Compositions and methods for stabilizing lipid nanoparticle mRNA vaccines

Abstract

The present invention provides technology related to the stabilization of lipid nanoparticle mRNA compositions (eg, vaccines).

Description

[Background technology]

[0001] background Messenger RNA (mRNA) has proven to be an interesting therapeutic modality, and has recently attracted considerable attention, particularly in the vaccine space. [Overview of the project]

[0002] summary The present invention provides technologies relating to RNA (e.g., mRNA) therapeutic formulations and, in particular, lipid nanoparticle (LNP) formulations containing RNA (e.g., mRNA) payloads. In particular, the present invention provides therapeutic RNA formulations (i.e., LNP formulations) that are suitable for storage and / or handling at temperatures above about -80°C or above about -70°C, about -60°C, about -50°C, about -40°C, about -30°C, or about -20°C (e.g., stable). In some embodiments, the formulations provided may be suitable for storage and / or handling at temperatures above freezing (e.g., above about 0°C), standard refrigeration temperatures (e.g., within the range of about 1°C to about 8°C or about 2°C to about 8°C or about 2°C to about 6°C or about 2°C to about 4°C) and / or room temperature (e.g., within the range of about 15°C to about 25°C or about 20°C to about 23°C).

[0003] In one embodiment, the present invention provides a formulation that is suitable for drying and / or is a dry product (for example, a lyophilized formulation).

[0004] The present invention provides a formulation that is particularly useful as a vaccine (and / or for its manufacture).

[0005] In one embodiment, the present invention provides formulations (and in particular LNP formulations) of RNA encoding a viral antigen (e.g., SARS-CoV2 antigen such as the S-protein or its epitope). Particularly exemplified formulations include RNA constructs that are BNT162 constructs (e.g., Walsh, E. et al. RNA-Based COVID-19 Vaccine BNT162b2 Selected for a Pivotal Efficacy Study. medRxiv (2020)), e.g., BNT162b2; and PCT application number PCT / EP2020 / 081981 filed November 12, 2020, titled “Coronavirus Vaccine” (the contents of each of these are incorporated herein by reference for the purposes described herein)).

[0006] In one embodiment, the formulation provided herein comprises (a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; (b) sucrose at a concentration of about 10% w / v in the formulation; and (c) Tris buffer at a concentration of about 10 mM in the formulation, substantially free of sodium chloride.

[0007] In one embodiment, the formulation provided herein is a frozen formulation comprising (a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; (b) sucrose at a concentration of about 10% w / v in the formulation; and (c) Tris buffer at a concentration of about 10 mM in the formulation, substantially free of sodium chloride.

[0008] In one embodiment, the formulation provided herein is a dried formulation comprising (a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; (b) sucrose at a concentration of about 10% w / v in the pre-dried formulation; and (c) Tris buffer at a concentration of about 10 mM in the pre-dried formulation, substantially free of sodium chloride.

[0009] A method for providing such a formulation is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs are i) A payload consisting of or containing one or more mRNA molecules; ii) (4-Hydroxybutyl)azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(Polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoyl phosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5 comprising; and b) A step of exchanging a first buffer system with a second buffer system, wherein the second buffer system i) A Tris buffer substantially free of sodium chloride and having a concentration of about 10 mM in the formulation; and ii) Sucrose at a concentration of about 10% w / v in the formulation comprising, the step comprising, a method.

[0010] In certain embodiments, a method comprising administering a dosage form of a formulation, wherein the formulation a) Is a lipid nanoparticle (LNP), wherein i) mRNA at a concentration of about 0.5 mg / ml; ii) (4-Hydroxybutyl)azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of about 7.17 mg / ml; iii) 2-[(Polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of about 0.89 mg / ml; iv) Distearoyl phosphatidylcholine (DSPC) at a concentration of about 1.56 mg / ml; v) Cholesterol at a concentration of about 3.1 mg / ml comprising LNP; b) Sucrose at a concentration of about 10% w / v; c) A Tris buffer substantially free of sodium chloride and having a concentration of about 10 mM in the formulation comprising; wherein the formulation is diluted to the dosage form before administration.

[0011] In one embodiment, provided herein is a formulation comprising (a) lipid nanoparticles (LNPs) (i) a payload of one or more mRNAs or containing thereof; (ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; (b) trehalose at a concentration of about 10% w / v in the formulation; and (c) Tris buffer at a concentration of about 10 mM in the formulation, substantially free of sodium chloride.

[0012] In one embodiment, the formulation provided herein is a frozen formulation comprising (a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; (b) trehalose at a concentration of about 10% w / v in the formulation; and c) Tris buffer at a concentration of about 10 mM in the formulation, substantially free of sodium chloride.

[0013] In one embodiment, the formulation provided herein is a dried formulation comprising (a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; (b) trehalose at a concentration of about 10% w / v in the pre-dried formulation; and (c) Tris buffer at a concentration of about 10 mM in the pre-dried formulation, substantially free of sodium chloride.

[0014] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is: i) Tris buffer, which is substantially sodium chloride-free and has a concentration of approximately 10 mM in the formulation; and ii) Trehalose at a concentration of approximately 10% w / v in the formulation The process includes This method includes [something].

[0015] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Trehalose at a concentration of approximately 10% w / v; c) Tris buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation. Includes; In this method, the preparation is diluted to the prescribed dosage form before administration.

[0016] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) Tris buffer at a concentration of approximately 10 mM in the formulation, substantially free of sodium chloride.

[0017] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) A frozen formulation containing Tris buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0018] In one embodiment, the formulation provided herein is a dried formulation comprising: a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 5% w / v in the pre-dried formulation; c) trehalose at a concentration of about 5% w / v in the pre-dried formulation; and d) Tris buffer at a concentration of about 10 mM in the pre-dried formulation, substantially free of sodium chloride.

[0019] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, (a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) Tris buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation; ii) Sucrose at a concentration of approximately 5% w / v in the formulation; and iii) Trehalose at a concentration of approximately 5% w / v in the formulation A process that includes This method includes [something].

[0020] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) Tris buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0021] In one embodiment, the formulations provided herein are (a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) Tris buffer containing approximately 6 mg / ml sodium chloride at a concentration of approximately 10 mM in the formulation.

[0022] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) A frozen formulation containing approximately 6 mg / ml sodium chloride and Tris buffer at a concentration of approximately 10 mM.

[0023] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 10% w / v in the pre-dried formulation; and c) Tris buffer containing about 6 mg / ml sodium chloride at a concentration of about 10 mM in the pre-dried formulation.

[0024] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing the formulation is provided herein. a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) Tris buffer containing approximately 6 mg / ml sodium chloride in the preparation and at a concentration of approximately 10 mM; and ii) Sucrose at a concentration of approximately 10% w / v in the formulation It is a method that includes a process, which includes the elements.

[0025] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) Tris buffer solution containing approximately 6 mg / ml of sodium chloride in the preparation and at a concentration of approximately 10 mM. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0026] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Trehalose at a concentration of approximately 10% w / v in the formulation; c) Tris buffer containing approximately 6 mg / ml sodium chloride at a concentration of approximately 10 mM in the formulation.

[0027] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Trehalose at a concentration of approximately 10% w / v in the formulation; c) A frozen formulation containing approximately 6 mg / ml sodium chloride and Tris buffer at a concentration of approximately 10 mM.

[0028] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) trehalose at a concentration of about 10% w / v in the pre-dried formulation; and c) Tris buffer containing about 6 mg / ml sodium chloride at a concentration of about 10 mM in the pre-dried formulation.

[0029] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) Tris buffer containing approximately 6 mg / ml sodium chloride in the preparation and at a concentration of approximately 10 mM; and ii) Trehalose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0030] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Trehalose at a concentration of approximately 10% w / v in the formulation; c) Tris buffer solution containing approximately 6 mg / ml of sodium chloride in the preparation and at a concentration of approximately 10 mM. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0031] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) Tris buffer containing approximately 6 mg / ml sodium chloride at a concentration of approximately 10 mM.

[0032] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) A frozen formulation containing approximately 6 mg / ml sodium chloride and Tris buffer at a concentration of approximately 10 mM.

[0033] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising: i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 5% w / v in the pre-dried formulation; c) trehalose at a concentration of about 5% w / v in the pre-dried formulation; and d) Tris buffer containing about 6 mg / ml sodium chloride at a concentration of about 10 mM in the pre-dried formulation.

[0034] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) Tris buffer solution containing approximately 6 mg / ml of sodium chloride in the preparation, with a concentration of approximately 10 mM; ii) Sucrose at a concentration of approximately 5% w / v in the formulation; and iii) Trehalose at a concentration of approximately 5% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0035] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) Tris buffer solution containing approximately 6 mg / ml of sodium chloride in the preparation and at a concentration of approximately 10 mM. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0036] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) Substantially sodium chloride-free and containing His buffer at a concentration of approximately 10 mM in the formulation.

[0037] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) A frozen formulation containing His buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0038] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 10% w / v in the pre-dry formulation; and c) His buffer at a concentration of about 10 mM in the pre-dry formulation, substantially free of sodium chloride.

[0039] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) His buffer, which is substantially sodium chloride-free and has a concentration of approximately 10 mM in the formulation; and ii) Sucrose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0040] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 10% w / v in the formulation; d) His buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0041] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Trehalose at a concentration of approximately 10% w / v in the formulation; c) His buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0042] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) A frozen preparation containing approximately 10% w / v trehalose; c) A preparation substantially free of sodium chloride and containing approximately 10 mM His buffer.

[0043] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) trehalose at a concentration of about 10% w / v in the pre-dry formulation; and c) His buffer at a concentration of about 10 mM in the pre-dry formulation, substantially free of sodium chloride.

[0044] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) His buffer, which is substantially sodium chloride-free and has a concentration of approximately 10 mM in the formulation; and ii) Trehalose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0045] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Trehalose at a concentration of approximately 10% w / v in the formulation; d) His buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0046] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) His buffer at a concentration of approximately 10 mM in the formulation, substantially free of sodium chloride.

[0047] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) A frozen formulation containing His buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0048] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of one or more mRNAs or containing thereof; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 5% w / v in the pre-dry formulation; c) trehalose at a concentration of about 5% w / v in the pre-dry formulation; and d) His buffer at a concentration of about 10 mM in the pre-dry formulation, substantially free of sodium chloride.

[0049] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) A His buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation; ii) Sucrose at a concentration of approximately 5% w / v in the formulation; and iii) Trehalose at a concentration of approximately 5% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0050] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) His buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0051] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) Contains HEPES buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0052] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) A frozen formulation containing HEPES buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0053] In one embodiment, the formulation provided herein is a dried formulation comprising: a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 10% w / v in the pre-dried formulation; and c) HEPES buffer at a concentration of about 10 mM in the pre-dried formulation, substantially free of sodium chloride.

[0054] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) HEPES buffer that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation; and ii) Sucrose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0055] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 10% w / v in the formulation; d) HEPES buffer that is substantially sodium chloride-free and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0056] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Trehalose at a concentration of approximately 10% w / v in the formulation; c) HEPES buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0057] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) A frozen preparation containing approximately 10% w / v trehalose; c) A preparation substantially free of sodium chloride and containing approximately 10 mM HEPES buffer.

[0058] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of one or more mRNAs or a payload containing the same; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) trehalose at a concentration of about 10% w / v in the pre-dry formulation; and c) HEPES buffer at a concentration of about 10 mM in the pre-dry formulation, substantially free of sodium chloride.

[0059] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) HEPES buffer that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation; and ii) Trehalose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0060] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Trehalose at a concentration of approximately 10% w / v in the formulation; d) HEPES buffer that is substantially sodium chloride-free and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0061] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) HEPES buffer at a concentration of approximately 10 mM in the formulation, substantially free of sodium chloride.

[0062] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) A frozen formulation containing HEPES buffer at a concentration of approximately 10 mM, substantially free of sodium chloride.

[0063] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of one or more mRNAs or containing thereof; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 5% w / v in the pre-dry formulation; c) trehalose at a concentration of about 5% w / v in the pre-dry formulation; and d) HEPES buffer at a concentration of about 10 mM in the pre-dry formulation, substantially free of sodium chloride.

[0064] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) HEPES buffer that is substantially free of sodium chloride and has a concentration of approximately 10 mM in the formulation; ii) Sucrose at a concentration of approximately 5% w / v in the formulation; and iii) Trehalose at a concentration of approximately 5% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0065] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation is a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 5% w / v in the formulation; c) Trehalose at a concentration of approximately 5% w / v in the formulation; d) HEPES buffer that is substantially sodium chloride-free and has a concentration of approximately 10 mM in the formulation. Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0066] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) A PBS buffer that is substantially free of sodium chloride.

[0067] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) A frozen formulation containing a PBS buffer that is substantially free of sodium chloride.

[0068] In one embodiment, the formulation provided herein is a dried formulation comprising: a) lipid nanoparticles (LNPs) i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 10% w / v in the pre-dried formulation; and c) a PBS buffer substantially free of sodium chloride.

[0069] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) PBS buffer substantially free of sodium chloride; and ii) Sucrose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0070] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 10% w / v; c) PBS buffer that is substantially free of sodium chloride Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0071] In one embodiment, the formulations provided herein are a) lipid nanoparticles (LNPs), i) a payload containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) PBS buffer containing approximately 6 mg / ml sodium chloride in the formulation.

[0072] In one embodiment, the formulation provided herein is a) a lipid nanoparticle (LNP), i) a payload containing one or more mRNAs; ii) an LNP containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) A frozen formulation containing PBS buffer with approximately 6 mg / ml sodium chloride.

[0073] In one embodiment, the formulation provided herein is a lipid nanoparticle (LNP) comprising i) a payload of or containing one or more mRNAs; ii) LNPs containing lipids including ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5; and b) sucrose at a concentration of about 10% w / v in the pre-dried formulation; and c) PBS buffer containing about 6 mg / ml sodium chloride in the pre-dried formulation.

[0074] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing the formulation is provided herein. a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) PBS buffer containing approximately 6 mg / ml sodium chloride in the preparation; and ii) Sucrose at a concentration of approximately 10% w / v in the formulation This is a method that includes a step that includes [a certain element].

[0075] In one embodiment, a method is provided herein that includes the step of administering a drug formulation, wherein the formulation a) Lipid nanoparticles (LNPs), i) mRNA at a concentration of approximately 0.5 mg / ml; ii) ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of approximately 7.17 mg / ml; iii) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of approximately 0.89 mg / ml; iv) Distearoylphosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg / ml; v) Cholesterol at a concentration of approximately 3.1 mg / ml LNPs including; b) Sucrose at a concentration of approximately 10% w / v in the formulation; c) PBS buffer containing approximately 6 mg / ml sodium chloride Includes; This method involves diluting the formulation to the appropriate dosage form before administration.

[0076] In one embodiment, a method for providing such a formulation as described herein is also described herein. In one embodiment, a method for preparing a formulation is provided herein, a) A step of preparing lipid nanoparticles (LNPs) in the first buffer system, wherein the LNPs i) A payload consisting of or containing one or more mRNA molecules; ii) Lipids including cholesterol, in a relative mass ratio in the range of approximately 8:1:1.5:3 to approximately 9:1:2:3.5: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); distearoylphosphatidylcholine (DSPC); and cholesterol. A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) PBS buffer containing approximately 6 mg / ml sodium chloride in the preparation; and ii) Sucrose at a concentration of approximately 10% w / v in the formulation The method includes a step in which the first buffer system contains sucrose at a concentration of approximately 10% w / v.

[0077] In one embodiment, provided herein is a method for delivering nucleic acids to cells in a subject, comprising administering one of the formulations described above.

[0078] In one embodiment, the method provided herein is a method for inducing an immune response in a subject, comprising administering one of the formulations described above.

[0079] definition In this specification, unless otherwise evident from the context, (i) singular expressions may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “include” and “inclusive” may be understood to include a bulleted component or process, whether it exists alone or with one or more further components or processes; and (iv) the terms “about” and “approximately” may be understood to allow a standard deviation as understood by those skilled in the art; and (v) when a range is given, the end is inclusive.

[0080] Administration: The term “administration” as used herein refers to the administration of the crude product to a subject. Exemplary routes of administration may include bronchial (including bronchial infusion), buccal, enteral, interdermal, intra-arterial, intradermal, gastric, intraspinal, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal infusion), percutaneous, vaginal, and vitreous. In many embodiments, the technology provided relates to an LNP composition (e.g., including the BNT162 construct) administered by intramuscular injection. In some embodiments, the LNP composition is administered in a first dose, followed by one or more doses (e.g., one or more booster doses). In some embodiments, the interval between each dose of the LNP composition, e.g., the first dose and the second dose, includes a period of time, e.g., about one week, two weeks, three weeks, four weeks or more, and is separated by about 24 hours, 48 ​​hours, 72 hours, 96 hours or more. In one embodiment, the interval between administrations is approximately 3 weeks (for example, about 21 days).

