WO2025202360A1 - Rna formulation - Google Patents

Abstract

The present invention relates to aqueous RNA compositions that are suitable for storage, comprising Tris, a saccharide, and phosphate anions. The present invention also relates to methods of producing such aqueous RNA compositions, as well as their use in therapy and prevention of infectious diseases.

Description

RNA formulation

FIELD OF THE INVENTION

The present invention relates to RNA aqueous formulations in which RNA stability is 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.

Delivery systems such as lipid nanoparticles (LNPs) are used to protect the RNA from degradation and help cellular uptake.

One challenge upon formulating RNA into LNPs is preserving RNA quality overtime. There is therefore a need in the field for improved RNA formulation.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an aqueous composition comprising an RNA molecule, a delivery system, Tris, and a saccharide. Suitably, the aqueous composition further comprises phosphate anions.

In another aspect, there is provided the aqueous 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 aqueous 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 aqueous composition of the invention to a patient in need thereof.

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

In another aspect, a method of producing the aqueous composition of the invention is provided, comprising a) providing an initial composition comprising an RNA molecule, suitably mRNA, and a delivery system, suitably LNPs, b) transferring the initial composition into a first buffer system to obtain an intermediate composition, c) diluting the intermediate composition with a dilution buffer to obtain the aqueous composition of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 - Formulations 1-6 (labelled 1-PSN, 2-Na_K_2, 3-Hist, 4-HistArg, 5-TSN, 6-TSG), LNP size (z-Average (nm)) and Pdl measured by DLS at TO, after 8 days at -20°C and after 14 days at +5°C.

FIG. 2 - Formulations 1-6 (labelled 1-PSN, 2-Na_K_2, 3-Hist, 4-HistArg, 5-TSN, 6-TSG), %LEP measured by HPLC at TO, after 8 days at -20°C, after 14 days at +5°C and after 14 days at +25°C.

FIG. 3 - Formulations 5 (TSN) and 12 (TSG’), % mRNA integrity after 21 days at +25°C.

FIG. 4 - Formulations 5 (TSN) and 12 (TSG’), % LEP after 21 days at +25°C.

FIG. 5 - Formulations 1 (PSN) and 7-10 (labelled 7-TTS, 8-TTS’, 9-TSN, 10-TSN’), encapsulation efficiency (%) over 90 days at -20°C, +5°C and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2,5 months at -20°C.

FIG. 6 - Formulations 1 (PSN) and 7-10 (labelled 7-TTS, 8-TTS’, 9-TSN, 10-TSN’), LNP size (z-Average (nm)) over 90 days at -20°C, +5°C and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2,5 months at -20°C. FIG. 7 - Formulations 1 (PSN) and 7-10 (labelled 7-TTS, 8-TTS’, 9-TSN, 10-TSN’), LNP Pdl over 90 days at -20°C, +5°C and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2,5 months at - 20°C.

FIG. 8 - Formulations 1 (PSN) and 7-10 (labelled 7-TTS, 8-TTS’, 9-TS, 10-TS’), In vitro expression at over 30 days at -20°C, +5°C and +25°C.

FIG. 9 - Formulations 1 (PSN pH 7.4), 9 (TSN pH 7.4) and 11 (TSN pH 8.0), LNP size (z- Average (nm) and Pdl) over 90 days at -20°C, +5°C and +25°C.

FIG. 10 - Formulations 1 (PSN pH 7.4), 9 (TSN pH 7.4) and 11 (TSN pH 8.0), encapsulation efficiency (%) over 90 days at -20°C, over 50 days at +5°C and over 50 days at +25°C.

FIG. 11 - Formulations 1 (PSN pH 7.4), 9 (TSN pH 7.4) and 11 (TSN pH 8.0), mRNA integrity (%) and LEP (%) over 50 days at -20°C, +5°C and +25°C.

FIG. 12 - Formulations 1 (PSN pH 7.4), 6 (TSG), 9 (TSN 7.4) and 11 (TSN 8), Potency (%) over 30 days at -20°C, +5°C and +25°C.

FIG. 13 - Formulations 6 (TSG), 9 (TSN) and 12 (TSG’), LNP size (z-Average (nm) and Pdl) over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days).

FIG. 14 - Formulations 6 (TSG), 9 (TSN) and 12 (TSG’), Encapsulation efficiency (%) over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days).

FIG. 15 - Formulations 6 (TSG), 9 (TSN) and 12 (TSG’), mRNA integrity (%) and LEP (%) over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days).

FIG. 16 - Mice immunized i.m. with formulations A (PSN), B (PSN/TSN), and C (PSN/TSG) comprising LNP formulated mRNA encoding influenza antigens. Muscle swelling scores at TO; after 21 days at 25°C, and after 40 days at 40°C (PSN only). Depicted is the mean score of each group with vertical lines indicating the SD.

FIG. 17 - Mice immunized i.m. with formulations A (PSN), B (PSN/TSN), and C (PSN/TSG) comprising LNP formulated mRNA encoding influenza antigens. Pro- inflammatory cytokine (IFN-y; CCL2, CXCL10) release at TO; after 21 days at 25°C, and after 40 days at 40°C (PSN only). Each dot represents an individual animal, lines depict the geometrical mean (GM) with 95% confidence interval (Cl). LLOQ= lower limit of quantification. Non-inferiority (Nl) between the induced responses is depicted, if the 90% lower limit of Cl of the geometric mean ratio (GMR) is >0.33 (Nl). ND = result inconclusive since the 90% lower limit of Cl< 0.33 and non-inferiority could not be demonstrated.

FIG. 18 - Mice immunized i.m. with formulations A (PSN), B (PSN/TSN), and C (PSN/TSG) comprising LNP formulated mRNA encoding influenza antigens. Humoral responses: HI and Nl titers 21 and 35 days after injection with formulations at TO; stored after 21 days at 25°C, and stored 40 days at 40°C (PSN only).

FIG. 19 - Mice immunized i.m. with formulations A (PSN), B (PSN/TSN), and C (PSN/TSG) comprising LNP formulated mRNA encoding influenza antigens. Cellular responses: CD4 and CD8 titers 21 and 35 days after injection with formulations at TO; stored after 21 days at 25°C, and stored 40 days at 40°C (PSN only).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides an aqueous composition comprising an RNA molecule, a delivery system, Tris, and a saccharide.

