HK40102562A - Compositions and methods for stabilization of lipid nanoparticle mrna vaccines - Google Patents

Description

Compositions and methods for stabilizing lipid nanoparticle mRNA vaccines

Background Technology

Messenger RNA (mRNA) has proven to be an exciting therapeutic approach and has recently gained significant attention, particularly in the field of vaccines.

Summary of the Invention

This invention provides techniques related to formulations for RNA (e.g., mRNA) therapy, particularly relating to lipid nanoparticle (LNP) formulations comprising an RNA (e.g., mRNA) payload. In other aspects, the invention provides therapeutic RNA formulations (i.e., LNP formulations) suitable for (e.g., stable) storage and/or handling at temperatures above about -80°C or even above about -70°C, about -60°C, about -50°C, about -40°C, about -30°C, or about -20°C. In some embodiments, the provided formulations may be suitable for storage and/or handling at temperatures above freezing (e.g., above about 0°C), at standard refrigeration temperatures (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), and/or at room temperature (e.g., in the range of about 15°C to about 25°C, or about 20°C to about 23°C).

In some embodiments, the present invention provides formulations suitable for drying and/or drying (e.g., lyophilized formulations).

This invention specifically provides certain formulations for use as (and/or preparation) vaccines.

In some embodiments, the present invention provides formulations (particularly LNP formulations) of RNA encoding viral antigens (e.g., SARS-CoV2 antigens, such as the S-protein or its epitopes). Specific exemplary formulations include: RNA constructs that are BNT162 constructs (e.g., described in Walsh, E. et al. RNA-Based COVID-19 Vaccine BNT162b2 Selected for a Pivotal Efficacy Study.medRxiv (2020)), such as BNT162b2; and PCT application No. PCT/EP2020/081981 entitled “Coronavirus Vaccine”, filed November 12, 2020, the contents of which are incorporated herein by reference for the purposes described herein.

In one aspect, the formulation provided herein comprises: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; (b) sucrose at a concentration of about 10% w/v in the formulation; and (c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; (b) sucrose at a concentration of about 10% w/v in the formulation; and (c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; (b) sucrose, having a concentration of about 10% w/v in the formulation prior to drying; and (c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

This document also describes methods for providing these formulations. In some embodiments, this document provides a method for preparing a formulation, which includes the following steps:

a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v.

In some implementations, a method includes the following steps:

A dosage form for a drug delivery formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, at a concentration of approximately 10% w/v;

c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation;

The preparation is diluted to the dosage form before administration.

In one aspect, this document provides a formulation comprising: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: (i) a payload, which is or contains one or more mRNAs; (ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; (b) trehalose in the formulation at a concentration of about 10% w/v; and (c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; (b) trehalose at a concentration of about 10% w/v in the formulation; and c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; (b) trehalose, having a concentration of about 10% w/v in the formulation prior to drying; and (c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and

ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Trehalose, at a concentration of approximately 10% w/v;

c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, which is present in the formulation at a concentration of about 5% w/v; c) trehalose, which is present in the formulation at a concentration of about 5% w/v; and d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is present in the formulation at a concentration of about 10 mM.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, having a concentration of about 5% w/v in the formulation prior to drying; c) trehalose, having a concentration of about 5% w/v in the formulation prior to drying; and d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation;

ii) sucrose, in the formulation at a concentration of about 5% w/v; and

iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 5% w/v;

c) Trehalose, which is present in the formulation at a concentration of about 5% w/v;

d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: (a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose at a concentration of about 10% w/v in the formulation; and c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose at a concentration of about 10% w/v in the formulation; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, having a concentration of about 10% w/v in the formulation prior to drying; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation;

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 10% w/v;

c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is at a concentration of approximately 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose, which is present in the formulation at a concentration of about 10% w/v; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is present in the formulation at a concentration of about 10 mM.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose at a concentration of about 10% w/v in the formulation; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose, having a concentration of about 10% w/v in the formulation prior to drying; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation;

ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v;

c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is at a concentration of approximately 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a dried formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[( [(a) Polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose, which is present in the formulation at a concentration of about 5% w/v before drying; (c) trehalose, which is present in the formulation at a concentration of about 5% w/v before drying; and (d) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is present in the formulation at a concentration of about 10 mM before drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation;

ii) sucrose, in the formulation at a concentration of about 5% w/v; and

iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 5% w/v;

c) Trehalose, which is present in the formulation at a concentration of about 5% w/v;

d) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is present at a concentration of approximately 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose at a concentration of about 10% w/v in the formulation; c) His buffer, wherein the His buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose at a concentration of about 10% w/v in the formulation; c) His buffer, wherein the His buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, having a concentration of about 10% w/v in the formulation prior to drying; and c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 10% w/v;

d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose, which is at a concentration of about 10% w/v in the formulation; c) His buffer, wherein the His buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose at a concentration of about 10% w/v in the formulation; c) His buffer, wherein the His buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose, having a concentration of about 10% w/v in the formulation prior to drying; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and

ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v;

d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) His buffer, wherein the His buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) His buffer, wherein the His buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, having a concentration of about 5% w/v in the formulation prior to drying; c) trehalose, having a concentration of about 5% w/v in the formulation prior to drying; and d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation;

ii) sucrose, in the formulation at a concentration of about 5% w/v; and

iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 5% w/v;

c) Trehalose, which is present in the formulation at a concentration of about 5% w/v;

d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose at a concentration of about 10% w/v in the formulation; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose at a concentration of about 10% w/v in the formulation; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, having a concentration of about 10% w/v in the formulation prior to drying; and c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 10% w/v;

d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose, which is at a concentration of about 10% w/v in the formulation; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose at a concentration of about 10% w/v in the formulation; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) trehalose, having a concentration of about 10% w/v in the formulation prior to drying; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and

ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v;

d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and (b) sucrose at a concentration of about 5% w/v in the formulation; c) trehalose at a concentration of about 5% w/v in the formulation; and d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and at a concentration of about 10 mM in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio ranging from about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, having a concentration of about 5% w/v in the formulation prior to drying; c) trehalose, having a concentration of about 5% w/v in the formulation prior to drying; and d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation prior to drying.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation.

ii) sucrose, in the formulation at a concentration of about 5% w/v; and

iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 5% w/v;

c) Trehalose, which is present in the formulation at a concentration of about 5% w/v;

d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, which, prior to drying, is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) a PBS buffer, wherein the PBS buffer is substantially free of sodium chloride; and

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, at a concentration of approximately 10% w/v;

c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride; wherein the formulation is diluted to the dosage form prior to administration.

In one aspect, the formulation provided herein comprises: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer contains about 6 mg/ml of sodium chloride in the formulation.

In some embodiments, the formulation provided herein is a cryogenic formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer contains about 6 mg/ml of sodium chloride in the formulation.

In some embodiments, the formulation provided herein is a drying formulation comprising: a) lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a payload, which is or contains one or more mRNAs; ii) lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC); and cholesterol; and b) sucrose, which, prior to drying, is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, prior to drying, wherein the PBS buffer contains about 6 mg/ml of sodium chloride in the formulation.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) PBS buffer, wherein in the formulation, the PBS buffer contains approximately 6 mg/ml of sodium chloride; and

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v.

In some embodiments, this document provides a method comprising the step of formulating a dosage form for administering a formulation, wherein the formulation comprises:

a) Lipid nanoparticles (LNPs), wherein the LNPs comprise:

i) mRNA, at a concentration of approximately 0.5 mg/ml;

ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml;

iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml;

iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml;

v) Cholesterol, with a concentration of approximately 3.1 mg/ml;

b) Sucrose, which is present in the formulation at a concentration of about 10% w/v;

c) PBS buffer, wherein the PBS buffer contains approximately 6 mg/ml of sodium chloride; wherein the formulation is diluted to the dosage form prior to administration.

In some embodiments, methods for providing these formulations described herein are also described. In some embodiments, a method for preparing a formulation is provided, comprising the following steps:

(a) Lipid nanoparticles (LNPs) are prepared in a first buffer system, wherein the LNPs comprise:

i) A payload, which is or contains one or more mRNAs;

ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and

b) Replace the first buffer system with a second buffer system, wherein the second buffer system comprises:

i) PBS buffer, wherein the PBS buffer contains approximately 6 mg/ml of sodium chloride in the formulation; and

ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v, wherein the first buffer system contains sucrose at a concentration of about 10% w/v.

In some embodiments, this document provides a method for delivering nucleic acids into the cells of a subject, comprising the step of administering an agent as defined in any of the preceding claims.

In some embodiments, this document provides a method for inducing an immune response in a subject, comprising the step of administering to the subject an agent as defined in any of the preceding claims.

definition

In this application, unless the context otherwise requires, (i) the term “a” is to be understood as “at least one”; (ii) the term “or” is to be understood as “and/or”; (iii) the terms “comprising” and “including” are to be understood as covering the listed components or steps, whether they appear alone or together with one or more other components or steps; (iv) the terms “about” and “approximately” are to be understood as permissible standard variations as understood by one of ordinary skill in the art; and (v) where the scope is provided, endpoints are included.

Administration: As used herein, the term "administration" means administering the composition to a subject. Exemplary routes of administration may include bronchial (including bronchial instillation), buccal, intraenteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intravenous, intraventricular, mucosal, nasal cavity, oral cavity, rectum, subcutaneous, sublingual, local, tracheal (including endotracheal instillation), percutaneous, vaginal, and vitreous. In many embodiments, the provided technology relates to LNP compositions (e.g., comprising a BNT162 construct) administered via intramuscular injection. In some embodiments, the LNP composition is administered once or multiple times following a first administration (e.g., one or more booster administrations). In some embodiments, the LNP composition is administered at intervals of time (e.g., between the first and second administrations), such as about 24, 48, 72, 96 hours or longer, including about 1, 2, 3, 4, or more weeks. In some embodiments, the interval between administrations is about 3 weeks (e.g., about 21 days).

Antibody agent: As used herein, the term "antibody agent" refers to a reagent that specifically binds to a particular antigen. In some embodiments, the term includes any polypeptide or polypeptide complex, including immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of antibodies from mice, rabbits, primates, or humans. In some embodiments, an antibody agent may include one or more humanized, primate-like, chimeric, or other sequence elements 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 that are used to utilize the structural and functional characteristics of antibodies in alternative presentations. For example, the antibody agents utilized in embodiments of the present invention are selected from, but not limited to: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments, such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fvs; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies (such as IgNAR or fragments thereof); camel antibodies; masking antibodies (e.g.); small modular immunopharmaceuticals (“SMIPs™”); single-chain or tandem biantibody VHH; minibody; ankyrin repeat protein or DART; TCR-like antibodies; microproteins; and s. In some embodiments, the antibody may lack the covalent modifications (e.g., glycan attachments) present in naturally occurring antibodies. In some embodiments, the antibody may contain covalent modifications (e.g., glycan attachment, payload [e.g., detectable portion, therapeutic portion, catalytic portion, etc.] or other side groups [e.g., polyethylene glycol, etc.]. In many embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements that are recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one CDR present in a reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., its sequence is the same as or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., it exhibits the same sequence as the reference CDR. The included CDR has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., compared to the reference CDR, at least one amino acid is deleted, added, or substituted in the included CDR, and the included CDR has the same amino acid sequence as the reference CDR in all other respects. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., compared to the reference CDR, 1-5 amino acids are deleted, added, or substituted in the included CDR. The included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., at least one amino acid in the included CDR is substituted compared to the reference CDR, and the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., 1-5 amino acids are deleted, added, or substituted compared to the reference CDR, and the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.

Antigen: The term "antigen," as used herein, refers to a reagent or portion that elicits an immune response; and/or a reagent or portion that specifically binds to an antibody or to a T-cell receptor (e.g., when presented by an MHC molecule). In some embodiments, the antigen elicits a humoral response (e.g., may involve or include the production of antigen-specific antibodies); in some embodiments, the antigen elicits a cellular response (e.g., it may involve or include T cells whose receptors specifically interact with the antigen). In some embodiments, the antigen binds to an antibody and may or may not induce a specific physiological response in vivo. Typically, an antigen may be or include any chemical entity, such as small molecules, nucleic acids, peptides, carbohydrates, lipids, polymers (in some embodiments, in addition to biopolymers [e.g., polymers other than nucleic acids or amino acids]), etc. In some embodiments, the antigen is or contains a peptide or its epitope. In some embodiments, the antigen is a recombinant antigen.