[0081] Antibody Agents: As used herein, the term “antibody agent” refers to a drug that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to enable specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may contain one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may contain one or more sequence elements such as humanized, primated, or chimeric, as known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional properties of antibodies in a separate presentation. For example, in the embodiments, the antibody agents used in the present invention include intact IgA, IgG, IgE, or IgM antibodies; two or multiple specific antibodies (e.g., Zybody®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-stranded Fvs; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probody®); small-module immunotherapies. S mall M odular I mmuno P harmaceuticals) ("SMIP TMThe forms include, but are not limited to, single-chain or tandem bispecific antibodies (TandAb®); VHH; Antikalin®; Nanobody® Minibody; BiTE®; Ankyrin repeat protein or DARPIN®; Abimer®; DART; TCR-like antibodies; Adnectin®; Affilin®; Transbody®; Affibody®; TrimerX®; Microproteins; Finomer®, Centilin®; and KALBITOR®. In some embodiments, antibodies may lack covalent modifications (e.g., glycan binding) that they would have if they occurred naturally. In some embodiments, antibodies may have covalent modifications (e.g., glycan binding, payload [e.g., detectable portion, therapeutic portion, catalytic portion, etc.] or other pendant groups [e.g., polyethylene glycol, etc.]). In many embodiments, the antibody agent is or comprises a polypeptide having an amino acid sequence comprising one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, the antibody agent is a polypeptide having an amino acid sequence comprising at least one CDR (e.g., at least one heavy-chain CDR and / or at least one light-chain CDR) substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDR in that their sequence is identical to or they contain 1 to 5 amino acid substitutions. In some embodiments, the included CDRs are substantially identical to the reference CDR in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In one embodiment, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.In one embodiment, the included CDR is substantially identical to the reference CDR in that at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In one embodiment, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In one embodiment, the included CDR is substantially identical to the reference CDR in that at least one amino acid in the included CDR is substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In one embodiment, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In one embodiment, the antibody agent is a polypeptide or comprises a polypeptide having an amino acid sequence that is recognized by those skilled in the art as an immunoglobulin variable domain. In another embodiment, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0082] Antigen: As used herein, the term “antigen” refers to a drug or part that induces an immune response and / or specifically binds to an antibody or T cell receptor (e.g., when presented to an MHC molecule). In some embodiments, the antigen induces a humoral response (e.g., may involve or include the production of antigen-specific antibodies); in some embodiments, the antigen triggers a cellular response (e.g., may involve or include T cells whose receptors specifically interact with the antigen); in some embodiments, the antigen may bind to an antibody and induce or not induce a specific physiological response in an organism. Generally, an antigen can be or include any chemical substance, such as small molecules, nucleic acids, polypeptides, carbohydrates, lipids, polymers (in some embodiments, except biopolymers [e.g., except nucleic acids or amino acid polymers]). In some embodiments, the antigen is or includes a polypeptide or its epitope. In some embodiments, the antigen is a recombinant antigen.

[0083] Related: Two events or objects are “related” to each other, to the extent used herein, if the existence, level, degree, type and / or form of one is related to the other. For example, a particular object (e.g., polypeptide, genetic trait, metabolite, microorganism, etc.) is considered related to a particular disease, disorder or condition if its existence, level and / or form is related to the incidence and / or morbidity of a disease, disorder or condition (e.g., across a related population). In some embodiments, two or more objects are physically “related” if they interact with each other, directly or indirectly, in such a way that they are physically proximal to each other and / or remain so. In some embodiments, two or more objects that are physically related to each other are covalently linked; in some embodiments, two or more objects that are physically related to each other are not covalently linked but are non-covalently related by means such as hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic forces and combinations thereof.

[0084] Combination Therapy: The term “combination therapy” or “combined” administration of drugs as used herein refers to a situation in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents or modalities). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., the entire “dose” of the first regimen is administered before any dose of the second regimen); in some embodiments, such drugs are administered in a duplicate dosing regimen. In some embodiments, “administration” of combination therapy may include the administration of one or more drugs or modalities to a subject receiving other drugs or modalities in combination. For clarity, combination therapy does not require (or even need to be administered at the same time) the individual drugs to be administered together as a single composition, but in some embodiments, two or more drugs or their active parts may be administered together as a combination composition or combination compound (e.g., as part of a single chemical complex or covalent compound).

[0085] Expression: As used herein, “expression” of a nucleic acid sequence means one or more of the following: (1) templated synthesis of complementary nucleic acids (e.g., production of an RNA template from a DNA sequence, e.g., by transcription); (2) processing of an RNA transcript to produce mRNA (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translation of RNA (e.g., mRNA) into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein. Those skilled in the art will recognize that in certain situations, “expression” may include multiple steps of templated synthesis (e.g., reverse transcription of RNA to produce a DNA strand, followed by transcription of such a DNA strand and / or optionally, synthesis of a complementary DNA strand to produce double-stranded DNA).

[0086] Formulation: “Formulation” is a composition prepared and / or provided as described herein. In many embodiments, the term “Formulation” means an LNP composition—that is, comprising RNA (in particular therapeutic RNA such as mRNA) and lipids as described herein.

[0087] Fragment: As used herein, a “fragment” of a substance or object means a structure that includes an individual part of the whole but lacks one or more parts present in the whole. In some embodiments, a fragment consists of such an individual part. In some embodiments, a fragment consists of or includes a characteristic structural element or part present in the whole. In one embodiment, the polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) found throughout the polymer (e.g., in a continuous relationship). In one embodiment, a polymer fragment contains or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more monomer units (e.g., residues) found throughout the polymer. In one embodiment, the entire substance or object may be referred to as the “parent” of the fragment.

[0088] Functional: As used herein, the term “functional” refers to a form or fragment of an object that exhibits certain properties and / or activity. In one embodiment, the properties and / or activity of such a “functional” fragment are comparable to those of the whole.

[0089] Identity: As used herein, the term “identity” refers to the overall relationship between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polymer molecules may be considered “substantially identical” to one another if their sequences are identical by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. As will be understood by those skilled in the art, a variety of algorithms are available that allow for the comparison of sequences to determine the degree of homology, including those that allow for a gap of a specified length in one sequence relative to the other when considering which residues “correspond” to each other in different sequences. The calculation of percentage identity between two nucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In one embodiment, the length of the sequences aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Then, the nucleotides at the corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then these molecules are identical at that position. The percentage identity between two sequences is a function of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that had to be introduced for optimal alignment of the two sequences. A representative algorithm and computer program useful for determining the percentage identity between two nucleotide sequences is, for example, the algorithm by Meyers and Miller (CABIOS, 1989, 4: 11-17), which is incorporated into the ALIGN program (version 2.0), and uses the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4.The percentage identity between two nucleotide sequences can be determined, for example, using the GAP program in the GCG software package, with the NWSgapdna.CMP matrix.

[0090] Nucleic acid: As used herein, the term “nucleic acid” means, in its broadest sense, any compound and / or substance that can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is evident from the context, in some embodiments, “nucleic acid” means an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid” means an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is or contains RNA; in some embodiments, “nucleic acid” is or contains DNA. In some embodiments, a nucleic acid is one or more native nucleic acid residues, or contains or consists of them. In some embodiments, a nucleic acid is one or more nucleic acid analogs, or contains or consists of them. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is one or more “peptide nucleic acids” known in the art, which contain peptide bonds rather than phosphodiester bonds in their backbone and are considered to be within the scope of the present invention. Separately or in addition thereto, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoamidite bonds rather than phosphodiester bonds. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In one embodiment, the nucleic acid is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynylcytidine, C5-propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), or comprises one or more nucleoside analogs. In one embodiment, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of a natural nucleic acid. In one embodiment, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or polypeptide; in one embodiment, such a nucleotide sequence may be codon-optimized for expression in a specific host (e.g., recipient target). In one embodiment, the nucleic acid containing the coding sequence also contains one or more introns. In one embodiment, the nucleic acid containing the coding sequence does not contain introns. In one embodiment, the nucleic acid is prepared by one or more of the following: isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in one embodiment, in vivo; in one embodiment, in vitro), replication in recombinant cells or systems, and chemosynthesis. In one embodiment, the nucleic acid has a residue length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more.

[0091] Specific: When the term “specific” is used herein in reference to an active drug, it will be understood by those skilled in the art that it means the drug distinguishes a potential target object or state. For example, in one embodiment, a drug may be said to bind “specifically” to a target if it preferentially binds to that target in the presence of one or more competing targets. In many embodiments, specific interaction depends on the presence of specific structural properties of the target object (e.g., epitopes, cavities, binding sites). It will be understood that specificity does not have to be absolute. In one embodiment, specificity may be assessed in comparison to the binders of one or more other potential target objects (e.g., competing agents). In one embodiment, specificity may be assessed in comparison to a reference specific binder. In one embodiment, specificity may be assessed in comparison to a reference nonspecific binder. In one embodiment, a drug or object does not detectably bind to another competing target under conditions in which it binds to the target object. In one embodiment, a binder binds to a target object with a high on-rate, low off-rate, increased affinity, decreased dissociation, and / or increased stability compared to another competing target.

[0092] Stable: When the term “stable” is applied herein to a composition, it means that the composition maintains one or more aspects of its physical structure and / or activity over a specified period of time under a specified set of conditions. In some embodiments, the period is at least about one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven months, eight months, nine months, ten months, eleven months, twelve months or more, including about one month, two months, three months, four months, five months, six months, seven months, eight weeks, nine months, ten months, eleven months, twelve months or more; in some embodiments, the specified set of conditions is a temperature above or including a low temperature threshold. In some embodiments, the low temperature threshold is about -80°C, -70°C, -50°C, -30°C, -20°C, 0°C, 2°C, 4°C, 8°C, 15°C, 20°C, 30°C, 40°C or higher. In some embodiments, the composition is considered stable based on the maintenance of colloidal contents containing lipid nanoparticles (LNPs). In some embodiments, a composition is considered stable based on the maintenance of one or more LNP features (e.g., including, but not limited to, its Z-mean and / or polydispersity index (PDI)). In some embodiments, a composition is considered stable based on the maintenance of nucleic acid integrity, degree of nucleic acid encapsulation (e.g., percent), and / or nucleic acid expression (e.g., expression level of the encoding polypeptide, which may be expressed as, for example, a percent of the relevant reference level). In some embodiments, a composition described herein is considered stable if, under a specified set of conditions, over a period of time, the lipid nanoparticles in such a composition exhibit a change of less than approximately 20 nm of the Z-mean compared to the relevant reference level (including changes of less than, for example, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm or less of the Z-mean). In one embodiment, the compositions described herein are considered stable if, under a specified set of conditions, over a period of time, the lipid nanoparticles in such compositions exhibit a change of less than approximately 10 nm in the Z mean compared to the relevant reference level (including changes of less than, for example, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less than 0.5 nm in the Z mean).In one embodiment, the compositions described herein are considered stable if, under a specified set of conditions, over a period of time, at least 50% of the nucleic acid encapsulation (e.g., including at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) is maintained in such compositions compared to the relevant reference level. In one embodiment, the compositions described herein are considered stable if, under a specified set of conditions, over a period of time, at least 50% of the expression level of the encoding polypeptide (e.g., including at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) is maintained compared to the relevant reference level.

[0093] Subject: The terms “subject” or “patient” as used herein refer to any living organism to which the provided composition is administered or may be administered for, for example, experimental, diagnostic, preventive, cosmetic and / or therapeutic purposes. Typical subjects are animals (e.g., mammals such as mice, rats, rabbits, non-human primates and / or humans). In some embodiments, the subject is human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. In some embodiments, the subject exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the subject is at risk of a viral infection or a disease or disorder associated with a viral infection.

[0094] Substantial: As used herein, the term “substantial” refers to a qualitative state indicating a complete or near-complete range or degree of the characteristic or property of the subject. Those skilled in the art of the biological field will understand that it is rare, if not rare, for biological and chemical phenomena to proceed to completion and / or move toward completeness or achieve or avoid absolute results. The term “substantial” is therefore used here to capture the possibility of an inherent lack of completeness in many biological and chemical phenomena.

[0095] Therapeutic Dose: As used herein, the term “therapeutic dose” means the amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition, according to a therapeutic dosing plan. In some embodiments, the therapeutic dose is the amount that reduces the frequency and / or severity of one or more symptoms of the disease, disorder, and / or condition, and / or delays their onset. Those skilled in the art will recognize that the term “therapeutic dose” does not necessarily require the achievement of successful treatment in a particular individual. Rather, the therapeutic dose may be the amount that, when administered to patients requiring such treatment, provides a particular desired pharmacological response in a significant number of subjects. It is understood that certain subjects may, in fact, be “refractory” to the “therapeutic dose.” For example, refractory subjects may have low bioavailability such that clinical efficacy cannot be obtained. In some embodiments, the reference to the therapeutic dose may refer to an amount measured in one or more specific tissues (e.g., tissues affected by the disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will recognize that, in some embodiments, a therapeutically effective dose may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective dose may be formulated and / or administered in multiple doses, for example, as part of a dosing plan.

[0096] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that exhibits substantial structural identity with a reference molecule but is structurally different from the reference molecule, for example, in the presence or absence or level of one or more chemical moieties. In some embodiments, a variant may also be functionally different from the reference molecule. Generally, whether a particular molecule can be considered a suitable “variant” of a reference molecule depends on the degree of structural identity with the reference molecule. As will be recognized by those skilled in the art, every biological or chemical reference molecule has certain characteristic structural elements. In some embodiments, a variant is another molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect.

[0097] To give some examples, polypeptides may have characteristic sequence elements consisting of multiple amino acids that have designated positions linearly or in three-dimensional space relative to each other and / or contribute to specific structural motifs and / or biological functions; nucleic acids may have characteristic sequence elements consisting of multiple nucleotide residues that have designated positions linearly or in three-dimensional space relative to each other. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequences and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., bound to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits overall sequence identity with a reference polypeptide or nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In one embodiment, the reference polypeptide or nucleic acid has one or more biological activities. In one embodiment, the variant polypeptide or nucleic acid shares one or more biological activities of the reference polypeptide or nucleic acid. In one embodiment, the variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In one embodiment, the variant polypeptide or nucleic acid exhibits a reduction of one or more levels of biological activity compared to the reference polypeptide or nucleic acid. In one embodiment, the polypeptide or nucleic acid of interest is considered a "variant" of the reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to the reference except for a few sequence modifications at specific positions.

[0098] In some embodiments, typically fewer than approximately 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains very few substitutions, insertions, or deletions, functional residues (i.e., residues that participate in specific biological activities) compared to the reference (e.g., fewer than approximately 5, 4, 3, 2, or 1). In some embodiments, the variant polypeptide or nucleic acid contains no more than approximately 5, 4, 3, 2, or 1 addition or deletion compared to the reference, and in some embodiments, no addition or deletion. In one embodiment, the variant polypeptide or nucleic acid contains fewer than approximately 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, or 6 additions or deletions compared to the reference polypeptide, generally fewer than approximately 5, 4, 3, or 2 additions or deletions. In one embodiment, the reference polypeptide or nucleic acid is one found in nature. In one embodiment, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0099] In one embodiment, the "variant" of an amino acid sequence (peptide, protein, or polypeptide) may be or include an amino acid insertion variant, an amino acid addition (i.e., terminal addition) variant, an amino acid deletion variant, and / or an amino acid substitution variant.

[0100] In some embodiments, “variant” may include mutants, splice variants, post-translational modification variants, conformations, isoforms, allele variants, species variants, and species homologs, particularly those occurring naturally. In some embodiments, the term “variant” includes, in particular, fragments of amino acid sequences.

[0101] In one embodiment, the amino acid insertion variant differs from the associated reference polypeptide by the insertion of one or more amino acids.

[0102] In one embodiment, the amino acid addition variant may include amino and / or carboxyl terminological fusions (i.e., extensions) of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0103] In one embodiment, an amino acid deletion variant is characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. In one embodiment, the deletion may be one or more N-terminal amino acids, one or more C-terminal amino acids, one or more internal amino acids, or a combination thereof.

[0104] In one embodiment, an amino acid substitution variant is characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. In one embodiment, the substitution is of a residue that is not highly conserved among the related polypeptides, sharing, for example, one or more common motifs (e.g., characteristic sequence elements) and / or functions. In one embodiment, the substitution is a “conservative” substitution in that the original residue and its substituted counterpart share one or more structural or functional attributes or properties (e.g., identity and / or type of charge or absence; hydrophobic or hydrophilic side chain, three-dimensional bulkiness of side chain, linear or branched characteristics of side chain, presence and / or type of heteroatoms in side chain, etc.). For example, in one embodiment, a substitution is conserved if it involves the exchange of residues within families such as acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), non-charged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine), and aromatic amino acids (phenylalanine, tryptophan, tyrosine). In one embodiment, the following group of conserved amino acid substitutions are considered conserved substitutions: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0105] In one embodiment, a variant may refer to a composition (e.g., a buffer) that is identical except for a few components being modified, for example, the presence or absence of a component or a different concentration of a component.

[0106] Wild-type: The terms “wild-type,” “WT,” or “natural” as used herein have the meaning understood in the art of being an object having the structure and / or activity found in nature in a “normal” state or situation (as opposed to mutated, diseased, modified, etc.). Those skilled in the art will recognize that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). In many embodiments, “wild-type” as used herein may refer to a naturally occurring amino acid sequence, including allele variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified. [Brief explanation of the drawing]

[0107] [Figure 1] Figures 1A-1F illustrate an exemplary workflow for the production of a formulation of the present invention. In an exemplary first buffer system, particle-forming lipids suspended in an organic solvent (e.g., ethanol) and nucleic acids (e.g., mRNA) suspended in an aqueous buffer (e.g., citrate buffer) are stirred for a certain period of time (A) until nucleic acid-containing lipid particles (e.g., LNPs) are formed (B). In one embodiment, such nucleic acid-containing lipid particles may be transferred to a second buffer system containing a concentration and / or protective agent (e.g., sucrose, trehalose, etc.) (C). In one embodiment, the lipid particles may then be diluted or dried (e.g., by lyophilization or other drying method) (D), or frozen (E), or dried and then frozen (F) for storage or use. In one embodiment, after drying and / or freezing, the lipid particles may be thawed and / or diluted for storage and / or use.

[0108] [Figure 2] Figures 2A and 2B show an exemplary formulation of the present invention (A) and an exemplary cycle designed for the formulation of the present invention (B).

[0109] [Figure 3] Figures 3A-3B show exemplary colloidal stability data for exemplary sucrose and trehalose formulations at various time points and temperatures.

[0110] [Figure 4] Figures 4A-4B show exemplary % inclusion data for exemplary sucrose and trehalose formulations at various time points and temperatures.

[0111] [Figure 5] Figure 5 shows illustrative graphs of the water content of exemplary sucrose and trehalose formulations.

[0112] [Figure 6] Figures 6A–6C show exemplary % expression data for exemplary sucrose and trehalose formulations at various time points and temperatures.

[0113] [Figure 7] Figures 7A to 7D show exemplary data characterizing the formulation of the present invention.