Suitably, the aqueous composition of the invention comprises an RNA molecule, a delivery system, Tris, a saccharide and phosphate anions.

The aqueous composition of the invention comprises Tris. As used herein, “T r is” refers to tris(hydroxymethyl)aminomethane, also known as “tromethamine” or “THAM”. Tris is an organic compound with the formula (HOCH2)₃CNH₂, and can be used as a buffering agent in pharmaceutical solutions.

Suitably, Tris is present at a concentration between 5 and 50 mM, suitably between 7.5 and 30 mM, for example at a concentration of about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 mM. Suitably, Tris is present at a concentration of 15 mM. In another embodiment, Tris is present at a concentration of 30 mM. In another embodiment, Tris is present at a concentration of 10 mM.The aqueous composition of the invention comprises a saccharide. A “saccharide”

(or “carbohydrate”) consists ofone or more unit(s) of general formula CnH2nOn. Saccharide units exist in either a ring or short chain conformation, and typically contain five or six carbon atoms. Saccharides can be divided into monosaccharides, disaccharides, oligosaccharides, and polysaccharides depending on the number of units. A monosaccharide consists of one saccharide unit. A disaccharide consists of two identical or non identical saccharide units. An oligosaccharide typically consists of 3 to 20 identical or non identical saccharide units. A polysaccharide typically consists of longer chains of identical or non identical saccharide units.

Suitably, the saccharide is a disaccharide (e.g. sucrose, trehalose). Suitably, the saccharide is a disaccharide selected from sucrose and trehalose or combinations thereof. In a particularly suitable embodiment, the saccharide is sucrose. In another embodiment, the saccharide is a combination of sucrose and trehalose. Suitably, the saccharide acts as a cryoprotectant and/or as a tonicity agent. As used herein, a “cryoprotectant” is an excipient which has the ability to preserve structural integrity, for example LNP structural integrity, upon freezing. As used herein, a “tonicity agent” is an excipient which has the ability to adjust the osmotic pressure via colligative properties.

Suitably, the saccharide is present in the aqueous composition at a concentration between 50 and 700 mM, suitably between 100 and 600 mM, for example at a concentration of about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, suitably at a concentration of about 300 mM.

Suitably, the saccharide is sucrose and is present at a concentration between 50 and 700 mM, suitably between 100 and 600 mM, for example at a concentration of about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM. More suitably sucrose is present at a concentration of about 300 mM.

In another suitable embodiment, the saccharide is a combination of sucrose and trehalose, and the combined concentration of sucrose and trehalose is between 50 and 700 mM, suitably between 100 and 600 mM, for example about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM. In one embodiment, sucrose and trehalose are both present at a concentration of about 150mM. In one embodiment, sucrose is present at a concentration of 50mM and trehalose is present at a concentration of 550mM.

The aqueous composition of the invention suitably comprises phosphate anions (PO4³). Typically, the phosphate anion is associated with cations to form a salt, for example NaK2 phosphate or Na/Na2 phosphate.

Suitably, the phosphate anions are present at a concentration from 0.5 to 15 mM, suitably from 1 to 10 mM, more suitably at a concentration from 1 to 5 mM, for example, at a concentration selected from about 1 , 2, 3, 4 or 5 mM. Suitably, the phosphate anions are present at a concentration of about 3 mM.

Suitably, the aqueous composition of the invention further comprises sodium chloride. In one embodiment, sodium chloride is present at a concentration between 5 and 50 mM, suitably between 10 and 40 mM, more suitably between 15 and 30 mM, for example at a concentration of about 15, 20, 21 , 22, 22.5, 23, 24, 25 or 30 mM. Suitably, sodium chloride is present at a concentration of about 22.5 mM. Suitably, the aqueous composition of the invention has a pH between 6 and 9, suitably between 6.5 and 8.5, for example about 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. Suitably, the aqueous composition has a pH of about 7.4 or about 8.0. More suitably, the aqueous composition has a pH of about 7.4.

In some embodiments, the aqueous composition of the invention has an osmolality of less than 450 mOsmol/kg or less than 400 mOsmol/kg.

In some embodiments, the aqueous composition of the invention has an osmolality of 100 mOsmol/kg to 400 mOsmol/kg, 150 mOsmol/kg to 400 mOsmol/kg, 200mOsmol/kg to 400mOsmol/kg, 300mOsmol/kg to 400mOsmol/kg.

The aqueous composition of the invention can optionally comprise glycerol. Suitably, when glycerol is present in the aqueous composition, it is at a concentration from 10 to 200 mM, suitably from 25 to 175 mM, for example, about 25, 50, 75, 100, or 150 mM, more suitably about 25 or about 100 mM.

In some embodiments, the aqueous composition of the invention comprises glycerol, and the combined concentration of saccharide and glycerol is between 50 and 700 mM, suitably between 100 and 600 mM, for example about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, and wherein for example the saccharide is sucrose and present at a concentration of about 150mM and glycerol is present at a concentration of about 100mM.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration ranging from 5 to 50 mM, suitably from 7.5 to 30 mM, b. the saccharide, suitably sucrose, is present at a concentration ranging from 50 to 700 mM, suitably from 100 to 600 mM, c. a phosphate anion is present at a concentration ranging from 0.5 to 15 mM, suitably from 1 to 5 mM, d. Sodium chloride is present at a concentration ranging from 5 to 50 mM, suitably from

15 to 30 mM, and the aqueous composition has a pH between 6 and 9, suitably between 6.5 and 8.5.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration ranging from 5 to 50 mM, suitably from 7.5 to 30 mM, b. the saccharide, suitably sucrose, is present at a concentration ranging from 50 to 700 mM, suitably from 100 to 600 mM, c. Glycerol is present at a concentration ranging from 10 to 200 mM, suitably from 25 to 175 mM, d. the phosphate anion is present at a concentration ranging from 0.5 to 15 mM, suitably from 1 to 5 mM, e. Sodium chloride that is present at a concentration ranging from 5 to 50 mM, suitably from 15 to 30 mM, and the aqueous composition has a pH between 6 and 9, suitably between 6.5 and 8.5.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 550 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 8.0.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 150 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, e. Glycerol is present at a concentration of about 100 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 10 mM, b. the saccharide is sucrose and is present at a concentration of about 225 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, e. Glycerol is present at a concentration of about 25 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 150 mM and trehalose present at a concentration of about 150 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 50 mM and trehalose present at a concentration of about 500 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In some embodiments, the aqueous composition of the invention is essentially free of phosphate anions. In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 550 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 8.0.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of 30 mM, b. the saccharide is sucrose and is present at a concentration of about 150 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, e. Glycerol is present at a concentration of about 100 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 10 mM, b. the saccharide is sucrose and is present at a concentration of about 225 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, e. Glycerol is present at a concentration of about 25 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 150 mM and trehalose present at a concentration of about 150 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4. In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 50 mM and trehalose present at a concentration of about 500 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

