-LNP of individual lipids. If more than one ionizable amino-lipid are present, the percentages above apply to the total amount of ionizable amino-lipids.

In addition to the ionizable amino-lipid, the composition according to the invention typically comprises one or more additional lipids selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g. PEGylated lipid) which together with the ionizable amino-lipid form an LNP.

Suitably, the composition according to the invention comprises a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” (or “PEG-modified lipid” or “PEG lipid”) refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-s-DMG) and the like.

A polymer conjugated lipid as defined herein, e.g. a PEGylated lipid, may serve as an aggregation reducing lipid.

Suitably, the LNP comprises a PEGylated lipid. Suitable PEGylated lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols. Representative PEGylated lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the PEGylated lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEGylated lipid is PEG-2000-DMG. In one embodiment, the PEGylated lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate.

In some embodiments, the PEGylated lipid comprises PEG-DMG or PEG-cDMA. In embodiments, the PEGylated lipid is suitably derived from formula (IV) of published PCT patent application W02018078053A1. Accordingly, PEGylated lipids derived from formula (IV) of published PCT patent application W02018078053A1 , and the respective disclosure relating thereto, are herewith incorporated by reference. ylated lipid has the formula IV:

wherein R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In one embodiment, R⁸ and R⁹ are saturated alkyl chains.

In some embodiments, the RNA, suitably mRNA, is complexed with one or more lipids thereby forming LNPs, wherein the LNP comprises a polymer conjugated lipid, suitably a PEGylated lipid, wherein the PEGylated lipid is suitably derived from formula (IVa) of published PCT patent application W02018078053A1. Accordingly, PEGylated lipid derived from formula (IVa) of published PCT patent application W02018078053A1 , and the respective disclosure relating thereto, is herewith incorporated by reference.

In some embodiments, the PEGylated lipid is of formula (IVa):

wherein n has a mean value ranging from 30 to 60, such as about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2, or 60±2. In an embodiment n is about 49. In another embodiment n is about 45. In further embodiments, the PEGylated lipid is of formula (IVa) wherein n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2000g/mol to about 3000 g/mol or about 2300g/mol to about 2700g/mol, suitably about 2500g/mol.

In some embodiments, the PEGylated lipid has the formula IVa:

wherein n has a mean value ranging from 30 to 60, suitably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, most suitably wherein n has a mean value of 49 or 45; or wherein n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2500g/mol.

The lipid of formula IVa as suitably used herein has the chemical term 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referred to as ALC-0159.

Further examples of PEGylated lipids suitable in that context are provided in LIS20150376115A1 and WO2015199952, each of which is incorporated by reference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 mole percent of PEG or PEGylated lipid, based on the total moles of lipid in the LNP.

In further embodiments, LNPs comprise from about 0.1% to about 20% of the PEGylated lipid on a molar basis, e.g., about 0.5 to about 15%, about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 3%, about 2,5%, about 2%, about 1.5%, about 1 %, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the LNP). In embodiments, LNPs comprise from about 1.0% to about 2.0% of the PEGylated lipid on a molar basis, e.g., about 1.2 to about 1.9%, about 1.2 to about 1.8%, about 1.3 to about 1.8%, about 1.4 to about 1.8%, about 1.5 to about 1.8%, about 1.6 to about 1.8%, in particular about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, most suitably 1.7% (based on 100% total moles of lipids in the LNP). In various embodiments, the molar ratio of the ionizable amino-lipid to the PEGylated lipid ranges from about 100: 1 to about 25: 1 .

In some embodiments, the LNP comprises a PEGylated lipid at around 0.5 to 10 molar %, optionally 0.5 to 5 molar % or 0.5 to 3 molar %.

Suitably, the antioxidant is not linked to the PEGylated lipid.

Suitably, the composition according to the invention comprises one or more stabilizing lipids (or helper lipids), which stabilize the formation of particles during their formulation or during the manufacturing process (e.g. neutral lipid and/or one or more steroid or steroid analogue).

Suitable stabilizing lipids (or helper lipids) include neutral lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides. In some embodiments, the composition according to the invention comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DM PE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearioyl-2- oleoylphosphatidyethanol amine (SOPE), and 1 ,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE), sphingomyelin (SM) or mixtures thereof.

In various embodiments, the molar ratio of the ionizable amino-lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.

Suitably, the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Suitably, the molar ratio of the ionizable amino-lipid to DSPC may be in the range from about 2:1 to about 8:1.

Suitable stabilizing lipids (or helper lipids) also include steroids or steroid analogues. In some embodiments, the steroid is a sterol, suitably cholesterol.

Suitably, the molar ratio of the ionizable amino-lipid to cholesterol may be in the range from about 2:1 to about 1 :1. In some embodiments, the cholesterol may be PEGylated.

The sterol can be about 10mol% to about 60mol% or about 25mol% to about 55mol% or about 25mol% to about 40mol% of the lipid particle. In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60mol% of the total lipid present in the lipid particle. In another embodiment, the LNPs include from about 5% to about 50% on a molar basis of the sterol, e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Suitably, in addition to the RNA, ionizable amino-acid and antioxidant, the composition according to the invention comprises a PEGylated lipid, a neutral lipid and a sterol. Suitably, the composition according to the invention comprises RNA, methionine, an ionizable amino-acid, a PEGylated lipid, a neutral lipid and a sterol. More suitably, the composition according to the invention comprises mRNA, methionine, an ionizable aminoacid, a PEGylated lipid, a neutral lipid and a sterol. In one embodiment, the composition comprises

(i) at least one ionizable amino-lipid as defined herein, suitably lipid of formula 111-3;

(ii) at least one neutral lipid as defined herein, suitably 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC);

(iii) at least one sterol as defined herein, suitably cholesterol; and

(iv) at least one a PEGylated lipid as defined herein, e.g. PEG-DMG or PEG-cDMA, suitably a PEGylated lipid that is or is derived from formula IVa.

In one embodiment, the ionizable amino-lipid has formula HI-3, the neutral lipid is DSPC, the steroid is cholesterol and the PEGylated lipid which has formula IVa.

Suitably, lipids (i) to (iv) form LNPs and are present in a molar ratio of about 20-60% ionizable amino-lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEGylated lipid. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 50:10:38.5:1.5. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 47.5:10.0:40.8:1.7. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 47.4:10.0:40.9:1.7.

In one embodiment, the composition according to the invention comprises the ionizable amino-lipid of formula HI-3, DSPC, cholesterol and the PEGylated lipid of formula Iva in a molar ratio of about 47.4:10.0:40.9:1.7.

Other useful LNP compositions are described in the following references:

WO2012/006376; WO2012/030901 ; WO2012/031046; W02012/031043;

WO2012/006378; WO2011/076807; WO2013/033563; WO2013/006825;

WO2014/136086; WO2015/095340; WO2015/095346; WO2016/037053; W02017/070620 which are incorporated herein by reference.

Suitably, the LNPs have a mean diameter of from about 50nm to about 200nm, from about 60nm to about 200nm, from about 70nm to about 200nm, from about 80nm to about 200nm, from about 90nm to about 200nm, from about 90nm to about 190nm, from about 90nm to about 180nm, from about 90nm to about 170nm, from about 90nm to about 160nm, from about 90nm to about 150nm, from about 90nm to about 140nm, from about 90nm to about 130nm, from about 90nm to about 120nm, from about 90nm to about 100nm, from about 70nm to about 90nm, from about 80nm to about 90nm, from about 70nm to about 80nm, or about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 160nm, 170nm, 180nm, 190nm, or 200nm and are substantially non-toxic. As used herein, the mean diameter may be represented by the z-average size as determined by dynamic light scattering as commonly known in the art. Suitably, the LNPs have a polydispersity index (Pdl) of 0.4 or less, suitably of 0.3 or less. Typically, the Pdl is determined by dynamic light scattering.

Suitably, at least 50%, more suitably at least 60%, 70% 80%, 85%, 90% or 95% of the RNA is encapsulated in the LNP. In this context, “encapsulated RNA” is understood as RNA (suitably mRNA) that is complexed with the lipids forming the LNP and/or that is contained within the interior space of the LNP. The proportion of encapsulated RNA can typically be determined using a RiboGreen assay.

Suitably, the composition contains less than about 30%, suitably less than 20%, 15%, 10% or 5% non-encapsulated RNA (or free RNA). In this context, the term “free RNA” or “non-encapsulated RNA” is understood as RNA (suitably mRNA) that is not encapsulated in the LNPs as defined herein. In a therapeutic composition, free RNA, may represent a contamination or an impurity.

The composition of the invention comprises an RNA molecule.

The term “RNA” is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosinemonophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogs thereof, which are connected to each other along a so-called backbone. The backbone is typically formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA sequence.

Suitably, the RNA molecule is selected from an antisense RNA, such as an antisense oligonucleotides (ASOs), a small interfering RNA (siRNA), a microRNA (miRNAs), a messenger RNA (mRNA) and an RNA forming part of a single-guide RNA (sgRNA)-mediated CRISPR-Cas system.

Suitably, the amount of RNA, suitably mRNA, in the composition according to the invention ranges from about 0.1 to about 1000 pg per dose, for example from about 1 to about 500 pg, especially from about 2 to about 500 pg, in particular from about 10 to about 250 pg, for example 10, 15, 20, 30 or 50 pg per dose.

Suitably, the RNA molecule has a length of at least 200, more suitably at least 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides.

In a preferred embodiment, the RNA molecule is a messenger RNA (mRNA).

An mRNA is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by ribosomes in the process of producing a protein. The mRNA may be selected from non-replicating mRNA and self-replicating mRNA (or self- amplifying mRNA or SAM). A non-replicating mRNA typically encodes a protein of interest and contains 5' and/or 3' untranslated regions (UTRs), a 5’ cap and a poly(A) tail. A selfamplifying mRNA also encodes a viral replication machinery that enables intracellular mRNA amplification.

In one embodiment, the RNA molecule is a non-replicating mRNA.

In one embodiment, the RNA molecule is a self-replicating mRNA.

Suitably, the antioxidant prevents or reduces loss of mRNA translation efficiency.