[0114] [Figure 8] Figures 8A-8B show exemplary colloidal stability data for exemplary sucrose and trehalose formulations at various time points and temperatures. [Modes for carrying out the invention]

[0115] Detailed description of one embodiment The present invention provides technologies relating, in particular, to nucleic acid / lipid nanoparticle (LNP) compositions and RNA / LNP compositions such as therapeutic RNA / LNP compositions.

[0116] Those skilled in the art recognize that one common obstacle encountered with nucleic acid / LNP formulations, and especially RNA / LNP formulations, is the need for low-temperature storage to maintain long-term stability. Various reports describe the need for temperatures as low as -90°C; others describe the need for temperatures lower than -80°C, -70°C, or -60°C. Higher temperatures, around -20°C, are often only tolerable for short periods (e.g., 1, 2, 3, 4 days to several days). Temperatures higher than freezing (e.g., above about 0°C) and / or temperatures achieved by refrigeration (e.g., in the range of about 1°C to about 8°C, or about 2°C to about 8°C, or about 2°C to about 6°C, or about 2°C to about 4°C) are often only tolerable for a few hours to 1-2 days. Room temperature storage and especially long-term room temperature storage (e.g., one week, two weeks, three weeks, four weeks, five weeks, six weeks or more, including at least one to two days and preferably one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months or more) remain targets.

[0117] In one embodiment, the present invention provides nucleic acid / LNP formulations and particularly RNA / LNP formulations that differ from a reference formulation and are modified (e.g., improved) in one or more properties compared to the reference formulation, and which include specific components (e.g., protective agents and / or buffer components) and / or are prepared according to a specific method. For example, in one embodiment, the formulation provided is improved compared to a reference formulation which contains the same lipids and nucleic acids but differs in protective agents and / or buffers and / or certain manufacturing or processing steps.

[0118] In one embodiment, the technology provided achieves the preparation of a dry formulation or a composition suitable for drying (e.g., stable).

[0119] In one embodiment, the provided composition can be efficiently dried using a shorter lyophilization cycle required for a reference formulation that is similarly dried, for example, an identical formulation produced using a buffer containing NaCl at a concentration in the range of about 5 to 10 mg / ml (e.g., about 6 mg / ml).

[0120] In one embodiment, the technology provided achieves the preparation of a cryogenic formulation or a composition suitable for freezing (e.g., stable).

[0121] In one embodiment, the technology provided achieves the preparation of a composition that is stable for storage for at least a specific period of time at a temperature above a low-temperature threshold. In one embodiment, the specific period may be at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more, including about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more. In one embodiment, the low-temperature threshold may be about -80°C, -70°C, -50°C, -30°C, -20°C, 0°C, 2°C, 4°C, 8°C, 15°C, 20°C, 30°C, 40°C or more.

[0122] In one embodiment, the technology provided is suitable for delivering a nucleic acid payload to a subject by administration of LNPs containing a payload encapsulated in the lipids described herein; in one embodiment, the lipids include cationic lipids, neutral lipids, polymer-conjugated lipids, and steroids. In one embodiment, the LNPs for use in the present invention are formed from ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), distearoylphosphatidylcholine (DSPC), and cholesterol, each combined in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5.

[0123] In one embodiment, the nucleic acid payload is or comprises RNA and / or DNA; in one embodiment, the nucleic acid payload polypeptide product may encode (e.g., a functional polypeptide that supplements or replaces, for example, the activity required or desired in a subject, or an immunomodulatory polypeptide that can induce or enhance, for example, a desired immune response or activity in a subject).

[0124] In one embodiment, the provided composition comprises LNP (i.e., nucleic acid / LNP), a protective agent, and a buffer. In one embodiment, the buffer does not contain sodium ions. In one embodiment, the buffer does not contain salts. In one embodiment, the buffer is the HEPES buffer, Tris buffer, or His buffer described herein. In one embodiment, the buffer is a phosphate-buffered saline variant prepared without the use of NaCl. In one embodiment, the buffer is a PBS variant with a lower sodium ion level compared to a reference PBS containing NaCl, KCl, Na2HPO4, and KH2PO4; in one embodiment, such a reference PBS is a “standard” PBS containing (or consisting of) 137 mM NaCl (i.e., 8 g / L NaCl), 2.7 mM KCl (i.e., 0.2 g / L KCl), 10 mM Na2HPO4 (i.e., 1.44 g / L Na2HPO4), and 1.8 mM KH2PO4 (i.e., 0.24 g / L KH2PO4). In one embodiment, the buffer used in the present invention is a PBS variant having lower levels of sodium ions than those found in such a reference standard PBS.

[0125] In one embodiment, the protective agent used in the present invention comprises a disaccharide. In another embodiment, the protective agent used in the present invention is or comprises sucrose and / or trehalose.

[0126] In one embodiment, the protective agent is or contains mannitol. In another embodiment, the protective agent is substantially free of mannitol.

[0127] In one embodiment, the present invention provides a technology for manufacturing, then storing, freezing, and / or drying LNP formulations (i.e., nucleic acid / LNP formulations and particularly RNA / LNP formulations). In one embodiment, the frozen composition is stored. In one embodiment, the dried composition is stored.

[0128] In one embodiment, the dried composition is resuspended and then administered to the subject. In one embodiment, the frozen composition is thawed and then administered to the subject. In one embodiment, the composition may be subjected to one or more freeze-and-thaw cycles, one or more dry-and-resuspend cycles and / or one or more freeze-and-thaw cycles and also one or more dry-and-resuspend cycles.

[0129] In one embodiment, the composition is diluted before administration.

[0130] nucleic acid payload In particular, the present invention provides LNP compositions containing nucleic acid payloads (i.e., nucleic acid-LNP compositions).

[0131] In one embodiment, the nucleic acid payload may include or encode functional nucleic acids such as, for example, antisense oligonucleotides (e.g., those that can promote RNAseH degradation and / or exon skipping), ribozymes, gRNAs, miRNAs and shRNAs, and siRNAs.

[0132] In one embodiment, the nucleic acid payload may encode one or more polypeptides (for example, as described further below).

[0133] In one embodiment, the nucleic acid payload used in the present invention is one or more natural nucleic acid residues or entirely natural nucleic acid residues or comprising them. In another embodiment, the nucleic acid is one or more non-natural nucleic acid residues (i.e., one or more nucleic acid analogs), comprises them, consists of them, or is entirely non-natural nucleic acid residues.

[0134] In one embodiment, the nucleic acid payload used in the present invention includes one or more internucleotide bonds that are not phosphodiester bonds. For example, in one embodiment, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In one embodiment, the nucleic acid includes several phosphodiester bonds and several non-phosphodiester bonds.

[0135] In one embodiment, the nucleic acid is or comprises one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In one embodiment, the nucleic acid is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynylcytidine, C5-propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), or comprises one or more nucleoside analogs.

[0136] In one embodiment, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to natural nucleic acids.

[0137] In one embodiment, the nucleic acid is one or more peptide nucleic acids.

[0138] In one embodiment of the present invention, nucleic acids are modified with the modifications described herein, which result in one or more desirable characteristics, such as enhanced stability, potency, etc.

[0139] RNA payload In one embodiment, the nucleic acid payload for use in the present invention is RNA (e.g., mRNA). In one embodiment, the RNA is produced by templated synthesis. In one embodiment, the RNA is produced by enzymatic synthesis, for example, by in vitro transcription (e.g., from a DN template). In one embodiment, the RNA is produced by chemical synthesis.

[0140] In one embodiment, the RNA is “replicon RNA” or simply “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In one embodiment, the replicon or self-replicating RNA is derived from or contains elements derived from ssRNA viruses, particularly positive-strand ssRNA viruses such as alphaviruses. Alphaviruses are a typical example of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856 for a review of the alphavirus life cycle). The total genome length of many alphaviruses is typically in the range of 11,000–12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (viral particle formation). The genome typically contains two open reading frames (ORFs). The four non-structural proteins (nsP1-nsP4) are typically encoded by a first ORF (Open Reading Frame) that begins near the 5' end of the genome, while the alphaviral structural proteins are encoded by a second ORF, located downstream of the first ORF and extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphavirus, only the nucleic acid sequences encoding non-structural proteins are translated from genomic RNA, while the genetic information encoding structural proteins is translatable from subgenomic transcripts, which are RNA molecules that mimic eukaryotic messenger RNA (mRNA) (Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly like messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or target organisms. In a simple approach, an open reading frame encoding an alphavirus structural protein is replaced with an open reading frame encoding the protein of interest. Alphavirus-based trans replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules, one encoding a viral replicase and the other being replicatable in trans by the replicase (hence the name trans replication system). Trans replication requires the presence of both of these nucleic acid molecules in a host cell. The nucleic acid molecule that can be replicatable in trans by the replicase must contain an alphavirus sequence element that enables recognition and RNA synthesis by the alphavirus replicase.

[0141] In one embodiment, the RNA for use in the present invention may contain one or more modified nucleosides. In one embodiment, the present invention provides RNA containing a modified nucleoside in place of at least one uridine. In one embodiment, the modified nucleoside is a substitute for all uridines in the RNA. In one embodiment, the modified nucleoside replacing at least one uridine includes, but is not limited to, pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine (m5U) or a combination thereof. In one embodiment, the modified nucleoside replacing at least one, for example, all uridines in the RNA may be any one or more of the following: 3-methyl-uridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s) 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxy-uridine (ho 5U), 5 - aminoallyl - uridine, 5 - halo - uridine (e.g., 5 - iodo - uridine or 5 - bromo - uridine), uridine 5 - oxyacetic acid (cmo 5 U), methyl uridine 5 - oxyacetate (mcmo 5 U), 5 - carboxymethyl - uridine (cm 5 U), 1 - carboxymethyl - pseudouridine, 5 - carboxyhydroxymethyl - uridine (chm 5 U), methyl 5 - carboxyhydroxymethyl - uridine (mchm 5 U), 5 - methoxycarbonylmethyl - uridine (mcm 5 U), 5 - methoxycarbonylmethyl - 2 - thio - uridine (mcm 5 s 2 U), 5 - aminomethyl - 2 - thio - uridine (nm 5 s 2 U), 5 - methylaminomethyl - uridine (mnm 5 U), 1 - ethyl - pseudouridine, 5 - methylaminomethyl - 2 - thio - uridine (mnm 5 s 2 U), 5 - methylaminomethyl - 2 - seleno - uridine (mnm 5 se 2 U), 5 - carbamoylmethyl - uridine (ncm 5 U), 5 - carboxymethylaminomethyl - uridine (cmnm 5 U), 5 - carboxymethylaminomethyl - 2 - thio - uridine (cmnm 5 s 2 U), 5 - propynyl - uridine, 1 - propynyl - pseudouridine, 5 - taurinomethyl - uridine (τm 5 U), 1 - taurinomethyl - pseudouridine, 5 - taurinomethyl - 2 - thio - uridine (τm5s2U), 1 - taurinomethyl - 4 - thio - pseudouridine), 5 - methyl - 2 - thio - uridine (m 5 s 2 U), 1 - methyl - 4 - thio - pseudouridine (m 1 s 4 ψ), 4 - thio - 1 - methyl - pseudouridine, 3 - methyl - pseudouridine (m 3ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psuduridine, 4-methoxy-2-thio-psuduridine, N1-methylpsuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudridine(ψm), 2-thio-2'-O-methyl-uridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in the art.

[0142] In one embodiment, the RNA used in the present invention includes a 5' cap. In another embodiment, the RNA of the present invention does not have an uncapped 5'-triphosphate. In another embodiment, it can be modified with an RNA 5' cap analog. The term “5' cap” refers to a structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide attached to mRNA via a 5'-→5'-triphosphate bond. In another embodiment, such guanosine is methylated at position 7. Attaching a 5' cap or 5' cap analog to RNA can be achieved by in vitro transcription in which the 5' cap or 5' cap analog is co-expressed on the RNA strand, or by attaching it to post-transcriptional RNA using a capping enzyme. In another embodiment, the 5' cap of RNA is m2 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3`O G(5')ppp(5')m 2’-O It is sometimes called ApG). In one embodiment, the 5' cap of the RNA of the present invention is an analog anti-reverse cap (ARCA cap (m2 7,3`O G(5')ppp(5')G)) In one embodiment, the 5' cap is beta-S-ARCA(m2 7,2`O G(5')ppSp(5')G) In one embodiment, the 5' cap is beta-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O It's ApG.

[0143] In one embodiment, the RNA for use in the present invention includes a 5'-UTR and / or a 3'-UTR. The terms “untranslated region” or “UTR” may refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule such as an mRNA molecule. The UTR may be located at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, if present, is located at the 5' end, upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap (if present), for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of the protein-coding region, but the term “3'-UTR” preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0144] The terms "poly(A) sequence" or "poly-A tail" as used herein refer to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3'-UTR of the RNAs described herein. Uninterrupted poly(A) sequences are characterized by a continuous sequence of adenylate residues. In fact, uninterrupted poly(A) sequences are typical. The RNAs disclosed herein may have a poly(A) sequence fused to the free 3' end of the RNA by a post-transcriptional template-independent RNA polymerase, or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. A poly(A) sequence of approximately 120 A nucleotides has been shown to have beneficial effects on RNA levels in transfected eukaryotic cells and on protein levels translated from an open reading frame located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0145] In different embodiments, the poly(A) sequence may vary in length. In one embodiment, the poly(A) sequence contains, essentially consists of, or comprises at least 20, at least 30, at least 40, at least 80, or at least 100 A nucleotides. In one embodiment, the poly(A) sequence contains, essentially consists of, or comprises up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides. In one embodiment, the poly(A) sequence contains about 120 A nucleotides. In this context, “essentially consists of” means that the majority of the nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly(A) sequence, are A nucleotides, but the remaining nucleotides may be non-A nucleotides such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consists of" means that all nucleotides in a poly(A) sequence, i.e., 100% of the nucleotides in a poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0146] In one embodiment, the poly(A) sequence is bound during RNA transcription, for example, during the preparation of in vitro transcription RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidylate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as the poly(A) cassette. In one embodiment, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by a random sequence of 4 nucleotides (dA, dC, dG, and dT). Such a random sequence may be 5–50, 10–30, or 10–20 nucleotides long. Such cassettes are disclosed in WO2016 / 005324A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO2016 / 005324A1 may be used in the present invention. A poly(A) cassette, essentially composed of dA nucleotides but interrupted by a random sequence in which the four nucleotides (dA, dC, dG, dT) are evenly distributed, and having a length of, for example, 5 to 50 nucleotides, exhibits beneficial properties in that, at the DNA level, it shows consistent proliferation of plasmid DNA in E. coli, and at the RNA level, it includes support for RNA stability and translation efficiency. As a result, in one embodiment, the poly(A) sequence contained in the RNA molecule described herein is essentially composed of A nucleotides but interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence may have a length of 5 to 50, 10 to 30, or 10 to 20 nucleotides.

[0147] In one embodiment, nucleotides other than A nucleotides do not adjoin the poly(A) sequence at their 3' end; that is, the poly(A) sequence is not masked and no nucleotides other than A follow at its 3' end.

[0148] In one embodiment, the poly(A) sequence may contain at least 20, at least 30, at least 40, at least 80 or at least 100 and up to 500, up to 400, up to 300, up to 200 or up to 150 nucleotides. In one embodiment, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80 or at least 100 and up to 500, up to 400, up to 300, up to 200 or up to 150 nucleotides. In one embodiment, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80 or at least 100 and up to 500, up to 400, up to 300, up to 200 or up to 150 nucleotides. In one embodiment, the poly(A) sequence contains at least 100 nucleotides. In one embodiment, the poly(A) sequence contains about 150 nucleotides. In one embodiment, the poly(A) sequence contains about 120 nucleotides.

[0149] In one embodiment, the nucleic acid for use in the present invention is codon-optimized and / or has an increased guanosine / cytosine (G / C) content compared to the wild-type coding sequence. Embodiments also include those in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence regions of the wild-type coding sequence. In one embodiment, codon optimization and / or increased G / C content do not alter the sequence of the encoded amino acid sequence.

[0150] G / C content In one embodiment of the present invention, the G / C content of the coding region (e.g., RNA) described herein is increased compared to the G / C content of the corresponding WT coding sequence, wherein the encoded amino acid sequence is not modified compared to such corresponding WT sequence. In one embodiment, the increase in G / C content may increase the translation efficiency of the RNA with such increased G / C content. Those skilled in the art will recognize that sequences with increased G / C content have been reported to be more stable than sequences with increased adenosine / uracil (A / U) content.

[0151] Given the fact that several codons encode identical amino acids (so-called genetic coding condensation), the most advantageous codon for stability can be determined (so-called alternative codon usage frequency). Depending on the desired amino acid to be encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to the wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by substituting these codons with other codons that encode the same amino acid but do not contain A and / or U, or have a lower A and / or U nucleotide content.

[0152] In various embodiments, the G / C content of the coding region of the RNA used by the present invention is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more compared to the G / C content of the coding region of wild-type RNA.

[0153] Coded polypeptide As described herein, in one embodiment, the nucleic acid payload (e.g., RNA) encodes a polypeptide.

[0154] In one embodiment, the encoded polypeptide is or comprises an antibody agent, a polypeptide chain, or a functional fragment thereof. In one embodiment, the antibody agent is or comprises a single-chain antibody agent such as scFC, camel antibody, etc.

[0155] In one embodiment, the encoded polypeptide is or includes cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell-surface receptors, ligands, hormones, etc.

[0156] In one example, the encoded polypeptide is an enzyme.

[0157] In one embodiment, the encoded polypeptide is a regulatory polypeptide such as a transcription factor or chaperone.

[0158] In one embodiment, the encoded polypeptide is or includes a polypeptide that replaces or activates an activity that is reduced or missing in the target.

[0159] In one embodiment, the encoded polypeptide is or comprises a polypeptide that induces and / or enhances an immune response in a subject. In one embodiment, the encoded polypeptide is or comprises at least one epitope that is specifically bound by an immunoglobulin agent (e.g., an antibody and / or a T cell receptor).