In some embodiments, the aqueous composition of the invention is essentially free of sodium chloride.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 550 mM, c. the phosphate anion is present at a concentration of about 3 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, and the aqueous composition has a pH of about 8.0.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 150 mM, c. the phosphate anion is present at a concentration of about 3 mM, e. Glycerol is present at a concentration of about 100 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 10 mM, b. the saccharide is sucrose and is present at a concentration of about 225 mM, c. the phosphate anion is present at a concentration of about 3 mM, e. Glycerol is present at a concentration of about 25 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 150 mM and trehalose present at a concentration of about 150 mM, c. the phosphate anion is present at a concentration of 3 mM, and the aqueous composition has a pH of about 7.4.

In one embodiment of the aqueous composition of the invention, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 50 mM and trehalose present at a concentration of about 500 mM, c. the phosphate anion is present at a concentration of about 3 mM, and the aqueous composition has a pH of about 7.4.

Suitably, the aqueous composition according to the invention is stable over time. Stability over time can be assessed by monitoring Quality Attributes of the aqueous composition over time. Physico-chemical QAs that can be used to monitor Quality Attributes of the aqueous composition over time include mRNA-LNP size (z-average) and polydispersity index (Pdl), encapsulation efficiency (EE), mRNA integrity and mRNA Late Eluting Peak (LEP), sometimes also referred to as Late Eluting sPecies.

Suitably, the aqueous composition according to the invention is stable after a thawing step. Stability after a thawing step can be assessed by monitoring Quality Attributes of the aqueous composition over time. Physico-chemical Qas that can be used to monitor Quality Attributes of the aqueous composition over time include mRNA-LNP size (z- average) and polydispersity index (Pdl), encapsulation efficiency (EE), mRNA integrity and mRNA Late Eluting Peak (LEP). mRNA-LNP size (z-average) and polydispersity index (Pdl) 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 is indicative of its dispersion. mRNA/LNP size and polydispersity index (Pdl) can be measured by methods well known in the art. For example, Dynamic Light Scattering (DLS) can be used to monitor mRNA-LNP size (Z-average) and polydispersity index (Pdl) as explained in example 1.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size does not increase or decrease by more than 10%, suitably 5%, over a period of 90 days at -20°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size remains between 60 and 100 nm, suitably between 70 and 90 nm, more suitably between 75 and 85 nm, over a period of 90 days at -20°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size does not increase or decrease by more than 10%, suitably 5%, over a period of 90 days at +5°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size remains between 60 and 100 nm, suitably between 70 and 90 nm, more suitably between 75 and 85 nm, over a period of 90 days at +5°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size does not increase or decrease by more than 10%, suitably 5%, over a period of 30 days, suitably 60 days, more suitably 90 days, at +25°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size remains between 60 and 100 nm, suitably between 70 and 90 nm, more suitably between 75 and 85 nm, over a period of 30 days, suitably 60 days, more suitably 90 days, at +25°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size does not increase or decrease by more than 10%, suitably 5%, over a period of 90 days at -20°C and a period of 90 days at +5°C or +25°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size remains between 60 and 100 nm, suitably between 70 and 90 nm, more suitably between 75 and 85 nm, over a period of 90 days at -20°C and a period of 90 days at +5°C or +25°C.

Encapsulation efficiency (EE) measures the percentage of mRNA which is encapsulated in LNP versus the total mRNA concentration in the composition. It is thought that encapsulation efficiency is closely related to mRNA activity and that a decrease in encapsulation may result in a reduced amount of mRNA delivered to cells and thus in a reduced protein expression efficiency. Encapsulation efficiency can be measured by methods well known in the art. For example, the Ribogreen assay can be used to monitor encapsulation efficiency (EE) as explained in example 1 .

Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is higher than 75%, suitably higher than 80%, more suitably higher than 85% over a period of 90 days at -20°C.

Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is higher than 75%, suitably higher than 80%, more suitably higher than 85% over a period of 90 days at +5°C.

Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is higher than 75%, suitably higher than 80%, more suitably higher than 85% over a period of 90 days at +25°C.

Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is higher than 75%, suitably higher than 80%, more suitably higher than 85% over a period of 90 days at -70°C.

Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is higher than 75%, suitably higher than 80%, more suitably higher than 85% over a period of 90 days at -20°C and a period of 90 days at +5°C or +25°C. mRNA integrity measures the relative amount (%) of mRNA integral molecules with respect to fragments which might generate from mRNA degradation. mRNA integrity can be monitored to detect mRNA degradation that can occur in time due to the labile nature of the molecule. It is considered that mRNA integrity impacts directly the mRNA efficacy since a reduced integrity may result in a reduced level of protein expression. Also, mRNA fragments might lead to increased reactogenicity. mRNA integrity can be measured by methods well known in the art. For example, Reversed Phase-Ion Pair High Performance Liquid Chromatography (RP-IP HPLC) can be used to monitor mRNA Integrity as explained in example 1 . Capillary electrophoresis (CGE) is another method that is suitable to evaluate mRNA integrity.

Suitably, in the aqueous composition according to the invention, the mRNA integrity is higher than 70%, suitably higher than 75% over a period of 90 days at -20°C.

Suitably, in the aqueous composition according to the invention, the mRNA integrity is higher than 70%, suitably higher than 75% over a period of 90 days at +5°C.

Suitably, in the aqueous composition according to the invention, the mRNA integrity is higher than 50%, suitably higher than 55% over a period of 30 days at +25°C. Suitably, in the aqueous composition according to the invention, the mRNA integrity is higher than 70%, suitably higher than 75% over a period of 90 days at -20°C and a period of 90 days at +5°C or +25°C. mRNA Late Eluting Peak (LEP) 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 phenomenon is thought to render the mRNA untranslatable, leading to loss of protein expression (M Packer at al., Nature Communications, 2021). The relative amount (%) of mRNA LEP can be measured by methods well known in the art. For example, RP-HPLC can be used to monitor mRNA LEP.