As used herein, the term “mRNA translation efficiency” refers to the ability of the mRNA to translate into a protein, suitably into a functional protein. The term “functional protein” is understood as a protein which performs a biological function, for example a protein that can serve as a vaccine antigen or as a therapeutic, eg. an antibody. mRNA translation efficiency may be determined eg. by measuring protein expression levels using methods commonly used in the art.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 25°C for a period of 6 weeks is at least 10% higher, suitably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% higher than for a corresponding composition in which the antioxidant is not present.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 25°C for a period of 11 weeks is at least 10% higher, suitably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% higher than for a corresponding composition in which the antioxidant is not present.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 40°C for a period of 4 weeks is at least 10% higher, suitably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% higher than for a corresponding composition in which the antioxidant is not present.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 40°C for a period of 8 weeks is at least 10% higher, suitably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% higher than for a corresponding composition in which the antioxidant is not present.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 50°C for a period of 2 weeks is at least 10% higher, suitably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% higher than for a corresponding composition in which the antioxidant is not present.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 25°C for a period of 6 weeks is at least 50%, suitably at least 60%, at least 70%, at least 80% or at least 90% equal to mRNA translation efficiency prior to storage.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 25°C for a period of 11 weeks is at least 50%, suitably at least 60%, at least 70%, at least 80% or at least 90% equal to mRNA translation efficiency prior to storage.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 40°C for a period of 4 weeks is at least 50%, suitably at least 60%, at least 70%, at least 80% or at least 90% equal to mRNA translatability prior to storage.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 40°C for a period of 8 weeks is at least 50%, suitably at least 60%, at least 70%, at least 80% or at least 90% equal to mRNA translation efficiency prior to storage.

Suitably, mRNA translation efficiency of the mRNA forming part of the composition according to the invention after storage at 50°C for a period of 2 weeks is at least 50%, suitably at least 60%, at least 70%, at least 80% or at least 90% equal to mRNA translation efficiency prior to storage.

The RNA, suitably mRNA, is suitably provided in a purified or substantially purified form i.e. substantially free from proteins (e.g., enzymes), other nucleic acids (e.g. DNA and nucleoside phosphate monomers), and the like, generally being at least about 50% pure (by weight), and usually at least 90% pure, such as at least 95% or at least 98% pure.

The RNA, suitably mRNA, may be prepared in many ways e.g. by chemical synthesis in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc. In particular, the mRNA may be prepared enzymatically using a DNA template.

The RNA, suitably mRNA, may be an artificial nucleic acid.

The term “artificial nucleic acid” as used herein is intended to refer to a nucleic acid that does not occur naturally. In other words, an artificial nucleic acid may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecules may be non-natural due to its individual sequence (e.g. G/C content modified coding sequence, UTRs) and/or due to other modifications, e.g. structural modifications of nucleotides. Typically, artificial nucleic acid may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, an artificial nucleic acid is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type or reference sequence/the naturally occurring sequence by at least one nucleotide (via e.g. codon modification as further specified below). The term “artificial nucleic acid” is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical nucleic acid molecules. Accordingly, it may relate to a plurality of essentially identical nucleic acid molecules.

In some embodiments, the mRNA may be a modified and/or stabilized mRNA, suitably a modified and/or stabilized artificial mRNA.

According to some embodiments, the mRNA may thus be provided as a “stabilized” artificial nucleic acid or stabilized coding nucleic acid that is to say a nucleic acid showing improved resistance to in vivo degradation and/or a nucleic acid showing improved stability in vivo, and/or a nucleic acid showing improved translation efficiency in vivo. In the following, specific suitable modifications/adaptations in this context are described which are suitable to stabilize the nucleic acid.

The mRNA may be codon optimized. In some embodiments, the mRNA comprises at least one codon modified coding sequence. In some embodiments, the coding sequence of the mRNA is a codon modified coding sequence. Suitably, the amino acid sequence encoded by the codon modified coding sequence is not modified compared to the amino acid sequence encoded by the corresponding wild type or reference coding sequence. Suitably, the mRNA may be codon optimized for expression in human cells.

By “codon optimized” is intended modification with respect to codon usage may increase translation efficacy and/or half-life of the nucleic acid. The term “codon modified coding sequence” relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type or reference coding sequence. Suitably, a codon modified coding sequence in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translation efficiency in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably (cf. Table 1 of W02020002525) to optimize/modify the coding sequence for in vivo applications as outlined herein.

In some embodiments, the mRNA may be modified, wherein the C content of the at least one coding sequence is increased, suitably maximized, compared to the C content of the corresponding wild type or reference coding sequence (herein referred to as “C maximized coding sequence”). The amino acid sequence encoded by the C maximized coding sequence of the mRNA is suitably not modified compared to the amino acid sequence encoded by the respective wild type or reference coding sequence. The generation of a C maximized nucleic acid sequences may suitably be carried out using a modification method according to WO2015/062738. In this context, the disclosure of WO2015/062738 is included herewith by reference.

In some embodiments, the mRNA may be modified, wherein the codons in the at least one coding sequence may be adapted to human codon usage (herein referred to as “human codon usage adapted coding sequence”). Codons encoding the same amino acid occur at different frequencies in humans. Accordingly, the coding sequence of the mRNA is suitably modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage. For example, in the case of the amino acid Ala, the wild type or reference coding sequence is suitably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with a frequency of 0.10 etc. (see e.g. Table 1 of W02020002525). Accordingly, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of the RNA to obtain sequences adapted to human codon usage.

In embodiments, the mRNA may be modified, wherein the codon adaptation index (CAI) may be increased or suitably maximised in the at least one coding sequence (herein referred to as “CAI maximized coding sequence”). In some embodiments, all codons of the wild type or reference nucleic acid sequence that are relatively rare in e.g. a human are exchanged for a respective codon that is frequent in the e.g. a human, wherein the frequent codon encodes the same amino acid as the relatively rare codon. Suitably, the most frequent codons are used for each amino acid of the encoded protein (see Table 1 of W02020002525, most frequent human codons are marked with asterisks). Suitably, the mRNA comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. In some embodiments, the codon adaptation index (CAI) of the at least one coding sequence is 1 (CAI=1). For example, in the case of the amino acid Ala, the wild type or reference coding sequence may be adapted in a way that the most frequent human codon “GCC” is always used for the amino acid. Accordingly, such a procedure (as exemplified for Ala) may be applied for each amino acid encoded by the coding sequence of the mRNA to obtain CAI maximized coding sequences.

In some embodiments, the mRNA may be modified, wherein the G/C content of the at least one coding sequence may be modified (or optimized) compared to the G/C content of the corresponding wild type or reference coding sequence (herein referred to as “G/C content modified

”)■ In this context, the terms “G/C optimization” or “G/C content modification” relate to a nucleic acid that comprises a modified suitably an increased number of guanosine and/or cytosine nucleotides as compared to the corresponding wild type or reference coding sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides. Suitably, nucleic acid sequences having an increased G /C content are more stable or show a better expression than sequences having an increased A/ll. The amino acid sequence encoded by the G/C content modified coding sequence of the mRNA is suitably not modified as compared to the amino acid sequence encoded by the respective wild type or reference sequence. In some embodiments, the G/C content of the coding sequence of the nucleic acid is increased by at least 10%, 20%, 30%, suitably by at least 40% compared to the G/C content of the coding sequence of the corresponding wild type or reference nucleic acid sequence. The generation of a G/C content optimized mRNA sequence may be carried out using a method according to W02002/098443. In this context, the disclosure of W02002/098443 is included in its full scope in the present invention.

In some embodiments, the mRNA may be modified by altering the number of A and/or II nucleotides in the nucleic acid sequence with respect to the number of A and/or II nucleotides in the original nucleic acid sequence (e.g. the wild type or reference sequence). In some embodiments, such an A/ll alteration is performed to modify the retention time of the individual nucleic acids in a composition, to (i) allow co-purification using a HPLC method, and/or to allow analysis of the obtained nucleic acid composition. Such a method is described in detail in published PCT application WO2019092153A1. Claims 1 to 70 of WO2019092153A1 herewith incorporated by reference.

In some embodiments, the modified RNA sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified (or optimized) sequence, A/ll alteration, or any combination thereof.

In some embodiments, the RNA sequence has a G/C content of at least about 45%, 50%, 55%, or 60%. In particular embodiments, the RNA sequence has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

Suitably, when transfected into mammalian host cells, the mRNA comprising a modified sequence has a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cell (e.g. a muscle cell).

Suitably, when transfected into mammalian host cells, the mRNA comprising a modified RNA sequence is translated into protein, wherein the amount of protein is at least comparable to, or suitably at least 10% more than, or at least 20% more than, or at least 30% more than, or at least 40% more than, or at least 50% more than, or at least 100% more than, or at least 200% or more than the amount of protein obtained by a naturally occurring or wild type or reference coding sequence transfected into mammalian host cells.

In some embodiments, the mRNA comprises at least one poly(N) sequence, e.g. at least one poly(A) sequence, at least one poly(ll) sequence, at least one poly(C) sequence, or combinations thereof.

In one embodiment, the mRNA comprises at least one poly(A) sequence. Suitably, a poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3' end of the RNA to increase its half-life.

The terms “poly(A) sequence”, “poly(A) tail” or “3’-poly(A) tail” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to be a sequence of adenosine nucleotides, typically located at the 3’-end of a linear RNA (or in a circular RNA), of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is essentially homopolymeric, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides has essentially the length of 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition the at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide).

The poly(A) sequence may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. In some embodiments, the length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides.

In some embodiments, the mRNA comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides. In some embodiments, the poly(A) sequence comprises about 64 adenosine nucleotides (A64). In other embodiments, the poly(A) sequence comprises about 100 adenosine nucleotides (A100). In other embodiments, the poly(A) sequence comprises about 150 adenosine nucleotides. In further embodiments, the mRNAs used herein comprise at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, suitably by 10 non-adenosine nucleotides (A30- N10-A70).

The poly(A) sequence as defined herein may be located directly at the 3’ terminus of the mRNA. In some embodiments, the 3’-terminal nucleotide (that is the last 3’-terminal nucleotide in the polynucleotide chain) is the 3’-terminal A nucleotide of the at least one poly(A) sequence. The term “directly located at the 3’ terminus” is to be understood as being located exactly at the 3’ terminus - in other words, the 3’ terminus of the nucleic acid consists of a poly(A) sequence terminating with an A nucleotide.

In one embodiment, the mRNA comprises a poly(A) sequence of at least 70 adenosine nucleotides, suitably consecutive at least 70 adenosine nucleotides, wherein the 3’-terminal nucleotide is an adenosine nucleotide.

In embodiments, the poly(A) sequence of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly(A) sequence is obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA template. In other embodiments, poly(A) sequences are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols known in the art, or alternatively, by using immobilized poly(A)polymerases e.g. using a methods and means as described in WO2016174271.

In one embodiment, the mRNA comprises at least one poly(C) sequence.

The term “poly(C) sequence” as used herein is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In an embodiment, the poly(C) sequence comprises about 30 cytosine nucleotides.

In some embodiments, the mRNA comprises a poly(A) tail sequence, preferably comprising 30 to 200 adenosine nucleotides and/or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides.

In some embodiments, the mRNA comprises at least one histone stem-loop (hSL) or histone stem loop structure.

The term “histone stem-loop” (abbreviated as “hSL” in e.g. the sequence listing) is intended to refer to nucleic acid sequences that form a stem-loop secondary structure predominantly found in histone mRNAs. Histone stem-loop sequences/structures may suitably be selected from histone stem-loop sequences as disclosed in WO2012019780, the disclosure relating to histone stem-loop sequences/histone stem-loop structures incorporated herewith by reference. A histone stem-loop sequence that may be used may be derived from formulae (I) or (II) of WO2012019780. According to a further embodiment, the mRNA comprises at least one histone stem-loop sequence derived from at least one of the specific formulae (la) or (Ila) of the patent application W02012019780.