[0160] In one embodiment, the encoded polypeptide is or contains an antigen (or its epitope). In one embodiment, the antigen may be characteristic of a particular disease, disorder, or condition. For example, the antigen may be or contain an antigen associated with a tumor antigen (e.g., a neoantigen) and / or an infectious agent (e.g., a virus or a microorganism such as bacteria or fungi). In one embodiment, an antigen associated with an infectious agent may be an antigen that is present on the surface of such infectious agent and / or can mediate infection by such agent (e.g., by participating in interactions with receptors on recipient cells).

[0161] In one embodiment, the antigen is or may include an antigen associated with a virus selected from the group consisting of, for example, adenovirus, cytomegalovirus, herpesvirus, human papillomavirus, measles virus, rubella virus, coronavirus, polynuclear respiratory virus, influenza virus, and mumps virus. In one embodiment, the antigen may be or may include a viral antigen associated with a virus selected from Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII viruses based on the Baltimore classification system. In one embodiment, the antigen may be or include a viral antigen associated with a virus selected from the virus families Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Aneroviridae, Pleolipoviridae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronavirusidae, Astroviridae, Bornaviridae, Arteriviridae, and Hepeviridae. In one particular embodiment, the viral antigen may be a coronavirus antigen.

[0162] In certain embodiments, the viral antigen may be an antigen derived from the SARS-CoV-2 protein sequence (for example, it may be or include such a sequence, a fragment thereof, or a variant thereof). In certain embodiments, the present invention provides a polypeptide having an antigen sequence derived from the SARS-CoV-2S protein sequence. In certain embodiments, the polypeptide is or includes an antigen sequence derived from the receptor-binding domain (RBD) of the SARS-CoV-2S protein sequence.

[0163] In certain embodiments, the payloads described herein are associated with or encapsulated within the lipid portion of the LNP. In certain embodiments, the payloads described herein are associated with the lipid portion of the LNP. In certain embodiments, the payloads described herein are encapsulated within the lipid portion of the LNP. In certain embodiments, such association (e.g., encapsulation) with the lipid portion reduces the susceptibility of the payload to degradation (e.g., enzymatic degradation) over a period of time and / or under certain conditions.

[0164] According to certain embodiments, the signal peptide is fused directly or via a linker to an antigen peptide or protein. In certain embodiments, the signal peptide for use in the present invention typically exhibits a length of about 15 to about 30 amino acids and is a sequence located at the N-terminus of an antigen peptide or protein, but is not limited thereto. In certain embodiments, the signal peptide as defined herein enables the antigen peptide or protein encoded by RNA to be transported to a defined cellular compartment, such as the cell surface, endoplasmic reticulum (ER) or endosome-lysosome compartment.

[0165] Signal peptide sequences that can be utilized according to certain embodiments of the present invention may be, for example, the signal peptide sequence of an immunoglobulin, such as the signal peptide sequence of a variable region of an immunoglobulin heavy chain, or may include the same, where the immunoglobulin may be a human immunoglobulin.

[0166] The signal peptide for use in the present invention is used to facilitate the secretion of the encoded antigen peptide or protein. In certain embodiments, the signal peptide as defined herein is fused to the encoded antigen peptide or protein as defined herein. In certain embodiments, the RNA described herein includes at least one coding region encoding an antigen peptide or protein and a signal peptide, where the signal peptide is fused to the N-terminus of the antigen peptide or protein, such as the antigen peptide or protein described herein.

[0167] In certain embodiments, the trimerization domain is fused to the antigen peptide or protein, either directly or via a linker, such as a glycine / serine linker. In certain embodiments, the trimerization domain is fused to the antigen peptide or protein, either directly or via a linker, such as a glycine / serine linker, and it is also fused to the signal peptide described herein.

[0168] In certain embodiments, such trimerization domains are located in, but not limited to, the antigen peptide or protein. The trimerization domains defined herein enable trimerization of antigen peptides or proteins encoded by RNA. Examples of trimerization domains defined herein include, but are not limited to, the foldon, which is the native trimerization domain of T4 fibritin. The C-terminal domain of T4 fibritin (foldon) is essential for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain.

[0169] In certain embodiments, the transmembrane domain is fused to the antigen peptide or protein, either directly or via a linker, such as a glycine / serine linker. Thus, in certain embodiments, the transmembrane domain is fused to the antigen peptide or protein, either directly or via a linker, such as a glycine / serine linker, and it is also fused to the signal peptide and / or trimerization domain defined herein.

[0170] In many embodiments, the transmembrane domain utilized in the present invention is located at, but not limited to, the C-terminus of the antigen peptide or protein. In certain embodiments, such transmembrane domains, if present, are located at, but not limited to, the C-terminus of the trimerization domain. In certain embodiments, the trimerization domain is present between the SARS-CoV-2 S protein, its variants or its fragments, i.e., the antigen peptide or protein and the transmembrane domain.

[0171] In one embodiment, the transmembrane domain used in the present invention may enable the immobilization of RNA-encoded antigen peptides or proteins to the cell membrane.

[0172] coronavirus Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+) ssRNA) viruses. They have the largest genome (26-32 kb) of all known RNA viruses and are phylogenetically divided into four genera (α, β, γ, and δ), with beta-coronaviruses further subdivided into four differentiation series (A, B, C, and D). Coronaviruses are associated with a wide range of bird and mammal species, including humans. Some human coronaviruses generally cause mild respiratory illness, but the severity can be greater in infants, the elderly, and immunocompromised individuals. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), belonging to beta-coronavirus differentiation series C and B, respectively, are highly pathogenic. Both viruses have emerged from pathogen-carrying animals into human populations within the last 15 years, causing epidemics with high case fatality rates. The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes atypical pneumonia (coronavirus disease 2019; COVID-19), began in China in mid-December 2019 and has developed into a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to the beta-coronavirus lineage B. It shares at least 70% sequence similarity with SARS-CoV.

[0173] Generally, coronaviruses possess four structural proteins: the envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins play crucial roles in viral assembly, while the N protein is necessary for viral RNA synthesis. The important glycoprotein S is responsible for viral binding and entry into target cells. The S protein is synthesized as a single-strand inactive precursor and cleaved in the producing cell by a Furin-like host protease into two non-covalently associated subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that recognizes the host-cell receptor. The S2 subunit contains a fusion peptide, two 7-amino acid repeats, and a transmembrane domain, all of which undergo large-scale structural rearrangement and are necessary for mediating viral and host-cell membrane fusion. The S1 and S2 subunits trimerize to form a large pre-fusion spike.

[0174] The SARS-CoV-2 S precursor protein is cleaved by proteolysis into S1(685 aa) and S2(588 aa) subunits. The S1 subunit consists of a receptor-binding domain (RBD), which mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0175] The SARS-CoV-2 coronavirus full-length spike (S) protein consists of 1273 amino acids (see Sequence ID No. 1).

[0176] In one embodiment, the present invention utilizes RNA encoding a peptide or protein containing at least the epitope SARS-CoV-2S protein to induce an immune response to the coronavirus S protein, particularly the SARS-CoV-2S protein, in a subject. In one embodiment, the RNA of the present invention encodes an amino acid sequence containing the SARS-CoV-2S protein, an immunogenic fragment of the SARS-CoV-2S protein, or an immunogenic variant thereof.

[0177] In one embodiment, the full-length spike (S) protein of SEQ ID NO: 1 is modified to stabilize the prototype pre-fusion structure. Stabilization of the pre-fusion structure can be achieved by introducing two consecutive proline substitutions at AS residues 986 and 987 in the full-length spike protein. Specifically, the spike (S) protein stabilization protein variant is obtained in such a way that the amino acid residue at position 986 is replaced with proline, and the amino acid residue at position 987 is also replaced with proline. In one embodiment, the SARS-CoV-2S protein variant contains the amino acid sequence shown in SEQ ID NO: 7.

[0178] In one embodiment, the vaccine antigen described herein comprises, essentially consists of, or consists of the SARS-CoV-2 spike protein (S), its variants, or fragments thereof.

[0179] In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO: 2, 8, or 9. The RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising amino acids 17-1273 of SEQ ID NO: 1 or 7; and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7; or an immunogenic fragment of the amino acid sequence comprising amino acids 17-1273 of SEQ ID NO: 1 or 7; or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7. In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising nucleotides 49-3819 of SEQ ID NO: 2, 8, or 9; and / or (ii) an amino acid sequence comprising amino acids 17-1273 of SEQ ID NO: 1 or 7.

[0180] In one embodiment, the vaccine antigen comprises, essentially consists of, or consists of, the SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the SARS-CoV-2 spike protein (S)), a variant thereof, or a fragment thereof.

[0181] In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO: 2, 8, or 9 The RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising amino acids 17-683 of SEQ ID NO: 1, and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence comprising amino acids 17-683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence comprising amino acids 17-683 of SEQ ID NO: 1. In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising nucleotides 49-2049 of SEQ ID NO: 2, 8, or 9; and / or (ii) an amino acid sequence comprising amino acids 17-683 of SEQ ID NO: 1.

[0182] In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO: 2, 8, or 9 The RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising amino acids 17-685 of SEQ ID NO: 1, and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence comprising amino acids 17-685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence comprising amino acids 17-685 of SEQ ID NO: 1. In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising nucleotides 49-2055 of SEQ ID NO: 2, 8, or 9; and / or (ii) an amino acid sequence comprising amino acids 17-685 of SEQ ID NO: 1.

[0183] In one embodiment, the vaccine antigen includes, essentially consists of, or comprises the receptor-binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike protein (S), its variants, or fragments thereof. The amino acid sequence of amino acids 327-528 of SEQ ID NO: 327, its variants, or fragments thereof are hereafter also referred to as the "RBD" or "RBD domain."

[0184] In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence 979-1584 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence 979-1584 of SEQ ID NO: 2, 8, or 9 The RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising amino acids 327-528 of SEQ ID NO: 1, and / or (ii) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence comprising amino acids 327-528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence comprising amino acids 327-528 of SEQ ID NO: 1. In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence comprising nucleotides 979-1584 of SEQ ID NO: 2, 8, or 9; and / or (ii) an amino acid sequence comprising amino acids 327-528 of SEQ ID NO: 1.

[0185] In one embodiment, the signal peptide is fused directly or via a linker to the SARS-CoV-2S protein, its variants or fragments, i.e., the antigen peptide or protein. Thus, in one embodiment, the signal peptide is fused to the above amino acid sequence derived from the SARS-CoV-2S protein or immunogenic fragments thereof (antigen peptide or protein) contained in the vaccine antigen described herein.

[0186] In some embodiments, the signal peptide for use in the present invention is typically about 15 to about 30 amino acids long and is a sequence located at the N-terminus of an antigen peptide or protein, but is not limited thereto. In some embodiments, the signal peptide defined herein enables the transport of an RNA-encoded antigen peptide or protein to a defined cellular compartment, such as the cell surface, the endoplasmic reticulum (ER), or the endosomal-lysosome compartment. In some embodiments, the signal peptide sequence defined herein includes, but is not limited to, the signal peptide sequence of the SARS-CoV-2S protein, particularly a sequence or functional variant thereof containing amino acids 1-16 or 1-19 of SEQ ID NO: 1.

[0187] A signal peptide sequence that may be used in one embodiment of the present invention may be, for example, a signal peptide sequence of an immunoglobulin, for example, a signal peptide sequence of a variable region of an immunoglobulin heavy chain, where the immunoglobulin may be a human immunoglobulin.

[0188] The signal peptide used in the present invention is used to promote the secretion of an encoded antigen peptide or protein. In one embodiment, the signal peptide as defined herein is fused to the encoded antigen peptide or protein as defined herein. In one embodiment, the RNA described herein comprises an antigen peptide or protein and at least one coding region encoding the signal peptide, wherein the signal peptide is fused to the N-terminus of the antigen peptide or protein, for example, the antigen peptide or protein described herein.

[0189] In certain embodiments, the trimerization domain is fused to a SARS-CoV-2 S protein, a variant thereof or a fragment thereof, i.e., an antigenic peptide or protein, either directly or via a linker, such as a glycine / serine linker. Thus, in certain embodiments, the trimerization domain is fused to the amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the vaccine antigen, which may optionally be fused to a signal peptide described herein.

[0190] In certain embodiments, such a trimerization domain is located in, but not limited to, an antigenic peptide or protein. The trimerization domain defined herein enables the trimerization of an antigenic peptide or protein encoded by RNA. Examples of trimerization domains defined herein include, but are not limited to, the foldon, the native trimerization domain of T4 fibritin. The C-terminal domain of T4 fibritin (foldon) is essential for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain.

[0191] In certain embodiments, the transmembrane domain is fused to a SARS-CoV-2 S protein, a variant thereof or a fragment thereof, i.e., an antigenic peptide or protein, either directly or via a linker, such as a glycine / serine linker. Thus, in certain embodiments, the transmembrane domain is fused to the amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the vaccine antigen, which may optionally be fused to a signal peptide and / or trimerization domain described herein.

[0192] In many embodiments, the transmembrane domain used in the present invention is located at the C-terminus of an antigen peptide or protein, but is not limited thereto. In some embodiments, such a transmembrane domain is located at the C-terminus of a trimerizing domain, if present, but is not limited thereto. In some embodiments, the trimerizing domain is located between the SARS-CoV-2S protein, its variants or fragments, i.e., the antigen peptide or protein, and the transmembrane domain.

[0193] In one embodiment, the transmembrane domain used in the present invention may enable the immobilization of RNA-encoded antigen peptides or proteins to the cell membrane.

[0194] In one embodiment, the transmembrane domain sequence defined herein includes, but is not limited to, the transmembrane domain sequence of the SARS-CoV-2S protein, particularly the sequence comprising amino acid sequence 1207-1254 of SEQ ID NO: 1, or functional variants thereof.

[0195] The trimerizing domains provided herein are used to facilitate the trimerization of an encoded antigen peptide or protein. In one embodiment, the trimerizing domain defined herein is fused to the antigen peptide or protein defined herein. In one embodiment, the RNA described herein comprises the antigen peptide or protein defined herein and at least one coding region encoding the trimerizing domain, which is fused to the C-terminus of the antigen peptide or protein, e.g., the antigen peptide or protein.

[0196] In one embodiment, the vaccine antigen described herein comprises a continuous sequence of SARS-CoV-2 coronavirus spike (S) protein consisting of or essentially consisting of the above amino acid sequence derived from the SARS-CoV-2 S protein or its immunogenic fragment (antigen peptide or protein) contained in the vaccine antigen described herein. In one embodiment, the vaccine antigen described herein comprises a continuous sequence of SARS-CoV-2 coronavirus spike (S) protein not exceeding 220 amino acids, 215 amino acids, 210 amino acids, or 205 amino acids.

[0197] In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) described herein as BNT162b2 (RBP020.1 or RBP020.2). In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) described herein as RBP020.2.

[0198] The various embodiments of nucleoside-modified messenger RNA (modRNA) described herein are as follows: BNT162b2;RBP020.1 (Sequence ID 19; Sequence ID 7) Structure: m27,3'-OGppp(m12'-O)ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoding antigen: SARS-CoV-2 viral spike protein (S1S2 protein) (S1S2 full-length protein, sequence variant) BNT162b2;RBP020.2 (Sequence ID 20; Sequence ID 7) Structure: m27,3'-OGppp(m12'-O)ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoding antigen: SARS-CoV-2 viral spike protein (S1S2 protein) (S1S2 full-length protein, sequence variant)

[0199] Nucleotide sequence of RBP020.1 The nucleotide sequence is shown with its individual sequence elements, indicated in bold. Furthermore, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (*=stop codon). [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0200] Nucleotide sequence of RBP020.2 The nucleotide sequence is shown with its individual sequence elements, indicated in bold. Furthermore, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (*=stop codon). [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0201] Lipid nanoparticles (LNPs) In one embodiment, one or more nucleic acids (e.g., RNA) described herein are formulated and / or administered in the form of LNPs. In one embodiment, the LNPs of the present invention comprise one or more lipids known in the art and / or established herein to produce lipid particles. In one embodiment, the LNPs of the present invention comprise one or more lipids selected from the group consisting of cationic lipids, neutral lipids, polymer-conjugated lipids and combinations thereof. In one embodiment, the LNPs of the present invention comprise a steroid such as cholesterol or a derivative thereof.

[0202] As used herein, “neutral lipids” refers to lipid species that exist in an uncharged or neutral zwitterionic form at a select pH. In some embodiments, the additional lipids include one of the following neutral lipid elements: (1) phospholipids, (2) cholesterol or its derivatives; or (3) a mixture of phospholipids and cholesterol or its derivatives. In some embodiments, the phospholipids may include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoseleoylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether The material comprises PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), and phosphatidylethanolamine, particularly diacylphosphatidylethanolamine, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids having various hydrophobic chains.

[0203] Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives and mixtures thereof.

[0204] The term "cationic lipid" refers to any of several lipid species that have a net positive charge at a selective pH, such as a physiological pH (e.g., a pH of approximately 7.0). Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1 ,2-Diacyloxy-3-dimethylammoniumpropane; 1,2-Dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-Dimiristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-Dimiristoyl-3-trimethylammonium Monium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamide gly Silspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadieneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl ru-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-di Methyl-2,3-bis(cis-9-tetradecenyloxy)-1-propaneaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy) C)-1-propaneaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amonium bromide (DMORIE), Di((Z)-non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)- This includes, but is not limited to, {2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N12-5) and 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (Lipidoid C12-200).

[0205] In one embodiment, the cationic lipid has a chemical structure disclosed in WO2017 / 075531, some of which are shown in Table A below: [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0206] Further examples of cationic lipids are shown in Table B below. [Table 6]

[0207] In one embodiment, the cationic lipid is an ionizable lipid-like substance (lipidoid). An exemplary lipidoid is C12-200, which has the following structure. [ka]

[0208] In one embodiment, the particles described herein include polymer-conjugated lipids such as PEGylated lipids. The term "PEGylated lipid" refers to a molecule containing both a lipid portion and a polyethylene glycol portion. PEGylated lipids are well known in the art.