Related: If the presence, level, extent, type, and/or form of one event or entity is related to the presence, level, extent, type, and/or form of another event or entity, then the two events or entities are "related" to each other, as used in this document. For example, if the presence, level, and/or form of a particular entity (such as a polypeptide, genetic trait, metabolite, microorganism, etc.) is related to the incidence and/or susceptibility to a particular disease, disorder, or condition (e.g., in a relevant population), then that particular entity is considered related to that particular disease, disorder, or condition. In some embodiments, two or more entities are physically "related" to each other if they interact directly or indirectly, thereby bringing them into physical proximity and/or keeping them in physical proximity. In some embodiments, two or more physically related entities are covalently linked; in some embodiments, two or more physically related entities are not covalently linked but are non-covalently related, for example, through hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

Combination therapy: As used herein, the term "combination therapy," or reference to "combined" administration of an agent, refers to those situations where a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents or modalities). In some embodiments, two or more treatment regimens may be administered simultaneously; in some embodiments, such treatment regimens may be administered sequentially (e.g., all "dose" of the first treatment regimen is administered before any dose of the second treatment regimen is administered); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may involve co-administering one or more agents or modalities to a subject receiving other agents or modalities. For clarity, combination therapy does not require that a single agent be co-administered in a single composition (or even simultaneously), although in some embodiments, two or more agents or their active portions may be co-administered in a composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Expression: As used herein, “expression” of a nucleic acid sequence means one or more of the following events: (1) template synthesis of complementary nucleic acids (e.g., generating an RNA template from a DNA sequence, e.g., by transcription); (2) processing of RNA transcripts (e.g., by splicing, editing, 5’ cap formation and/or 3’ end formation), e.g., to generate mRNA; (3) translation of RNA (e.g., mRNA) into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein. Those skilled in the art will understand that in some cases, “expression” may involve multi-step template synthesis (e.g., reverse transcription of RNA to generate a DNA strand, followed by transcription of that DNA strand and/or optionally synthesis of a complementary DNA strand, e.g., to generate double-stranded DNA).

Formulation: A “formulation” is a composition prepared and/or provided as described herein. In many instances, the term “formulation” is used to refer to an LNP composition—that is, one that comprises RNA (particularly therapeutic RNA, such as mRNA) and lipids as described herein.

Fragment: A "fragment" of a substance or entity as described herein, whose structure comprises discrete parts of a whole but lacks one or more parts found in the whole. In some embodiments, a fragment consists of such discrete parts. In some embodiments, a fragment includes or consists of characteristic structural elements or parts found in the whole. In some embodiments, 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 monomer units (e.g., residues) found throughout the polymer (e.g., sequentially related). In some embodiments, the polymer fragment comprises 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 of monomeric units (e.g., residues) found throughout the polymer. In some embodiments, the entire substance or entity may be referred to as the “parent” of the fragment.

Functionality: As used herein, the term "functionality" refers to a form or segment in which an entity exhibits a particular property and/or activity. In some embodiments, the nature and/or activity of such a "functional" segment is equivalent to that of the whole.

Identity: As used herein, the term "identity" refers to the overall relevance between polymer molecules (e.g., between nucleic acid molecules such as DNA and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, a polymer molecule is considered "substantially identical" to another if its sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. As those skilled in the art will understand, various algorithms allow for sequence comparisons to determine their degree of homology, including allowing a gap of a specified length relative to another sequence when considering which residues in different sequences "correspond" to each other. For example, calculating the percentage of identity between two nucleic acid sequences can be done by aligning the two sequences for optimal comparison purposes (e.g., a gap can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be disregarded for comparison purposes). In some embodiments, the length of the sequences aligned for comparison purposes is a reference... The sequence length is considered to be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or essentially 100%. Nucleotides at corresponding nucleotide positions are then compared. The two molecules are considered identical at that position when a position in the first sequence is occupied by the same nucleotide at the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared positions, taking into account the number of gaps and the length of each gap, which requires incorporating optimal alignment of the two sequences. Representative algorithms and computer programs useful for determining the percentage of identity between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table with a gap length penalty of 12 and a gap penalty of 4. The percentage of identity between two nucleotide sequences can also be determined, for example, using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

Nucleic acid: As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain via a phosphodiester linker. As will become clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain containing a single nucleic acid residue. In some embodiments, "nucleic acid" is RNA or contains RNA; in some embodiments, "nucleic acid" is DNA or contains DNA. In some embodiments, a nucleic acid is one or more natural nucleic acid residues, contains one or more natural nucleic acid residues, or is composed of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is one or more nucleic acid analogs, contains one or more nucleic acid analogs, or is composed of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that the nucleic acid analog does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is one or more "peptide nucleic acids," contains one or more "peptide nucleic acids," or is composed of one or more "peptide nucleic acids," which are known in the art and whose backbone contains peptide bonds instead of phosphodiester bonds, and are considered to be within the scope of the invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more thiophosphates and/or 5'-N-phosphamide linkages instead of phosphodiester bonds. In some embodiments, the nucleic acid is one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), contains one or more natural nucleosides, or is composed of one or more natural nucleosides. In some embodiments, the nucleic acid is one or more nucleoside analogs, contains one or more nucleoside analogs, or is composed of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazoguanosine, 8-oxadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (e.g., RNA or polypeptide); in some embodiments, this nucleotide sequence may be a codon for optimizing expression in a specific host (e.g., in a recipient subject). In some embodiments, the nucleic acid including the coding sequence also includes one or more introns. In some embodiments, the nucleic acid including the coding sequence does not include introns. In some embodiments, the nucleic acid is prepared by one or more of the following methods: isolation from a natural source, enzymatic synthesis via complementary template-based polymerization (in some embodiments, in vivo; in some embodiments, in vitro), replication in recombinant cells or systems, and chemical synthesis. In some implementations, the nucleic acid has a 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 residues.

Specificity: The term "specificity" is used herein to mean, when referring to an active reagent, that a person skilled in the art understands as meaning that the reagent distinguishes a potential target entity or state. For example, in some embodiments, a reagent is said to bind "specifically" to its target if it preferentially binds to that target in the presence of one or more competing alternative targets. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitopes, clefts, binding sites). It should be understood that specificity is not necessarily absolute. In some embodiments, specificity can be evaluated relative to the specificity of the binding reagent against one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding reagent. In some embodiments, specificity is evaluated relative to the specificity of a reference non-specific binding reagent. In some embodiments, the reagent or entity, when bound to its target entity, cannot be detected as binding to competing alternative targets. In some embodiments, the binding reagent binds to its target entity with a higher binding rate (on-rate), a lower dissociation rate (off-rate), increased affinity, reduced dissociation, and/or increased stability compared to competing alternative targets.

Stable: When applied to the compositions herein, the term "stable" means that, under a specified set of conditions, the composition retains one or more aspects of its physical structure and/or activity over a period of time. In some embodiments, this period of time is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or longer, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer; in some embodiments, the specified set of conditions is or includes temperatures above a low-temperature threshold. In some embodiments, the low-temperature threshold is above 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 the colloidal content containing lipid nanoparticles (LNPs). In some embodiments, the composition is considered stable based on the maintenance of one or more characteristics of the LNP (including, but not limited to, its Z-mean and/or polydispersity index (PDI)). In some embodiments, the composition is considered stable based on the maintenance of nucleic acid integrity, the degree of nucleic acid encapsulation (e.g., percentage), and/or nucleic acid expression (e.g., expression level of the encoded peptide, as can be expressed as a percentage of a relevant reference level). In some embodiments, the composition described herein is considered stable if, over a specified set of conditions over a period of time, the lipid nanoparticles in such compositions exhibit a Z-mean change of less than about 20 nm (including, for example, a Z-mean change of less than 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, or less) compared to a relevant reference level. In some embodiments, the composition is considered stable if, over a specified set of conditions and compared to relevant reference levels, the lipid nanoparticles in such compositions exhibit a Z-mean change of less than about 10 nm (including, for example, Z-mean changes of less than 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, or less). In some embodiments, the composition is considered stable if, over a specified set of conditions and compared to relevant reference levels, the nucleic acid encapsulation in such compositions is maintained at at least 50% (including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) over a specified set of conditions and compared to relevant reference levels. In some embodiments, the composition described herein is considered stable if the expression level of the encoded polypeptide remains at least 50% (including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more) compared to a relevant reference level under a specified set of conditions over a certain period of time.

Subject: As used herein, the terms "subject" or "patient" refer to any organism that is administered (e.g., for experimental, diagnostic, preventative, cosmetic, and/or therapeutic purposes) or may be administered the provided composition. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the subject is a human. In some embodiments, the subject has or is susceptible to one or more disorders or conditions. In some embodiments, the subject exhibits symptoms of one or more disorders or conditions. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the subject is at risk of viral infection or a disease or disorder associated with a viral infection.

Essentially: As used herein, the term “essentially” refers to a qualitative condition of the degree or extent to which all or nearly all of the characteristics or related attributes are exhibited. Those skilled in the art of biotechnology will understand that biological and chemical phenomena rarely (if at all) complete and/or proceed to a complete degree or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to capture the inherent potential incompleteness in many biological and chemical phenomena.

Therapeutic Effective Amount: As used herein, the term "therapeutic effective amount" means an amount sufficient to treat a disease, disorder, and/or condition when administered to a population having or susceptible to such a disease, disorder, and/or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutic effective amount is an amount capable of reducing the incidence and/or severity and/or delaying the onset of one or more symptoms of a disease, disorder, and/or condition. Those skilled in the art will understand that the term "therapeutic effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutic effective amount can be an amount that provides a specific, desired pharmacological response in a substantial number of subjects when administered to a patient requiring such treatment. It is understood in particular that a particular subject may be "refractory" to a "therapeutic effective amount." For example, a refractory subject may have low bioavailability to achieve a clinical benefit. In some embodiments, reference to a therapeutic effective amount may refer to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, the therapeutically effective amount may be formulated and/or administered as a single dose. In some embodiments, the therapeutically effective amount may be formulated and/or administered as multiple doses, for example, as part of a dosing regimen.

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 significant structural similarity to a reference molecule but differs structurally from it, for example, in the presence or absence of one or more chemical components compared to the reference entity. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is considered a "variant" of a reference molecule is based on the degree of its structural similarity to the reference molecule. As those skilled in the art will understand, any biological or chemical reference molecule has certain characteristic structural elements. In some embodiments, a variant is a unique molecule that has one or more of these characteristic structural elements but differs from the reference molecule in at least one respect.

To cite just a few examples, a polypeptide may have a characteristic sequence element consisting of multiple amino acids at designated positions relative to each other in linear or three-dimensional space and/or contributing to a specific structural motif and/or biological function; a nucleic acid may have a characteristic sequence element consisting of multiple nucleotide residues at designated positions relative to each other in linear or three-dimensional space. In some embodiments, variant polypeptides or nucleic acids may differ from reference polypeptides or nucleic acids due to one or more differences in the amino acid or nucleotide sequences and/or one or more differences in the chemical portions (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., linked to the polypeptide or nucleic acid backbone). In some embodiments, variant polypeptides or nucleic acids exhibit an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% with the reference polypeptide or nucleic acid. In some embodiments, variant polypeptides or nucleic acids do not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant peptide or nucleic acid shares one or more biological activities with the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid lacks one or more biological activities of the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid exhibits a reduction in one or more biological activities compared to the reference peptide or nucleic acid. In some embodiments, if the relevant peptide or nucleic acid has the same amino acid or nucleotide sequence as the reference peptide or nucleic acid, but with minor sequence changes at specific positions, the relevant peptide or nucleic acid is considered a "variant" of the reference peptide or nucleic acid.

In some embodiments, typically, compared to the reference, the variant contains fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of residue substitutions, insertions, or deletions. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid includes about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue. In some embodiments, relative to the reference, the variant polypeptide or nucleic acid contains a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a specific biological activity). In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and in some embodiments, no addition or deletion is included. In some embodiments, the variant polypeptide or nucleic acid, compared to the reference, contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and generally fewer than about 5, about 4, about 3, or about 2 additions or deletions. In some embodiments, the reference polypeptide or nucleic acid is found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

In some implementations, the “variants” of the amino acid sequence (peptide, protein, or polypeptide) can be or include amino acid insertion variants, amino acid addition (i.e., terminal addition) variants, amino acid deletion variants, and/or amino acid substitution variants.

In some embodiments, "variant" can be or includes mutants, splicing variants, post-translational modification variants, conformations, isomers, allele variants, species variants, and species homologs, particularly those naturally occurring variants. In some embodiments, the term "variant" specifically includes fragments of amino acid sequences.

In some implementations, amino acid insertion variants are made different from the relevant reference polypeptide by inserting one, two, or more amino acids.

In some implementations, the amino acid addition variant may comprise an amino-terminal and/or carboxyl-terminal fusion (i.e., extension) of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

In some embodiments, the 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 some embodiments, the deleted amino acid 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.

In some embodiments, the amino acid substitution variant is characterized by the removal of at least one residue in the sequence and the insertion of another residue at its position. In some embodiments, the substituted residues are not highly conserved in the relevant polypeptide, for example, sharing one or more common motifs (e.g., characteristic sequence elements) and/or functions. In some embodiments, the substitution is a “conservative” substitution, meaning that the original residue and its substitute share one or more structural or functional properties or characteristics (e.g., the characteristics and/or type of charge, or its absence; hydrophobicity or hydrophilicity of the side chain, three-dimensional volume of the side chain, linear or branched properties of the side chain, presence and/or type of heteroatoms in the side chain, etc.). For example, in some embodiments, if the substitution involves residues within an exchange family, the substitution is considered conserved, such as: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine), and aromatic amino acids (phenylalanine, tryptophan, tyrosine). In some embodiments, conserved amino acid substitutions within the following group are considered conserved substitutions: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

In some implementations, a variant may refer to a composition (e.g., a buffer solution) that is identical to a reference composition except for minor changes in composition (e.g., the presence or absence of certain components, or different concentrations of certain components).