In other embodiments, the mRNA does not comprise a hsL as defined herein.

The mRNA may be modified by the addition of a 5’-cap structure, which suitably stabilizes the RNA and/or enhances expression of the encoded antigen and/or reduces the stimulation of the innate immune system (after administration to a subject).

The term “5’-cap structure” as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a 5’ modified nucleotide, particularly a guanine nucleotide, positioned at the 5’-end of an RNA, e.g. an mRNA.

For example, the 5' end of the mRNA may be capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl-transferase and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the mRNA molecule may be further modified by a 2'-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy.

In some embodiments, the 5’-cap structure is connected via a 5’-5’-triphosphate linkage to the RNA.

5’-cap structures which may be suitable are capO (methylation of the first nucleobase, e.g. m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2’-fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the mRNA comprises a 5’ cap, preferably m7G, capO, cap1 , cap2, a modified capO or a modified cap1 structure, suitably a 5’-cap1 structure.

A 5’-cap (such as capO or cap1) structure may be formed in chemical RNA synthesis or in RNA in vitro transcription (co-transcriptional capping) using cap analogues.

The term “cap analogue” as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a non-polymerizable dinucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of a nucleic acid molecule, particularly of an RNA molecule, when incorporated at the 5’-end of the nucleic acid molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3’-direction by a template-dependent polymerase, particularly, by template-dependent RNA polymerase. Examples of cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g. GpppG); dimethylated cap analogue (e.g. m2,7GpppG), trimethylated cap analogue (e.g. m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g. m7Gpppm7G), or anti reverse cap analogues (e.g. ARCA; m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, m7,3’dGpppG and their tetraphosphate derivatives). Further cap analogues have been described previously (W02008016473, WO2008157688, WG2009149253, WO2011015347, and WO2013059475). Further suitable cap analogues in that context are described in WO2017066793, WO2017066781 , WO2017066791 , WO2017066789, WO2017/053297, WO2017066782, WO2018075827 and

WO2017066797 wherein the disclosures referring to cap analogues are incorporated herewith by reference.

In some embodiments, a modified cap1 structure is generated using tri-nucleotide cap analogue as disclosed in WO2017053297, WO2017066793, WO2017066781 , WO2017066791 , WO2017066789, WO2017066782, WO2018075827 and

WO2017066797. In particular, any cap structures derivable from the structure disclosed in claim 1-5 of WO2017053297 may be suitably used to co-transcriptionally generate a modified cap1 structure. Further, any cap structures derivable from the structure defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate a modified cap1 structure.

In some embodiments, the mRNA comprises a cap1 structure.

In some embodiments, the 5’-cap structure may be added co-transcriptionally using tri-nucleotide cap analogue as defined herein, suitably in an RNA in vitro transcription reaction as defined herein. In some embodiments, the cap1 structure of the mRNA is formed using co- transcriptional capping using tri-nucleotide cap analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG. A suitable cap1 analogues in that context is m7G(5’)ppp(5’)(2’OMeA)pG.

In other embodiments, the cap1 structure of the mRNA is formed using co- transcriptional capping using tri-nucleotide cap analogue 3’0Me- m7G(5’)ppp(5’)(2’OMeA)pG.

In other embodiments, a capO structure of the mRNAs used herein is formed using co-transcriptional capping using cap analogue 3’0Me-m7G(5’)ppp(5’)G.

In other embodiments, the 5’-cap structure is formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases) to generate capO or cap1 or cap2 structures. The 5’-cap structure (capO or cap1) may be added using immobilized capping enzymes and/or cap-dependent 2’-0 methyltransferases using methods and means disclosed in WO2016193226.

For determining the presence/absence of a capO or a cap1 structure, a capping assays as described in published PCT application W02015101416, in particular, as described in claims 27 to 46 of published PCT application W02015101416 can be used. Other capping assays that may be used to determine the presence/absence of a capO or a cap1 structure of an RNA are described in PCT/EP2018/08667, or published PCT applications WO2014152673 and WO2014152659.

In some embodiments, the mRNA comprises an m7G(5’)ppp(5’)(2’OMeA) cap structure. In such embodiments, the mRNAs comprise a 5’-terminal m7G cap, and an additional methylation of the ribose of the adjacent nucleotide of m7GpppN, in that case, a 2’0 methylated Adenosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises such a cap1 structure as determined using a capping assay.

In other embodiments, the mRNAs used herein comprise an m7G(5’)ppp(5’)(2’OMeG) cap structure. In such embodiments, the mRNAs comprise a 5’- terminal m7G cap, and an additional methylation of the ribose of the adjacent nucleotide, in that case, a 2’0 methylated guanosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the coding RNA (species) comprises such a cap1 structure as determined using a capping assay.

Accordingly, the first nucleotide of the mRNA sequence, that is, the nucleotide downstream of the m7G(5’)ppp structure, may be a 2’0 methylated guanosine or a 2’0 methylated adenosine. Suitably, the mRNAs used herein comprise a ribosome binding site, also referred to as Kozak sequence. In some embodiments, the A/ll (A/T) content in the environment of the ribosome binding site of the mRNAs used herein may be increased compared to the A/ll (A/T) content in the environment of the ribosome binding site of its respective wild type or reference nucleic acid. This modification (an increased A/ll (A/T) content around the ribosome binding site) increases the efficiency of ribosome binding to the mRNA. An effective binding of the ribosomes to the ribosome binding site in turn has the effect of an efficient translation the mRNA.

In some embodiments, the mRNA comprises at least one heterologous untranslated region (UTR), e.g. a 5’ UTR and/or a 3’ UTR.

The term “untranslated region” or “UTR” or “UTR element” will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a part of a nucleic acid molecule typically located 5’ or 3’ of a coding sequence. An UTR is not translated into protein. An UTR may be part of a nucleic acid, e.g. a DNA or an RNA. An UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, promotor elements etc.

In some embodiments, the mRNA comprises a protein-coding region (“coding seguence” or “cds”), and 5’-UTR and/or 3’-UTR. Notably, UTRs may harbor regulatory sequence elements that determine nucleic acid, e.g. RNA turnover, stability, and localization. Moreover, UTRs may harbor sequence elements that enhance translation. In medical application of nucleic acid sequences (including DNA and RNA), translation of the nucleic acid into at least one peptide or protein is of paramount importance to therapeutic efficacy. Certain combinations of 3’-UTRs and/or 5’-UTRs may enhance the expression of operably linked coding sequences encoding peptides or proteins of the invention. Nucleic acid molecules harboring the UTR combinations advantageously enable rapid and transient expression of antigenic peptides or proteins after administration to a subject, suitably after intramuscular administration. Accordingly, the mRNA comprising certain combinations of 3’-UTRs and/or 5’-UTRs as provided herein is particularly suitable for administration as a vaccine or therapeutic, in particular, suitable for administration into the muscle, the dermis, or the epidermis of a subject.

In some embodiments, the mRNA comprises at least one heterologous 5’-UTR and/or at least one heterologous 3’-UTR. The heterologous 5’-UTRs or 3’-UTRs may be derived from naturally occurring genes or may be synthetically engineered. In embodiments, the mRNA comprises at least one coding sequence as defined herein operably linked to at least one (heterologous) 3’-UTR and/or at least one (heterologous) 5’-UTR. In some embodiments, the mRNA comprises at least one heterologous 3’-UTR.

The term “3’-untranslated region” or “3’-UTR” or “3’-UTR element” will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a part of a nucleic acid molecule located 3’ (i.e. downstream) of a coding sequence and which is not translated into protein. A 3’-UTR may be part of a nucleic acid, e.g. a DNA or an RNA, located between a coding sequence and an (optional) terminal poly(A) sequence. A 3’-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.

In some embodiments, the mRNA comprises a 3’-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).

In some embodiments, a 3’-UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.

In embodiments, the mRNA comprises at least one heterologous 3’-UTR, wherein the at least one heterologous 3’-UTR comprises a nucleic acid sequence is derived or selected from a 3’-UTR of a gene selected from PSMB3, ALB7, alpha-globin (referred to as “muag”), CASP1 , COX6B1 , GNAS, NDLIFA1 and RPS9, or from a homolog, a fragment or variant of any one of these genes.

In some embodiments, the mRNA comprises a 3’ UTR comprising or consisting of a nucleic acid sequence derived from a 3’-UTR of a gene selected from PSMB3, ALB7, CASP1 , COX6B1 , GNAS, NDLIFA1 and RPS9, or from a homolog, a fragment or a variant of any one of these genes.

Nucleic acid sequences in that context can be derived from published PCT application WO2019077001 A1 , in particular, claim 9 of WO2019077001 A1. The corresponding 3’-UTR sequences of claim 9 of WO2019077001 A1 are herewith incorporated by reference.

In some embodiments, the mRNA comprises a 3’-UTR as described in WO2016107877, the disclosure of WO2016107877 relating to 3’-UTR sequences herewith incorporated by reference. Suitable 3’-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49- 318 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 3’-UTR as described in WO2017036580, the disclosure of WO2017036580 relating to 3’-UTR sequences herewith incorporated by reference. Suitable 3’-UTRs are SEQ ID NOs: 152-204 of WO2017036580, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 3’-UTR as described in WO2016022914, the disclosure of WO2016022914 relating to 3’-UTR sequences herewith incorporated by reference. Particularly suitable 3’-UTRs are nucleic acid sequences according to SEQ ID NOs: 20-36 of WO2016022914, or fragments or variants of these sequences.

In some embodiments, the mRNA comprises at least one heterologous 5’-UTR.

The terms “5’-untranslated region” or “5’-UTR” or “5’-UTR element” will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a part of a nucleic acid molecule located 5’ (i.e. “upstream”) of a coding sequence and which is not translated into protein. A 5’-UTR may be part of a nucleic acid located 5’ of the coding sequence. Typically, a 5’-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence. A 5’-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc. The 5’-UTR may be post- transcriptionally modified, e.g. by enzymatic or post-transcriptional addition of a 5’-cap structure (e.g. for mRNA as defined herein).

In some embodiments, the mRNA comprises a 5’-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).

In some embodiments, a 5’-UTR comprises one or more of a binding site for proteins that affect an RNA stability or RNA location in a cell, or one or more miRNA or binding sites for miRNAs.

In embodiments, the mRNA comprises at least one heterologous 5’-UTR, wherein the at least one heterologous 5’-UTR comprises a nucleic acid sequence is derived or selected from a 5’-UTR of gene selected from HSD17B4, RPL32, ASAH1 , ATP5A1 , MP68, NDUFA4, NOSIP, RPL31 , SLC7A3, TUBB4B, and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.