[0209] In one embodiment, the LNP of the present invention comprises ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). In one embodiment, the LNP of the present invention comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In one embodiment, the present invention provides an LNP comprising distearoylphosphatidylcholine (DSPC). In one embodiment, the LNP of the present invention comprises cholesterol. In one embodiment, the LNP of the present invention comprises lipids including ALC-0315, ALC-0159, DSPC, and cholesterol.

[0210] In one embodiment, the LNP of the present invention comprises about 40 to about 55 mole percent, about 40 to about 50 mole percent, about 41 to about 49 mole percent, about 41 to about 48 mole percent, about 42 to about 48 mole percent, about 43 to about 48 mole percent, about 44 to about 48 mole percent, about 45 to about 48 mole percent, about 46 to about 48 mole percent, about 47 to about 48 mole percent, or about 47.2 to about 47.8 mole percent of ALC-0315. In one embodiment, the LNP comprises about 47.0, about 47.1, about 47.2, about 47.3, about 47.4, about 47.5, about 47.6, about 47.7, about 47.8, about 47.9, or about 48.0 mole percent of ALC-0315.

[0211] In one embodiment, the LNP of the present invention contains ALC-0315 in concentrations of approximately 6 mg / ml to approximately 9 mg / ml, approximately 6 mg / ml to approximately 8 mg / ml, approximately 6 mg / ml to approximately 7 mg / ml, approximately 7 mg / ml to approximately 9 mg / ml, approximately 8 mg / ml to approximately 9 mg / ml, or approximately 7 mg / ml to approximately 8 mg / ml. In one embodiment, the LNP contains ALC-0315 in concentrations of approximately 7 mg / ml to approximately 8 mg / ml. In one embodiment, ALC-0315 is present at a concentration of approximately 7.17 mg / ml.

[0212] In one embodiment, the LNP of the present invention contains about 5 to about 15 mole percent, about 7 to about 13 mole percent, or about 9 to about 11 mole percent of DSPC. In one embodiment, the DSPC is present at a concentration of about 9.5, about 10, or about 10.5 mole percent.

[0213] In one embodiment, the LNP of the present invention contains DSPC in concentrations of approximately 1 mg / ml to approximately 2.5 mg / ml, approximately 1 mg / ml to approximately 2 mg / ml, or approximately 1 mg / ml to approximately 1.5 mg / ml. In another embodiment, the LNP contains DSPC in concentrations of approximately 1.5 mg / ml to approximately 2 mg / ml. In yet another embodiment, ALC-0315 is present at a concentration of approximately 1.56 mg / ml.

[0214] In one embodiment, cholesterol is present at concentrations ranging from about 30 to about 50 mole percent, about 35 to about 45 mole percent, or about 38 to about 43 mole percent. In another embodiment, cholesterol is present at concentrations of about 40, about 41, about 42, about 43, about 44, about 45, or about 46 mole percent.

[0215] In one embodiment, cholesterol is present at concentrations of approximately 2 mg / ml to 4 mg / ml, 2 mg / ml to 3.5 mg / ml, 2 mg / ml to 3 mg / ml, 2 mg / ml to 2.5 mg / ml, 2.5 mg / ml to 4 mg / ml, 3 mg / ml to 4 mg / ml, or 3.5 mg / ml to 4 mg / ml. In another embodiment, cholesterol is present at a concentration of approximately 3 mg / ml to 3.5 mg / ml. In yet another embodiment, cholesterol is present at a concentration of approximately 3.1 mg / ml.

[0216] In one embodiment, ALC-0159 is present in concentrations ranging from about 1 to about 10 mole percent, about 2 to about 8 mole percent, about 4 to about 8 mole percent, about 4 to about 6 mole percent, about 1 to about 5 mole percent, or about 1 to about 3 mole percent.

[0217] In one embodiment, ALC-0159 is present in concentrations ranging from approximately 0.5 mg / ml to approximately 2.5 mg / ml, approximately 1 mg / ml to approximately 2.5 mg / ml, approximately 1.5 mg / ml to approximately 2.5 mg / ml, approximately 2 mg / ml to approximately 2.5 mg / ml, approximately 0.5 mg / ml to approximately 2 mg / ml, approximately 0.5 mg / ml to approximately 1.5 mg / ml, or approximately 0.5 mg / ml to approximately 1 mg / ml. In one embodiment, ALC-0159 is present in concentrations ranging from approximately 0.5 mg / ml to approximately 1 mg / ml. In one embodiment, ALC-0159 is present in concentrations ranging from approximately 0.89 mg / ml.

[0218] In one embodiment, the mole percent is determined based on the total moles of lipids present in the LNP described herein.

[0219] In one embodiment, the present invention provides LNPs containing lipids including ALC-0315, ALC-0159, DSPC, and cholesterol, which exist in mass ratios in the range of about 8:1:1.5:3 to about 9:1:2:3.5.

[0220] In one embodiment, the lipid particles of the present invention (e.g., LNPs) may have an average diameter of at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, or at least 1000 nm. In one embodiment, the lipid particles of the present invention (e.g., LNPs) may have an average diameter of up to 30 nm, up to 40 nm, up to 50 nm, up to 60 nm, up to 70 nm, up to 80 nm, up to 90 nm, up to 100 nm, up to 200 nm, up to 300 nm, up to 400 nm, up to 500 nm, up to 1000 nm, or up to 1200 nm. In one embodiment, the lipid particles (e.g., LNPs) of the present invention may have an average diameter in the range of about 30 nm to about 1000 nm, about 50 nm to about 1000 nm, about 70 nm to about 1000 nm, about 30 nm to about 500 nm, about 30 nm to about 100 nm, or about 30 nm to about 80 nm.

[0221] The nucleic acids described herein may be packaged in lipids (e.g., RNA / LNP) using a wide range of methods, such as film hydration, reverse-phase evaporation, and ethanol injection techniques, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, and mixing the colloid with the nucleic acid to obtain lipid particles (e.g., RNA / LNP).

[0222] In one embodiment, RNA is packaged into lipid particles (e.g., LNPs) using an ethanol injection technique, in which a lipid-containing ethanol solution is rapidly injected into an aqueous solution via a needle. Thus, in one embodiment, nucleic acid-containing lipid particles (e.g., RNA / LNPs) are prepared as follows: an ethanol solution containing lipids, such as cationic lipids and additional lipids (e.g., the lipid compositions described herein), is injected into an aqueous solution containing nucleic acids (e.g., RNA) under stirring or shaking of the combined solution. The nucleic acids in the prepared lipid particles prepared by this method can be further processed, for example, by concentration, or by transferring to one or more different buffer systems.

[0223] In one embodiment, the RNA described herein is packaged into lipid particles (e.g., LNPs) by mixing the RNA with particle-forming lipids (e.g., those described herein) using the LNP formation method described herein. In one embodiment, the RNA-containing LNPs (RNA / LNPs) are prepared in a first buffer system and then replaced with a second buffer system for storage and / or use.

[0224] In one embodiment, the first buffer system includes an aqueous buffer, such as PBS buffer, Tris buffer, HEPES buffer, His buffer, etc. In one embodiment, the first buffer system includes PBS buffer. In one embodiment of the present invention, the first buffer system includes about 5 mg / ml to about 7 mg / ml, about 6 mg / ml to about 7 mg / ml, or about 5 mg / ml to about 6 mg / ml of sodium chloride. In one embodiment, the first buffer system includes about 6 mg / ml of sodium chloride. In one embodiment, the first buffer system is substantially sodium chloride-free. In this context, substantially sodium chloride-free means that sodium chloride is not added, and those skilled in the art will understand that sodium and / or chloride ions may still be present due to other elements in such a formulation. Accordingly, in one embodiment, the PBS buffer of the present invention is a PBS buffer that is substantially sodium chloride-free and contains 0.15 g / L KCl, 1.08 g / L Na2HPO4, and 0.15 g / L KH2PO4. In one embodiment, the PBS of the present invention comprises 6 g / L NaCl, 0.15 g / L KCl, 1.08 g / L Na2HPO4, and 0.15 g / L KH2PO4.

[0225] In one embodiment, the first buffer system includes a protective agent, such as sucrose, trehalose, or a combination thereof. In one embodiment, the protective agent of the first buffer system is sucrose and / or trehalose. In one embodiment, the sucrose is at a concentration of about 10% w / v. In one embodiment, the sucrose is at a concentration of about 5%. In one embodiment, the trehalose is at a concentration of about 10% w / v. In one embodiment, the trehalose is at a concentration of about 5%.

[0226] In one embodiment, the second buffer system of the present invention includes an aqueous buffer, such as PBS buffer, Tris buffer, HEPES buffer, His buffer, etc. In one embodiment, the second buffer system includes PBS. In one embodiment, the PBS of the present invention includes 6 g / L NaCl, 0.15 g / L KCl, 1.08 g / L Na2HPO4, and 0.15 g / L KH2PO4. In one embodiment, the PBS of the present invention is a PBS buffer that is substantially sodium chloride-free (as defined herein) and contains 0.15 g / L KCl, 1.08 g / L Na2HPO4, and 0.15 g / L KH2PO4. In one embodiment, the second buffer system includes Tris buffer. In one embodiment, the second buffer system includes Tris buffer at a concentration of about 10 mM. In one embodiment, Tris buffer is substantially sodium chloride-free. In one embodiment, Tris buffer contains about 6 mg / ml sodium chloride. In one embodiment, the second buffer system includes His buffer. In one embodiment, the second buffer system contains His buffer at a concentration of about 10 mM. In one embodiment, the His buffer is substantially sodium chloride-free. In one embodiment, the His buffer contains about 6 mg / ml sodium chloride. In one embodiment, the second buffer system contains HEPES buffer. In one embodiment, the second buffer system contains HEPES buffer at a concentration of about 10 mM. In one embodiment, the HEPES buffer is substantially sodium chloride-free. In one embodiment, the HEPES buffer contains about 6 mg / ml sodium chloride.

[0227] In one embodiment, RNA-LNP contains approximately 0.4 mg / ml to approximately 0.6 mg / ml, approximately 0.4 mg / ml to approximately 0.5 mg / ml, or approximately 0.5 mg / ml to approximately 0.6 mg / ml mRNA. In another embodiment, RNA-LNP contains approximately 0.5 mg / ml mRNA.

[0228] formulation The present invention provides, in particular, technologies relating to RNA therapeutic formulations and, more specifically, LNP formulations comprising a nucleic acid (e.g., mRNA) payload. Such RNA / LNP formulations, unlike reference formulations, include and / or are manufactured by specific components (e.g., protective agents and / or buffer components) that modify (e.g., improve) one or more properties of the reference formulation. For example, in one embodiment, the provided formulation exhibits improvement over a reference formulation that contains the same lipids and nucleic acids but differs in protective agents and / or buffers and / or certain manufacturing or processing steps.

[0229] In one embodiment, the present invention provides a composition suitable for drying and / or already dried. In one embodiment, the composition described herein is dried by freeze-drying.

[0230] In one embodiment, the composition described herein is dried until it is substantially water-free or substantially water-free. In one embodiment, the composition contains less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, or less than 0.3% w / w of water. In one embodiment, the compositions described herein maintain water content of less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, or less than 0.3% w / w for a certain period of time, for example, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more, including about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more, and above a certain low temperature threshold, for example, about -80°C, -70°C, -50°C, -30°C, -20°C, 0°C, 2°C, 4°C, 8°C, 15°C, 20°C, 30°C, 40°C or above.

[0231] In one embodiment of the present invention, the composition is annealed during drying (e.g., freeze-drying). In another embodiment, the composition is not annealed during drying.

[0232] In one embodiment, a composition is provided that is stable for storage for at least a specific period of time at temperatures above a low-temperature threshold. In one embodiment, the composition provided herein is stable for storage for at least a certain period of time, including about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more, including about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more. In one embodiment, the composition provided herein is stable for storage for at least about 12 weeks. In one embodiment, the composition is stable for storage at temperatures above a low-temperature threshold, which may be about -80°C, -70°C, -50°C, -30°C, -20°C, 0°C, 2°C, 4°C, 8°C, 15°C, 20°C, 30°C, 40°C or higher. In one embodiment, the composition is stable for storage at temperatures of about 0°C, 2°C, 5°C, 8°C, 25°C, 40°C or higher. In one embodiment, the compositions provided herein are stable for storage for a certain period of at least about 12 weeks in a temperature range of about 2°C to about 40°C, 2°C to about 30°C, about 2°C to about 20°C, about 2°C to about 10°C, about 8°C to about 40°C, about 20°C to about 40°C, or about 30°C to about 40°C.

[0233] In some embodiments, the compositions described herein are considered stable based on the maintenance of colloidal contents containing lipid nanoparticles (LNPs). In some embodiments, the compositions described herein are considered stable based on the maintenance of LNP characteristics (including, but not limited to, their Z-mean and / or polydispersity index (PDI)). In some embodiments, the compositions described herein are considered stable based on the maintenance of nucleic acid integrity, the degree of nucleic acid encapsulation (e.g., percent) and / or nucleic acid expression (e.g., the expression level of the encoding polypeptide, which may be expressed as, for example, a percentage of the relevant reference level). In some embodiments, the compositions described herein are considered stable if, under a specified set of conditions over a period of time, the lipid nanoparticles in such compositions exhibit a change of less than approximately 20 nm of the Z-mean (including, for example, a change of less than or equal to 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, or 11 nm of the Z-mean) compared to the relevant reference level. In one embodiment, a composition provided herein is considered stable if, compared to a relevant reference level, the lipid nanoparticles in such composition exhibit a change of less than approximately 10 nm in the Z mean over a period of time under a specified set of conditions (including changes of less than, for example, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm or less in the Z mean). In one embodiment, a composition provided herein is considered stable if, compared to a relevant reference level, the lipid nanoparticles in such composition exhibit a change of less than 0.1 in the polydispersity index (PDI) over a period of time under a specified set of conditions (including changes of less than, for example, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or less in the PDI). In one embodiment, a composition provided herein is considered stable, compared to the relevant reference level, if, under a specified set of conditions, such composition maintains at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) nucleic acid encapsulation over a period of time.In one embodiment, the compositions described herein are considered stable if, under a specified set of conditions, at least 50% (e.g., including at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) of the expression level of the encoding polypeptide is maintained over a period of time, compared to the relevant reference level.

[0234] In one embodiment, the composition described herein (e.g., an LNP composition) is prepared in a first buffer system and then replaced with the second buffer system described herein.

[0235] In one embodiment, the composition described herein (LNP) comprises one or more particle-forming lipids. In one embodiment, the particle-forming lipids include ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), distearoylphosphatidylcholine (DSPC), and cholesterol.

[0236] In one embodiment, the LNP composition comprises ALC-0315, ALC-0159, DSPC, and cholesterol, each present in a relative mass ratio ranging from approximately 8:1:1.5:3 to approximately 9:1:2:3.5.

[0237] In one embodiment, the composition described herein contains ALC-0315, ALC-0159, DSPC, and cholesterol at concentrations of 7.17 mg / ml, 0.89 mg / ml, 1.56 mg / ml, and 3.1 mg / ml, respectively.

[0238] In some embodiments of the present invention, one or more protective agents are utilized. In some embodiments, the protective agents are sucrose, trehalose, or a combination thereof, or comprise the same. In some embodiments, sucrose is present in a composition or method of the present invention at a concentration of about 10% w / v. In some embodiments, trehalose is present in a composition or method of the present invention at a concentration of about 10% w / v. In some embodiments, sucrose is present in a composition or method of the present invention at a concentration of about 5% w / v, and trehalose is present at a concentration of about 5% w / v.

[0239] In one embodiment, a cryoprotectant is added to the composition before the freezing or drying process to a desired concentration (e.g., as described herein).

[0240] In one embodiment, the protective agent is added to the first buffer system in which the LNP is prepared, for example, as described herein. In another embodiment, the protective agent is added to both the first and second buffer systems. In the embodiment in which the protective agent is added to both the first and second buffer systems, different protective agents may be used for each buffer system, or the same protective agent may be used. In the embodiment in which the protective agent is added to both the first and second buffer systems, different concentrations of the protective agent may be used, or the same concentration may be used.

[0241] One embodiment of the present invention utilizes one or more buffer systems. In one embodiment, a first and a second buffer system are used.

[0242] In one embodiment, the preparation and / or use of the provided composition may include a dilution step by adding a buffer system, which may be the same as, for example, the buffer system used previously, such as the buffer system contained in the diluted LNP composition, in one embodiment, and may be different in other embodiments.

[0243] In one embodiment, the buffer used (for example, the buffer used in the buffer system described herein) is substantially sodium chloride-free. Those skilled in the art will understand that, in one embodiment, "substantially free" means that sodium chloride is not added, even if sodium and / or chloride ions may still be present due to other elements in such a formulation.

[0244] In one embodiment, the provided composition comprises LNP (i.e., nucleic acid / LNP), a protective agent, and a buffer. In one embodiment, the buffer is sodium-free. In one embodiment, the buffer is salt-free. In one embodiment, the buffer is HEPES buffer, Tris buffer, or His buffer as described herein. In one embodiment, the buffer is a phosphate-buffered saline variant prepared without the use of NaCl. In one embodiment, the buffer is a PBS variant with a lower sodium ion level compared to a reference PBS containing NaCl, KCl, Na2HPO4, and KH2PO4; in one embodiment, such a reference PBS is a “standard” PBS containing (or consisting of) 137 mM NaCl (i.e., 8 g / L NaCl), 2.7 mM KCl (i.e., 0.2 g / L KCl), 10 mM Na2HPO4 (i.e., 1.44 g / L Na2HPO4), and 1.8 mM KH2PO4 (i.e., 0.24 g / L KH2PO4). In one embodiment, the buffer used in the present invention is a PBS variant having lower levels of sodium ions than those found in such reference standard PBS. In one embodiment, the buffer used in the present invention is approximately 10 mM Tris buffer. In one embodiment, the buffer used in the present invention is approximately 10 mM His buffer. In one embodiment, the buffer used in the present invention is approximately 10 mM HEPES buffer. In one embodiment, the buffer used in the present invention is supplemented with 6 mg / ml sodium chloride.