Wild-type: As used herein, the terms "wild-type," "WT," or "natural" have their meaning as understood in the art, referring to an entity having a structure and/or activity found in nature in a "normal" state or context (as opposed to mutation, disease, alteration, etc.). Those skilled in the art will understand that wild-type genes and peptides typically exist in many different forms (e.g., alleles). In many embodiments, as used herein, "wild-type" can refer to an amino acid sequence found in nature, including allele variants. Wild-type amino acid sequences, peptides, or proteins have an unmodified amino acid sequence.

Attached Figure Description

Figures 1A-1F illustrate an exemplary workflow for producing a formulation of the present invention. In an exemplary first buffer system, lipids forming particles suspended in an organic solvent (e.g., ethanol) and nucleic acids (e.g., mRNA) suspended in an aqueous buffer (e.g., citrate buffer) are mixed for a period of time (A) until nucleic acid-containing lipid particles (e.g., LNPs) are formed (B). In some embodiments, such nucleic acid-containing lipid particles may be concentrated and/or transferred to a second buffer system (which contains a protective agent (e.g., sucrose, trehalose, etc.)) (C). In some embodiments, the lipid particles may subsequently be stored or diluted for use, or dried (e.g., by lyophilization or other drying methods) (D), or frozen (E), or dried and then frozen (F). In some embodiments, after drying and/or freezing, the lipid particles may be stored and/or thawed and/or diluted for use.

Figures 2A-2B illustrate an exemplary formulation (A) of the present invention and an exemplary cycle (B) designed for the formulation of the present invention.

Figures 3A-3B show exemplary colloidal stability data for exemplary sucrose and trehalose formulations at different time points and temperatures.

Figures 4A-4B show exemplary encapsulation % data at different time points and temperatures for exemplary sucrose and trehalose formulations.

Figure 5 shows an exemplary graph of water content for exemplary sucrose and trehalose formulations.

Figures 6A-6C show data on exemplary expression % at different time points and temperatures for exemplary sucrose and trehalose formulations.

Figures 7A-7D show exemplary data characterizing exemplary formulations of the present invention.

Figures 8A-8B show exemplary colloidal stability data for exemplary sucrose and trehalose formulations at different time points and temperatures.

Detailed Implementation

This invention provides, among other things, techniques relating to nucleic acid/lipid nanoparticle (LNP) compositions, particularly RNA/LNP compositions (e.g., therapeutic RNA/LNP compositions).

Those skilled in the art will recognize that a common problem with nucleic acid/LNP formulations, particularly RNA/LNP formulations, is their need for cryogenic storage to maintain long-term stability. Various reports describe temperature requirements as low as -90°C; others mention temperatures below -80°C, -70°C, or -60°C. Temperatures up to -20°C are typically only tolerated for short periods (e.g., 1, 2, 3, 4 to several days). Temperatures above freezing (e.g., above approximately 0°C) and/or temperatures achieved through refrigeration (e.g., in the range of approximately 1°C to approximately 8°C, or approximately 2°C to approximately 8°C, or approximately 2°C to approximately 6°C, or approximately 2°C to approximately 4°C) are typically tolerated for only a few hours to 1-2 days. Room temperature storage, especially long-term room temperature storage (e.g., at least 1-2 days, preferably 1, 2, 3, 4, 5, 6 weeks or longer, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer), remains a target.

In some embodiments, the present invention provides nucleic acid/LNP formulations, particularly RNA/LNP formulations, which include specific components (e.g., protectants and/or buffer components) and/or are prepared according to specific methods, differing from a reference formulation, and modifying (e.g., improving) one or more properties relative to the reference formulation. For example, in some embodiments, the provided formulations exhibit improvements relative to a reference formulation containing the same lipids and nucleic acids but differing in protectants and/or buffers and/or in manufacturing or processing steps.

In some embodiments, the provided technology enables the preparation of compositions that are drying formulations or suitable for drying (e.g., stable after drying).

In some embodiments, lyophilization cycles can be used to efficiently dry the provided composition in a shorter manner compared to the lyophilization cycles required to dry an equivalent reference formulation (e.g., a formulation produced using a buffer containing NaCl in a concentration range of about 5 to 10 mg/ml (e.g., about 6 mg/ml) and otherwise identical).

In some embodiments, the provided technology enables the preparation of compositions that are cryogenic formulations or suitable for freezing (e.g., stable to freezing).

In some embodiments, the provided technology enables the preparation of a composition that is stably stored at a temperature above a low-temperature threshold for at least a specific period of time. In some embodiments, this specific period of time can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. In some embodiments, the low-temperature threshold can 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 some embodiments, the provided technology can be used to deliver nucleic acid payloads to subjects, for example, by administering an LNP containing a payload encapsulated by lipids as described herein; in some embodiments, the lipids comprise cationic lipids, neutral lipids, polymer-conjugated lipids, and steroids. In some embodiments, the LNP used according to the invention is formed from ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(hexyl 2-decanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), distearate phosphatidylcholine (DSPC), and cholesterol, and combined in relative mass ratios ranging from about 8:1:1.5:3 to about 9:1:2:3.5.

In some embodiments, the nucleic acid payload is or contains RNA and/or DNA; in some embodiments, the nucleic acid payload may encode a polypeptide product (e.g., a functional polypeptide, which may complement or replace an activity required or desired in a subject; or an immunomodulatory polypeptide, which may induce or enhance a desired immune response or activity in a subject).

In some embodiments, the provided composition comprises LNP (i.e., nucleic acid/LNP), a protectant, and a buffer. In some embodiments, the buffer does not contain sodium ions. In some embodiments, the buffer does not contain salt. In some embodiments, the buffer is HEPES buffer, Tris buffer, or His buffer as described herein. In some embodiments, the buffer is a phosphate-buffered saline variant prepared without NaCl. In some embodiments, the buffer is a PBS variant having a reduced sodium ion level relative to a reference PBS containing NaCl, KCl, _Na₂HPO₄ , and _{KH₂PO₄ ;} in some embodiments, such a reference PBS is _a “standard” PBS containing (or composed 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 some embodiments, the buffer used according to the invention is a variant of PBS with a sodium ion level lower than that found in such reference standard PBS.

In some embodiments, the protective agent used according to the invention comprises a disaccharide. In some embodiments, the protective agent used according to the invention is or comprises sucrose and/or trehalose.

In some embodiments, the protective agent is or contains mannitol. In some embodiments, the protective agent substantially does not contain mannitol.

In some embodiments, the present invention provides techniques for producing LNP formulations (i.e., nucleic acid/LNP formulations, and particularly RNA/LNP formulations), followed by storage, freezing, and/or drying. In some embodiments, the frozen composition is stored. In some embodiments, the dried composition is stored.

In some embodiments, the dried composition is resuspended and then administered to a subject. In some embodiments, the frozen composition is thawed and then administered to a subject. In some embodiments, the composition may undergo one or more rounds of freezing and thawing, one or more rounds of drying and resuspension, and/or one or more rounds of freezing and thawing, and further one or more rounds of drying and resuspension.

In some embodiments, the composition is diluted before administration.

Nucleic acid payload

In other respects, the present invention provides an LNP composition comprising a nucleic acid payload (i.e., a nucleic acid-LNP composition).

In some implementations, the nucleic acid payload may contain or encode functional nucleic acids such as antisense oligonucleotides (e.g., those that can promote RNAeH degradation and/or exon skipping), ribozymes, gRNA, miRNA and shRNA, siRNA, etc.

In some implementations, the nucleic acid payload may encode one or more polypeptides (as further described below, for example).

In some embodiments, the nucleic acid payload used according to the present invention is or comprises one or more natural nucleic acid residues or completely natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more non-natural nucleic acid residues (i.e., one or more nucleic acid analogs), or completely non-natural nucleic acid residues.

In some embodiments, the nucleic acid payload used according to the invention comprises one or more internucleotide linkages that are not phosphodiester bonds. For example, in some embodiments, the nucleic acid has one or more thiophosphate and/or 5'-N-phosphamide linkages instead of phosphodiester bonds. In some embodiments, the nucleic acid comprises some phosphodiester bonds and some non-phosphodiester bonds.

In some implementations, the nucleic acid is or contains one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is one or more nucleoside analogs, contains one or more nucleoside analogs, or is composed of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazoguanosine, 8-oxadenosine, 8-oxazoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).

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

In some implementations, the nucleic acid is or contains one or more peptide nucleic acids.

In some embodiments of the present invention, nucleic acids are modified with the modifications described herein to impart one or more desired properties, such as enhanced stability, potency, etc.

RNA payload

In some embodiments, the nucleic acid payload used according to the invention is RNA (e.g., mRNA). In some embodiments, the RNA is produced by template synthesis. In some embodiments, the RNA is produced by enzymatic synthesis, for example by in vitro transcription (e.g., from a DNA template). In some embodiments, the RNA is produced by chemical synthesis.

In some implementations, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In some implementations, the replicon or self-replicating RNA is derived from or contains elements derived from ssRNA viruses, particularly positive-sense ssRNA viruses, such as alphaviruses. Alphaviruses are typical representatives of positive-sense RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, vol. 4, pp. 837–856). The total genome length of many alphaviruses is typically between 11,000 and 12,000 nucleotides, and the genomic RNA usually has a 5'-cap and a 3'-poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in viral RNA transcription, modification, and replication, as well as protein modification) and structural proteins (forming viral particles). The genome typically contains two open reading frames (ORFs). Four non-structural proteins (nsP1-nsP4) are typically encoded by a first ORF starting near the 5' end of the genome, while alphavirus structural proteins are encoded by a second ORF located downstream of the first ORF and extending near the 3' end of the genome. Generally, the first ORF is larger than the second ORF, in a ratio of approximately 2:1. In alphavirus-infected cells, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111–124). Post-infection, in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly as messenger RNA, translating the open reading frame encoding the non-structural multiprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simplified approach, the open reading frame encoding the relevant protein replaces the open reading frame encoding the alphavirus structural protein. 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 capable of being trans-replicated by that replicase (hence the name trans-replication system). Trans-replication requires the simultaneous presence of both nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the trans-replicase must contain some alphavirus sequence elements to allow the alphavirus replicase to recognize and synthesize RNA.

In some embodiments, the RNA used according to the present invention may include one or more modified nucleosides. In some embodiments, the present invention provides RNA comprising a modified nucleoside replacing at least one uridine. In some embodiments, the modified nucleoside replaces all uridines in the RNA. In some embodiments, the modified nucleoside replaces at least one uridine, said uridine including but not limited to pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U) or combinations thereof. In some embodiments, the modified nucleoside replaces at least one (e.g., all) uridine in the RNA, said modified nucleoside may be any one or more of the following: 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- Uranyluridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine-5-oxyacetic acid (cmo5U), methyl uridine-5-oxyacetic acid (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethyl pseudouridine, 5-carboxyhydroxymethyluridine (chm5U), methyl 5-carboxyhydroxymethyluridine (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methylaminomethyluridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U) 5-Carboxymethylaminomethyluridine (cmnm5U), 5-Carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-Propynouridine, 1-Propynouridine, 5-Taurine methyluridine (τm5U), 1-Taurine methyl pseudouridine, 5-Taurine methyl-2-thiouridine (τm5s2U), 1-Taurine methyl-4-thiopseudouridine), 5-Methyl-2-thiouridine (m5s2U), 1- Methyl-4-thiopseudouridine (m1s4ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m3ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydrouridine, 2-methoxy 2-Methoxy-4-thiouridine, 4-Methoxy-2-thiouridine, N1-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)-2-thiouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U) ), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methylpseudouridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2’-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2’-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2’-F-aradiuridine, 2’-F-uridine, 2’-OH-aradiuridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other uridine with any other modifications known in the art.

In some embodiments, the RNA used according to the invention comprises a 5'-cap. In some embodiments, the RNA of the invention does not have an uncapped 5'-triphosphate. In some embodiments, the RNA may be modified with a 5'-cap analog. The term "5'-cap" refers to the structure found at the 5' end of an mRNA molecule and is typically composed of a guanosine nucleotide linked to the mRNA via a 5'-to-5'-triphosphate. In some embodiments, such guanosine is methylated at the 7-position. Providing the RNA with a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription, wherein the 5'-cap or 5'-cap analog is co-transcribed into the RNA chain, or by post-transcriptional ligation using a capping enzyme. In some embodiments, the 5'-cap used for the RNA is m27,3'-OGppp(m12'-O)ApG (sometimes also referred to as m27,3'OG(5')ppp(5')m2'-OApG). In some embodiments, the 5'-cap of the RNA used in this invention is an anti-reverse cap analog (ARCA cap (m27,3'OG(5')ppp(5')G)). In some embodiments, the 5'-cap is Beta-S-ARCA (m27,2'OG(5')ppSp(5')G). In some embodiments, the 5'-cap is beta-S-ARCA(D1)(m27,2'-OGppSpG) or m27,3'-OGppp(m12'-O)ApG.