Nucleic acid sequences in that context can be selected from published PCT application W02019077001A1 , in particular, claim 9 of WO2019077001 A1. The corresponding 5’-UTR sequences of claim 9 of W02019077001A1 are herewith incorporated by reference (e.g., SEQ ID NOs: 1-20 of WO2019077001 A1 , or fragments or variants thereof).

In some embodiments, the mRNA comprises a 5’-UTR as described in WQ2013143700, the disclosure of WQ2013143700 relating to 5’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of WQ2013143700, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in WQ2016107877, the disclosure of WQ2016107877 relating to 5’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in WO2017036580, the disclosure of WO2017036580 relating to 5’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 of WO2017036580, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in WO2016022914, the disclosure of WO2016022914 relating to 5’-UTR sequences herewith incorporated by reference. Particularly suitable 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 of WQ2016022914, or fragments or variants of these sequences.

In some embodiments, the mRNA comprises an heterologous 5’-UTR that comprises or consists of a nucleic acid sequence derived from a 5’-UTR from HSD17B4 and at least one heterologous 3’-UTR comprises or consists of a nucleic acid sequence derived from a 3’-UTR of PSMB3.

In one embodiment, the mRNA comprises from 5’ to 3’: i) 5’-cap1 structure; ii) 5’-UTR derived from a 5’-UTR of a HSD17B4 gene; iii) the coding sequence; iv) 3’-UTR derived from a 3’-UTR of a PSMB3 gene; v) optionally, a histone stem-loop sequence; and vi) poly(A) sequence comprising about 100 A nucleotides, wherein the 3’ terminal nucleotide of said RNA is an adenosine.

Chemical Modifications

In some embodiments, the RNA, suitably mRNA, comprises a coding sequence that consists only of G, C, A and II nucleotides and therefore does not comprise modified nucleotides (except of the 5’ terminal cap structure (capO, cap1 , cap2)).

In some embodiments, the RNA, suitably mRNA, is a modified RNA, suitably mRNA, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.

A modified RNA, suitably mRNA, may comprise one or more nucleotide analogs or modified nucleotides (nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications). As used herein, "nucleotide analog" or "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g. cytosine (C), thymine (T) or uracil (II)), adenine (A) or guanine (G)) and/or one or more chemical modifications in or one the phosphates of the backbone. A nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g. ribose, modified ribose, sixmembered sugar analog, or open-chain sugar analog), or the phosphate. The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, see the following references: US Patent Numbers 4373071 , 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.

A backbone modification as described herein is a modification, in which phosphates of the backbone of the nucleotides of the RNA, suitably the mRNA, are chemically modified. A sugar modification as described herein is a chemical modification of the sugar of the nucleotides of the RNA, suitably mRNA. Furthermore, a base modification as described herein is a chemical modification of the base moiety of the nucleotides of the RNA, suitably mRNA. In this context, nucleotide analogues or modifications are suitably selected from nucleotide analogues which are applicable for transcription and/or translation.

In some embodiments, the RNA, suitably the mRNA, comprises at least one chemical modification.

Modified nucleobases (chemical modifications) which can be incorporated into modified nucleosides and nucleotides and be present in the RNA, suitably mRNA, molecules include: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6- methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1 -methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (I ,2'-O-dimethylinosine); m3C (3- methylcytidine); Cm (2’-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2-O- methylcytidine); k2C (lysidine); m1G (1 -methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2- dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O- trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl- 2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5- aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5- methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L-O-methyl uridine); cmnm5s2U (5-carboxymethylaminomethyl-2- thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4- methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); rn62Am (N6,N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2’-O-dimethyluridine); m5D (5- methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); mIGm (I ,2'-0- dimethylguanosine); m'Am (1 ,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S- taurinomethyl-2-thiouridine)); iniG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Ci-Ce)- alkyluracil, 5-methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 5- (hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (Ci-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-Ce)- alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2- Ce)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6- diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7- deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O- methyl-U. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. In some embodiments, the nucleotide analogues/modifications which may be incorporated into a modified RNA, suitably mRNA, are selected from 2-amino-6- chloropurineriboside-5’-triphosphate, 2-Aminopurine-riboside-5’-triphosphate; 2- aminoadenosine-5’-triphosphate, 2’-Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5’- triphosphate, 2-thiouridine-5’-triphosphate, 2’-Fluorothymidine-5’-triphosphate, 2’-O- Methyl-inosine-5’-triphosphate 4-thiouridine-5’-triphosphate, 5-aminoallylcytidine-5’- triphosphate, 5-aminoallyluridine-5’-triphosphate, 5-bromocytidine-5’-triphosphate, 5- bromouridine-5’-triphosphate, 5-Bromo-2’-deoxycytidine-5’-triphosphate, 5-Bromo-2’- deoxyuridine-5’-triphosphate, 5-iodocytidine-5’-triphosphate, 5-lodo-2’-deoxycytidine-5’- triphosphate, 5-iodouridine-5’-triphosphate, 5-lodo-2’-deoxyuridine-5’-triphosphate, 5- methylcytidine-5’-triphosphate, 5-methyluridine-5’-triphosphate, 5-Propynyl-2’- deoxycytidine-5’-triphosphate, 5-Propynyl-2’-deoxyuridine-5’-triphosphate, 6-azacytidine- 5’-triphosphate, 6-azauridine-5’-triphosphate, 6-chloropurineriboside-5’-triphosphate, 7- deazaadenosine-5’-triphosphate, 7-deazaguanosine-5’-triphosphate, 8-azaadenosine-5’- triphosphate, 8-azidoadenosine-5’-triphosphate, benzimidazole-riboside-5’-triphosphate, N 1 -methyladenosine-5’-triphosphate, N 1 -methylguanosine-5’-triphosphate, N6- methyladenosine-5’-triphosphate, O6-methylguanosine-5’-triphosphate, pseudouridine-5’- triphosphate, or puromycin-5’-triphosphate, xanthosine-5’-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of basemodified nucleotides consisting of 5-methylcytidine-5’-triphosphate, 7-deazaguanosine-5’- triphosphate, 5-bromocytidine-5’-triphosphate, and pseudouridine-5’-triphosphate, pyridin- 4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxy methyluridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl- pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4- methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza- guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7- methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2, N2-dimethyl-6-thio-guanosine, 5’-O-(1 -thiophosphate)-adenosine, 5’-O-(1 - thiophosphate)-cytidine, 5’-O-(1 -thiophosphate)-guanosine, 5’-O-(1 -thiophosphate)- uridine, 5’-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio- cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl- pseudouridine, 5,6-dihydrouridine, alpha -thio-uridine, 4-thio-uridine, 6-aza-uridine, 5- hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha -thio- guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso- cytidine, 6-Chloro-purine, N6-methyl-adenosine, alpha -thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

In some embodiments, the chemical modification is selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5- methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methoxyuridine and 2'-O-methyl uridine.

Particularly suitable in that context are pseudouridine (qj), N1 -methylpseudouridine (m1i ), 5-methylcytosine, and 5-methoxyuridine, more suitably pseudouridine (i ) and N1- methylpseudouridine (m1i ), still more suitably N1-methylpseudouridine (m1 i ).

In some embodiments, essentially all, e.g. essentially 100% of the uracil in the coding sequence of the RNA, suitably mRNA, have a chemical modification, suitably a chemical modification is in the 5-position of the uracil. In some embodiments, the RNA, suitably mRNA, comprises a chemical modification being a uridine modification, preferably wherein 100% of the uridine positions in the mRNA are modified.

Incorporating modified nucleotides such as e.g. pseudouridine (qj), N1- methylpseudouridine (m1 i ), 5-methylcytosine, and/or 5-methoxyuridine into the coding sequence of the RNA, suitably mRNA, used herein may be advantageous as unwanted innate immune responses (upon administration of the coding mRNA or the vaccine) may be adjusted or reduced (if required).

In some embodiments, the coding sequence of the RNA, suitably mRNA, comprises at least one modified nucleotide selected from pseudouridine (i ) and N1- methylpseudouridine (m1 i ), suitably wherein all uracil nucleotides are replaced by pseudouridine (i ) nucleotides and/or N1 -methylpseudouridine (m1ip) nucleotides, optionally wherein all uracil nucleotides are replaced by pseudouridine (^P) nucleotides and/or N1 -methylpseudouridine (ml ^P) nucleotides.

In some embodiments, the RNA, suitably mRNA, does not comprise N1- methylpseudouridine (ml^P) substituted positions. In further embodiments, the RNAs, suitably mRNAs, used herein do not comprise pseudouridine (ip), N1 -methylpseudouridine (m1ip), 5-methylcytosine, and 5-methoxyuridine substituted position.

In some embodiments, the chemical modification is N1 -methylpseudouridine and/or pseudouridine. In some embodiments, the chemical modification is N1- methylpseudouridine.

In the context of nucleic acid-based vaccine or therapeutic production, it may be required to provide GMP-grade nucleic acid, e.g. a GMP grade RNA or DNA. GMP-grade RNA or DNA may be produced using a manufacturing process approved by regulatory authorities. Accordingly, in some embodiments, RNA production is performed under current good manufacturing practice (GMP), implementing various quality control steps on DNA and RNA level, suitably according to WO2016180430. In embodiments, the RNA, suitably mRNA of the invention is a GMP-grade RNA.

RNA synthesis

In some embodiments, the RNA, suitably mRNA, may be prepared using any method known in the art, including chemical synthesis such as e.g. solid phase RNA synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions.

Suitably, the RNA, suitably mRNA, used herein is in vitro transcribed RNA.

The terms “RNA in vitro transcription” or “in vitro transcription” relate to a process wherein RNA is synthesized in a cell-free system in vitro). RNA may be obtained by DNA- dependent in vitro transcription of an appropriate DNA template, which may be a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases. In an embodiment of the present invention the DNA template is linearized with a suitable restriction enzyme, before it is subjected to RNA in vitro transcription. Reagents used in RNA in vitro transcription typically include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein; optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate, which may inhibit RNA in vitro transcription; MgCI2, which supplies Mg2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine at optimal concentrations, e.g. a buffer system comprising TRIS-Citrate as disclosed in W02017109161.

In embodiments, the nucleotide mixture used in RNA in vitro transcription may additionally comprise modified nucleotides as defined herein. In that context, suitable modified nucleotides may in particular be selected from pseudouridine (qj), N1- methylpseudouridine (m1i ), 5-methylcytosine, and 5-methoxyuridine. In embodiments, uracil nucleotides in the nucleotide mixture are replaced (either partially or completely) by pseudouridine (i ) and/or N1 -methylpseudouridine (m1i ) to obtain a modified RNA.

In other embodiments, the nucleotide mixture used in RNA in vitro transcription does not comprise modified nucleotides as defined herein. In embodiments, the nucleotide mixture used in RNA in vitro transcription only comprises G, C, A and II nucleotides, and, optionally, a cap analog as defined herein.