Suitably, in the aqueous composition according to the invention, the relative amount of mRNA LEP is lower than 2%, suitably lower than 1.5%, more suitably lower than 1% over a period of 90 days at -20°C.

Suitably, in the aqueous composition according to the invention, the relative amount of mRNA LEP is lower than 2%, suitably lower than 1.5%, more suitably lower than 1% over a period of 90 days at +5°C.

Suitably, in the aqueous composition according to the invention, the relative amount of mRNA LEP is lower than 2%, suitably lower than 1.5%, more suitably lower than 1% over a period of 30 days at +25°C.

Potency can be considered as the expression of the encoded peptide or protein upon administration of the aqueous composition to a cell. If the aqueous composition is a vaccine, potency can be considered as the induction of specific antibody titers upon administration of the aqueous composition to a cell, and/or the induction of neutralizing antibody titers upon administration of aqueous composition to a cell, and/or the induction of antigen-specific T-cell responses upon administration of aqueous composition to a cell. Potency is typically determined using a cell-based protein expression assay.

Suitably, the potency of the aqueous composition according to the invention, decreases less than 30%, suitably less than 20%, more suitably lower than 10% over a period of 90 days at -20°C.

Suitably, the potency of the aqueous composition according to the invention, decreases less than 30%, suitably less than 20%, more suitably lower than 10% over a period of 90 days at +5°C. Suitably, the potency of the aqueous composition according to the invention, decreases less than 30%, suitably less than 20%, more suitably lower than 10% over a period of 90 days at +25°C.

Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is higher than 75%, suitably higher than 80%, more suitably higher than 85% after a thawing step from e.g. -20°C to 5°C.

Suitably, in the aqueous composition according to the invention, the z-average mRNA/LNP size does not increase or decrease by more than 10%, suitably 5%, after a thawing step from e.g. -20°C to 5°C.

Suitably, the potency of the aqueous composition according to the invention, decreases less than 30%, suitably less than 20%, more suitably lower than 10% after a thawing step from e.g. -20°C to 5°C.

The aqueous composition of the invention comprises 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 the LNPs have a spherical morphology. LNPs are typically suitable for intramuscular, intradermal and/or intravenous administration.

Suitably, the LNP comprises (i) an ionizable amino-lipid, (ii) a neutral lipid, suitably DSPC, (iii) a sterol, suitably cholesterol; and (iv) a PEGylated 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 aqueous composition s and methods of the invention include those described in international patent publications WO2010053572 (and particularly, Cl 2-200 described at paragraph [00225]) and W02012170930, 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 WO2017117530A1 (/.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 HI-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 -(0=0)0-;

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, ON, -C(=0)0R⁴, -0C(=0)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl;

R⁵ is H or Ci-C₆ alkyl.

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 -0(0=0)- or -(0=0)0-;

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:

In some embodiments, the ionizable (cationic) lipid has the formula 111-3: , , , , , 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 aqueous composition has an N/P ratio from about 0.1 to about 20. In one embodiment, the aqueous 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 aqueous 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.

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 LNP 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 aminolipids.

In addition to the ionizable amino-lipid, the LNP 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 LNP 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 polyethylene 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 4-0-(2’,3’-di(tetradecanoyloxy)propyl-1-0-(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.

In some embodiments, the PEGylated 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 US20150376115A1 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 LNP 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 LNP 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), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (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, the LNP comprises ionizable an amino-acid, a PEGylated lipid, a neutral lipid and a sterol. Suitably, the LNP comprises an ionizable amino-acid, a PEGylated lipid, a neutral lipid and a sterol. More suitably, the LNP comprises, an ionizable amino-acid, a PEGylated lipid, a neutral lipid and a sterol.

In one embodiment, the LNP comprises

(i) at least one ionizable amino-lipid as defined herein, suitably lipid of formula III-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 III-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 LNP comprises the ionizable amino-lipid of formula III-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; WO2012/031043;

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

WO2014/136086; WO2015/095340; WO2015/095346; WO2016/037053; WO2017/070620 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 aqueous 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 aqueous 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 aqueous 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, in particular from about 2 to about 250 pg, for example 2, 3, 4, 5, 10, 15, 20, 25, 30 or 50 pg per dose.

As used herein, a “dose” refers to the amount of the aqueous composition that is to be administered to a subject, suitably a human subject.

In one embodiment, the volume of a dose is from 0.1 to 1 ml, suitably from 0.2 to 0.8 ml, more suitably from 0.3 to 0.6 ml. Typically, the volume of a dose is selected from 0.3, 0.4 and 0.5 ml. In one embodiment, the volume of the dose is 0.3 ml. In one embodiment, the volume of the dose is 0.5 ml.

In embodiments, the therapeutic composition is a ready-to-use composition. A ready-to-use composition according to the invention is configured for direct administration into a subject and does not require any step of handling I preparation I dilution (as for e.g. a multidose composition) and/or tonicity adjustment (e.g. by dilution with NaCI solution etc.).

Suitably, the amount of RNA in the therapeutic composition is at about 100ng to about 500pg, 1 pg to about 200pg, 5pg to about 100pg, 5pg to about 50pg. Accordingly, the therapeutic composition may contain one dose of RNA that is at about 100ng to about 500pg, 1 pg to about 200pg, 5pg to about 100pg, 5pg to about 50pg.

In embodiments in that context, the amount of lipid in the therapeutic composition is at about 2500ng to about 12.5mg, 25pg to about 5mg, 125pg to about 2,5mg, 250pg to about 1.25mg.

In embodiments, the therapeutic composition has a total volume of about 50pl to 2ml, about 10OpI to 1 ml, about 250p I to 1 ml, preferably about 500p I .

In embodiments in that context, the therapeutic composition has a total volume of about 50pl to about 2ml, 10OpI to 1 ml, 250pl to 1 ml, preferably about 500pl and comprises one dose of RNA that is at about 10Ong to about 500pg, 1 pg to about 200pg, 5pg to about 100pg, 5pg to about 50pg.

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 preferred embodiments, the RNA molecule has a length of 1000 to 3000 nucleotides, suitable 1500 to 2000 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 selfamplifying 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.

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/n 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 (CAM). 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 coding sequence”). 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/U. 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 U nucleotides in the nucleic acid sequence with respect to the number of A and/or U nucleotides in the original nucleic acid sequence (e.g. the wild type or reference sequence). In some embodiments, such an A/U alteration is performed to modify the retention time of the individual nucleic acids in an aqueous 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/U 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(U) 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 WO2012019780.