[0245] In one embodiment, the composition of the present invention is prepared into a drug-dosage form by dilution with a buffer solution.

[0246] use The technologies provided by the present invention described herein relate to and / or are useful for the formulation and / or administration of nucleic acid / LNP (e.g., RNA / LNP) compositions.

[0247] In one embodiment, the technology described herein provides an LNP composition (e.g., an LNP / RNA composition) that is stable for storage for a set period of time, at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 months, 11 months, 12 weeks or more, including about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more. In one embodiment, the technology of the present invention provides an LNP composition that is stable for storage for at least about 12 weeks. In one embodiment, the composition provided is stable for storage at temperatures above a low threshold which may be about -80°C, -70°C, -50°C, -30°C, -20°C, 0°C, 2°C, 4°C, 8°C, 15°C, 20°C, 30°C, 40°C or higher. In one embodiment, the composition provided is stable for storage at temperatures of about 0°C, 2°C, 5°C, 8°C, 25°C, 40°C or higher. In one embodiment, the composition provided herein is stable for storage for a certain period of at least about 12 weeks in a temperature range of about 2°C to about 40°C, 2°C to about 30°C, about 2°C to about 20°C, about 2°C to about 10°C, about 8°C to about 40°C, about 20°C to about 40°C, or about 30°C to about 40°C.

[0248] The compositions provided herein are considered stable based on the maintenance of colloidal contents containing lipid nanoparticles (LNPs). In some embodiments, the compositions provided herein are considered stable based on the maintenance of LNP characteristics (including, but not limited to, their Z-mean and / or polydispersity index (PDI)). In some embodiments, the compositions provided herein are considered stable based on the maintenance of nucleic acid integrity, the degree of nucleic acid encapsulation (e.g., percent) and / or nucleic acid expression (e.g., the expression level of the encoding polypeptide, which may be expressed as, for example, a percentage of the relevant reference level). In some embodiments, the compositions provided herein are considered stable if, over a period of time under a specified set of conditions, the lipid nanoparticles in such compositions exhibit a change of less than approximately 20 nm of the Z-mean (including, for example, a change of less than or equal to 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm of the Z-mean) compared to the relevant reference level. In one embodiment, a composition provided herein is considered stable if, compared to a relevant reference level, the lipid nanoparticles in such composition exhibit a change of less than approximately 10 nm in the Z mean over a period of time under a specified set of conditions (including changes of less than, for example, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm or less in the Z mean). In one embodiment, a composition provided herein is considered stable if, compared to a relevant reference level, the lipid nanoparticles in such composition exhibit a change of less than 0.1 in the polydispersity index (PDI) over a period of time under a specified set of conditions (including changes of less than, for example, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or less in the PDI). In one embodiment, a composition provided herein is considered stable, compared to the relevant reference level, if, under a specified set of conditions, such composition maintains at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) nucleic acid encapsulation over a period of time.In one embodiment, the compositions described herein are considered stable if, under a specified set of conditions, at least 50% (e.g., including at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) of the expression level of the encoding polypeptide is maintained over a period of time, compared to the relevant reference level.

[0249] In one embodiment, the technology provided herein utilizes an antigen that is or may include an antigen associated with a virus selected from the group consisting of, for example, adenovirus, cytomegalovirus, herpesvirus, human papillomavirus, measles virus, rubella virus, coronavirus, polynuclear respiratory virus, influenza virus, and mumps virus. In one embodiment, the antigen may be or may include a viral antigen associated with a virus selected from Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII viruses based on the Baltimore classification system. In one embodiment, the technology described herein provides a subject with immunity from a virus selected from the virus families Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Aneroviridae, Pleolipoviridae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronavirusidae, Astroviridae, Bornaviridae, Arteriviridae, or Hepeviridae. In one embodiment, the technology described herein provides a subject with immunity to viral infection. In one embodiment, the technology described herein provides a subject with immunity to coronavirus, coronavirus infection, or coronavirus-related disease or disorder. Therefore, the present invention provides compositions and methods for the treatment or prevention of coronavirus-related infection, disease, or disorder.

[0250] In one embodiment, the technology described herein provides an LNP composition to be administered to a subject having an infection, disease, or disorder associated with coronavirus. In one embodiment, the technology described herein provides an LNP composition for administration to a subject at risk of developing an infection, disease, or disorder associated with coronavirus. For example, the technology described herein provides an LNP composition that can be administered to a subject at risk of coming into contact with coronavirus. In one embodiment, the technology described herein provides an LNP composition to be administered to a subject living in, traveling to, or expected to travel to a geographic area where coronavirus is prevalent. In one embodiment, the technology described herein provides an LNP composition to be administered to a subject living in, traveling to, or expected to travel to a geographic area where contact with or ingestion of others is expected. In one embodiment, the technology described herein provides an LNP composition to be administered to a subject known to be exposed to coronavirus through occupation or other contact. In one embodiment, the coronavirus is SARS-CoV-2.

[0251] In one embodiment, the technology described herein provides a composition that can be administered prophylactically (i.e., for the prevention of disease or disability) or therapeutically (i.e., for the treatment of disease or disability) to subjects who have or are at risk of developing (or are susceptible to) a disease or disability. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration is performed before the manifestation of obvious clinical symptoms of the disease so that the disease or disability is prevented (e.g., by reducing the burden of disease mortality or morbidity) or, separately, by delaying its progression.

[0252] Administration Provided herein are compositions (e.g., pharmaceutical compositions) and methods for delivering a payload (e.g., mRNA) to target cells requiring such a payload. In one embodiment, the compositions provided are administered for prophylactic purposes against viral infection and / or for therapeutic purposes to treat viral infection. In one embodiment, the technology of the present invention provides compositions that can be used as therapeutic or prophylactic agents for the treatment of coronaviruses, e.g., SARS-CoV-2.

[0253] The pharmaceutical compositions of the present invention may be administered for prophylactic purposes to subjects who have not been diagnosed with a particular disease, disorder, or condition and / or who do not exhibit one or more specific symptoms or characteristics. In one embodiment, the pharmaceutical compositions provided herein are administered to the cells or tissues of the subject in an immunopreventive dose. The pharmaceutical compositions provided herein may be administered together with other therapeutic or prophylactic compounds.

[0254] In one embodiment, the pharmaceutical composition is administered therapeutically to a subject who has been diagnosed with a particular disease, disorder, or condition and / or who exhibits one or more specific symptoms or characteristics. In one embodiment, the pharmaceutical composition provided herein is administered to the cells or tissues of the subject in a therapeutically effective amount. Such a pharmaceutical composition provided herein may be administered together with other therapeutic or prophylactic compounds.

[0255] The precise amount of pharmaceutical composition (e.g., RNA / LNP composition) required for preventive and / or therapeutic purposes will vary from subject to subject, depending on numerous considerations, including the species, age, and general condition of the subject, the severity of the disease, the method of administration, and the mode of activity. However, it is understood that the use of the composition provided may be determined by the treating physician within the bounds of reasonable medical judgment. Therefore, a specific therapeutic and / or prophylactic effective dose for a particular patient will depend on a variety of factors, including the disorder being treated and its severity, the activity or potency of the specific composition used, the patient's age, weight, general health, sex, and dietary habits, the time of administration, the route of administration, and the rate of excretion of the specific compound used, the duration of treatment; and similar factors known in the medical field and drugs used in combination with or simultaneously with the specific compound used.

[0256] In some embodiments, the provided pharmaceutical composition is administered to a subject who is receiving, will receive, or will receive other treatments. In some embodiments, the other treatments are administered, for example, simultaneously or in alternating regimens, with other treatments that address one or more symptoms or characteristics of the disease, disorder, or condition treated by the provided treatment. Separately or in addition thereto, in some embodiments, the other treatments address one or more symptoms or characteristics of different diseases. For example, in various embodiments, it may be desirable to administer multiple prophylactic treatments (e.g., prophylactic vaccines) substantially simultaneously.

[0257] The pharmaceutical compositions described herein may contain one or more adjuvants or may be administered in combination with one or more adjuvants (i.e., may be administered to subjects who are receiving, receiving, or planning to receive them). The adjuvants utilized in this invention may relate to any compound that prolongs, enhances, or accelerates the immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (e.g., alum), bacterial products (e.g., pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. The cytokines utilized in this invention may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a, or combinations thereof. Further known adjuvants that may be used in the present invention include aluminum hydroxide, Freund's adjuvant, or oils, such as Montanide® ISA51. Other suitable adjuvants for use in the present invention include lipopeptides such as Pam3Cys.

[0258] The pharmaceutical compositions described herein may be provided, for example, as frozen concentrates for injectable solutions at a concentration of about 0.50 mg / mL. In one embodiment, for formulation of an injectable solution, the formulation is thawed and diluted and / or rehydrated and diluted with an isotonic sodium chloride solution (e.g., 0.9% NaCl, saline) by, for example, a one-step dilution method. The concentration of the final injectable solution will vary depending on each dose level administered.

[0259] In one embodiment, the amount of RNA described herein may be administered per dose in amounts of 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, for example, about 1 μg, about 3 μg, about 10 μg, about 30 μg, about 50 μg, or about 100 μg. In one embodiment, the disclosed composition described herein is administered in a single dose. In one embodiment, the composition described herein is administered in a priming dose, followed by one or more booster doses. In one embodiment, a booster dose or a first booster dose may be administered 7 to 28 days or 14 to 24 days after administration of the priming dose.

[0260] In one embodiment, the amount of RNA listed herein may be administered per dose in amounts of 60 μg or less, 50 μg or less, 40 μg or less, 30 μg or less, 20 μg or less, 10 μg or less, 5 μg or less, 2.5 μg or less, or 1 μg or less.

[0261] In one embodiment, the amount of RNA described herein can be administered per dose as at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 20 μg, at least 30 μg, or at least 40 μg.

[0262] In one embodiment, the amounts of RNA listed herein, such as 0.25 μg to 60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg, may be administered per dose.

[0263] In one embodiment, approximately 30 μg of the RNA described herein is administered per dose. In another embodiment, such a dose is administered at least twice. For example, the second dose may be administered approximately 21 days after the first dose.

[0264] In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) described herein as BNT162b2 (RBP020.1 or RBP020.2). In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) described herein as RBP020.2.

[0265] In one embodiment, the immunogenic composition or vaccine of the present invention may be administered as a single dose or boosted by multiple doses.

[0266] Sequence List Sequence ID 1 Met Phe Val Phe Leu Val Leu Pro Leu Val Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270

[0267] Sequence No. 2 auguuugugu uucuugugcu gcugccucuu gugucuucuc agugugugaa uuugacaaca 60 agaacacagc ugccaccagc uuauacaaau ucuuuuacca gaggagugua uuauccugau 120 aaaguguuua gaucuucugu gcugcacagc acacaggacc uguuucugcc auuuuuuagc 180 aaugugacau gguuucaugc aauucaugug ucuggaacaa auggaacaaa aagauuugau 240 aauccugugc ugccuuuuaa ugauggagug uauuuugcuu caacagaaaa gucaaauauu 300 auuagaggau ggauuuuugg aacaacacug gauucuaaaa cacagucucu gcugauugug 360 aauaaugcaa caaauguggu gauuaaagug ugugaauuuc aguuuguaa ugauccuuuuu 420 cugggagugu auuaucacaa aaauaauaaaa ucuuggaugg aaucugaau uagaguguau 480 uccucugcaa auaauuguac auuugaauau gugucucagc cuuuucgau ggaucuggaa 540 ggaaaacagg gcaauuuuaa aaaucugaga gauuugugu uuaaaauau ugauggauau 600 uuuaaaauuu auucuaaaca cacaccaauu aauuuaguga gagaucugcc ucaggguauu 660 ucugcucugg aaccucuggu ggaucugcca auuggcauua auuuacaag auuucagaca 720 cugcuggcuc ugcacagauc uuaucugaca ccuggagauu cucuucugg auggacagcc 780 ggagcugcag cuuauuaugu gggcuaucug cagccaagaa cauuucugcu gaauauaau 840 gaaauggaa caauuacaga ugcuguggau uggcucugg auccuguc ugaaacaaaa 900 ugacauuaa aaucuuuuac aguggaaaaa ggcauuuauc agacaucuua uuuuagagug 960 cagccaacag aaucuauugu gagauuucca aauuuacaa aucugugucc auuuggagaa 1020 guguuuaaug caacaagauu ugcauucugug uaugcaugga auagaaaaag aauuuucuaau 1080 uguguggcug auuauucugu gcuguauaau agugcuucuu uuuccacauu uaaauguuau 1140 ggagugucuc caacaaaauu aaaugauuua uguuuuaacaa auguguaugc ugauucuuuu 1200 gugauucagag gugaugaagu gagacagauu gccccccggac agacaggaaa aauugcugau 1260 uacaauuaca aacugccuga ugauuuuaca ggauguguga uugcuuggaa uucuaauaau 1320 uuagauucua aagugggagg aaauuacaau uaucuguaca gacuguuuag aaaaucaaau 1380 cugaaaccuu uugaaagaga uauuucaaca gaauuuuauc aggcuggauc aacaccuugu 1440 aauggagugg aaaguuuaa uuguuauuuu ccaauuacaga gcuauggauu ucagccaacc 1500 aauggugugg gauaucagcc auauagagug gugugcugu cuuuugaacu gguccaugca 1560 ccugcaacag uguguggacc uaaaaaaucu acaaauuuag ugaaaaauaa augugugaau 1620 uuuaauuuua auggauuaac agaacagga gugcugacag aaucuaauaa aaaauuucug 1680 1740 acauuagaaa uucuggaau uacaccuugu ucuuuugggg gugugucugu gauacaccu 1800 Ggaacaaua Caucaauca Guggcugug Cuguacagg Augugaauug Ouacagaagug 1860 ccaguggcaa shakeshaga ccacauggca gaguguauuc shakeshaga 1920 aoogooooc agahagagagc aoooooooooooooooooooooo 1980 gaaugugaua uuccaauugg agcaggcauu ugugcaucuu aucagacaca vakaaauucc 2040 ccaaggagag caagaucugu ggcaucucag ucuauuauug cauacaccau gucucuggga 2100 gcagaaaauu cuguggcaua ucuaauaau ucuauugcua uuccaaaa uuuuaccauu 2160 ucugugacaa cagaaaaaaa caucugugga uuguaccaug 2220 uacauuuugug gagauucuac agauuguucu auucugcugc ugcaguaugg aucuuuuuugu 2280 accagcuga auagagcuuu aacaggaauu gcuguggaac aggauaaaa uacacaggaa 2340 guguuugcuc aggugaaca gauuuacaaa acaccaa uuaagauuu uggaggauuu 2400 aauuuuagcc agaucugcc ugauccuucu aaaccuucua aaaugauuuu uauugagau 2460 cugcuguuua auaaagugac acuggcagau gcaggauuua uuaaacagua uggagauugc 2520 cugggugaua uugcugcaag aguaucugauu ugugcucaga aauuuaaugg acugacagug 2580 2640 acaauuacaa gcggauggac auuuggagcu ggagcugcuc ugcagauucc uuuugcaaug 2700 cagauggcuu acagauuuaa uggaauugga gugacacaga auguguaua ugaaaaucag 2760 aaacugauug caaaucaguu uaauucugca auuggcaaaa uucaggauuc ucugucuucu 2820 acagcuucug cucugggaaa acugcaggau guggugaauc agaaugcaca ggcacugaau 2880 acucugguga aacagcuguc uagcaauuuu ggggcaauuu cuucugugcu gaaugauauu 2940 cugucuagac uggauaaagu ggaagcugaa gugcagauug auagacugau cacaggaa 3000 3060 ucugcuaauc uggcugcuac aaaaaugucu gaaugugugc ugggacaguc aaaaaagagug 3120 gauuuuugug gaaagguaa ucaucugaug ucauuuuccac aguucugcucc acauggagug 3180 guguuuuuac augugacaua ugugccagca caggaaaaga auuuuaccac agcaccagca 3240 auuugucaug auggaaaagc acauuuucca agagaaggag uguuuguguc uaauggaaca 3300 cauugguuug ugacacagag aaauuuuuau gaaccucaga uuauuacaac agauaauaca 3360 uuugugucag gaaauuguga uguggugauu ggaauuguga auaauacagu guaugaucca 3420 cugcagccag aacuggauuc uuuuaaagaa gaacuggaua aauauuuuaa aaaucacaca 3480 ucuccugaug uggauuuagg agauauuucu ggaaucaaug caucuguggu gaauauucag 3540 aaagaaauug auagacugaa ugaaguggcc aaaaaucuga augaaucucu gauugaucug 3600 caggaacuug gaaaauauga acaguacauu aaauggccuu gguacauuug gcuuggauuu 3660 auugcaggau uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu 3720 uguucuuguu uaaaaggaug uuguucuugu ggaagcuguu guaaauuuga ugaagaugau 3780 ucugaaccug uguuaaaagg agugaaauug cauuacaca 3819

[0268] SEQ ID NO: 7 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Contains Thr Glu and Tyr Gln Only Gly Serves Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270