In some embodiments, the nucleotide mixture (i.e. the fraction of each nucleotide in the mixture) used for RNA in vitro transcription reactions may be optimized for the given RNA sequence, suitably as described in WO2015188933.

In this context, the in vitro transcription has been performed in the presence of a sequence optimized nucleotide mixture and optionally a cap analog. RNA purification

Suitably, the RNA (or mRNA), is a purified RNA (or mRNA).

The term “purified RNA (or mRNA)” as used herein has to be understood as RNA which has a higher purity after certain purification steps (e.g. HPLC, TFF, Oligo d(T) purification, precipitation steps) than the starting material (e.g. in vitro transcribed RNA). Typical impurities that are essentially not present in purified RNA comprise peptides or proteins (e.g. enzymes derived from DNA dependent RNA in vitro transcription, e.g. RNA polymerases, RNases, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double stranded RNA fragments, abortive sequences etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogue), template DNA fragments, buffer components (HEPES, TRIS, MgCI2) etc. Other potential impurities that may be derived from e.g. fermentation procedures comprise bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents etc.). Accordingly, it is desirable in this regard for the “degree of RNA purity” to be as close as possible to 100%. It is also desirable for the degree of RNA purity that the amount of full-length RNA transcripts is as close as possible to 100%. Accordingly, “purified RNA (or mRNA)” as used herein has a degree of purity of more than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% or more. The degree of purity may for example be determined by an analytical HPLC, wherein the percentages provided above correspond to the ratio between the area of the peak for the target RNA and the total area of all peaks representing the byproducts. Alternatively, the degree of purity may for example be determined by an analytical agarose gel electrophoresis or capillary gel electrophoresis.

In some embodiments, the RNA is purified using RP-HPLC, suitably using Reversed-Phase High pressure liquid chromatography (RP-HPLC) with a macroporous styrene/divinylbenzene column (e.g. particle size 30pm, pore size 4000 A) and additionally using a filter cassette with a cellulose based membrane with a molecular weight cutoff of about 100kDa. The RNA may in particular be purified using PUREMESSENGER (CureVac, Tubingen, Germany; RP-HPLC according to W02008077592) and/or tangential flow filtration (as described in WO2016193206) and/or oligo d(T) purification (see WO2016180430).

In some embodiments, the RNA, suitably mRNA, is purified by RP-HPLC and/or TFF to remove double-stranded RNA, non-capped RNA and/or RNA fragments.

The formation of double stranded RNA as side products during e.g. RNA in vitro transcription can lead to an induction of the innate immune response, particularly IFNalpha which is the main factor of inducing fever in vaccinated subjects, which is of course an unwanted side effect. Current techniques for immunoblotting of dsRNA (via dot Blot, serological specific electron microscopy (SSEM) or ELISA for example) are used for detecting and sizing dsRNA species from a mixture of nucleic acids.

In embodiments, the RNA, suitably mRNA, comprises about 5%, 10%, or 20% less double stranded RNA side products as an RNA, suitably mRNA, that has not been purified with RP-HPLC and/or TFF.

In some embodiments, the RP-HPLC and/or TFF purified RNA, suitably mRNA, comprises about 5%, 10%, or 20% less double stranded RNA side products as an RNA, suitably mRNA, that has been purified with Oligo dT purification, precipitation, filtration and/or AEX.

Suitably, the composition is a therapeutic composition. In one embodiment, the therapeutic composition is an immunogenic composition, suitably a vaccine composition.

The therapeutic composition according to the invention may be administered via various suitable routes, including parenteral, such as intramuscular, intradermal, intranasal, or subcutaneous administration. Suitably, the therapeutic composition is suitable for intramuscular administration to a subject.

Subjects to which administration of the therapeutic composition is contemplated include, but are not limited to mammals, including humans and/or other primates, as well as commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys. Suitably, the therapeutic composition is for administration to human subjects.

Suitably, the therapeutic composition further comprises at least one pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein suitably includes the liquid or non-liquid basis of the composition for administration. If the composition is provided in liquid form, the carrier may be water, e.g. pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions. Water or suitably a buffer, more suitably an aqueous buffer, may be used, containing a sodium salt, suitably at least 50mM of a sodium salt, a calcium salt, suitably at least 0.01 mM of a calcium salt, and optionally a potassium salt, suitably at least 3mM of a potassium salt. According to some embodiments, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Examples of sodium salts include NaCI, Nal, NaBr, Na2COs, NaHCCh, Na2SC>4, examples of the optional potassium salts include KCI, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include CaCb, Cal2, CaBr2, CaCCh, CaSC , Ca(0H)2.

Furthermore, organic anions of the afore mentioned cations may be in the buffer. Accordingly, in embodiments, the immunogenic composition may comprise pharmaceutically acceptable carriers or excipients using one or more pharmaceutically acceptable carriers or excipients to e.g. increase stability, increase cell transfection, permit the sustained or delayed, increase the translation of encoded antigenic peptides or proteins in vivo, and/or alter the release profile of encoded antigenic peptides or proteins protein in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics and combinations thereof. In embodiments, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well, which are suitable for administration to a subject. The term “compatible” as used herein means that the constituents of the composition are capable of being mixed with the at least one nucleic acid of component A and/or component B and, optionally, a plurality of nucleic acids of the composition, in such a manner that no interaction occurs, which would substantially reduce the biological activity or the pharmaceutical effectiveness of the composition under typical use conditions (e.g., intramuscular or intradermal administration). Pharmaceutically acceptable carriers or excipients must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated. Compounds which may be used as pharmaceutically acceptable carriers or excipients may be sugars, such as, for example, lactose, glucose, trehalose, mannose, and sucrose; starches, such as, for example, corn starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

In another aspect, there is provided the composition of the invention for use in therapy, suitably for use as a vaccine.

In another aspect, there is provided a method for treating or preventing a disease, comprising administering the composition of the invention to a patient in need thereof. In another aspect, there is provided a method for treating or preventing an infectious disease, comprising administering the composition of the invention to a patient in need thereof.

In another aspect, there is provided the use of an antioxidant to stabilize a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid.

In one embodiment of the use according to the invention, the antioxidant is an amino acid. Suitably, the amino acid is selected from methionine, tryptophan, histidine, lysine, cysteine, arginine and tyrosine. In a preferred embodiment, the antioxidant is methionine.

Suitably, the antioxidant is present in the composition in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose. In one embodiment, the antioxidant is present in the composition in an amount of 0.45 mg per dose.

Suitably, the antioxidant is present in the composition at a concentration between 0.1 and 5 mg per ml, more suitably at a concentration between 1 and 3 mg per ml, for example at a concentration selected from 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 mg per ml. In one embodiment, the antioxidant is present in the composition at a concentration of 1.5 mg per ml.

Suitably, methionine is present in the composition in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose. In one embodiment, methionine is present in the composition in an amount of 0.45 mg per dose.

Suitably, methionine is present in the composition at a concentration between 0.1 and 5 mg per ml, more suitably at a concentration between 1 and 3 mg per ml, for example at a concentration selected from 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 mg per ml. In one embodiment, methionine is present in the composition at a concentration of 1.5 mg per ml.

Suitably, the antioxidant prevents or reduces the formation of RNA adduct. More suitably, the antioxidant prevents or reduces the formation of lipid-RNA adduct.

In one embodiment of the use according to the invention, the composition is a liquid composition. Suitably, the liquid composition does not comprise a surfactant. Suitably, the liquid composition does not comprise a non-ionic surfactant. Suitably, the liquid composition does not comprise a non-ionic surfactant selected from polysorbate-20 (PS20), polysorbate- 60 (PS60), polysorbate-80 (PS80) and poloxamers.

In one embodiment of the use according to the invention, the composition is a dry composition.

In one embodiment of the use according to the invention, the composition is a lyophilized, freeze-dried or spray-dried dry composition comprising one or more further excipients selected from cryoprotectants, plasticizers and polymers. Suitably, the lyophilized, freeze-dried or spray-dried dry composition is mixed with a liquid, suitably an aqueous liquid such as sterile water or saline, to form a “reconstituted liquid formulation” prior to administration to a patient.

Suitably, the plasticizer is selected from glycerol, sorbitol and PEG4000, more suitably from sorbitol and PEG4000.

In one embodiment of the use according to the invention, the lyophilized, freeze- dried or spray-dried dry composition comprises glycerol in an amount between 0.3 and 3 mg per dose, for example in an amount selected from 0.3, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 2.5, 2.75 and 3.0 mg per dose. In one embodiment, glycerol is present in the composition in an amount of 1 .5 mg per dose.

In one embodiment of the use according to the invention, the lyophilized, freeze- dried or spray-dried dry composition comprises sorbitol in an amount between 0.3 and 9 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.25, 1.5, 1.75, 2, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 and 9 mg per dose. In one embodiment, sorbitol is present in the composition in an amount of 3 mg per dose. In one embodiment of the use according to the invention, the lyophilized, freeze- dried or spray-dried dry composition comprises PEG4000 in an amount between 0.03 and 3 mg per dose, for example in an amount selected from 0.03, 0.05, 0.075, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 and 3 mg per dose. In one embodiment, PEG4000 is present in the composition in an amount of 0.3 mg per dose.

In one embodiment, the lyophilized, freeze-dried or spray-dried dry composition does not comprise a plasticizer. In one embodiment, the lyophilized, freeze-dried or spray- dried dry composition does not comprise glycerol.

Suitably, the polymer is polyvinyl pyrrolidone (PVP).

In one embodiment of the use according to the invention, the composition comprises PPV in an amount between 0.3 and 3 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg per dose. In one embodiment, PVP is present in the composition in an amount of 1.5 mg per dose.

In another aspect, there is provided a method for stabilizing a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid, comprising adding an antioxidant to said composition.

In one embodiment of the method according to the invention, the antioxidant is an amino acid. Suitably, the amino acid is selected from methionine, tryptophan, histidine, lysine, cysteine, arginine and tyrosine. In a preferred embodiment, the antioxidant is methionine.

Suitably, methionine is present in the composition in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose. In one embodiment, methionine is present in the composition in an amount of 0.45 mg per dose. Suitably, methionine is present in the composition at a concentration between 0.1 and 5 mg per ml, more suitably at a concentration between 1 and 3 mg per ml, for example at a concentration selected from 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 mg per ml. In one embodiment, methionine is present in the composition at a concentration of 1.5 mg per ml.

In one embodiment of the method according to the invention, the composition is a liquid composition. Suitably, the liquid composition does not comprise a surfactant. Suitably, the liquid composition does not comprise a non-ionic surfactant. Suitably, the liquid composition does not comprise a non-ionic surfactant selected from polysorbate-20 (PS20), polysorbate-60 (PS60), polysorbate-80 (PS80) and poloxamers.