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 (eg. 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, W02008157688, WO2009149253, 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’OMe- 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 orcapl 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/U (A/T) content in the environment of the ribosome binding site of the mRNAs used herein may be increased compared to the A/U (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/U (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 sequence” 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 “muaq’”), CASP1 , C0X6B1 , GNAS, NDUFA1 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, NDUFA1 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 WO2019077001 A1 , in particular, claim 9 of WO2019077001 A1 . The corresponding 5’-UTR sequences of claim 9 of WO2019077001 A1 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 WO2013143700, the disclosure of WO2013143700 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 WO2013143700, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5’-UTR as described in WO2016107877, the disclosure of WO2016107877 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 WO2016022914, 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 U 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 (U)), 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, six-membered 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.

In some embodiments, the chemical modification is selected from pseudouridine (i ), N1 -methylpseudouridine (ml i ), 5-methylcytosine, and 5-methoxyuridine, more suitably pseudouridine (i ) and N1 -methylpseudouridine (m1 i ), 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 (i ), N1- methylpseudouridine (m1 ip), 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 (ip) and N1- methylpseudouridine (m1 ip), suitably wherein all uracil nucleotides are replaced by pseudouridine (ip) nucleotides and/or N1 -methylpseudouridine (m1 ip) nucleotides, optionally wherein all uracil nucleotides are replaced by pseudouridine (¹) nucleotides and/or N1-methylpseudouridine (ml¹) 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 (m1 ip), 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- m ethylpseudouridine.

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 W02016180430. 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 (i ), N1- methylpseudouridine (ml i ), 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 (m1 i ) to obtain a modified RNA. In other embodiments, the nucleotide mixture used in RNA /n 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 U 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 aqueous composition is a therapeutic composition. In one embodiment, the therapeutic composition is an immunogenic composition, suitably a vaccine composition.

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.

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.

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 RNA molecule is an mRNA encoding a protein of interest. In one embodiment, the mRNA encoding a protein of interest is a non-replicating mRNA. In one embodiment, the mRNA encoding a protein of interest is a non-replicating mRNA a selfreplicating mRNA. Suitably, the protein of interest is an 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.

Suitably, the protein of interest is an antigen derived from a respiratory virus, such as an influenza virus, a SARS-CoV-2 virus, respiratory syncytial (RSV) a human pneumovirus (HMPV) or a human parainfluenza virus (PIV) .

In one embodiment the protein of interest is an influenza virus antigen selected from Haemagglutinin (HA) antigens and Neuraminidase (NA) antigens.

In one embodiment the protein of interest is a SARS-CoV-2 spike protein (S) antigen.

In one embodiment the protein of interest is an RSV fusion protein (F) antigen.

In one embodiment the protein of interest is an HMPV fusion protein (F) antigen.

In one embodiment the protein of interest is a PIV fusion protein (F) antigen.

In some embodiments, the aqueous composition of the invention is obtained by first formulating the RNA, suitably mRNA, and the delivery system, suitably LNPs, in a first buffer system, followed by a buffer exchange to obtain the aqueous composition as defined herein (e.g. comprising Tris, a saccharide, phosphate anions, sodium chloride and optionally glycerol).

The intermediate buffer is suitably a phosphate buffer. Suitably, the intermediate buffer is a PBS-sucrose buffer that suitably comprises sucrose in a concentration of about 150mM, NaCI in a concentration of about 75mM and Na3PO4 in a concentration of about 10mM.

The buffer exchange from the intermediate buffer to the aqueous composition may be performed by dilution. The dilution factor may be in a range of 1 :1 to 1 :100, suitably 1 :1 to 1 :10, more suitably 1 :2 to 1 :5, e.g. 1 :3.

In another aspect, there is provided the aqueous 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 aqueous 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 aqueous composition of the invention to a patient in need thereof.

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

In another aspect, a method of producing the aqueous composition of the invention is provided, comprising a) providing an initial composition comprising an RNA molecule, suitably mRNA, and a delivery system, suitably LNPs, b) transferring the initial composition into a first buffer system to obtain an intermediate composition, c) diluting the intermediate composition with a dilution buffer to obtain the aqueous composition of the invention.

The first buffer system may be a phosphate buffer, for example a PBS buffer. Suitably, the PBS buffer comprises sucrose. Suitably the PBS Sucrose buffer comprises sucrose preferably in a concentration of about 150mM; NaCI, preferably in a concentration of about 75mM; and NaPO4, preferably in a concentration of about 10mM; and has a pH of preferably about pH 7.4.

The transferring step b) of the method may be carried out using filtration such as ultrafiltration or tangential flow filtration (TFF). The diluting of step c) of the method may be performed with a dilution factor that is in a range of 1 :1 to 1 :100, suitably 1 :1 to 1 :10, more suitably 1 :2 to 1 :5, e.g. 1 :3.

The dilution buffer comprises a saccharide as defined herein and Tris. In some embodiments, the dilution buffer comprises glycerol. In some embodiments, the dilution buffer may comprise phosphate anions and/or sodium chloride.

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, an aqueous 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. an aqueous 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.

Coding sequence/codinq 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 (U) 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 U 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.

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 sequence, DNA sequence, RNA sequence: 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 an aqueous 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.

Example 1 - mRNA-LNP aqueous composition screen

Liquid formulations were prepared by diluting a concentrated mRNA-LNP bulk (initially in PBS sucrose buffer) in formulations comprising different excipient contents.

The concentrated mRNA/LNP bulk comprised mRNA expressing 4 influenza virus antigens (HA and NA from Wisconsin H1 N1 , HA and NA from Darwin H3N2), encapsulated in lipid nanoparticles (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). In addition to the antigen coding sequence, each mRNA construct comprised an mRNA cap (Cap1), a HSD17B4 5’UTR, a PSMB3 3’UTR, a histone stem loop (HSL) and a polyA tail (A100). The antigen coding sequence was GC enriched, and all uridine residues were replaced by N1-methylpseudouridine (ml i ). The mRNA constructs had a length of about 1700 nucleotides (NA) and 1900 nucleotides (HA).

The excipient content of each of the tested aqueous compositions is shown in Table 1.