In some embodiments, the RNA used according to the invention comprises a 5'-UTR and/or a 3'-UTR. The terms "untranslated region" or "UTR" may refer to a region in a DNA molecule that has been transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule (such as an mRNA molecule). A UTR may be present at the 5' (upstream) end of an open reading frame (5'-UTR) and/or the 3' (downstream) end of an open reading frame (3'-UTR). The 5'-UTR (if present) is located at the 5' end upstream of the start codon in a protein-coding region. The 5'-UTR is located 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 in a protein-coding region, but the term "3'-UTR" preferably does not include the poly-(A) sequence. Thus, the 3'-UTR is located upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

As used herein, the term "poly(A) sequence" or "poly-A tail" refers to a continuous or discontinuous sequence of adenosine residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may be located after the 3'-UTR in the RNA described herein. A continuous poly(A) sequence is characterized by a continuous sequence of adenosine residues. In nature, continuous poly(A) sequences are typical. The RNAs disclosed herein may have a free 3' poly(A) sequence that is ligated to the RNA post-transcriptionally by a 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 nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells and on protein levels translated from an open reading frame present upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

In different embodiments, the poly(A) sequence can be of different lengths. In some embodiments, the poly(A) sequence comprises, is substantially composed of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises, is substantially composed of, or consists of up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 A nucleotides. In this document, “substantially composed 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 number of nucleotides in the poly(A) sequence, are A nucleotides, but the remaining nucleotides are permitted to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "composed of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the nucleotides in the poly(A) sequence are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

In some embodiments, the poly(A) sequence is linked during RNA transcription, for example, during the preparation of in vitro transcribed RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence (coding strand) encoding the end of the poly(A) sequence is called a poly(A) box. In some embodiments, the poly(A) box present in the DNA coding strand consists essentially of dA nucleotides but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). The length of this random sequence can be 5 to 50, 10 to 30, or 10 to 20 nucleotides. Such a box is disclosed in WO 2016/00524A1, which is incorporated herein by reference. Any poly(A) box disclosed in WO 2016/00524A1 can be used in this invention. A poly(A) cassette, essentially composed of dA nucleotides but interrupted by random sequences of four nucleotides (dA, dC, dG, dT) of equal length and for example 5 to 50 nucleotides, demonstrates the sustained propagation of plasmid DNA in *E. coli* at the DNA level and is also associated with beneficial properties at the RNA level, supporting RNA stability and translation efficiency. Therefore, in some embodiments, the poly(A) sequence contained in the RNA molecule described herein is essentially composed of A nucleotides but interrupted by random sequences of four nucleotides (A, C, G, U). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides.

In some implementations, no other nucleotides besides A are flanked at the 3' end of the poly(A) sequence, meaning that the poly(A) sequence is not masked or followed by any nucleotide other than A at its 3' end.

In some embodiments, the poly(A) sequence may comprise 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 some embodiments, the poly(A) sequence may consist substantially 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 50 nucleotides. In some embodiments, 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 some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

In some embodiments, the nucleic acid used according to the invention is codon-optimized and/or has an increased guanosine/cytosine (G/C) content compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and/or have increased G/C content compared to the corresponding sequence regions of the wild-type coding sequence. In some embodiments, the codon optimization and/or increased G/C content does not alter the sequence of the encoded amino acid sequence.

G/C content

In some embodiments of the invention, the G/C content of the coding region (e.g., of 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 a corresponding WT sequence. In some embodiments, the increased G/C content can increase the translation efficiency of RNA including such an 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.

The fact that some codons encode the same amino acid (so-called degeneracy of the genetic code) allows for the identification of codons most favorable for stability (so-called alternative codon usage). Compared to the wild-type sequence, there are multiple possibilities for modifying the RNA sequence based on the desired amino acid encoded by the RNA. In particular, codons containing A and/or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and/or U, or contain a lower concentration of A and/or U nucleotides.

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

Encoded polypeptide

As described herein, in some implementations, the nucleic acid payload (e.g., RNA) encodes a polypeptide.

In some embodiments, the encoded polypeptide is or comprises an antibody agent or a polypeptide chain or a functional fragment thereof. In some embodiments, the antibody agent is or comprises a single-chain antibody agent, such as scFC, camel antibody, etc.

In some implementations, the encoded polypeptide is or contains cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors, ligands, hormones, etc.

In some implementations, the encoded polypeptide is an enzyme.

In some implementations, the encoded polypeptide is a regulatory polypeptide, such as a transcription factor or molecular chaperone.

In some implementations, the encoded polypeptide is or contains a polypeptide whose expression substitutes for or activates an activity that is reduced or absent in the subject.

In some embodiments, the encoded polypeptide is or is contained in a polypeptide that induces and/or enhances an immune response in a subject. In some embodiments, the encoded polypeptide is or contains at least one epitope that specifically binds to an immunoglobulin reagent (e.g., an antibody and/or a T-cell receptor).

In some embodiments, the encoded polypeptide is or contains an antigen (or an epitope thereof). In some embodiments, the antigen may be a characteristic of a specific disease, disorder, or condition. For example, the antigen may be or contain a tumor antigen (e.g., a neoantigen) and/or an antigen associated with an infectious pathogen (e.g., a virus or microorganism, such as bacteria or fungi). In some embodiments, the antigen associated with an infectious pathogen may be an antigen displayed on the surface of such an infectious pathogen and/or an antigen that can mediate infection by such pathogens (e.g., through interaction with receptors on receptor cells).

In some embodiments, the antigen may be or contain viral antigens, such as antigens associated with viruses selected from: adenovirus, cytomegalovirus, herpesvirus, human papillomavirus, measles virus, rubella virus, coronavirus, respiratory syncytial virus, influenza virus, and mumps virus. In some embodiments, the antigen may be or contain viral antigens associated with viruses selected from categories I, II, III, IV, V, VI, or VII (based on the Baltimore classification system). In some implementations, the antigen may be or include viral antigens associated with viruses selected from the following families: Adenoviridae, Papaverviridae, Parvovirdiae, Herpesviridae, Poxviridae, Anelloviridae, Pleolipoviridae, Reoviridae, Picornaviridae, Caliciviridae, Clonorviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, Arteriviridae, and Hepatitis Viridae. In some specific implementations, the viral antigen may be a coronavirus antigen.

In some specific embodiments, the viral antigen may be an antigen derived from a SARS-CoV-2 protein sequence (e.g., a variant comprising or containing such a sequence, fragments thereof, or both). In some embodiments, the present invention provides a polypeptide having an antigenic sequence derived from a SARS-CoV-2S protein sequence. In some embodiments, the polypeptide is an antigenic sequence comprising or containing a receptor-binding domain (RBD) derived from a SARS-CoV-2S protein sequence.

In some embodiments, the payload, as described herein, is associated with or encapsulated within the lipid portion of the LNP. In some embodiments, the payload, as described herein, is associated with the lipid portion of the LNP. In some embodiments, the payload, as described herein, is encapsulated within the lipid portion of the LNP. In some embodiments, association with such a lipid portion (e.g., encapsulation therein) reduces susceptibility to payload degradation (e.g., enzymatic degradation), such as over a given time period and/or under specific conditions.

According to some embodiments, the signal peptide is fused to the antigenic peptide or protein directly or via a linker. In some embodiments, the signal peptide used according to the invention is a sequence that typically exhibits a length of about 15 to about 30 amino acids and may be located at the N-terminus of the antigenic peptide or protein, but is not limited thereto. In some embodiments, the signal peptide, as defined herein, allows the RNA-encoded antigenic peptide or protein to be transported into a defined cellular region, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome region.

The signal peptide sequence that can be used according to certain embodiments of the present invention may be, or include, for example, a signal peptide sequence of an immunoglobulin, such as a signal peptide sequence of the variable region of the immunoglobulin heavy chain, wherein the immunoglobulin may be human immunoglobulin.

The signal peptide used according to the present invention is used to promote the secretion of encoded antigenic peptides or proteins. In some embodiments, the signal peptide as defined herein is fused to an encoded antigenic peptide or protein as defined herein. In some embodiments, the RNA described herein comprises at least one coding region for an antigenic peptide or protein and a signal peptide, wherein the signal peptide is fused to the antigenic peptide or protein, for example, fused to the N-terminus of an antigenic peptide or protein as described herein.

In some embodiments, the trimerizing domain is fused to the antigenic peptide or protein directly or via a linker (e.g., a glycine/serine linker). In some embodiments, the trimerizing domain is fused to the antigenic peptide or protein directly or via a linker (e.g., a glycine/serine linker), and also to a signal peptide as described herein.

In some implementations, such trimerizing domains are located at the C-terminus of an antigenic peptide or protein, but are not limited thereto. Trimerizing domains as defined herein allow for the trimerization of antigenic peptides or proteins encoded by RNA. Examples of trimerizing domains as defined herein include, but are not limited to, the folded structure and the native trimerizing domain of T4 fibritin. The C-terminal domain of T4 fibritin (folded structure) is essential for the formation of fibritin trimer structures and can be used as an artificial trimerizing domain.

In some embodiments, the transmembrane domain is fused to the antigenic peptide or protein directly or via a linker (e.g., a glycine/serine linker). Accordingly, in some embodiments, the transmembrane domain is fused to the antigenic peptide or protein directly or via a linker (e.g., a glycine/serine linker), and it is also fused to a signal peptide and/or trimerizing domain as described herein.

In many embodiments, the transmembrane domain used according to the invention is located at the C-terminus of the antigenic peptide or protein, but is not limited thereto. In some embodiments, such a transmembrane domain (if present) is located at the C-terminus of the trimerizing domain, but is not limited thereto. In some embodiments, the trimerizing domain is present between the SARS-COV-2S protein, its variants, or fragments thereof (i.e., the antigenic peptide or protein) and the transmembrane domain.

In some embodiments, the transmembrane domains used according to the present invention may allow antigenic peptides or proteins encoded by RNA to anchor into the cell membrane.

coronavirus

Coronaviruses are enveloped, positive-sense, single-stranded RNA (ssRNA) viruses. They have the largest genomes (26–32 kb) among known RNA viruses and are phylogenetically divided into four lineages (α, β, γ, and δ), with β coronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some coronaviruses typically cause mild respiratory illness, although severity may be higher in infants, the elderly, and those with weakened immune systems. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), belonging to the β coronavirus lineages C and B, respectively, are highly pathogenic. Both viruses have entered humans from animal hosts over the past 15 years, causing outbreaks with high mortality rates. SARS-CoV-2 (MN908947.3) belongs to the β coronavirus lineage B. It shares at least 70% sequence similarity with SARS-CoV.

Coronaviruses typically possess four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike (S). E and M proteins play crucial roles in viral assembly, while the N protein is essential for viral RNA synthesis. The critical glycoprotein S is responsible for viral binding and entry into target cells. S protein synthesis occurs as a single-stranded, inactive precursor, which is cleaved in the producing cell by furin-like host proteases into two non-covalently related 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 heptap repeats, and a transmembrane domain, all of which are necessary for undergoing a large conformational rearrangement to mediate viral-host cell membrane fusion. The S1 and S2 subunits trimerize to form a large prefusion spike.

The SARS-CoV-2 S precursor protein can be 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.

The full-length spike (S) protein of the SARS-CoV-2 coronavirus consists of 1273 amino acids (see SEQ ID NO: 1).

In some embodiments, the present invention uses RNA encoding a peptide or protein comprising at least the epitope SARS-CoV-2S protein to induce an immune response against coronavirus S protein, particularly SARS-CoV-2S protein, in subjects. In some embodiments, the RNA of the present invention encodes an amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic fragment of the SARS-CoV-2S protein, or an immunogenic variant thereof.

In some embodiments, the full-length spike (S) protein according to SEQ ID NO: 1 is modified in a stable prototypical prefusion conformation. Stabilization of the prefusion conformation can be obtained by introducing two consecutive proline substitutions at AS residues 986 and 987 of the full-length spike protein. Specifically, the spike (S) protein stabilized variant is obtained by exchanging the amino acid residue at position 986 for proline and also exchanging the amino acid residue at position 987 for proline. In some embodiments, the SARS-COV-2S protein variant comprises the amino acid sequence shown in SEQ ID NO: 7.

In some implementations, the vaccine antigen described herein comprises, is substantially composed of, or consists of the spike protein (S) of SARS-CoV-2, its variants, or fragments thereof.

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

In some implementations, the vaccine antigen comprises, is substantially composed of, or consists of the following: a SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the spike protein (S) of SARS-CoV-2), a variant thereof, or a fragment thereof.