[0269] SEQ ID NO:8 auguuugugu uucuugugcu gcugccucuu gugucuucuc agugugugaa uuugacaaca 60 agaacacagc ugccaccagc uuauacaaau ucuuuuacca gaggagugua uuauccugau 120 aaaguguuua gaucuucugu gcugcacagc acacaggacc uguuucugcc auuuuuuagc 180 aaugugacau gguuucaugc aauucaugug ucuggaacaa auggaacaaa aagauuugau 240 aauccugugc ugccuuuuaa ugauggagug uauuuugcuu caacagaaaa gucaaauauu 300 auuagaggau ggauuuuugg aacaacacug gauucuaaaa cacagucucu gcugauugug 360 aauaaugcaa caaauguggu gauuaaagug ugugaauuuc aguuuuguaa ugauccuuuu 420 cugggagugu auuaucacaa aaauaauaaa ucuuggaugg aaucugaauu uagaguguau 480 uccucugcaa auaauuguac auuugaauau gugucucagc cuuuucugau ggaucuggaa 540 ggaaaacagg gcaauuuuaa aaaucugaga gaauuugugu uuaaaaauau ugauggauau 600 uuuaaaauuu auucuaaaca cacaccaauu aauuuaguga gagaucugcc ucaggguauu 660 ucugcucugg aaccucuggu ggaucugcca auuggcauua auuuacaag auuucagaca 720 cugcuggcuc ugcacagauc uuaucugaca ccuggagauu cucuucugg auggacagcc 780 ggagcugcag cuuauuaugu gggcuaucug cagccaagaa cauuucugcu gaauauaau 840 gaaauggaa caauuacaga ugcuguggau uggcucugg auccuguc ugaaacaaaa 900 ugacauuaa aaucuuuuac aguggaaaaa ggcauuuauc agacaucuua uuuuagagug 960 cagccaacag aaucuauugu gagauuucca aauuuacaa aucugugucc auuuggagaa 1020 guguuuaaug caacaagauu ugcauucugug uaugcaugga auagaaaaag aauuuucuaau 1080 uguguggcug auuauucugu gcuguauaau agugcuucuu uuuccacauu uaaauguuau 1140 ggagugucuc caacaaaauu aaaugauuua uguuuuaacaa auguguaugc ugauucuuuu 1200 gugauucagag gugaugaagu gagacagauu gccccccggac agacaggaaa aauugcugau 1260 uacaauuaca aacugccuga ugauuuuaca ggauguguga uugcuuggaa uucuaauaau 1320 uuagauucua aagugggagg aauaaaaaaau uaucuguaca gacuguuag aaaaaaaaau 1380 cugaaccuu uugaagaga uauuucaca gaauuuuauc aggcuggauc aacaccuugu 1440 aooggagogg nooooooooooooooooooooooooooooooooooooooooooooooum Me auugggugg gaaucagcc auauagagug guggugcugu cuuugaacu gcugcaugca 1560 ccugcacag uguguggacc uaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ugugugaau 1620 uuuaauuua auggauuaac aggacagga gugcugacag aucuaaaa aaaauuucg 1680 ccuuuucagc aguuuggcag agauuugca gauaccacag augcagugag agauccucag 1740 acauuagaaa uucuggaau uacaccuugu ucuuuugggg gugugucugu gauacaccu 1800 Ggaacaaua Caucaauca Guggcugug Cuguacagg Augugaauug Ouacagaagug 1860 ccaguggcaa shakeshaga ccacauggca gaguguauuc shakeshaga 1920 aoogooooc agahagagagc aoooooooooooooooooooooo 1980 gaaugugaua uuccaauugg agcaggcauu ugugcaucuu aucagacaca vakaaauucc 2040 ccaaggagag caagaucugu ggcaucucag ucuauuauug cauacaccau gucucuggga 2100 gcagaaaauu cuguggcaua uucuaauaau ucuauugcua uuccaacaaa uuuuaccauu 2160 ucugugacaa cagaaauuuu accugucu augacaaaaa caucugugga uuguaccaug 2220 uacauuugug gagauucuac agaauguucu aaucugcugc ugcaguaugg aucuuuuugu 2280 acacagcuga auagagcuuu aacaggaau gcuguggaac aggauaaaaa uacagaggaa 2340 guguugcuc aggugaaaca gauuuacaaa acaccaccaa uuaaagauuu uggaggauuuu 2400 aauuuuaagcc agauucugcc ugauccuucu aaaccuucua aaagaucuu uauugaagau 2460 cugcuguuua auaaagugac acuggcagau gcaggauuua uuaaacagua uggagauugc 2520 cugggugaua uugcugcaag aguaucugauu ugugcucaga aauuuaaugg acugacagug 2580 2640 acaauuacaa gcggauggac auuuggagcu ggagcugcuc ugcagauucc uuuugcaaug 2700 cagauggcuu acagauuuaa uggaauugga gugacacaga auguguaua ugaaaaucag 2760 aaacugauug caaaucaguu uaauucugca auuggcaaaa uucaggauuc ucugucuucu 2820 acagcuucug cucugggaaa acugcaggau guggugaauc agaaugcaca ggcacugaau 2880 acucugguga aacagcuguc uagcaauuuu ggggcaauuu cuucugugcu gaaugauauu 2940 cugucuagac uggaucccucc ugaagcugaa gugcagauug auagacugau cacaggaa 3000 3060 ucugcuaauc uggcugcuac aaaaaugucu gaaugugugc ugggacaguc aaaaaagagug 3120 gauuuuugug gaaagguaa ucaucugaug ucauuuuccac aguucugcucc acauggagug 3180 3240 auuugucaug auggaaaagc acauuuucca agagaaggag uguuuugugc uaauggaaca 3300 cauugguuug ugacacagag aaauuuuuau gaaccucaga uuauuacaac aguaauaca 3360 uuugugucag gaauuguga ugguguguuu ggaauuguga auaauacagu guaugaucca 3420 cugcagccag aacuggauuc uuuuaaagaa gaacuggaua aauuuuuaa aaaucacaca 3480 ucuccugaug uggauuuuagg agauauuucu ggaaucaaug caucuguggu gaauauucag 3540 aaagaaauug auagacugaa ugaaguggcc aaaaaucuga augaaucucu gauugaucug 3600 caggaacuug gaaaaauga acaguacauu aaauggccuu gguacauuug gcuuggauuu 3660 auugcaggau uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu 3720 uguucuuguu uaaaaggaug uuguucuugu ggaagcuguu guaaaauuga ugaagaugau 3780 ucugaaccug uguuaaaagg agugaaauug cauuacaca 3819

[0270] sequence number 9 auguucgugu uccuggugcu ggucccucug guguccagcc agugugugaa ccugaccacc 60 agaacacagc ugccuccagc cuacaccaac agcuuuacca gaggcgugua cuaccccgac 120 aagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcc uuucuucagc 180 aacgugaccu gguuccagc cauccacgug uccggcacca auggcaccaa gagauucgac 240 aaccccgugc ugcccuucaa cgacggggug uacuuugcca gcaccgagaa guccaacauc 300 aucagaggcu ggaucuucgg caccacacug gacagcaaga cccagagccu gcugaucgug 360 aacaacgcca ccaacguggu caucaaagug ugcgaguucc aguucugcaa cgacccccuuc 420 cugggcgucu acuaccacaa gaacaacaag agcuggaugg aaagcgaguu ccggguguac 480 540 ggcaagcagg caacuucaa gaaccugcgc gaguucgugu uuaagaacau cgacggcuac 600 uucaagaucu acagcaagca caccccuauc aaccucgugc gggauucugcc ucagggcuuc 660 ucugcucugg aaccccuggu ggaucugccc aucggcauca acaucacccg guuucagaca 720 780 ggugccgccg cuuacuaugu gggcuaccug cagccuaagaa ccuuccugcu gaaguacaac 840 gagaacggca ccaucaccga cgccguggau uggcucugg auccugag cgagacaaag 900 ugcacccuga aguccuucac cguggaaaag ggcaucuacc agaccagcaa cuuccgggug 960 cagcccaccg aauccaucgu gcgguucccc aauacacca aucugugccc cuucggcgag 1020 guguucaaug ccaccagauu cgccucugug uacgccugga accggaagcg gaucagcaau 1080 ugcguggccg acuacuccgu gcuguacaac uccgccagcu ucagcaccuu caagugcuac 1140 ggcguguccc cuaccaagcu gaacgaccug ugcuucacaa acguguacgc cgacagcuuc 1200 gugauccggg gagaugaagu gcggcagauu gccccuggac agacaggcaa gaucgccgac 1260 uacaacuaca agcugcccga cgacuucacc ggcuguguga uugccuggaa cagcaacaac 1320 cuggacucca aagucggcgg caacuacaau uaccuguacc ggcuguuccg gaaguccaau 1380 cugaagcccu ucgagcggga caucuccacc gagaucuauc aggccggcag caccccuugu 1440 aacggcgugg aaggcuucaa cugcuacuuc ccacugcagu ccuacggcuu ucagcccaca 1500 aauggcgugg gcuaucagcc cuacagagug guggugcuga gcuucgaacu gcugcaugcc 1560 ccugccacag ugugcggccc uaagaaaagc accaaucucg ugaagaacaa augcgugaac 1620 uucaacuuca acggccugac cggcaccggc gugcugacag agagcaacaa gaaguuccug 1680 ccauuccagc aguuuggccg ggauaucgcc gauaccacag acgccguuag agauccccag 1740 acacuggaaa uccuggacau caccccuugc agcuucggcg gagugucugu gaucaccccu 1800 ggcaccaaca ccagcaauca gguggcagug cuguaccagg acgugaacug uaccgaagug 1860 cccguggcca uucacgccga ucagcugaca ccuacauggc ggguguaacuc caccggcagc 1920 aauguguuuc agaccagagc cggcugucug aucggagccg agcacgugaa caauagcuac 1980 gagugcgaca uccccaucgg cgcuggaauc ugcgccagcu accagacaca gacaaacagc 2040 ccucggagag ccagaagcgu ggccagccag agcaucuug ccuacacaau gucucuggcgc 2100 gccgagaaca gcguggccua cuccaacaac ucuaucgcua uccccaccaa cuucaccauc 2160 agcgugacca cagagauccu gccugugucc augaccaaga ccagcgugga cugcaccaug 2220 uacaucugcg gcgauuccac cgagugcucc aaccugcugc ugcaguacgg cagcuucugc 2280 acccagcuga auagagcccu gacagggauc gccguggaac agcaagaa cacccaagag 2340 guguucgccc aagugaagca gaucuacaag acccccuccua ucaaggacuu cggcggcuuc 2400 aauuucagcc agauucugcc cgauccuagc aagcccagca agcggagcuu caucgaggac 2460 cugcuguuca acaaagugac acuggccgac gccggcuuca ucaagcagua uggcgauugu 2520 cugggcgaca uugccgccag gguacugauu ugcgcccaga aguuuaaacgg acugacagug 2580 2640 acaaucacaa gcggcuggac auuuggagca ggcgccgcuc ugcagauccc cuuugcuaug 2700 cagauggccu accgguucaa cggcaucgga gugacccaga augugcugua cgagaaccag 2760 2820 acagcaagcg cccugggaaa gcugcaggac guggucaacc agaaugccca ggcacugaac 2880 acccugguca agcagcuguc cuccaauc ggcgccauca gcucugugcu gaacgauauc 2940 3000 cugcagagcc uccagacaua cgugacccag cagcugauca gagccgccga gauuagagcc 3060 ucugccaauc uggccgccac caagaugucu gagugugugc ugggccagag caagagagug 3120 gacuuuugcg gcaagggcua ccaccuugaug agcuucccuc agucugcccc ucacggcgug 3180 guguuucugc acgugacaua ugugcccgcu caagagaaga auuucaccac cgcuccagcc 3240 aucugccacg acggcaagc ccacuuuccu agaaaggcg uguucguguc caacggcacc 3300 cauugguucg ugacacagcg gaacuucuac gagccccaga ucaucaccac cgacaacacc 3360 uucgugucug caacugcga cgucgugauc ggcauuguga acaauaccgu guacgacccu 3420 cugcagcccg agcuggacag cuucaaagag gaacugca aguacuuuaa gaacccacaca 3480 agccccgacg uggacggg cgauauacagc ggaaucaaug ccagcgucgu gaacauccag 3540 aaagagaucg accggcugaa cgagguggcc aagaaucuga acgagagcccu gaucgaccug 3600 caagaacugg ggaaguacga gcaguacauc aaguggcccu gguacaucug gcugggcuuu 3660 3720 ugaagcugcc ugaagggcug uuguagcugu ggcagcugcu caaguucga cgaggacgau 3780 ucugagcccg ugcugaaggg cgugaaacug cacuacaca 3819

[0271] sequence number 19 agaauaaacu aguauucuuc uggucccccac agacucagag agaacccgcc accauguuug 60 uguuucugu gcugcugccu cugugucuu cucagugugu gaauuugaca acaagacac 120 agcugccacc agcuuauaca auuucuuuua ccagaggagu guauauccu gauaaagugu 180 uuagaucuuc ugugcugcac agcacacagg accuguuucu gccauuuuuu agcaaguga 240 caugguuca ugcaauucau gugucuggaa caauggaac aaaagauuu gauaauccug 300 ugcugccuuu uaauugaugga guguauuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuagag 360 gauggauuu uuggaaaca neighbors aaacacaguc ucugcugau gugauaaug 420 siaaaaugu ggugauaaaaaaaaugu oocagouuuuuuuuuuuuuccuuuuuucugggag 480 uguauuaaaaaaaaaaaaacuugga uggaucuga auuuagagog uauuccucug 540 caaaaoug uacauuuuuuuuuuucuc gauggaucug gagaaaac 600 agggcaauuu uaaaaaaaacug aggaauugg uuuuaaaaa uaougaugga uauuuaaaaa 660 uuuauucuaa acacacca auuaauuuag ugagaucu gccucaggga uuuucugcuc 720 uggaaccucu gguggaucg ccaauuggca uuaauauuac aagauuucag acacugcugg 780 840 cagcuuauua ugugggcuau cugcagccaa gaacauuucu gcugaaau aaugaaaaug 900 gaacaauuac agaugcugug gauugugcuc uggauuccucu gucugaaaca aaauguacau 960 uaaaaucuuu uacaguggaa aaaggcauuu aucagacauc uaauuuuaaga gugcagccaa 1020 cagaaucuau ugugaguuuu ccaauauua caaaucugug uccauuugga gaaguguuua 1080 augcaacaag auuugcaucu guguaugcau ggaauagaaa aagaauuucu aauugugg 1140 cugauuauuc ugugcuguau aauagugcuu cuuuuuccac auuuaaaugu uauggagugu 1200 cuccaacaaa auuaaaugau uuauguuuua caaugugua ugcugauucu uuugugauca 1260 gaggugauga agugagacag auugccccg gacagacagg aaaaauugcu gauuacaauu 1320 acaaacugcc ugaugauuuu acaggaugug ugauugcuug gaauucuaau aauuuagauu 1380 cuaaaguggg aggaauuac aauuaucugu acagacuguu uagaaaauca aaucugaaac 1440 cuuuugaaag agauauuuca acagaaauuu aucaggcugg aucaacaccu uguaauggag 1500 uuggaggau uaauuguuau uuuccauac agagcuaugg auuucagcca accauggg 1560 ugggauauca gccauauaga guggugc ugucuuuuga acugcugcau gcaccugcau 1620 cagugoogg accuaaaaa ucuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaw 1680 uaaaoggau aaaaggaaa ggagugcuga cagaaucuaa uaaaaaauuu cugccuuuuc 1740 agcaguuugg cagagauauu gcagauacca cagaugcagu gagagauccu cagacauuag 1800 aaauucugga uauuacaccu uguucuuuug gggguguguc ugugauuaca ccuggaacaa 1860 auacaucuaa ucagguggcu gugcuguauc aggauguga uguacaga gugccagugg 1920 caaucaugc agaucagcug acacaucaucaucaucaucaucaugue 1980 oucagacaag aggagoug cugauuggag cagaauugu gaauaauucu oauugaougug 2040 auauuccaau uggagcaggc auuugugcau cuaucagac acagacaau ucccaagga 2100 2160 2160 auucuguggc auauucuauau auucuauug auuuccac aauuuuacc auucuguga 2220 caacagaaau uuuaccugug ucuaugacaa aaacaucugu ggauuguacc auguacauu 2280 guggagauuc uacagaaugu ucuaaucugc ugcugcagua uggaucuuuu uguacacagc 2340 ugaauagagc uuuaacagga auugcugugg aacaggauaa aaaauacacag gaaguguuuug 2400 cucaggugaa acagauuac aaacaccac caauuaaaga uuuuggagga uuuaauuuua 2460 gccagauucu gccugauccu ucuaaaccuu cuaaaaagauc uuuuuugaa gaucugcugu 2520 uuauaaagu gacacuggca gaugcaggau uuauuaaaca guauggagau ugccugggug 2580 auugcugc aagagaucug auuugugcuc agaaauuuaa uggacugaca gugcugccuc 2640 cugcugac agaugaaaug auugcucagu acacaucugc uuuacuggcu ggaacaauua 2700 caagcggaug gacauuugga gcuggagcug cucugcagau uccuuuugca augcagaugg 2760 cuuacagauu uaauggaauu ggagugacac agaauguguu auugaaaau cagaaacuga 2820 uugcaaauca guuuauaucu gcaauuggca aaauucagga uucucugucu ucuacagcuu 2880 cugcucugg aaaacugcag gaugugguga aucagaaugc acaggcacug aauacucugg 2940 ugaacagcu gucuagcaau uuuggggcau uucucugu gcugaauugau auucugucua 3000 gacuggaacc uccugaagcu gaguagacu gaucaquaga agaquaga 3060 cucugcagac uuaugugaca cagcagcuga uuagagcugc ugaaauuaga gcucugcua 3120 aucuggcugc uacaaaaug ucugaaugug ugcugggaca whaaaaga guggauuuuu 3180 guggaaagg auaucaucug augucuuuuc cacagucugc uccacaugga gugguuuuu 3240 uacaugugac auaugugcca gcacaggaaa agauuuuac cacagcacca gcaauuuguc 3300 augauggaaa acacauuuu ccaagagaag gagugouugu accauugga acacauuggu 3360 uugugacaca gagaaauuuu uuugaaccuc agauuuuac acagauaau acauuugugu 3420 caggaaoug ugauggug auoggauug ugauaauac aguguaugau ccacugcagc 3480 cagaaacugga oocuuuuaaaaaaaaaaaaaaaaaaaaaaacucug 3540 auguggauu aggagaauu ucuggauca auugcaucugu ggugaauau cagaaagaaa 3600 uugauagacu gaugaagug gccaaaaauc ugaugaauuc ucugauugau neighborhood 3660 uuggaaaaua ugacaguac auuaaauggc cugguacau uuggcuugga uuuuuugcag 3720 gauuaauugc auugugaug gugacaua uguuauguug uuugacauca uguucuu 3780 Gouaaaagg auguaoocu uguggaagcu gouaaaoo ugaugagau gaucugaac 3840 cuguguaaaa aggagugaa uugcauaca caugaugacu cgagcuggua cugcaugcac 3900 gcaugcuag cugccccuuu cccguccugg guacccgag cucccccga cucgggucc 3960 squirrel squirrel squirrel squirrel squirrel squirrel squirrel squirrel 4020 caagcacgca gcaugcagc ucaaacgcu uagccuagcc acacccccac gggaacagc 4080 agugauuaac cuuagcaau aaacgaaagu uuaacuaacc uauacuaacc ccagggougg 4140 ucauuucgu gccagccaca cccuggagcu agcaaaaaaaaaaaaaaaaaaaaaaaaa 4200 aaagcaauug acuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 4260 aaaaaaaaaaaaaaaaaaa 4283