Table 1. Composition of each tested aqueous composition

The following analytical methods, briefly described below, were used to monitor quality attributes of the tested formulations: mRNA-LNPsize (Z-averac/e) and polydispersity index (Pdl)

Dynamic Light Scattering (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.

Encapsulation efficiency (EE) 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.

.mRNA Integrity and mRNA LEP (Late Eluting Peak)

Reversed Phase-Ion Pair High Performance Liquid Chromatography (RP-IP HPLC) was used to assess mRNA integrity and to detect potential adduct events (mRNA LEP) 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).

In vitro relative expression assay

HEK293T cells were incubated with various quantities of mRNA-LNP. Cells were harvested 24h post incubation and analyzed for surface expression of the HA and NA protein by flow cytometry. For this purpose, cells were stained using sheep anti-HA and anti-NA influenza serum followed by secondary anti-sheep FITC-conjugated antibody, acquired on a MACSQuant® VYB Flow Cytometer and analyzed using FlowJo Software Version 10.9.0.

Results

1. Aqueous compositions N° 1 to 6

Aqueous compositions N° 1 to 6 comprising mRNA encoding 4 influenza antigens formulated in LNPs as described above and excipients shown in table 1. The total mRNA concentration was 0.3 g/L. LNP size (z-Average (nm)) and polydispersity index (Pdl) were measured at TO, after 8 days at -20°C and after 14 days at +5°C. Results are shown in Fig. 1. Tris based matrices outperformed phosphate and histidine-based compositions.

The proportion of LEP was measured by HPLC at TO, after 8 days at -20°C, after 14 days at +5°C and after 14 days at +25°C. Results are shown in Fig. 2. Tris based matrices outperformed phosphate and histidine-based compositions.

2. Aqueous compositions N° 5 and 12

Aqueous compositions N° 5 and 12 comprising mRNA encoding 4 influenza antigens formulated in LNPs as described above and excipients shown in table 1. The total mRNA concentration was 0.3 g/L. mRNA integrity and the proportion of LEP were monitored over 21 days at +25°C. Results are shown in Fig. 3 (% mRNA integrity) and Fig. 4 (% LEP).

3. Aqueous compositions N° 1 and 7-10

Aqueous compositions N° 1 and 7-10 comprising mRNA encoding 4 influenza antigens formulated in LNPs as described above and excipients shown in table 1 . The total mRNA concentration was 0.032 g/L.

Encapsulation efficiency (%) was monitored over 90 days at -20°C, +5°C and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2,5 months at -20°C. Results are shown in Fig. 5.

LNP size (z-Average (nm)) was monitored over 90 days at -20°C, +5°C and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2,5 months at -20°C. Results are shown in Fig. 6.

LNP Pdl was monitored over 90 days at -20°C, +5°C and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2,5 months at -20°C. Results are shown in Fig. 7.

In vitro relative expression (%) was monitored at over 30 days at -20°C, +5°C and +25°C. Results are shown in Fig. 8.

4. Aqueous compositions N° 1 , 9 and 11 Aqueous compositions N° 1 , 9 and 11 comprising mRNA encoding 4 influenza antigens formulated in LNPs as described above and excipients shown in table 1. The total mRNA concentration was 0.032 g/L.

LNP size (z-Average (nm) and Pdl) was monitored over 90 days at -20°C, +5°C and +25°C. Results are shown in Fig. 9.

Encapsulation efficiency (%) was monitored over 90 days at -20°C, over 50 days at +5°C and over 50 days at +25°C. Results are shown in Fig. 10. mRNA integrity (%) and LEP (%) were monitored over 50 days at -20°C, +5°C and +25°C. Results are shown in Fig. 11.

In vitro relative expression (%) was monitored over 30 days at -20°C, +5°C and +25°C. Results are shown in Fig. 12.

5. Aqueous compositions N° 6, 9 and 12

Aqueous compositions N° 6, 9 and 12 comprising 0.1 g/L mRNA encoding 4 influenza antigens formulated in LNPs as described above and excipients shown in table 1 . The total mRNA concentration was 0.1 g/L.

LNP size (z-Average (nm) and Pdl) were monitored over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days). Results are shown in Fig. 13.

Encapsulation efficiency (%) was monitored overtime at -20°C (120 days), +5°C (90 days) and +25°C (90 days). Results are shown in Fig. 14. mRNA integrity (%) and LEP (%) were monitored over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days). Results are shown in Fig. 15. is of formulations stored at 25°C mRNA-LNP formulations were produced that comprise mRNA constructs encoding four different influenza antigens (HA and NA from Wisconsin H1 N1 , HA and NA from Darwin H3N2), encapsulated in lipid nanoparticles (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). In addition to the antigen coding sequence, each mRNA construct comprised an mRNA cap (Cap1), a HSD17B45’UTR, a PSMB3 3’UTR, a histone stem loop (HSL) and a polyA tail (A100). The antigen coding sequence was GC enriched, and all uridine residues were replaced by N1 -methylpseudouridine (ml i ). The mRNA constructs had a length of about 1700 nucleotides (NA) and 1900 nucleotides (HA).

The excipient content of each of the tested aqueous compositions are provided in

Table 2.

Table 2. Composition of each tested aqueous compositions

Mice were immunized twice i.m. on day 0 and 21 with the respective mRNA-LNP formulations at 0.5pg total mRNA dose. Animals in the control groups were injected with PSN only (lacking mRNA-LNPs).

The following analytical methods were used:

Muscle swelling score

Muscle swelling was used as indicator for local tolerability. The injected muscle was monitored by visual inspection after each injection for swelling and redness. The swelling was scored as follows compared to the non-injected muscle: absent (score=0), mild (score=1), moderate (score=2), pronounced (score=3).

Mice were immunized i.m. on day 0 and 21 with 0.5pg of the indicated formulations (A (PSN), B (PSN/TSN), and C (PSN/TSG)) stored at -70°C (TO) or at 25°C for 21 days (21d25°C) (n= 14). Control animals received PSN matrix (lacking LNP mRNA) (n= 5) via the i.m. route on day 0 and 21 . Post each immunization, muscle swelling was scored and compared to the non-injected muscle: absent (score=0), mild (score=1), moderate (score=2), pronounced (score=3).

Quantification of pro-inflammatory cytokines and chemokines in mouse serum Cytokines and chemokines were quantified in serum samples using the cytometric bead assay LEGENDplex™ mouse anti-virus response panel (13-plex including CCL2, CCL5, CXCL1 , CXCL10, GM-CSF, IFNa, IFN|3, IFNy, IL-1 , IL-6, IL-10, IL-12p70, TNF) according to manufacturer’s instructions. The beads were measured using a flow cytometer (Biorad) and analyzed using a software (BioLegend).