In some embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9; or, the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9 or having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9. The fragment encodes an amino acid sequence containing amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence containing amino acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence containing amino acids 17 to 683 of SEQ ID NO: 1. In some embodiments, the RNA encoding the vaccine antigen (i) contains a nucleotide sequence containing nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9; and/or (ii) encodes an amino acid sequence containing amino acids 17 to 683 of SEQ ID NO: 1.

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

In some implementations, the vaccine antigen comprises, is substantially composed of, or consists of the receptor-binding domain (RBD) of the S1 subunit of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof. The amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, a variant thereof, or a fragment thereof is also referred to herein as “RBD” or “RBD domain”.

In some embodiments, the RNA (i) encoding the vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 2, 8, or 9, nucleotides 979 to 1584, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequences of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of SEQ ID NO: 2, 8, or 9, nucleotides 979 to 1584, or the nucleotide sequence of SEQ ID NO: 2, 8, or 9, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequences of SEQ ID NO: 2, 8, or 9, nucleotides 979 to 1584. Fragments of homologous nucleotide sequences; and/or (ii) amino acid sequences encoding amino acid sequences comprising amino acid sequences 327 to 528 of SEQ ID NO: 1, amino acid sequences having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with amino acid sequences 327 to 528 of SEQ ID NO: 1, or immunogenic fragments of amino acid sequences 327 to 528 of SEQ ID NO: 1 or amino acid sequences having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with amino acid sequences 327 to 528 of SEQ ID NO: 1. In some embodiments, the RNA encoding the vaccine antigen (i) comprises a nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9; and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.

According to some embodiments, the signal peptide is fused directly or via a linker to the SARS-CoV-2 S protein, its variants, or fragments thereof (i.e., antigenic peptides or proteins). Therefore, in some embodiments, the signal peptide is fused to the aforementioned amino acid sequence derived from the novel coronavirus S protein or its immunogenic fragments (antigenic peptides or proteins) contained in the vaccine antigen described herein.

In some embodiments, the signal peptide used according to the invention is a sequence that typically exhibits a length of about 15 to about 30 amino acids and is located at the N-terminus of an antigenic peptide or protein, but is not limited thereto. In some embodiments, the signal peptide, as defined herein, allows the transport of an RNA-encoded antigenic peptide or protein into a defined cellular region, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome region. In some embodiments, the signal peptide sequence, as defined herein, includes, but is not limited to, the signal peptide sequence of the SARS-CoV-2S protein, particularly the amino acid sequence comprising amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1, or a functional variant thereof.

The signal peptide sequence that can be used according to certain embodiments of the present invention may be, or include, for example, a signal peptide sequence of an immunoglobulin, such as a signal peptide sequence of the variable region of the immunoglobulin heavy chain, wherein the immunoglobulin may be human immunoglobulin.

The signal peptide used according to the present invention is used to promote the secretion of encoded antigenic peptides or proteins. In some embodiments, the signal peptide as defined herein is fused to an encoded antigenic peptide or protein as defined herein. In some embodiments, the RNA described herein comprises at least one coding region for an antigenic peptide or protein and a signal peptide, wherein the signal peptide is fused to the antigenic peptide or protein, for example, fused to the N-terminus of an antigenic peptide or protein as described herein.

In some embodiments, the trimerizing domain is fused directly or via a linker (e.g., a glycine/serine linker) to the SARS-COV-2S protein, its variants, or fragments thereof (i.e., antigenic peptides or proteins). Therefore, in some embodiments, the trimerizing domain is fused with the aforementioned amino acid sequence of the SARS-COV-2S protein or its immunogenic fragments (antigenic peptides or proteins) contained in the aforementioned vaccine antigen (which may optionally be fused with a signal peptide as described herein).

In some implementations, such trimerizing domains are located at the C-terminus of an antigenic peptide or protein, but are not limited thereto. Trimerizing domains as defined herein allow for the trimerization of antigenic peptides or proteins encoded by RNA. Examples of trimerizing domains as defined herein include, but are not limited to, the folded structure and the native trimerizing domain of T4 fibritin. The C-terminal domain of T4 fibritin (folded structure) is essential for the formation of fibritin trimer structures and can be used as an artificial trimerizing domain.

In some embodiments, the transmembrane domain is fused directly or via a linker (e.g., a glycine/serine linker) to the SARS-COV-2S protein, its variants, or fragments thereof (i.e., antigenic peptides or proteins). Therefore, in some embodiments, the transmembrane domain is fused with the aforementioned amino acid sequence of the SARS-COV-2S protein or its immunogenic fragments (antigenic peptides or proteins) contained in the aforementioned vaccine antigen (which may optionally be fused with a signal peptide and/or trimerizing domain as described herein).

In many embodiments, the transmembrane domain used according to the invention is located at the C-terminus of the antigenic peptide or protein, but is not limited thereto. In some embodiments, such a transmembrane domain (if present) is located at the C-terminus of the trimerizing domain, but is not limited thereto. In some embodiments, the trimerizing domain is present between the SARS-COV-2S protein, its variants, or fragments thereof (i.e., the antigenic peptide or protein) and the transmembrane domain.

In some embodiments, the transmembrane domains used according to the present invention may allow antigenic peptides or proteins encoded by RNA to anchor into the cell membrane.

In some implementations, the transmembrane domain sequence defined herein includes, but is not limited to, the transmembrane domain sequence of the SARS-COV-2S protein, particularly the amino acid sequence containing amino acids 1207 to 1254 of SEQ ID NO:1 or a functional variant thereof.

As presented herein, a trimerizing domain is used to facilitate the trimerization of encoded antigenic peptides or proteins. In some embodiments, the trimerizing domain, as defined herein, is fused to an antigenic peptide or protein, as defined herein. In some embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a trimerizing domain, as defined herein, fused to the antigenic peptide or protein, for example, fused to its C-terminus.

In some embodiments, the vaccine antigen described herein comprises a continuous sequence of the SARS-CoV-2 coronavirus spike (S) protein, consisting of or substantially consisting of the aforementioned amino acid sequence derived from the SARS-CoV-2 S protein or its immunogenic fragment (antigenic peptide or protein) contained in the vaccine antigen described herein. In some embodiments, the vaccine antigen described herein comprises a continuous sequence of the SARS-CoV-2 coronavirus spike (S) protein of no more than 220 amino acids, 215 amino acids, 210 amino acids, or 205 amino acids.

In some embodiments, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) of BNT162b2 (RBP020.1 or RBP020.2) as described herein. In some embodiments, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) of RBP020.2 as described herein.

As described in this article, different implementation schemes for nucleoside-modified messenger RNA (modRNA) are as follows:

BNT162b2; RBP020.1 (SEQ ID NO: 19; SEQ ID NO: 7)

Structure: m27,3’-OGppp(m12’-O)ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

Encoded antigen: The viral spike protein (S1S2 protein) of SARS-CoV-2 (full-length S1S2 protein, sequence variant).

BNT162b2; RBP020.2 (SEQ ID NO: 20; SEQ ID NO: 7)

Structure: m27,3’-OGppp(m12’-O)ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

Encoded antigen: The viral spike protein (S1S2 protein) of SARS-CoV-2 (full-length S1S2 protein, sequence variant).

Nucleotide sequence of RBP020.1

Nucleotide sequences are represented by individual sequence elements in bold. Furthermore, the sequence of the translated protein is represented in italics below the coding nucleotide sequence (* = stop codon).

Nucleotide sequence of RBP020.2

Nucleotide sequences are represented by individual sequence elements in bold. Furthermore, the sequence of the translated protein is represented in italics below the coding nucleotide sequence (* = stop codon).

Lipid nanoparticles (LNP)

In some embodiments, one or more nucleic acids (e.g., RNA) as described herein are formulated and/or administered in the form of an LNP. In some embodiments, the LNP of the present invention comprises one or more lipids known in the art and/or established herein to generate lipid particles. In some embodiments, the LNP of the present invention comprises one or more lipids selected from cationic lipids, neutral lipids, polymer-conjugated lipids, and combinations thereof. In some embodiments, the LNP of the present invention comprises a steroid, such as cholesterol or a derivative thereof.

As used herein, “neutral lipid” refers to a type of lipid that exists as an uncharged or neutral zwitterion at a selected pH. In some embodiments, additional lipids comprise one of the following neutral lipid components: (1) phospholipids; (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. In some embodiments, phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Specific examples of such phospholipids include diacylphosphatidylcholine, such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and diilignoceroylphosphatidylcholine. e, DLPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0Diether PC), 1-oleoyl-2-cholestenyl hemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), and phosphatidylethanolamines, particularly diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearate phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilaurylphosphatidylethanolamine (DLPE) and diphytylphosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

Examples of cholesterol derivatives include, but are not limited to, cholesterol alcohols, cholesterol ketones, cholesterol ketones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

The term "cationic lipid" refers to any number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH approximately 7.0). Examples of cationic lipids include, but are not limited to, 1,2-dioleoyl-3-trimethylpropane ammonium (DOTAP); N,N-dimethyl-2,3-dioleoyloxypropane (DODMA), 1,2-di-O-octadecenyl-3-trimethylpropane ammonium (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethylbis(octadecyl)ammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; bis(octadecyl)dimethylammonium chloride (DODAC), and 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDM). A) 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazonium (DMRIE), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylpropaneammonium (DMTAP), 1,2-dioleoylpropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate ammonium (DOSPA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), and dioctadecane 3-Dimethylamino-2-(cholest-5-en-3-β-oxybutyl-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5’-(cholest-5-en-3-β-oxy)-3’-oxaproloxy)-3-dimethyl-1-(cis,cis-9’,12’-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N’-dioleocarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N’ -Dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptadecyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin- MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propanium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (βAE-DMRIE), N-(4-carboxyphenyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium (DOBAQ), 2-({8-[(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecene-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-aminopropyl)amino]butylformylamino)ethyl]-3,4-di[oleoyloxy] [Methyl]-Benzamide (MVL5), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-bromine (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-bromine (DMORIE), di((Z)-non-2-en-1-yl)-8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azadiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propane-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amine (DMDMA), Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanoic acid ester (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecane-2-ol (lipidoid C12-200).

In some embodiments, the cationic lipids have chemical structures as shown in WO 2017/075531, some of which are described in Table A below:

Table A: Exemplary cationic lipids

Further examples of cationic lipids are shown in Table B below.

Table B: Other exemplary cationic lipids

In some embodiments, the cationic lipid is an ionizable lipid-like substance (lipidoid). An exemplary lipidoid is C12-200, which has the following structure:

In some embodiments, the particles described herein comprise polymer-conjugated lipids, such as polyethylene glycol-modified lipids. The term "polyethylene glycol-modified lipid" refers to a molecule that simultaneously comprises a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art.

In some embodiments, the LNP of the present invention comprises ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(hexyl 2-decanoate) (ALC-0315). In some embodiments, the LNP of the present invention comprises 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159). In some embodiments, the present invention provides an LNP comprising distearate phosphatidylcholine (DSPC). In some embodiments, the LNP of the present invention comprises cholesterol. In some embodiments, the LNP of the present invention comprises lipids comprising: ALC-0315, ALC-0159, DSPC, and cholesterol.

In some embodiments, the LNP of the present invention comprises about 40 to about 55 mole percentages, about 40 to about 50 mole percentages, about 41 to about 49 mole percentages, about 41 to about 48 mole percentages, about 42 to about 48 mole percentages, about 43 to about 48 mole percentages, about 44 to about 48 mole percentages, about 45 to about 48 mole percentages, about 46 to about 48 mole percentages, about 47 to about 48 mole percentages, or about 47.2 to about 47.8 mole percentages of ALC-0315. In some embodiments, 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 percentages of ALC-0315.

In some embodiments, the LNP of the present invention comprises ALC-0315 at a concentration of about 6 mg/ml to about 9 mg/ml, about 6 mg/ml to about 8 mg/ml, about 6 mg/ml to about 7 mg/ml, about 7 mg/ml to about 9 mg/ml, about 8 mg/ml to about 9 mg/ml, or about 7 mg/ml to about 8 mg/ml. In some embodiments, the LNP comprises ALC-0315 at a concentration of about 7 mg/ml to about 8 mg/ml. In some embodiments, ALC-0315 is present at a concentration of about 7.17 mg/ml.

In some embodiments, the LNP of the present invention comprises 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 some embodiments, the DSPC is present at a concentration of about 9.5, about 10, or about 10.5 mole percent.

In some embodiments, the LNP of the present invention comprises about 1 mg/ml to about 2.5 mg/ml, about 1 mg/ml to about 2 mg/ml, or about 1 mg/ml to about 1.5 mg/ml of DSPC. In some embodiments, the LNP comprises about 1.5 mg/ml to about 2 mg/ml of DSPC. In some embodiments, ALC-0315 is present at a concentration of about 1.56 mg/ml.

In some embodiments, cholesterol is present at a concentration of about 30 to about 50 mole percent, about 35 to about 45 mole percent, or about 38 to about 43 mole percent. In some embodiments, cholesterol is present at a concentration of about 40, about 41, about 42, about 43, about 44, about 45, or about 46 mole percent.