In one embodiment of the method according to the invention, the composition is a dry composition.

In one embodiment of the method according to the invention, the composition is a lyophilized, freeze-dried or spray-dried dry composition comprising one or more further excipients selected from cryoprotectants, plasticizers and polymers. Suitably, the lyophilized, freeze-dried or spray-dried dry composition is mixed with a liquid, suitably an aqueous liquid such as sterile water or saline, to form a “reconstituted liquid formulation” prior to administration to a patient.

Suitably, the plasticizer is selected from glycerol, sorbitol and PEG4000, more suitably from sorbitol and PEG4000. In one embodiment of the method according to the invention, the lyophilized, freeze- dried or spray-dried dry composition comprises glycerol in an amount between 0.3 and 3 mg per dose, for example in an amount selected from 0.3, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 2.5, 2.75 and 3.0 mg per dose. In one embodiment, glycerol is present in the composition in an amount of 1 .5 mg per dose.

In one embodiment of the method according to the invention, the lyophilized, freeze- dried or spray-dried dry composition comprises sorbitol in an amount between 0.3 and 9 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.25, 1.5, 1.75, 2, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 and 9 mg per dose. In one embodiment, sorbitol is present in the composition in an amount of 3 mg per dose.

In one embodiment of the method according to the invention, the lyophilized, freeze- dried or spray-dried dry composition comprises PEG4000 in an amount between 0.03 and 3 mg per dose, for example in an amount selected from 0.03, 0.05, 0.075, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 and 3 mg per dose. In one embodiment, PEG4000 is present in the composition in an amount of 0.3 mg per dose.

Suitably, the polymer is polyvinyl pyrrolidone (PVP).

In one embodiment of the methods according to the invention, the composition comprises PPV in an amount between 0.3 and 3 mg per dose, for example in an amount selected from 0.3, 0.4, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg per dose. In one embodiment, PVP is present in the composition in an amount of 1.5 mg per dose.

Further definitions

For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.

Throughout the specification, including the claims, where the context permits, the term “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element (e.g., integer) or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers). Thus, a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y. The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus, components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

Percentages in the context of numbers should be understood as relative to the total number of the respective items. In other cases, and unless the context dictates otherwise, percentages should be understood as percentages by weight (wt.-%).

About: The term “about” is used when determinants or values do not need to be identical, i.e. 100% the same. Accordingly, “about” means, that a determinant or values may diverge by 1% to 20%, for example by 1 % to 10%; in particular, by 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. The skilled person knows that e.g. certain parameters or determinants can slightly vary based on the method how the parameter has been determined. For example, if a certain determinants or value is defined herein to have e.g. a length of “about 100 nucleotides”, the length may diverge by 1 % to 20%. Accordingly, the skilled person knows that in that specific example, the length may diverge by 1 to 20 nucleotides. Accordingly, a length of “about 100 nucleotides” may encompass sequences ranging from 80 to 120 nucleotides.

Antigen: The term “antigen” as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a substance which may be recognized by the immune system, for example by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. Also fragments, variants and derivatives of peptides or proteins comprising at least one epitope are understood as antigens.

Coding seguence/coding region: The terms “coding sequence” or “coding region” and the corresponding abbreviation “cds” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a sequence of several nucleotide triplets, which may be translated into a peptide or protein. A coding sequence in the context of the present invention may be an RNA sequence consisting of a number of nucleotides that may be divided by three, which starts with a start codon and which for example terminates with a stop codon.

Derived from: The term “derived from” as used throughout the present specification in the context of a nucleic acid, i.e. for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares e.g. at least 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived. The skilled person is aware that sequence identity is typically calculated for the same types of nucleic acids, i.e. for DNA sequences or for RNA sequences. Thus, it is understood, if a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (II) by thymines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by II throughout the sequence). Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined. For example, a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g. in order to increase RNA stability even further and/or to prolong and/or increase protein production. In the context of amino acid sequences (e.g. antigenic peptides or proteins) the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g. at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.

Fragment: The term “fragment” as used throughout the present specification in the context of a nucleic acid sequence (e.g. RNA or a DNA) or an amino acid sequence may typically be a shorter portion of a full-length sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment typically consists of a sequence that is identical to the corresponding stretch within the full-length sequence. A particular fragment of a sequence in the context of the present invention, consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e. full-length) molecule from which the fragment is derived (e.g. a virus protein). The term “fragment” as used throughout the present specification in the context of proteins or peptides may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence, N- terminally and/or C-terminally truncated compared to the amino acid sequence of the original protein. The term “fragment” as used throughout the present specification in the context of RNA sequences may, typically, comprise an RNA sequence that is 5’-terminally and/or 3’-terminally truncated compared to the reference RNA sequence. Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore for example refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide. Fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides.

Heterologous: The terms “heterologous” or “heterologous sequence” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence refers to a sequence (e.g. RNA, DNA, amino acid) has to be understood as a sequence that is derived from another gene, another allele, or e.g. another species or virus. Two sequences are typically understood to be “heterologous” if they are not derivable from the same gene or from the same allele. I.e., although heterologous sequences may be derivable from the same organism or virus, in nature, they do not occur in the same nucleic acid or protein.

Identity (of a sequence): The term “identity” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to the percentage to which two sequences are identical. To determine the percentage to which two sequences are identical, e.g. nucleic acid sequences or amino acid (aa) sequences as defined herein, for example the aa sequences encoded by the nucleic acid sequence as defined herein or the aa sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same residue as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using an algorithm, e.g. an algorithm integrated in the BLAST program. Sequence identity can be determined by using the EMBOSS Water sequence alignment tool at the EMBL-EBI website https://www.ebi.ac.uk/Tools/psa/emboss_water/ with the parameters gap open=12, gap extend=1 and matrix=BLOSUM62 for protein sequences or matrix=fullDNA for DNA/RNA sequences, or by using the EMBOSS Needle sequence alignment tool at the EMBL-EBI website https://www.ebi.ac.uk/Tools/psa/emboss_needle/ with default parameters (e.g. gap open=10, gap extend=0.5, end gap penalty=false, end gap open=10 and end gap extend=0.5 and matrix=BLOSUM62 for protein sequences or matrix=fullDNA for DNA/RNA sequences). Unless specified otherwise, where the application refers to sequence identity to a particular reference sequence, the identity is intended to be calculated over the entire length of that reference sequence.

Immunogen, Immunogenic: The terms “immunogen” or “immunogenic” will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a compound that is able to stimulate/induce an (adaptive) immune response. An immunogen may be a peptide, polypeptide, or protein.

Immune response: The term “immune response” will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response), or a combination thereof.

Lipidoid compound: A lipidoid compound, also simply referred to as lipidoid, is a lipid-like compound, i.e. an amphiphilic compound with lipid-like physical properties. In the context of the present invention, the term lipid is considered to encompass lipidoid compounds.

Nucleic acid, nucleic acid molecule: The terms “nucleic acid” or “nucleic acid molecule” as used herein, will be recognized and understood by the person of ordinary skill in the art. The terms “nucleic acid” or “nucleic acid molecule” particularly refers to DNA (molecules) or RNA molecules). The term is used synonymously with the term polynucleotide. For example, a nucleic acid or a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers that are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. The terms “nucleic acid” or “nucleic acid molecule” also encompasses modified nucleic acid (molecules), such as basemodified, sugar-modified or backbone-modified DNA or RNA (molecules) as defined herein.

Nucleic acid seguence, DNA seguence, RNA seguence: The terms “nucleic acid sequence”, “DNA sequence”, “RNA sequence” will be recognized and understood by the person of ordinary skill in the art, and e.g. refer to a particular and individual order of the succession of its nucleotides. Stabilized RNA: The term “stabilized RNA” refer to an RNA that is modified such, that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digest, such as by exo- or endonuclease degradation, compared to an RNA without such modification. Preferably, a stabilized RNA in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell. The stabilization effect may also be exerted outside of cells, e.g. in a buffer solution etc., e.g., for storage of a composition comprising the stabilized RNA.

Variant (of a sequence): The term “variant” as used throughout the present specification in the context of a nucleic acid sequence will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence. E.g., a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from. The variant is a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from. A “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotides of such nucleic acid sequence.

The term “variant” as used throughout the present specification in the context of proteins or peptides is e.g. intended to refer to a proteins or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(s)/substitution(s), such as one or more substituted, inserted and/or deleted amino acid(s). Suitably, these fragments and/or variants have the same, or a comparable specific antigenic property (immunogenic variants, antigenic variants). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three- dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra). A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Alternatively, a “variant” of a protein or polypeptide may have from 1 to 20, for example from 1 to 10 single amino acid mutations compared to such protein or peptide, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19 or 20 single amino acid mutations. For mutations we mean or include substitution, insertion or deletion. In one embodiment, a variant of a protein comprises a functional variant of the protein, which means, in the context of the invention, that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity as the protein it is derived from.

EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods, which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

for a lyophilized thermostable presentation of an mRNA-LNP Covid-19 vaccine

• Study setup

For the formulation development study of a lyohpilized mRNA-LNP presentation, 42 formulations combining different excipients were freeze-dried and subjected to three independent thermal stresses at 25, 40 and 50°C.

Selected excipients were then tested by diluting a concentrated mRNA-LNP bulk in different formulations. The concentrated mRNA/LNP bulk comprised mRNA expressing a SARS-CoV-2 Spike antigen at a concentration of 1.04 mg/mL, encapsulated in LNPs comprising DSPC, ALC-0315, cholesterol and ALC-0159 at a molar ratio of about 47.4:10.0:40.9:1.7 (DSPC:ALC-0315:cholesterol:ALC-0159), and buffered in Sodium Phosphate 10mM, NaCI 75mM, Sucrose 150mM at pH 7.4.

For the preparation of the different formulations, the starting mRNA-LNP bulk was diluted to a final mRNA concentration of 50pg/mL (in the pre-lyophlized bulk). The selected excipients as well as the concentration tested for each of them are shown in Table 2.

Table 2. Excipients list and tested concentration

Table 3 below indicates all the different excipient combinations corresponding to the different obtained formulations.

Table 3. Excipient combinations corresponding to the different obtained formulations.

In order to prepare the different formulations, different dilution buffers were prepared to reach the specific excipient combination and composition in the final drug product after dilution of the mRNA-LNP bulk. All formulations were prepared in a final buffer composition of Potassium Phosphate 10mM, pH 7.4, leading to a 20 times dilution of the starting concentrated bulk buffer (Sodium Phosphate 10mM, NaCI 75mM, Sucrose 150mM, pH 7.4) in the final product. For excipient combinations including sucrose as cryoprotectant, the final sucrose concentration in the final product was calculated taking into account the sucrose contribution coming from the buffer of the concentrated bulk.