Hemagglutinin inhibition (HI) assay

A HI assay was used to assess functional anti-HA antibody responses induced by the administered formulations. The basis of the HI assay is the prevention of attachment of the virus to red blood cells in the presence of antibodies to influenza virus. Therefore, hemagglutination can be inhibited when antibodies are present in the serum of vaccinated mice. The HI assay was performed on individual serum samples obtained three weeks post first and two weeks post second vaccination. Before the assay, to eliminate non-specific inhibitors of hemagglutination, serum samples were treated with receptor destroying enzyme (RDE), followed by heat inactivation for 30 min and pre-adsorption to red blood cells (RBCs). Serum samples from mice receiving NaCI solution served as negative control. Sheep serum anti-HA of influenza viruses were used as positive controls in the respective HI assay. Samples were read visually as either agglutinated in which RBCs formed a pattern whereas non-agglutinated RBCs formed a teardrop in the center of a well. The HI titer was defined as the reciprocal of the last serum dilution that inhibited agglutination.

Enzyme linked lectin assay (ELLA)

To assess the immunogenicity of the mRNA compositions regarding NA antigens, an enzyme linked lectin assay (ELLA) was carried out. The assay allows the measurement of antibodies inhibiting the enzymatic activity of the NA. In brief, 96-well plates were coated with the carbohydrate fetuin, which was then exposed to NA through NA-bearing single cycle pseudoviruses (PV) used as a surrogate virus. The NA enzyme cleaves terminal sialic acid residues from the fetuin, exposing galactose that is then bound by peanut agglutinin, conjugated to horseradish peroxidase (PNA-HRPO). This reagent then forms the basis for colorimetric reading of NA activity. This activity can be inhibited by antibodies present in the serum of vaccinated mice. To measure the ELLA titers, each serum sample was heat- treated and then diluted serially in PBS-BSA. 50 pl of each dilution was added to duplicate wells of a fetuin-coated plate. An equal volume (50 pl) of the selected pseudovirus dilution was added to all serum-containing wells in addition to at least 4 wells containing diluent without serum that served as a positive (pseudovirus only) control. The plates were incubated for 16-18 h at 37°C. As described for the virus titration, the plates were washed and PNA-HRPO was added to all wells. After 2 h of incubation, the plates were washed, and an o-phenylenediamine dihydrochloride (OPD) (Sigma, St. Louis, MO, USA) substrate was added. The reaction was stopped by addition of chloric acid, and the absorbance was read at 490 nm. The ELLA titers were defined as the reciprocal of the last dilution that resulted in at least 50% inhibition.

Detection of cellular immune responses by ICS

T cell immune responses were analyzed two weeks post second vaccination by intracellular cytokine staining (ICS) in isolated splenocytes stimulated with custom-made 15-mer overlapping peptide libraries spanning the full-length HA and NA proteins. Splenocytes were thawed and seeded in 96-well plates. The cells were stimulated with 0.5pg/ml/peptide of the HA or NA peptide libraries in the presence of anti-CD28 antibody. After 1 h incubation at 37°C, GolgiPlug containing brefeldin A was added and the cells were incubated for another 5-6 h at 37°C. Afterwards the splenocytes were washed twice in PBS and incubated with LIVE/DEAD fixable Aqua Dye at 4°C for 30 min. Cells were stained with antibodies against CD90.2 (Thy1.2)-FITC, CD8-APC-Cy7 and CD4-BD Horizon V450 in the presence of Fcy-blocking reagent for 30 min at 4°C in PBS/0.5% BSA. Subsequently, the cells were washed and fixed. Finally, the cells were incubated with anti-IFNy-APC and anti- TNF-PE at 4°C for 30 min. For the FACS analysis, splenocytes were resuspended in PBS/0.5% BSA/2 mM EDTA. The cells were acquired on a ZE5 flow cytometer (Biorad), and data was analyzed using a software.

Results

• Clinical observations and local effects

Mice were immunized i.m. on day 0 and 21 with 0.5pg of the indicated formulations (A (PSN), B (PSN/TSN), and C (PSN/TSG)) stored at -70°C (TO) or at 25°C for 21 days (21d25°C) (n= 14). Control animals received PSN matrix (lacking LNP mRNA) (n= 5) via the i.m. route on day 0 and 21 . Post each immunization, muscle swelling was scored and compared to the non-injected muscle: absent (score=0), mild (score=1), moderate (score=2), pronounced (score=3). lAt most mild muscle swelling was observed after the first and second administration of the formulations at TO and at 21d25°C (see FIG. 16) that was comparable between the groups. Induction of cytokines and chemokines in the serum

Mice were immunized i.m. on day 0 and 21 with 0.5 g of the indicated formulations (A (PSN), B (PSN/TSN), and C (PSN/TSG)) stored at -70°C (TO) or at 25°C for 21 days (21d25°C) (n= 14). Control animals received PSN matrix (lacking LNP mRNA) (n= 5) via the i.m. route on day 0 and 21. The levels of IFNy, CCL2 and CXCL10 were determined using cytometric bead array (CBA) in serum samples collected 18 h after the first immunization.

Early innate immune responses were assessed by determining levels of pro- inflammatory cytokines and chemokines in the serum 18 h after the first immunization. As shown in FIG. 17, administration of formulations A, B and C led to induction of IFNy, CCL2 and CXCL10 responses. Overall, formulation “PSN/TSN” (B) showed the lowest induction of cytokines.

• Induction of humoral and cellular responses

Mice were immunized i.m. on day 0 and 21 with 0.5pg of the indicated formulations (A (PSN), B (PSN/TSN), and C (PSN/TSG)) stored at -70°C (TO) or at 25°C for 21 days (21d25°C) (n= 14).

The induction of functional antibodies was analyzed in the serum collected three weeks post first and two weeks post second immunization using the HI assay for the anti- HA responses and the ELLA for the anti-NA responses. The results are presented in FIG 18.

• Induction of humoral and cellular responses

Mice were immunized i.m. on day 0 and 21 with 0.5pg of the indicated formulations (A (PSN), B (PSN/TSN), and C (PSN/TSG)) stored at -70°C (TO) or at 25°C for 21 days (21d25°C) (n= 14).