In some embodiments, cholesterol is present at concentrations of about 2 mg/ml to about 4 mg/ml, about 2 mg/ml to about 3.5 mg/ml, about 2 mg/ml to about 3 mg/ml, about 2 mg/ml to about 2.5 mg/ml, about 2.5 mg/ml to about 4 mg/ml, about 3 mg/ml to about 4 mg/ml, or about 3.5 mg/ml to about 4 mg/ml. In some embodiments, cholesterol is present at a concentration of about 3 mg/ml to about 3.5 mg/ml. In some embodiments, cholesterol is present at a concentration of about 3.1 mg/ml.

In some embodiments, ALC-0159 is present at concentrations of about 1 to about 10 mol percent, about 2 to about 8 mol percent, about 4 to about 8 mol percent, about 4 to about 6 mol percent, about 1 to about 5 mol percent, or about 1 to about 3 mol percent.

In some embodiments, ALC-0159 is present at concentrations of about 0.5 mg/ml to about 2.5 mg/ml, about 1 mg/ml to about 2.5 mg/ml, about 1.5 mg/ml to about 2.5 mg/ml, about 2 mg/ml to about 2.5 mg/ml, about 0.5 mg/ml to about 2 mg/ml, about 0.5 mg/ml to about 1.5 mg/ml, or about 0.5 mg/ml to about 1 mg/ml. In some embodiments, ALC-0159 is present at a concentration of about 0.5 mg/ml to about 1 mg/ml. In some embodiments, ALC-0159 is present at a concentration of about 0.89 mg/ml.

In some implementations, the molar percentage is determined based on the total molar amount of lipids present in the LNP described herein.

In some embodiments, the present invention provides an LNP comprising lipids including ALC-0315, ALC-0159, DSPC, and cholesterol, which are present in a mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5.

In some embodiments, the lipid particles (e.g., LNPs) of the present invention 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 some embodiments, the lipid particles (e.g., LNPs) of the present invention may have an average diameter of at most 30 nm, at most 40 nm, at most 50 nm, at most 60 nm, at most 70 nm, at most 80 nm, at most 90 nm, at most 100 nm, at most 200 nm, at most 300 nm, at most 400 nm, at most 500 nm, at most 1000 nm, or at most 1200 nm. In some embodiments, the lipid particles (e.g., LNPs) of the present invention may have an average diameter ranging from 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.

Nucleic acids described herein can be packaged into lipids (e.g., RNA/LNP) using various methods (e.g., thin-film hydration, reverse-phase evaporation, ethanol injection techniques). These methods may involve obtaining a colloid from at least one cation or cationic ionizable lipid or lipid-like substance and/or at least one cationic polymer, and mixing the colloid with nucleic acids to obtain lipid particles (e.g., RNA/LNP).

In some embodiments, RNA is packaged into lipid particles (e.g., LNPs) using an ethanol injection technique, wherein an ethanol solution containing lipids is rapidly injected into an aqueous phase solution via a needle. Therefore, in some embodiments, nucleic acid-containing lipid particles (e.g., RNA/LNPs) are prepared as follows: an ethanol solution containing lipids (e.g., cationic lipids and other lipid compositions as described herein) is injected into an aqueous phase solution containing nucleic acids (e.g., RNA) while the mixture is stirred or agitated. The nucleic acids prepared from the lipid particles obtained by this method can be further processed, such as concentrated, transferred to one or more different buffer systems, etc.

In some embodiments, according to the LNP formation method described herein, the RNA is packaged into lipid particles (e.g., LNPs) by mixing the RNA with lipids that form particles (e.g., those described herein). In some embodiments, RNA-containing LNPs (RNA/LNPs) are prepared in a first buffer system and then exchanged into a second buffer system for storage and/or use.

In some embodiments, the first buffer system comprises an aqueous buffer, such as PBS buffer, Tris buffer, HEPES buffer, His buffer, etc. In some embodiments, the first buffer system comprises PBS buffer. In some embodiments of the invention, the first buffer system comprises sodium chloride at 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. In some embodiments, the first buffer system comprises about 6 mg/ml of sodium chloride. In some embodiments, the first buffer system is substantially sodium chloride-free. Those skilled in the art will understand that "substantially sodium chloride-free" herein means that no sodium chloride is added, while sodium and/or chloride ions may still be present due to other components in such formulations. Accordingly, in some embodiments, the PBS buffer of the present invention is a substantially sodium chloride-free PBS buffer and comprises 0.15 g/L KCl, 1.08 g/L _{Na₂HPO₄ ,} and 0.15 g/L _{KH₂PO₄ .} In some embodiments, 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 .}

In some embodiments, the first buffer system comprises a protective agent, such as sucrose, trehalose, or a combination thereof. In some embodiments, the protective agent in the first buffer system is sucrose and/or trehalose. In some embodiments, the concentration of sucrose is about 10% w/v. In some embodiments, the concentration of sucrose is about 5%. In some embodiments, the concentration of trehalose is about 10% w/v. In some embodiments, the concentration of trehalose is about 5%.

In some embodiments, the second buffer system of the present invention comprises an aqueous buffer, such as PBS buffer, Tris buffer, HEPES buffer, His buffer, etc. In some embodiments, the second buffer system comprises PBS. In some embodiments, 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 . In some embodiments, the PBS of the present invention is a substantially sodium chloride-free (as defined herein) PBS buffer and comprises 0.15 g/L KCl, 1.08 g/L _{Na₂HPO₄ ,} and _0.15 g/L _KH₂PO₄ . In some embodiments, the second buffer system comprises Tris buffer. In some embodiments, the second buffer system comprises a Tris buffer at a concentration of about 10 mM. In some embodiments, the Tris buffer is substantially sodium chloride-free. In some embodiments, the Tris buffer contains about 6 mg/ml of sodium chloride. In some embodiments, the second buffer system comprises His buffer. In some embodiments, the second buffer system comprises a His buffer at a concentration of approximately 10 mM. In some embodiments, the His buffer is substantially free of sodium chloride. In some embodiments, the His buffer contains approximately 6 mg/ml of sodium chloride. In some embodiments, the second buffer system comprises a HEPES buffer. In some embodiments, the second buffer system comprises a HEPES buffer at a concentration of approximately 10 mM. In some embodiments, the HEPES buffer is substantially free of sodium chloride. In some embodiments, the HEPES buffer contains approximately 6 mg/ml of sodium chloride.

In some embodiments, RNA-LNP comprises about 0.4 mg/ml to about 0.6 mg/ml, about 0.4 mg/ml to about 0.5 mg/ml, or about 0.5 mg/ml to about 0.6 mg/ml of mRNA. In some embodiments, RNA-LNP comprises about 0.5 mg/ml of mRNA.

preparation

In other aspects, the present invention provides techniques relating to formulations for RNA therapy, and particularly LNP formulations comprising a nucleic acid (e.g., mRNA) payload. Such RNA/LNP formulations include specific components (e.g., protectants and/or buffer components) and/or are prepared according to specific methods, differing from a reference formulation and modifying (e.g., improving) one or more properties relative to the reference formulation. For example, in some embodiments, the provided formulations exhibit improvements relative to a reference formulation comprising the same lipids and nucleic acids but differing in protectants and/or buffers and/or in manufacturing or processing steps.

In some embodiments, the present invention provides compositions suitable for drying and/or for use with drying. In some embodiments, the compositions described herein are dried by lyophilization.

In some embodiments, the compositions described herein are substantially anhydrous or dried until substantially anhydrous. In some embodiments, the compositions contain 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 some embodiments, the compositions described herein are maintained at 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 for a certain period of time, such as about 1, 2, 3, 4, 5, 6 weeks or longer, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer, and above a certain low temperature threshold, such as above 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 compositions of the present invention are annealed during drying (e.g., lyophilization). In some embodiments, the compositions are not annealed during drying.

In some embodiments, the provided compositions are stored stably at temperatures above a cryogenic threshold for at least a specific period of time. In some embodiments, the stably stored period of the compositions provided herein is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. In some embodiments, the compositions provided herein are stored stably for at least about 12 weeks. In some embodiments, the compositions are stored stably at temperatures above a cryogenic 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 some embodiments, the compositions are stored stably at temperatures about 0°C, 2°C, 5°C, 8°C, 25°C, 40°C, or higher. In some embodiments, the compositions provided herein are stably stored for at least about 12 weeks in temperature ranges of about 2°C to about 40°C, about 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.

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

In some embodiments, the composition as described herein (e.g., the LNP composition) is prepared in a first buffer system and then exchanged into a second buffer system as described herein.

In some embodiments, the LNP compositions described herein comprise one or more lipids that form particles. In some embodiments, the lipids that form particles include: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(hexyl 2-decanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), distearate phosphatidylcholine (DSPC), and cholesterol.

In some embodiments, the LNP composition includes ALC-0315, ALC-0159, DSPC and cholesterol, present in relative mass ratios of about 8:1:1.5:3 to about 9:1:2:3.5.

In some embodiments, the LNP composition described herein includes ALC-0315, ALC-0159, DSPC, and cholesterol, present at concentrations of 7.17 mg/ml, 0.89 mg/ml, 1.56 mg/ml, and 3.1 mg/ml, respectively.

Some embodiments of the present invention use one or more protective agents. In some embodiments, the protective agent is or comprises sucrose, trehalose, or a combination thereof. In some embodiments, the concentration of sucrose in the composition or method of the present invention is about 10% w/v. In some embodiments, the concentration of trehalose in the composition or method of the present invention is about 10% w/v. In some embodiments, the concentration of sucrose in the composition or method of the present invention is about 5% w/v and the concentration of trehalose is about 5% w/v.

In some embodiments, a lyoprotectant is added to the composition and brought to the desired concentration (e.g., those described herein) prior to the freezing or drying step.

In some embodiments, a protective agent is added to a first buffer system for preparing LNPs, such as those described herein. In some embodiments, a protective agent is added simultaneously to both a first buffer system and a second buffer system. In some embodiments, a protective agent is added simultaneously to both a first buffer system and a second buffer system, and different protective agents or the same protective agent may be used in each buffer system. In embodiments where a protective agent is added simultaneously to both a first buffer system and a second buffer system, different concentrations of the protective agent or the same concentration may be used.

Some embodiments of the present invention use one or more buffer systems. In some embodiments, first and second buffer systems are used.

In some embodiments, the preparation and/or use of the provided composition may involve a dilution step, such as by adding a buffer system, which in some embodiments may be the same as the previously used buffer system, and in other embodiments may be different from the previously used buffer system, such as a buffer system included in the LNP composition undergoing dilution.

In some embodiments, the buffer solution used (e.g., the buffer solution used in the buffer system described herein) is substantially free of sodium chloride. Those skilled in the art will understand that substantially free of sodium chloride herein means no sodium chloride salt is added, although in some embodiments, sodium and/or chloride ions may still be present due to other components in such compositions or formulations.

In some embodiments, the provided composition comprises LNP (i.e., nucleic acid/LNP), a protectant, and a buffer. In some embodiments, the buffer does not contain sodium ions. In some embodiments, the buffer does not contain salt. In some embodiments, the buffer is HEPES buffer, Tris buffer, or His buffer as described herein. In some embodiments, the buffer is a phosphate-buffered saline variant prepared without NaCl. In some embodiments, the buffer is a PBS variant having a reduced sodium ion level relative to a reference PBS containing NaCl, KCl, _Na₂HPO₄ , and _{KH₂PO₄ ;} in some embodiments, such a reference PBS is _a “standard” PBS containing (or composed 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 some embodiments, the buffer used according to the invention is a variant of PBS with a sodium ion level lower than that found in such reference standard PBS. In some embodiments, the buffer used according to the invention is approximately 10 mM Tris buffer. In some embodiments, the buffer used according to the invention is approximately 10 mM His buffer. In some embodiments, the buffer used according to the invention is approximately 10 mM HEPES buffer. In some embodiments, the buffer used according to the invention is supplemented with 6 mg/ml sodium chloride.

In some embodiments, the compositions of the present invention are prepared into dosage forms by diluting them with a buffer solution.

use

As described herein, the technology provided by this invention relates to and/or can be used to prepare and/or administer one or more nucleic acid/LNP (e.g., RNA/LNP) compositions.

In some embodiments, the techniques described herein provide LNP compositions (e.g., LNP/RNA compositions) that are stably stored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. In some embodiments, the LNP compositions provided by the techniques of the present invention are stably stored for at least about 12 weeks. In some embodiments, the provided compositions are stably stored 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 some embodiments, the provided compositions are stably stored at temperatures of about 0°C, 2°C, 5°C, 8°C, 25°C, 40°C, or higher. In some embodiments, the LNP compositions provided herein are stable for at least about 12 weeks in a temperature range of about 2°C to about 40°C, about 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.