The following Critical Quality Attributes (CQAs) were identified:

Physico-chemical QAs

- Residual moisture: it can impact the lyophilized product stability when stored in the dried state, since the remaining water at the end of the Lyophilization process could potentially be involved in hydrolysis reactions of mRNA and lipids, leading to the product degradation.

- mRNA-LNP size and polydispersity index (Pdl): both of them allow to characterize and monitor LNP particle size distribution during thermal stress. In particular, size is indicative of the average of the particle size distribution while Pdl of its dispersion. They could be stability indicating since both size and Pdl could increase overtime, expecially under stress conditions.

- mRNA total content: the total mRNA amount is monitored to ensure that the fixed target of mRNA concentration is maintained.

- mRNA encapsulation %: it measures the percentage of mRNA which is encapsulated in LNP versus the total mRNA concentration in the drug product. It is closely connected to the mRNA activity since an encapsulation decrease could result in a reduced amount of mRNA delivered to cells and thus in a reduced protein expression efficiency.

- mRNA integrity: it measures the relative amount of mRNA integral molecules with respect to fragments which might generate from mRNA degradation. It is monitored to detect mRNA degradation that can occur in time due to the labile nature of the molecule. mRNA integrity impacts directly the dug product efficacy since a reduced integrity may result in a reduced protein expression. Also mRNA fragments might lead to increased reactogenicity.

Product-related impurities

- Impurities (Late Eluting sPecies - LEP): it accounts for lipid mRNA adducts which can be generated by electrophilic impurities derived from the ionizable cationic lipid component through the covalent addition to the mRNA nucleobases. This seems to make the mRNA untranslatable, leading to loss of protein expression (M Packer at al., Nature Communications, 2021).

In Table 1 , selected analytical methods for monitoring all the afore mentioned CQAs are listed. Table 1. CQAs and analytical methods

• Material & Methods mRNA covid vaccine

An mRNA covid vaccine was prepared and formulated in LNPs as described in ADD REF. Dynamic Light Scattering (DLS)

DLS was the selected technique to characterize and monitor in stability both LNPs size and polydispersity (size distribution) by measuring the diffusion of particles moving under Brownian motion, and converting this to size and a size distribution using the Stokes-Einstein relationship. Analyses were performed by the Zetasizer Ultra Pro using the software ZS EXPLORER.

Data are reported as diameters size (expressed in nm) and the Polydispersity Index of each sample.

RiboGreen assay mRNA encapsulation efficiency was measured by the RiboGreen assay, which relies on a dye that fluoresces upon binding to single-stranded mRNA. Dye accessibility is low with intact LNPs, so only unencapsulated mRNA is detected. To determine the total mRNA concentration, entrapped mRNA is released by addition of a detergent (Triton X- 100) to lyse the LNPs. The ratio of fluorescence intensity before and after addition of Triton allows for the calculation of the proportion of encapsulated mRNA payload.

The same method is used to quantify mRNA total content (after the treatment of the sample in Triton X-100 to release mRNA from LNPs). Microfluidic electrophoresis

The integrity of nucleoside-modified mRNA was assessed by Microfluidic electrophoresis after mRNA extraction from LNP. RNA integrity was evaluated by capillary electrophoresis separation through LabChip GX Touch nucleic acid analyzer’s microfluidic technology by selecting the high throughput methodology and standard sensitivity, as per instruction of the supplier (Perkin Elmer).

Reversed Phase-Ion Pair High Performance Liquid Chromatography (RP-IP HPLC)

RP-IP HPLC was used to detect potential adduct events on intact mRNA, after mRNA extraction from LNPs. The method at RP-IP HPLC for mRNA-lipids adducts has been adapted based on the paper from Packer at al. (2021 , Nature Communications).

Karl-Fischer titration

A Colorimetric Karl Fischer Titrator, equipped with a Metrohm 831 oven, was used to determine residual moisture content in lyophilized cakes.

• Filling and lyophilisation

Before lyophilization, the different formulations have been filled in 2R vials (2ml Schott Standard line). The vials have been preliminarily washed and depyrogenized at 240°C for 3hr. For each of the 42 formulations, 30 vials of DP and 12 vials of the corresponding placebo (e.g. same excipients/buffer composition without mRNA LNP) were filled with a filling volume of 0.3ml/vial.

After lyophilization, the lyophilized samples for each formulation have been split in different thermal stress conditions, and the CQAs monitored at time zero (after the lyophilization process end) and at different time points with the aim to understand the behavior of the different formulations in stability and to compare them, with the main goal of discarding excipients detrimental or not useful to preserve/improve the product. Vials with the lyophilized drug product have then been put in climate chambers at 25°C/60%RH, 40°C/75%RH and 50°C (humidity not controlled). The timepoints considered for this thermal stress are reported in Table 4, namely the product CQAs have been quantified, after 2 weeks at 50°C, after 4 and 8 weeks at 40°C, and after 6 and 11 weeks at 25°C.

Table 4. Applied thermal stress

• Pre-Lyo/Post-Lyo samples characterization (time 0)

A first analysis on the liquid formulated bulks prior to lyophilization (pre-lyo) was carried out with the aim of observing a potential effect of excipients addition regardless of the subsequent freeze-drying step. Additionally, the characterization of the pre-lyo bulks allows to set the level of the CQAs before lyophilization, in order to understand any potential protective effect of excipients during freeze-drying. In FIG. 1 , panels A and B, both mRNA encapsulation % and LNP size of all the pre-lyo formulations are shown. Formulations have been grouped according to the used cryo-protectant (i.e., HP-p-cyclodextrin, sucrose and trehalose). As noticeable, all HP-p-cyclodextrin formulations show mRNA leakage and LNP size increase since the pre-lyo bulk, indicating a potential effect of the considered cryoprotectant on LNPs since the liquid phase. For this reason, HP-p-cyclodextrin formulations were not further considered for both lyo product characterization and thermal stress. No major differences were found instead betweeen sucrose- and trehalose-based formulations.

After lyohpilization, the lyophilized products were analyzed and compared to the pre- lyo samples in order to detect any potential effect of the freeze-drying process on the products and especially any specific effect of the excipients used in the excipient screening study. All CQAs previously selected (Table 1) were analyzed. The Lyo products were resuspended with 0.3mL of WFI prior to the analysis.

Hereafter the results obtained for each CQA are reported, by comparing for each formulation the level pre- and post-lyophilization. mRNA encapsulation

In FIG. 2, Panels A and B, the mRNA encapsulation percentage results are shown. Notably, lyophilized samples have been split in 2 different analytical sessions, according to the presence or absence of methionine in the formulations. Of note, in both Panels of FIG. 2 also the encapsulation % of control samples has been reported. The control samples correspond to the original mRNA-LNP bulk, diluted in its original matrix (10 mM sodium phosphate pH 7.4 + 75 mM NaCI + 150 mM sucrose) to a target concentration of 100 mcg/ml and have been added to the Ribogreen sessions (and to the plots) to demonstrate potential session effect in the analyses of samples. Notably, the control samples in FIG. 2 have been analyzed in the same sessions in which samples have been split (pre-lyo formulations without methionine and with methionine, lyo formulations without and with methionine).

Three main conclusions can be captured by this analysis: o the lyophilization process impacts the encapsulation level (lower on lyo samples than on pre-lyo ones); o lyohpilized sucrose-based formulations show on average better encapsulation than trehalose-based ones; o 2-components formulations show on average better encapsulation levels upon lyophilization than 3-components ones (except for formulations involving PVP); o No significant differences can be found between formulation with or without methionine (regardless if 2-components or 3-components).

LNPs Size and Pdl.

In FIG.s 3 and 4, Panels A and B, results of LNP size and PDI are shown, respectively. In general, the lyophilization process impacts both the Size and Pdl of LNPs, which are higher on the lyo samples, independently of the excipient/s presence in the formulation. However, sucrose-based formulations show a lower increase of Size and Pdl than trehalose-based formulations. As observed for the encapsulation percentage, the lower size increases can be observed for the simplest formulations involving only the cryoprotectant or the cryoprotectant with a plasticizer or methionine. 3-component formulations show limited size increase only when one of the component is methionine. The differences are less pronounced with regards to Pdl. mRNA Integrity

Samples for the Integrity characterization in microfluidic electrophoresis were divided into sessions in a similar way as for the quantification of the encapsulation percentage, with the same control samples inserted in each session. FIG. 5, Panels A and B reports the Integrity results for each formulation, comparing pre-lyo and lyophilized samples. A slight loss of integrity for most of the formulations upon lyophilization was observed, with a slight loss increase for formulations including methionine. No major differences between sucrose and trehalose formulations were observed. Late Eluting sPecies (LEP)

In FIG. 6, Panels A and B, results related to LEP are shown. For this attribute, an important role in the prevention of LEP formation is played by the presence of methionine. As a matter of fact, for most of the formulations not including methionine a slight increase in the LEP level is observed after lyophilization. This is not the case when using methionine, for which lyophilized samples show very similar levels as pre-lyo ones. In general, sucrose- and trehalose-based formulations show a similar behavior in terms of LEP, which seems to increase for all formulations not including methionine.

After the CQAs characterization of pre-Lyo and Lyo samples at time 0, sucrose- based formulations showed better mRNA encapsulation percentage than trehalose-based ones and, in general, the presence of trehalose did not demonstrate any superiority with respect to sucrose as cryoprotectant. For this reason, only sucrose-based formulations were considered for thermal stress studies at all previously selected temperatures (25°C, 40°C and 50°C).

After removal of samples with HP-p-ciclodextrin and trehalose as excipients for the reasons explained above, 14 excipients combinations were considered for the subsequent thermal stress study (Table 5), by monitoring two vials of the same formulations for each excipients combination (28 vials in total). The formulations were firstly subdivided in two blocks (14 samples/block) and each samples block was put in thermal stress shifted by one week with respect to the other one.

Table 5. Excipients combinations considered for the thermal stress study

Of note both replicates of the same formulation were put in stability in the same block. The 2 blocks samples were analyzed in two different analytical sessions (one session/samples block) at each stability point, for each CQA. Thermal stress at 50°C

Hereafter results related to selected CQAs after 2 weeks of thermal stress at 50°C are reported. Each plot reports for each formulation the comparison of the CQAs values measured at time 0 versus the ones measured after 2 weeks at 50°C. mRNA encapsulation %

Results are shown in FIG. 7 and summarized hereafter:

Encapsulation loss was observed for all formulations except the ones including sucrose and sorbitol;

Mono- and bi- components formulations showed better performances than 3- and 4- component ones (except for the one includeing PEG4000);

Formulations with sorbitol showed better results, but presented melted lyo cakes after 2 weeks at 50°C, most probably linked to the high water content in the cake as consequence of the presence of sorbitol;

No beneficial nor detrimental effect due to methionine was observed.

LNP Size.

Results are shown in FIG. 8:

In general, a size increase upon the applied thermal stress was observed for all formulations.

Formulations with 3- and 4- excipient components showed to be more stable, however they already start from a high size of LNPs since time 0.