T cell immune responses (CD4 and CD8) were analyzed two weeks post second vaccination by ICS in isolated splenocytes stimulated with custom-made 15-mer overlapping peptide libraries spanning the full-length HA and NA proteins. The results are presented in FIG 19.

Claims

1. An aqueous composition comprising an RNA molecule, a delivery system, Tris, a saccharide and phosphate anions.

2. The aqueous composition of claim 1 , wherein Tris is present at a concentration between 5 and 50 mM, suitably between 7.5 and 30 mM, for example at a concentration of about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 mM, suitably at a concentration of about 15 mM.

3. The aqueous composition of claim 1 or 2, wherein the saccharide is a disaccharide, suitably selected from sucrose, trehalose and combinations thereof.

4. The aqueous composition of claim 3, wherein the disaccharide is sucrose, and wherein sucrose is present at a concentration between 50 and 700 mM, suitably between 100 and 600 mM , for example at a concentration of about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, suitably at a concentration of about 300 mM.

5. The aqueous composition of claim 3, wherein the disaccharide is a combination of sucrose and trehalose, and wherein the combined concentration of sucrose and trehalose is between 50 and 700 mM, suitably between 100 and 600 mM, for example 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, and wherein for example sucrose and trehalose are both present at a concentration of about 150mM, or sucrose is present at a concentration of 50mM and trehalose is present at a concentration of about 550mM.

6. The aqueous composition of any one of claims 1 to 5, wherein phosphate anions are present at a concentration from 0.5 to 15 mM, suitably at a concentration from 1 to 10 mM, more suitably at a concentration from 1 to 5 mM, for example at a concentration of about 1 , 2, 3, 4 or 5 mM, suitably at a concentration of 3 mM.

7. The aqueous composition of any one of claims 1 to 6, further comprising sodium chloride, suitably present at a concentration between 5 and 50 mM, suitably between 10 and 40 mM, more suitably between 15 and 30 mM, for example at a concentration of about 15, 20, 21 , 22, 22.5, 23, 24, 25 or 30 mM, suitably at a concentration of about 22.5 mM. 8. The aqueous composition of any one of claims 1 to 7, wherein said aqueous composition has a pH between 6.5 and 8.5, for example about 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.

8, 7.9 or 8.0, suitably about 7.4 or about 8.0.

9. The aqueous composition of any one of claims 1 to 8 further comprising glycerol, suitably present at a concentration from 10 to 200 mM, for example, about 25, 50, 75, 100, or 150 mM, more suitably about 25 or about 100 mM.

10. The aqueous composition of claim 9, wherein the combined concentration of saccharide and glycerol is between 50 and 700 mM, suitably between 100 and 600 mM, for example about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, and wherein for example the saccharide is sucrose and present at a concentration of about 150mM and glycerol is present at a concentration of about 100mM.

11 . The aqueous composition of any one of claims 1 to 10, wherein a. Tris is present at a concentration ranging from 5 to 50 mM, suitably from 7.5 to 30 mM, b. the saccharide, suitably sucrose, is present at a concentration ranging from 50 to 700 mM, suitably from 100 to 600 mM, c. the phosphate anion is present at a concentration ranging from 0.5 to 15 mM, suitably from 1 to 5 mM, d. Sodium chloride is present at a concentration ranging from 5 to 50 mM, suitably from 15 to 30 mM, and wherein said aqueous composition has a pH between 6 and 9, suitably between 6.5 and 8.5.

12. The aqueous composition of claim 11 , wherein glycerol is present at a concentration ranging from 10 to 200 mM, suitably from 25 to 175 mM.

13. The aqueous composition of any one of claims 1 to 12, wherein a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. Phosphate is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

14. The aqueous composition of any one of claims 1 to 12, wherein a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

15. The aqueous composition of any one of claims 1 to 12, wherein, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 550 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

16. The aqueous composition of any one of claims 1 to 12, wherein, a. Tris is present at a concentration of about 15 mM, b. the saccharide is sucrose and is present at a concentration of about 300 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 8.0.

17. The aqueous composition of any one of claims 1 to 12, wherein, a. Tris is present at a concentration of about 30 mM, b. the saccharide is sucrose and is present at a concentration of about 150 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, e. Glycerol is present at a concentration of about 100 mM, and the aqueous composition has a pH of about 7.4.

18. The aqueous composition of any one of claims 1 to 12, wherein, a. Tris is present at a concentration of about 10 mM, b. the saccharide is sucrose and is present at a concentration of about 225 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, e. Glycerol is present at a concentration of about 25 mM, and the aqueous composition has a pH of about 7.4.

19. The aqueous composition of any one of claims 1 to 12, wherein, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 150 mM and trehalose present at a concentration of about 150 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

20. The aqueous composition of any one of claims 1 to 12, wherein, a. Tris is present at a concentration of about 15 mM, b. the saccharide is a combination of sucrose present at a concentration of about 50 mM and trehalose present at a concentration of about 500 mM, c. the phosphate anion is present at a concentration of about 3 mM, d. Sodium chloride is present at a concentration of about 22.5 mM, and the aqueous composition has a pH of about 7.4.

21 . The aqueous composition of any one of claims 1 to 20, wherein the RNA molecule is an mRNA encoding a protein of interest, suitably an mRNA selected from a non-replicating mRNA and a self-replicating mRNA.

22. The aqueous composition of claim 21 , wherein the protein of interested is an antigen, suitably a respiratory virus antigen selected from an influenza HA antigen, an influenza NA antigen, a SARS-CoV-2 S antigen, an RSV F antigen, an HMPV F antigen an a PIV F antigen.

23. The aqueous composition of any one of claims 1 to 22, wherein the delivery system is a lipid nanoparticle (LNP), suitably comprising (i) an ionizable amino-lipid, (ii) a neutral lipid, suitably DSPC, (iii) a sterol, suitably cholesterol; and (iv) a PEGylated lipid.

24. The aqueous composition of any one of claims 1 to 23 for use in therapy, suitably for use as a vaccine.

25. A method of producing the aqueous composition of claims 1 to 23, comprising a) providing an initial composition comprising an RNA molecule, suitably mRNA, and a delivery system, suitably LNPs, b) transferring the initial composition into a first buffer system to obtain an intermediate composition, c) diluting the intermediate composition with a dilution buffer to obtain the aqueous composition as defined in claims 1 to 23.