[0272] 20 years old agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguucg 60 uguuccuggu gcugcugccu cuggugucca gccagugugu gaaccugacc accagaacac 120 180 ucagauccag cgugcugcac ucuacccagg accuguuccu gccuucuuc agcaacguga 240 ccugguucca cgccauccac guguccggca ccaauggcac caagagauuc gacaaccccg 300 ugcugcccuu caacgacggg guguacuuug ccagcaccga gaaguccaac aucaucagag 360 cguggaucuu cggcaccaca cuggacagca agacccagag ccugcugauc gugaacaacg 420 ccaccaacgu ggucaucaaa gugugcgagu uccaguucug caacgacccc uuccugggcg 480 ucuacuacca caagaacaac aagagcugga uggaaagcga guuccgggug uacagcagcg 540 ccaacaacug caccuucgag uacguguccc agccuuuccu gauggaccug gaaggcaagc 600 agggcaacuu caagaaccug cgcgaguucg uguuuaaagaa caucgacggc uacuucaaga 660 ucuacagcaa gcacacccccu aucaaccucg ugcgggacu gccucagggc uucucugcuc 720 uggaaccccu gguggaucug cccaucggca ucaacaucac ccgguuucag acacugcugg 780 840 900 gcacaucac cgacgccgug gauugugcuc uggauccu gagcgagaca aagugcaccc 960 ugaaguccuu caccguggaa aagggcaucu accagaccag caacuuccgg gugcagccca 1020 ccgaauccau cgugcgguuc cccaauaca ccaaucugug ccccuucggc gagguguuca 1080 1140 ccgacuacuc cgugcuguac aacuccgcca gcuucagcac cuucaagugc uacggcgugu 1200 ccccuaccaa gcugaacgac cugugcuuca caaacgugua cgccgacagc uucgugaucc 1260 ggggagauga agugcggcag auugccccug gacagacagg caagaucgcc gacuacaacu 1320 acaagcugcc cgacgacuuc accggcugug ugauugccug gaacagcaac aaccuggacu 1380 ccaaagucgg cggcaacuac aauuaccugu accggcuguu ccggaagucc aaucugaagc 1440 1500 uggaaggcuu caacugcuac uucccacugc aguccuacgg cuuucagccc acaaauggcg 1560 ugggcuauca gcccuacaga ggguggugc ugagcuucga acugcugcau gccccugcca 1620 cagugugcgg cccuaagaaa agcaccaauc ucgugaagaa caaaugcgug aacuucaacu 1680 ucaacggccu gaccggcacc ggcgugcuga cagagagcaa caagaaguuc cugccauucc 1740 agcaguuugg ccgggauauc gccgauacca cagacgccgu uagagauccc cagacacugg 1800 aaauccugga caucacccccu ugcagcuucg gcggaguguc uggugaucacc ccuggcacca 1860 acaccagcaa ucagguggca gugcuguacc aggacgugaa cuguaccgaa gugccccgugg 1920 ccauucacgc cgaucagcug acaccuacau ggcgggugua cuccaccggc agcaugugu 1980 uucagaccag agccggugu cugaucggag ccgagcacgu gaacaauagc uacgagugcg 2040 acauccccau cggcgcugga aucugcgcca gcuaccagac agacaaac agcccucgga 2100 gagccagaag cguggccagc cagagcauca uugccuacac aaugucucug ggcgccgaga 2160 acagcguggc cuacuccaac aacucuaucg cuauccccac caacuucacc aucagcguga 2220 ccacagagau ccugccugug uccaugacca agaccagcgu ggacugcacc auguacaucu 2280 gcggcgauuc caccgagugc uccaaccugc ugcugcagua cggcagcuuc ugcacccagc 2340 ugaauagagc ccugacaggg aucgccgugg aacaggacaa gaacacccaa gagguguucg 2400 cccaagugaa gcagaucuac aagaccccuc cuaucaagga cuucggcggc uucaauuuca 2460 gccagauucu gcccgauccu agcaagccca gcaagcggag cuucaucgag gaccugcugu 2520 ucaacaaagu gacacuggcc gacgccggcu ucaucaagca guauggcgau ugucugggcg 2580 acauugccgc cagggaucug auuugcgccc agaaguuuaa cggacugaca gugcugccuc 2640 cucugcugac cgaugagaug aucgcccagu acacaucugc ccugcuggcc ggcacaauca 2700 caagcggcug gacauuugga gcaggcgccg cucugcagau ccccuuugcu augcagaugg 2760 ccuaccgguu caacggcauc ggagugaccc agaaugugcu guacgagaac cagaagcuga 2820 ucgccaacca guucaacagc gccaucggca agauccagga cagccugagc agcacagcaa 2880 gcgcccuggg aaagcugcag gacgugguca accagaaugc ccaggcacug aacacccugg 2940 ucaagcagcu guccuccac ucggcgcca ucagcugu gcugaacgau auccugagca 3000 gacuggacccc uccugaggcc gagguggga gaucacaggc gacuggc 3060 gccuccagac auacgugacc cagcagcuga ucagagccgc cgagauuaga gccucugcca 3120 aucuggccgc caccaagaug ucugagugug ugcugggcca gaggagaga guggacuuuu 3180 gcggcaaggg cuccaccug augagcuucc cucagucugc cccucacggc gugguguuuc 3240 ugcacgugac auauugugccc gcucaagaga agauuucac caccgcucca gccaucugcc 3300 acgacggcaa agcccacuuu ccuagagaag gcguguucgu guccaacggc acccauuggu 3360 ucgugacaca gcggaacuuc uacgagcccc agaucaucac caccgacac accuucgugu 3420 cuggcacug cgacgucgug aucggcauug ugacaauac cguguacgac ccucugcagc 3480 ccgagcugga cagcuucaaa gaggaacugg acaaguacuu uagaaccac acaagccccg 3540 acguggaccu gggcgaauuc agcggaauc augccaggcgu cgugaacuc cagaagaga 3600 ucgaccggcu gaacgaggug gccagauc ugaacgagag ccugaucgac cugacgac 3660 ugggaagua cgagcaguac aucaaguggc ccugguacau cuggcugggc uuuauucgccg 3720 gacugauugc caucgugaug gacauca ugcugugug caugaccagc ugcuguagcu 3780 gccugaaggg cuguguagc ugugghagcu gcughau cgacgaggac gaucugagc 3840 ccgugcugaa gggcgugaaa cugcacuca caugaugacu cgagcuggua cugcaugcac 3900 gcaugcuag cugccccuuu cccguccugg guacccgag cucccccga cucgggucc 3960 squirrel squirrel squirrel squirrel squirrel squirrel squirrel squirrel 4020 caagcacgca gcaugcagc ucaaacgcu uagccuagcc acacccccac gggaacagc 4080 agugauuaac cuuagcaau aaacgaaagu uuaacuaacc uauacuaacc ccagggougg 4140 ucauuucgu gccagccaca cccuggagcu agcaaaaaaaaaaaaaaaaaaaaaaaaa 4200 aaagcaauug acuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 4260 aaaaaaaaaaaaaaaaaaa 4283

Overnight

[0273] The following embodiments are provided for illustrative purposes only and do not limit the scope of the invention in any way. Those skilled in the art will recognize that certain designs and selection criteria described herein may vary in accordance with common practice in the art.

[0274] Example 1: Exemplary composition and characterization This example describes the development and / or characterization of a certain RNA / LNP composition according to the present invention.

[0275] The RNA payload used in this embodiment was a modified RNA payload containing 4283 nucleotide residues. The RNA payload used in this embodiment encoded a viral antigen, specifically the SARS-CoV-2S protein. Specifically, the RNA payload used in this embodiment was the BNT162b2 construct represented by RBP020.2(v9) described herein.

[0276] This embodiment protected certain protective agents (particularly the disaccharide protective agents sucrose and trehalose) and specific buffers (e.g., non-phosphate buffers such as Tris and histidine buffers and / or NaCl-free buffers).

[0277] While not wishing to be bound by any particular theory, isotonicity was considered desirable; one evaluated composition contained a protective agent at a 10% w / v concentration because it produced a nearly isotonic solution.

[0278] The buffer concentration was selected to be sufficient to maintain the pH of the composition.

[0279] The compositions evaluated did not contain mannitol.

[0280] In this example, the composition was not frozen before drying (for example, it was maintained at a temperature in the range of approximately 2°C to approximately 8°C). In this example, drying was carried out by freeze-drying (specifically, lyophilization).

[0281] The electrical conductivity of the composition was measured, and low-temperature DSC experiments were performed before the start of freeze-drying. LNP size and polydispersity were determined using dynamic light scattering after maintaining a temperature of 2°C to 8°C and before freeze-drying.

[0282] Table 1 below shows some of the compositions that were evaluated; as can be seen, (i) the type and concentration of protective agents were varied; different manufacturing procedures were evaluated during manufacturing (particularly for sucrose-containing formulations, the RNA stock solution was diluted in sucrose-citric acid buffer instead of citrate-only buffer); (ii) buffers lacking NaCl were evaluated; and (iii) non-phosphate buffers (e.g., Tris, His, HEPES) were evaluated. [Table 7] *Two 15 mL aliquots each must be prepared and stored separately for lyophilization cycles with and without annealing during freezing.

[0283] The freeze-drying procedure used included a cooling and heating gradient during the freezing process, carried out at 0.5°C / min. The formulation was frozen at a temperature below the Tg of the relevant formulation. While we do not wish to be bound by any particular theory, we selected an annealing temperature of -10°C to maximize Ostwald aging during isothermal holding (and thereby increase ice crystal size) and reduce cake resistance while maintaining the product at a temperature below the melting point of the formulation. The gradient rate to secondary drying was 0.2°C / min.

Claims

1. a) Lipid nanoparticles (LNPs), i) A payload containing one or more mRNA molecules; ii) Cationic lipids, neutral lipids, polymer-conjugated lipids, and lipids containing steroids LNP including; and b) Sucrose at a concentration of approximately 10% w / v; Trehalose at a concentration of approximately 10% w / v; or Sucrose at a concentration of approximately 5% w / v and trehalose at a concentration of approximately 5% w / v; c) Tris buffer, i) substantially free of sodium chloride; or ii) containing 6 mg / ml sodium chloride, with a concentration of approximately 10 mM. A formulation containing the above.

2. The formulation according to claim 1, wherein component b) is sucrose at a concentration of about 10% w / v; and component c) is a Tris buffer that is substantially sodium chloride-free and at a concentration of about 10 mM.

3. a) Lipid nanoparticles (LNPs), i) A payload containing one or more mRNA molecules; ii) Cationic lipids, neutral lipids, polymer-conjugated lipids, and lipids containing steroids LNP including; and b) Sucrose at a concentration of approximately 10% w / v; Trehalose at a concentration of approximately 10% w / v; or Sucrose at a concentration of approximately 5% w / v and trehalose at a concentration of approximately 5% w / v; c) A His buffer solution that is substantially free of sodium chloride and has a concentration of about 10 mM; or HEPES buffer solution that is virtually sodium chloride-free and has a concentration of approximately 10 mM. A formulation containing the above.

4. a) Lipid nanoparticles (LNPs), i) A payload containing one or more mRNA molecules; ii) Cationic lipids, neutral lipids, polymer-conjugated lipids, and lipids containing steroids LNP including; and b) Sucrose at a concentration of approximately 10% w / v; c) PBS buffer, i) substantially free of sodium chloride; or ii) containing 6 mg / ml sodium chloride. A formulation containing the above.

5. The formulation according to claim 4, wherein component c) is a PBS buffer containing 6 mg / ml sodium chloride.

6. A formulation according to any one of claims 1 to 5, which is a frozen formulation.

7. A formulation according to any one of claims 1 to 5, which is diluted before administration for therapeutic use, wherein component a) LNP is i) 1 or more mRNA molecules at a concentration of approximately 0.5 mg / ml; ii) neutral lipids; iii) Polymer-conjugated lipids; iv) Cationic lipids; and v) Steroids A formulation containing the above.

8. A formulation according to any one of claims 1 to 7 for delivering nucleic acids to cells in a target.

9. A formulation according to any one of claims 1 to 8 for inducing an immune response in a target.

10. The formulation according to claim 9, wherein the immune response is against a viral antigen or its epitope encoded by mRNA.

11. The formulation according to claim 10, wherein the viral antigen is a coronavirus antigen.

12. The formulation according to claim 11, wherein the coronavirus is the SARS-CoV-2 virus.

13. The formulation according to claim 11, wherein the antigen contains an S protein.

14. A formulation according to any one of claims 1 to 13, wherein one or more mRNAs encode one or more polypeptides.

15. The formulation according to claim 14, wherein one or more polypeptides comprise an epitope that induces an immune response to an antigen in a target.

16. A formulation according to any one of claims 1 to 15, wherein one or more mRNAs comprise RNA encoding an epitope that induces an immune response to an antigen in a target.

17. A formulation according to any one of claims 1 to 16, wherein one or more RNA molecules are self-amplifying RNA molecules.

18. A formulation according to any one of claims 1 to 17, wherein one or more RNA molecules are modified RNA molecules or unmodified RNA molecules.

19. The formulation according to claim 18, wherein one or more RNA molecules are unmodified uridine RNA molecules.

20. The formulation according to claim 18, wherein one or more RNA molecules include a nucleoside-modified RNA molecule.

21. (i) At least one polypeptide is derived from the SARS-CoV-2S protein of SEQ ID NO: 1 or 7; (ii) At least one polypeptide has at least 85% sequence identity with the SARS-CoV-2S protein of SEQ ID NO: 1 or 7; (iii) At least one polypeptide comprises one or more full-length SARS-CoV-2S proteins of SEQ ID NO: 1 or 7; (iv) At least one polypeptide has at least 85% sequence identity with the receptor-binding domain (RBD) of the SARS-CoV-2S protein of SEQ ID NO: 1 or 7; or (v) At least one polypeptide comprising one or more polypeptides includes the receptor-binding domain (RBD) of the SARS-CoV-2S protein of SEQ ID NO: 1 or 7, The formulation according to claim 14.

22. A formulation according to any one of claims 1 to 21, wherein one or more mRNAs are associated with or encapsulated in an LNP.

23. (i) 1 μg to 100 μg of mRNA; or (ii) mRNA less than 60 μg A formulation according to any one of claims 1 to 22, comprising the above.

24. A formulation according to any one of claims 1 to 23, wherein the cationic lipid is ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); the neutral lipid is distearoylphosphatidylcholine (DSPC); the polymer conjugate lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and the steroid is cholesterol.

25. ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)(ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); A formulation according to claim 24, comprising distearoylphosphatidylcholine (DSPC); and a lipid containing cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.

5.

26. A method for preparing the formulation according to claim 1, a) A step of preparing lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs are i) A payload containing one or more mRNA molecules; ii) Cationic lipids, neutral lipids, polymer-conjugated lipids, and lipids containing steroids A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) Tris buffer, i) substantially free of sodium chloride; or ii) Tris buffer containing 6 mg / ml sodium chloride at a concentration of about 10 mM; and ii) Sucrose at a concentration of approximately 10% w / v; Trehalose at a concentration of approximately 10% w / v; or Sucrose at a concentration of approximately 5% w / v and trehalose at a concentration of approximately 5% w / v The process includes Methods that include...

27. In step b), the second buffer system i) Tris buffer solution that is substantially free of sodium chloride and has a concentration of approximately 10 mM; ii) Sucrose at a concentration of approximately 10% w / v The method of claim 26, including the method of claim 26.

28. A method for preparing the formulation according to claim 3, a) A step of preparing lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs are i) A payload containing one or more mRNA molecules; ii) Cationic lipids, neutral lipids, polymer-conjugated lipids, and lipids containing steroids A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) A His buffer solution that is substantially free of sodium chloride and has a concentration of about 10 mM; or A HEPES buffer that is substantially free of sodium chloride and has a concentration of approximately 10 mM; and ii) Sucrose at a concentration of approximately 10% w / v; Trehalose at a concentration of approximately 10% w / v; or Sucrose at a concentration of approximately 5% w / v and trehalose at a concentration of approximately 5% w / v The process includes Methods that include...

29. A method for preparing the formulation according to claim 4, a) A step of preparing lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs are i) A payload containing one or more mRNA molecules; ii) Cationic lipids, neutral lipids, polymer-conjugated lipids, and lipids containing steroids A process that includes; and b) A step of replacing the first buffer system with a second buffer system, wherein the second buffer system is i) PBS buffer, i) substantially free of sodium chloride; or ii) containing 6 mg / ml sodium chloride; and ii) Sucrose at a concentration of approximately 10% w / v The process includes Methods that include...

30. In step b), the second buffer system i) PBS buffer containing 6 mg / ml sodium chloride The method of claim 29, including the method of claim 29.

31. The method of claim 30, wherein the first buffer system contains sucrose at a concentration of approximately 10% w / v.

32. The method according to any one of claims 26 to 31, wherein the cationic lipid is ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); the neutral lipid is distearoylphosphatidylcholine (DSPC); the polymer conjugate lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and the steroid is cholesterol.

33. ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)(ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); The method of claim 32, comprising a lipid containing distearoylphosphatidylcholine (DSPC) and cholesterol in a relative mass ratio in the range of about 8:1:1.5:3 to about 9:1:2:3.5.

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