No beneficial nor detrimental effect due to the addition of Methionine was observed.

LNP Pdl

Results are shown in FIG. 9. In general, Pdl showed a decrease for most of the formulations upon thermal stress at 50°C, likely due to the fact that the size increase for the formulations after the thermal stress is larger than the increase in the particle size distribution variance, thus impacting the calculation of Pdl. No major differences can be seen between formulations, besides some formulations show larger variability among samples. mRNA Integrity.

A loss of mRNA integrity at different levels with respect to the samples analyzed at time 0 was observed for all formulations (FIG. 10). No significant differences can be seen among formulations, meaning that no specific excipient showed a superior performance in decreasing the integrity loss at 50°C.

LEP

A LEP increase was observed upon thermal stress for all formulations. However, formulations with methionine show a lower increase, indicating that methionine, when present as further excipient, can help in minimizing the LEP formation or reducing the LEP growth (FIG. 11).

• Thermal stress at 40°C

Formulations were stored in thermal stress conditions at 40°C and the CQAs monitored after 4 and 8 weeks of storage. Hereafter results obtained for each CQA are reported: mRNA encapsulation %

In general encapsulation decreases during thermal stress at 40°C. However, it seems to be more stable for the formulation with sucrose and sorbitol, with or without methionine. Beneficial effects coming from the other excipients were not observed (FIG. 12). Encapsulation percentage seems to stabilize and leveling off for formulations with PVP, 3-component formulations without methionine and 4-component formulations with methionine, however these formulations did show a lower level of encapsulation since time 0 (i.e. after the lyophilization cycle).

LNPs Size.

Size was observed to increase during thermal stress at 40°C. It seems to be more stable for bi-component sucrose-based formulations, and tri-component formulations which include methionine, except when PVP is included in the excipient combinations (FIG. 13). For the other formulations the increase is also less evident, however they start from a higher size since the end of the lyophilization cycle. LNPs Pdl

Pdl slightly decreases during thermal stress at 40°C for most of the formulations. This effect might be linked to the fact that the size (FIG. 13) is increasing relatively more than how much the dispersion of the nanoparticle size distribution is increasing, giving this effect of decrease. It keeps a lower and less variable level for formulations including sucrose, sucrose+sorbitol and sucrose+PEG (FIG. 14). mRNA integrity.

Integrity was observed to decrease during thermal stress at 40°C for all the formulations. However, the addition of methionine seems to slow down/leveling off the integrity loss for sucrose and sucrose+PEG formulations (FIG. 15). Despite the variability between data related to the same formulation, no superior performances can be clearly identified between the different tested formulations.

LEP.

In general LEP tends to increase in all formulation conditions and seemingly reaches a plateau at 8 weeks for most of them. The use of methionine seems to reduce the increase in LEP in all formulations (FIG. 16), especially formulations with methionine show that a different lower plateau level for LEP is reached with respect to formulations without methionine.

• Thermal stress at 25°C

Formulations were stored at 25°C up to 11 weeks of thermal stress and their CQAs analyzed at 4 and 11 weeks. Hereafter obtained results for each CQA are reported: mRNA encapsulation %

Overall, all formulations show better stability for mRNA encapsulation when stored at 25°C. As a matter of fact, the encapsulation values are mostly constant for each formulation at the different time points, or more affected by method variability. Mono- and bi-component formulations as well as tri-component formulations which include methionine (excluding formulations with PVP) show the better values of encapsulation in stability, as they also started from higher level at time 0 (i.e. after lyophilization). Sucrose, sucrose+sorbitol and sucrose+PEG4K formulations seem to be the best combinations for this CQA. In particular, sorbitol when used in combination with sucrose seems to prevent encapsulation loss during the thermal stress at 25°C (FIG. 17).

LNPs size

Also for LNP size the values at 25°C are more stable than what observed at higher temperatures (FIG. 18). No increasing trend can be observed for any formulation. As for mRNA encapsulation, the formulations with sucrose and a plasticizer show the best values of LNP size, since time 0 and in stability. The same applies for the same formulations in presence of methionine, which in turn does not appear to impact LNP size. All the other formulations show a stable trend in LNP size along time, although they start from higher values at time 0, in general more variable in stability.

PDI

Pdl shows a stable trend for mostly all the formulations (FIG. 19). More variable results have been obtained for formulations with 3 components without methionine or with 4 components including the methionine.

Also at 25°C the Pdl decrease that was observed at higher temperatures is not such evident as for the higher temperatures. Rather, some formulations (as the sucrose-based or the sucrose+PEG) the trend is decreasing while for some others (like sucrose+methionine or sucrose+sorbitol) is increasing. mRNA integrity.

Integrity was observed to decrease during thermal stress at 25°C for all the formulations (FIG. 20). In absolute terms, the decrease is similar to the one observed at higher temperatures (ranging between 30% and 40% at the end of the storage at 25°C), however the rate of decrease is lower, as very likely consequence of the lower storage temperature for which the degradation kinetic is decreased. Differently from the 40°C data it is less clear if a leveling-off of the integrity levels is achieved: some formulations (like the sucrose-based one or sucrose+sorbitol) seems to indicate a slowing-down of the integrity loss, however it is not clear if this could be imputed to the variability among sessions, which is actually confounded with the time points effects. Of note, also the control used in the sessions (corresponding to the same lot diluted in the frozen product matrix to 100 mcg/ml) show a decrease in the integrity level during time, indicating a potential session effect and thus a potential underestimation of the real integrity levels of the formulations. LEP.

The increase in LEP was observed upon storage at 25°C for all the formulations as observed in FIG. 21. The behavior of the different formulations seem quite similar. As observed for the data at 40°C, formulations with methionine show a lower increase, confirming that methionine, when present as further excipient, seem to minimize the LEP formation or reducing the LEP growth (FIG. 11). As a matter of fact, formulations without methionine reaches a LEP level between approximately 12% and 16%, while formulations with methionine levels off at a level of 6%. Interestingly enough, the LEP levels reached a plateau during the applied thermal stress, possibly meaning that an equilibrium is reached in the LEP generation reaction.

• Summary

Obtained results, after lyophilization and during stability studies, highlight:

- A general worsening of all cQAs upon lyophilization.

The superiority of sucrose as cryoprotectant with respect to trehalose and HP-p-cyclodextrin for the specific product (encapsulated Covid mRNA-LNP).

- An improved behavior in thermostability for formulations which include sucrose alone, sucrose+sorbitol and sucrose+PEG4K ,

The beneficial effect of sorbitol when used in combination with sucrose to prevent encapsulation loss and size increase in thermal stress (at 40°C and 25°C),

The strong beneficial effect of methionine to reduce LEP increase (half increase with respect to the formulations without methionine).

Claims

1. A composition comprising an RNA molecule, an ionizable amino-lipid and an antioxidant.

2. The composition according to claim 1, wherein said antioxidant prevents or reduces the formation of RNA adduct, suitably of lipid-RNA adduct.

3. The composition according to claim 1 or 2, wherein said RNA molecule is an mRNA, suitably selected from a non-replicating mRNA and a self-replicating mRNA.

4. The composition according to claim 1 , 2 or 3, wherein said antioxidant is methionine.

5. The composition according to claim 4, wherein said composition comprises an amount of methionine between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose.

6. The composition according to any one of claims 1 to 4, wherein said antioxidant is not linked via a linker to a PEG moiety.

7. The composition according to any one of claims 1 to 6, wherein said composition further comprises at least one neutral lipid, suitably DSPC; at least one sterol, suitably cholesterol; and (iv) at least one a PEGylated lipid.

8. The composition according to any one of claims 1 to 7, wherein said composition is a liquid composition.

9. The composition according to claim 8, wherein said composition does not comprise a non-ionic surfactant selected from polysorbate-20 (PS20), polysorbate-60 (PS60), polysorbate-80 (PS80) and poloxamers.

10. The composition according to any one of claims 1 to 7, wherein said composition is a dry composition, suitably a lyophilized, freeze-dried or spray-dried dry composition.

11. The composition of claim 10, wherein said composition comprises one or more further excipients selected from cryoprotectants, plasticizers and polymers.

12. The composition of claim 11, wherein said composition comprises a cryoprotectant selected from trehalose, sucrose or a combination of trehalose and sucrose, a plasticizer selected from glycerol, sorbitol and PEG4000, and/or a polymer which is PVP.

13. The composition of claim 10, wherein said composition does not comprise a plasticizer or does not comprise glycerol

14. The composition of any one of claim 1 to 14 for use in therapy, suitably for use as a vaccine.

15. Use of an antioxidant to stabilize a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid.

16. Use according to claim 15, wherein said antioxidant prevents or reduces the formation of lipid-RNA adduct.

17. Use according to claim 15 or 15, wherein said antioxidant is methionine.

18. Use according to claim 17, wherein said methionine is present in said composition in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose.

19. Use according to any one of claims 16 to 18, wherein said antioxidant is not linked via a linker to a PEG moiety.

20. Use according to any one of claims 16 to 19, wherein said composition is a dry composition, suitably a lyophilized, freeze-dried or spray-dried dry composition.

21. Use according to claim 20, wherein said composition comprises one or more further excipients selected from cryoprotectants, plasticizers and polymers.

22. Use according to claim 21 , wherein said composition comprises a cryoprotectant selected from trehalose, sucrose or a combination of trehalose and sucrose, a plasticizer selected from glycerol, sorbitol and PEG4000, and/or a polymer which is PVP.

23. Use according to claim 20, wherein said composition does not comprise a plasticizer or does not comprise glycerol

24. Method for stabilizing a composition comprising RNA, suitably mRNA, and an ionizable amino-lipid, comprising adding an antioxidant to said composition.

25. Method according to claim 24, wherein said antioxidant prevents or reduces the formation of lipid-RN A adduct.

26. Method according to claim 24 or 25, wherein said antioxidant is methionine.

27. Method according to claim 26, wherein said methionine is present in said composition in an amount between 0.03 and 1.5 mg per dose, more suitably in an amount between 0.15 and 1 mg per dose, for example in an amount selected from 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg per dose.

28. Method according to any one of claims 24 to 27, wherein said antioxidant is not linked via a linker to a PEG moiety.

29. Method according to any one of claims 24 to 28, wherein said antioxidant is not linked via a linker to a PEG moiety.

30. Method according to any one of claims 24 to 29, wherein said composition is a dry composition, suitably a lyophilized, freeze-dried or spray-dried dry composition.

31. Method according to claim 30, wherein said composition comprises one or more further excipients selected from cryoprotectants, plasticizers and polymers.

32. Method according to claim 31 , wherein said composition comprises a cryoprotectant selected from trehalose, sucrose or a combination of trehalose and sucrose, a plasticizer selected from glycerol, sorbitol and PEG4000, and/or a polymer which is PVP.

33. Method according to claim 30, wherein said composition does not comprise a plasticizer or does not comprise glycerol.