The compositions provided herein are considered stable based on the maintenance of the colloidal content containing lipid nanoparticles (LNPs). In some embodiments, the compositions provided herein are considered stable based on the maintenance of one or more characteristics of the LNPs, 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., percentage), and/or nucleic acid expression (e.g., expression level of the encoded peptide, as can be expressed as a percentage of a relevant reference level). In some embodiments, the compositions provided herein are considered stable if, over a specified set of conditions over a period of time, the lipid nanoparticles in such compositions exhibit a Z-mean change of less than about 20 nm (including, for example, a Z-mean change of less than 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, or less). In some embodiments, the provided composition is considered stable if, over a specified set of conditions and compared to relevant reference levels within a certain time period, the lipid nanoparticles in such compositions exhibit a Z-mean change of less than about 10 nm (including, for example, a Z-mean change of less than 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, or less). In some embodiments, the provided composition is considered stable if, over a specified set of conditions and compared to relevant reference levels within a certain time period, the lipid nanoparticles in such compositions exhibit a change of less than 0.1 in the polydispersity index (PDI) (including, for example, a change of less than 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, or less). In some embodiments, the provided composition is considered stable if, over a specified set of conditions, the nucleic acid encapsulation in such a composition is maintained at at least 50% (including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) compared to relevant reference levels over a specified period of time. In some embodiments, the provided composition is considered stable if, over a specified set of conditions, the expression level of the encoded polypeptide is maintained at at least 50% (including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) compared to relevant reference levels over a specified period of time.

In some embodiments, the antigens used in the techniques provided herein may be or contain viral antigens, such as antigens associated with viruses selected from: adenovirus, cytomegalovirus, herpesvirus, human papillomavirus, measles virus, rubella virus, coronavirus, respiratory syncytial virus, influenza virus, and mumps virus. In some embodiments, the antigens may be or contain viral antigens associated with viruses selected from categories I, II, III, IV, V, VI, or VII (based on the Baltimore classification system). In some embodiments, the techniques described herein provide immunity to a virus selected from the following families of viruses: adenoviridae, papilloviridae, parvovirdiae, herpesviruses, poxviruses, anelloviridae, pleolipoviridae, reoviruses, microRNAviruses, caliciviruses, clovenviridae, arenaviridae, flaviviridae, orthomyxoviridae, paramyxoviridae, Bunyaviridae, rhabdoviridae, filoviridae, coronaviruses, astroviridae, Bornaviridae, arteriviridae, or hepatitisviridae. In some embodiments, the techniques described herein provide immunity to a viral infection in a subject. In some embodiments, the techniques described herein provide immunity to a coronavirus, coronavirus infection, or coronavirus-related disease or disorder in a subject. Therefore, the present invention provides compositions and methods for treating or preventing coronavirus-related infections, diseases, or disorders.

In some embodiments, the technology described herein provides an LNP composition for administration to a subject having a coronavirus-related infection, disease, or disorder. In some embodiments, the technology described herein provides an LNP composition for administration to a subject at risk of developing a coronavirus-related infection, disease, or disorder. For example, the technology described herein provides an LNP composition that can be administered to a subject at risk of exposure to a coronavirus. In some embodiments, the technology described herein provides an LNP composition for administration to a subject residing in, traveling to, or expecting to travel to a geographic area where a coronavirus is prevalent. In some embodiments, the technology described herein provides an LNP composition for administration to a subject who has been in contact with, or expects to be in contact with, another person residing in, traveling to, or expecting to travel to a geographic area where a coronavirus is prevalent. In some embodiments, the technology described herein provides an LNP composition for administration to a subject who has been intentionally exposed to a coronavirus through their occupation or other contact. In some embodiments, the coronavirus is SARS-CoV-2.

In some embodiments, the compositions provided by the technology described herein can be administered prophylactically (i.e., to prevent disease or disorder) or therapeutically (i.e., to treat disease or disorder) to subjects who have or are at risk (or susceptible) to develop a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of this invention, prophylactic administration occurs before the obvious clinical symptoms of a disease manifest, thereby preventing the disease or disorder (e.g., reducing the burden of mortality or morbidity from the disease) or alternatively delaying its progression.

Dosage

The compositions (e.g., pharmaceutical compositions) and methods provided herein are for delivering a payload (e.g., mRNA) to the cells of a subject who requires such a payload. In some embodiments, the provided compositions are administered for the purpose of preventing and/or treating viral infections. In some embodiments, the technology of the present invention provides compositions that can be used as therapeutic or prophylactic agents for treating coronaviruses (e.g., SARS-CoV-2).

The pharmaceutical compositions of the present invention can be administered for prophylactic purposes, such as in subjects who are undiagnosed and/or do not exhibit one or more specific symptoms or features of a particular disease, disorder, or condition. In some embodiments, the pharmaceutical compositions provided herein are administered to the cells or tissues of a subject in an immunoprophylacticly effective amount. The pharmaceutical compositions provided herein can be administered together with other therapeutic or prophylactic compounds.

In some embodiments, the pharmaceutical composition is administered therapeutically, for example, to a subject who has been diagnosed with and/or has exhibited one or more specific symptoms or characteristics of a particular disease, disorder, or condition. In some embodiments, the pharmaceutical compositions provided herein are administered to the cells or tissues of a subject in a therapeutically effective amount. These pharmaceutical compositions provided herein may be administered together with other therapeutic or prophylactic compounds.

The exact dosage of a pharmaceutical composition (e.g., an RNA/LNP composition) provided for preventative and/or therapeutic purposes will vary from subject to subject, depending on the subject's species, age and general condition, severity of disease, route of administration and mode of activity, and other considerations. However, it is understood that the use of the provided composition may be determined by the attending physician within reasonable medical judgment. Accordingly, the specific therapeutically and/or preventatively 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 condition, sex and diet, timing of administration, route of administration and excretion rate of the specific compound used, duration of treatment; drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field.

In some embodiments, the provided pharmaceutical composition is administered to a subject who has received, is receiving, or will receive other therapies. In some embodiments, other therapies, for example, administered concurrently or alternately, address one or more symptoms or features of a disease, disorder, or condition treated by the provided therapy. Alternatively, or additionally, in some embodiments, other therapies address one or more symptoms or features of different diseases. As just one example, in various embodiments, it is desirable to administer multiple preventative therapies (such as preventative vaccines) substantially simultaneously.

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 have received, will receive, or are receiving). Adjuvants used in this invention may involve any compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a group of heterogeneous compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligonucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines. Cytokines used according to this invention may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-α, or combinations thereof. Further known adjuvants that may be used according to this invention may be aluminum hydroxide, Freund's adjuvant, or oils such as ISA51. Other adjuvants suitable for use in this invention include lipopeptides, such as Pam3Cys.

The pharmaceutical compositions described herein may be provided as frozen concentrates of injectable solutions, for example at a concentration of about 0.50 mg/mL. In some embodiments, to prepare the injectable solution, the pharmaceutical product is thawed and diluted with an isotonic sodium chloride solution (such as 0.9% sodium chloride, saline), and/or rehydrated and diluted, for example, by a one-step dilution process. The final concentration of the injectable solution varies depending on the respective dosage level.

In some embodiments, each dose of the RNA described herein may be from 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg (e.g., about 1 μg, about 3 μg, about 10 μg, about 30 μg, about 50 μg, or about 100 μg). In some embodiments, the disclosed compositions described herein are administered as a single dose. In some embodiments, the compositions described herein are administered at an initial dose, followed by one or more booster doses. In some embodiments, the booster dose or the first booster dose may be administered 7 to 28 days or 14 to 24 days after the initial dose.

In some embodiments, the amount of RNA described herein administered per dose may be 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.

In some embodiments, the amount of RNA described herein administered per dose may be 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.

In some embodiments, the amount of RNA described herein administered per dose may be 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.

In some embodiments, each dose of the RNA described herein may be about 30 μg. In some embodiments, at least two such doses may be administered. For example, the second dose may be administered about 21 days after the first dose.

In some embodiments, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) of BNT162b2 (RBP020.1 or RBP020.2) as described herein. In some embodiments, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) of RBP020.2 as described herein.

In some embodiments, administration of the immunogenic composition or vaccine of the present invention may be performed by a single dose or by multiple doses to enhance the effect.

sequence list

SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 7

SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 19

SEQ ID NO: 20

Example

The following embodiments are provided for illustration but do not limit the scope of the invention in any way. Those skilled in the art will understand that certain design and selection criteria described in this invention can be modified according to conventional practice in the art.

Example 1: Exemplary Composition and Characterization

This embodiment describes the development and/or characterization of a certain RNA/LNP composition according to the present invention.

The RNA payload used in this embodiment is a modified RNA payload comprising 4283 nucleotide residues. The RNA payload used in this embodiment encodes a viral antigen, specifically the SARS-CoV-2S protein. Specifically, the RNA payload used in this embodiment is the BNT162b2 construct represented by RBP020.2(v9) as described herein.

This embodiment evaluates certain protective agents (particularly disaccharide protective agents sucrose and trehalose) and specific buffers (e.g., non-phosphate buffers, such as Tris and histidine buffers and/or buffers that do not contain NaCl).

Not wanting to be bound by any particular theory, it is assumed that isotonicity may be ideal; some evaluated compositions include a 10% w/v protective agent because it produces a nearly isotonic solution.

Choose a buffer concentration sufficient to maintain the pH of the composition.

The compositions evaluated did not include mannitol.

In this embodiment, the composition is not frozen before drying (e.g., maintained at a temperature in the range of about 2°C to about 8°C). In this embodiment, drying is performed by freeze-drying (specifically, lyophilization).

The electrical conductivity of the composition was measured and low-temperature DSC experiments were performed before freeze-drying. The size and polydispersity of the LNP were determined by dynamic light scattering after holding at 2°C–8°C and before freeze-drying.

Table 1 below shows some of the compositions evaluated; it can be seen that (i) the types and concentrations of the protective agents are different; during the manufacturing process, alternative manufacturing processes are evaluated (especially for formulations containing sucrose, diluting the RNA stock solution into a sucrose-citrate buffer instead of a citrate-only buffer); (ii) buffers lacking NaCl are evaluated; and (iii) non-phosphate buffers (e.g., Tris, His, HEPES) are evaluated.

Table 1. Composition of formulations used for lyophilization evaluation

*Two 15 mL aliquots should be prepared and stored separately for lyophilization cycles during freezing, with and without annealing.

The freeze-drying process used involves cooling and heating at a rate of 0.5 °C/min during the freezing step. The formulation is frozen to a temperature below the Tg' of the relevant formulation. Not wanting to be bound by any particular theory, an annealing temperature of -10 °C was chosen to maximize Ostwald ripening (thus increasing ice crystal size) during isothermal holding and reduce cake resistance, while keeping the product below the melting point of the formulation. The slope of the secondary drying is 0.2 °C/min.

Claims (178)

1. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 2. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 3. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 10% w/v in the formulation; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 4. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v. 5. The method of claim 4, further comprising the step of: c) Freeze the preparation. 6. The method of claim 4, further comprising the step of: c) Dry the preparation. 7. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 1; b) Thaw and dilute the formulation of claim 2; and/or c) Resuspend and dilute the formulation of claim 3. 8. The method of claim 7, further comprising administering the dosage form to a subject in need of it. 9. The method of claim 8, wherein the subject requires the expression product of the mRNA. 10. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 11. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 12. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 13. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which, before drying, is at a concentration of approximately 10% w/v in the formulation; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 14. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v. 15. The method of claim 14, further comprising the step of: c) Freeze the preparation. 16. The method of claim 14, further comprising the step of: c) Dry the preparation. 17. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 11; b) Thaw and dilute the formulation of claim 12; and/or c) Resuspend and dilute the formulation of claim 13. 18. The method of claim 17, further comprising administering the dosage form to a subject in need of it. 19. The method of claim 18, wherein the subject requires the expression product of the mRNA. 20. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Trehalose, at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 21. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is present in the formulation at a concentration of about 10 mM. 22. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is present in the formulation at a concentration of about 10 mM. 23. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 5% w/v in the formulation; c) Trehalose, which, before drying, is at a concentration of approximately 5% w/v in the formulation; d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 24. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is at a concentration of about 10 mM in the formulation; ii) sucrose, in the formulation at a concentration of about 5% w/v; and iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v. 25. The method of claim 24, further comprising the step of: c) Freeze the preparation. 26. The method of claim 24, further comprising the step of: c) Dry the preparation. 27. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 21; b) Thaw and dilute the formulation of claim 22; and/or c) Resuspend and dilute the formulation of claim 23. 28. The method of claim 27, further comprising administering the dosage form to a subject in need of it. 29. The method of claim 28, wherein the subject requires the expression product of the mRNA. 30. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) Tris buffer, wherein the Tris buffer is substantially free of sodium chloride and is present in the formulation at a concentration of about 10 mM; The preparation is diluted to the dosage form before administration. 31. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is present at a concentration of approximately 10 mM in the formulation. 32. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is present at a concentration of approximately 10 mM in the formulation. 33. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 10% w/v in the formulation; c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and has a concentration of approximately 10 mM in the formulation prior to drying. 34. A method for preparing a formulation, comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation; ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v. 35. The method of claim 34, further comprising the step of: c) Freeze the preparation. 36. The method of claim 34, further comprising the step of: c) Dry the preparation. 37. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 31; b) Thaw and dilute the formulation of claim 32; and/or c) Resuspend and dilute the formulation of claim 33. 38. The method of claim 37, further comprising administering the dosage form to a subject who requires it. 39. The method of claim 38, wherein the subject requires the expression product of the mRNA. 40. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 41. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is present at a concentration of approximately 10 mM in the formulation. 42. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and is present at a concentration of approximately 10 mM in the formulation. 43. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which, before drying, is at a concentration of approximately 10% w/v in the formulation; c) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and has a concentration of approximately 10 mM in the formulation prior to drying. 44. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation; ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v. 45. The method of claim 44, further comprising the step of: c) Freeze the preparation. 46. The method of claim 44, further comprising the step of: c) Dry the preparation. 47. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 41; b) Thaw and dilute the formulation of claim 42; and/or c) Resuspend and dilute the formulation of claim 43. 48. The method of claim 47, further comprising administering the dosage form to a subject who requires it. 49. The method of claim 48, wherein the subject requires the expression product of the mRNA. 50. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 51. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation. 52. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation. 53. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 5% w/v in the formulation; c) Trehalose, which, before drying, is at a concentration of approximately 5% w/v in the formulation; d) Tris buffer, wherein the Tris buffer contains approximately 6 mg/ml of sodium chloride and has a concentration of approximately 10 mM in the formulation prior to drying. 54. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation; ii) sucrose, in the formulation at a concentration of about 5% w/v; and iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v. 55. The method of claim 54, further comprising the step of: c) Freeze the preparation. 56. The method of claim 54, further comprising the step of: c) Dry the preparation. 57. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 51; b) Thaw and dilute the formulation of claim 52; and/or c) Resuspend and dilute the formulation of claim 53. 58. The method of claim 57, further comprising administering the dosage form to a subject who requires it. 59. The method of claim 58, wherein the subject requires the expression product of the mRNA. 60. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) Tris buffer, wherein the Tris buffer contains about 6 mg/ml of sodium chloride and is at a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 61. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 62. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 63. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 10% w/v in the formulation; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 64. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v. 65. The method of claim 64, further comprising the step of: c) Freeze the preparation. 66. The method of claim 64, further comprising the step of: c) Dry the preparation. 67. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 61; b) Thaw and dilute the formulation of claim 62; and/or c) Resuspend and dilute the formulation of claim 63. 68. The method of claim 67, further comprising administering the dosage form to a subject who requires it. 69. The method of claim 68, wherein the subject requires the expression product of the mRNA. 70. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 71. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 72. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 73. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which, before drying, is at a concentration of approximately 10% w/v in the formulation; c) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 74. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v. 75. The method of claim 74, further comprising the step of: c) Freeze the preparation. 76. The method of claim 74, further comprising the step of: c) Dry the preparation. 77. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 71; b) Thaw and dilute the formulation of claim 72; and/or c) Resuspend and dilute the formulation of claim 73. 78. The method of claim 77, further comprising administering the dosage form to a subject who requires it. 79. The method of claim 78, wherein the subject requires the expression product of the mRNA. 80. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 81. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 82. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 83. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 5% w/v in the formulation; c) Trehalose, which, before drying, is at a concentration of approximately 5% w/v in the formulation; d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 84. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; ii) sucrose, in the formulation at a concentration of about 5% w/v; and iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v. 85. The method of claim 84, further comprising the step of: c) Freeze the preparation. 86. The method of claim 84, further comprising the step of: c) Dry the preparation. 87. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 81; b) Thaw and dilute the formulation of claim 82; and/or c) Resuspend and dilute the formulation of claim 83. 88. The method of claim 87, further comprising administering the dosage form to a subject in need of it. 89. The method of claim 88, wherein the subject requires the expression product of the mRNA. 90. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) His buffer, wherein the His buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 91. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 92. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 93. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 10% w/v in the formulation; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 94. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v. 95. The method of claim 94, further comprising the step of: c) Freeze the preparation. 96. The method of claim 94, further comprising the step of: c) Dry the preparation. 97. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 91; b) Thaw and dilute the formulation of claim 92; and/or c) Resuspend and dilute the formulation of claim 93. 98. The method of claim 97, further comprising administering the dosage form to a subject who requires it. 99. The method of claim 98, wherein the subject requires the expression product of the mRNA. 100. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 101. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 102. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation. 103. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Trehalose, which, before drying, is at a concentration of approximately 10% w/v in the formulation; c) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 104. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; and ii) Trehalose, which is present in the formulation at a concentration of about 10% w/v. 105. The method of claim 104, further comprising the step of: c) Freeze the preparation. 106. The method of claim 104, further comprising the step of: c) Dry the preparation. 107. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 101; b) Thaw and dilute the formulation of claim 102; and/or c) Resuspend and dilute the formulation of claim 103. 108. The method of claim 107, further comprising administering the dosage form to a subject who requires it. 109. The method of claim 108, wherein the subject requires the expression product of the mRNA. 110. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Trehalose, which is present in the formulation at a concentration of approximately 10% w/v; d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 111. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and is present in the formulation at a concentration of about 10 mM. 112. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and is present in the formulation at a concentration of about 10 mM. 113. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 5% w/v in the formulation; c) Trehalose, which, before drying, is at a concentration of approximately 5% w/v in the formulation; d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation before drying. 114. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; ii) sucrose, in the formulation at a concentration of about 5% w/v; and iii) Trehalose, which is present in the formulation at a concentration of about 5% w/v. 115. The method of claim 114, further comprising the step of: c) Freeze the preparation. 116. The method of claim 114, further comprising the step of: c) Dry the preparation. 117. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 111; b) Thaw and dilute the formulation of claim 112; and/or c) Resuspend and dilute the formulation of claim 113. 118. The method of claim 117, further comprising administering the dosage form to a subject in need of it. 119. The method of claim 118, wherein the subject requires the expression product of the mRNA. 120. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 5% w/v; c) Trehalose, which is present in the formulation at a concentration of about 5% w/v; d) HEPES buffer, wherein the HEPES buffer is substantially free of sodium chloride and has a concentration of about 10 mM in the formulation; The preparation is diluted to the dosage form before administration. 121. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride. 122. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride. 123. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 10% w/v in the formulation; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride. 124. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) a PBS buffer, wherein the PBS buffer is substantially free of sodium chloride; and ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v. 125. The method of claim 124, further comprising the step of: c) Freeze the preparation. 126. The method of claim 124, further comprising the step of: c) Dry the preparation. 127. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 121; b) Thaw and dilute the formulation of claim 122; and/or c) Resuspend and dilute the formulation of claim 123. 128. The method of claim 127, further comprising administering the dosage form to a subject who requires it. 129. The method of claim 128, wherein the subject requires the expression product of the mRNA. 130. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, at a concentration of approximately 10% w/v; c) PBS buffer, wherein the PBS buffer is substantially free of sodium chloride; The preparation is diluted to the dosage form before administration. 131. A formulation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer contains approximately 6 mg/ml of sodium chloride in the formulation. 132. A cryogenic preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer contains approximately 6 mg/ml of sodium chloride in the formulation. 133. A drying preparation comprising: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Sucrose, which, before drying, is at a concentration of about 10% w/v in the formulation; c) PBS buffer, wherein, prior to drying, the PBS buffer in the formulation contains approximately 6 mg/ml of sodium chloride. 134. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) PBS buffer, wherein in the formulation, the PBS buffer contains approximately 6 mg/ml of sodium chloride; and ii) Sucrose, which is present in the formulation at a concentration of about 10% w/v. 135. The method of claim 134, further comprising the step of: c) Freeze the preparation. 136. The method of claim 134, further comprising the step of: c) Dry the preparation. 137. A method for preparing a dosage form, the method comprising the following steps: a) Dilute the formulation of claim 131; b) Thaw and dilute the formulation of claim 132; and/or c) Resuspend and dilute the formulation of claim 133. 138. The method of claim 137, further comprising administering the dosage form to a subject who requires it. 139. The method of claim 138, wherein the subject requires the expression product of the mRNA. 140. A method comprising the following steps: A dosage form for a drug delivery formulation, wherein the formulation comprises: a) Lipid nanoparticles (LNPs), wherein the LNPs comprise: i) mRNA, at a concentration of approximately 0.5 mg/ml; ii)((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315), with a concentration of approximately 7.17 mg/ml; iii) 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), with a concentration of approximately 0.89 mg/ml; iv) Distearate phosphatidylcholine (DSPC) at a concentration of approximately 1.56 mg/ml; v) Cholesterol, with a concentration of approximately 3.1 mg/ml; b) Sucrose, which is present in the formulation at a concentration of about 10% w/v; c) PBS buffer, wherein the PBS buffer contains approximately 6 mg/ml of sodium chloride; The preparation is diluted to the dosage form before administration. 141. A method for preparing a formulation, the method comprising the following steps: a) Prepare lipid nanoparticles (LNPs) in a first buffer system, wherein the LNPs comprise: i) A payload, which is or contains one or more mRNAs; ii) Lipids comprising: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester) (ALC-0315) in a relative mass ratio of about 8:1:1.5:3 to about 9:1:2:3.5; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); distearate phosphatidylcholine (DSPC) and cholesterol; and b) Replacing the first buffer system with a second buffer system, wherein the second buffer system comprises: i) PBS buffer, wherein in the formulation, the PBS buffer contains approximately 6 mg/ml of sodium chloride; and ii) Sucrose, in the formulation at a concentration of about 10% w/v The first buffer system contains sucrose at a concentration of approximately 10% w/v. 142. The method of claim 141, further comprising the step of: c) Freeze the preparation. 143. The method of claim 141, further comprising the step of: c) Dry the preparation. 144. A method for delivering nucleic acids into the cells of a subject, comprising the step of administering an agent as defined in any of the preceding claims. 145. A method for inducing an immune response in a subject, comprising the step of administering to the subject an agent as defined in any of the preceding claims. 146. The method of claim 145, wherein the immune response is directed against a viral antigen or epitope encoded by mRNA. 147. The method of claim 146, wherein the viral antigen is an antigen of a coronavirus. 148. The method of claim 147, wherein the coronavirus is the SARS-CoV2 virus. 149. The method of claim 147 or claim 148, wherein the antigen is or comprises an S protein. 150. The formulation of any one of claims 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 or 133, or the method of any one of claims 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136 or 143, wherein the formulation is dried until it is substantially anhydrous. 151. The formulation of any one of claims 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133 or 150, or the method of any one of claims 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 143, 150, wherein the formulation is dried by lyophilization. 152. The formulation of any one of claims 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 150, or 151, or the method of any one of claims 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 143, 150, or 151, wherein the formulation is dried until it 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. 153. The formulation of any one of claims 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 150, 151 or 152, or the method of any one of claims 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 143, 150, 151 or 152, wherein the formulation is annealed during drying. 154. The formulation of any one of claims 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 150, 151 or 152, or the method of any one of claims 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 143, 150, 151 or 152, wherein the formulation is not annealed during drying. 155. The formulation of any one of claims 150-154, or the method of any one of claims 150-154, wherein the formulation is maintained at a temperature range of about 2°C to about 25°C with 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 for at least 12 weeks. 156. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein the one or more mRNAs encode one or more polypeptides. 157. The formulation of claim 156, wherein the one or more polypeptides are or contain epitopes for inducing an immune response against an antigen in a subject. 158. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, wherein the one or more mRNAs are or contain epitopes for inducing an immune response against an antigen in a subject. 159. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, 131-133 or 158, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139 or 141-149, wherein the one or more RNAs are or comprise self-amplifying RNA molecules. 160. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, 131-133, 158 or 159, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, 141-149 or 159, wherein the one or more RNAs are or comprise modified RNA molecules or unmodified RNA molecules. 161. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, 131-133, or 158-160, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, 141-149, 159, or 160, wherein the one or more RNAs are or comprise unmodified uridine RNA molecules. 162. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, 131-133 or 158-161, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, 141-149 or 159-161, wherein the one or more RNAs are or comprise nucleoside-modified RNA molecules. 163. The formulation of claim 156 or the method of claim 156, wherein at least one of the one or more polypeptides is derived from the SARS-CoV-2S protein of SEQ ID NO: 1 or 7. 164. The formulation of claim 156 or the method of claim 156, wherein at least one of the one or more polypeptides comprises at least 85% sequence identity with the SARS-CoV-2S protein of SEQ ID NO: 1 or 7. 165. The formulation of claim 156 or the method of claim 156, wherein at least one of the one or more polypeptides is or comprises the full-length SARS-CoV-2S protein of SEQ ID NO: 1 or 7. 166. The formulation of claim 156 or the method of claim 156, wherein at least one of the one or more polypeptides comprises at least 85% sequence identity with the receptor-binding domain (RBD) of the SARS-CoV-2S protein of SEQ ID NO: 1 or 7. 167. The formulation of claim 156 or the method of claim 156, wherein at least one of the one or more polypeptides is or contains a receptor-binding domain (RBD) of the SARS-CoV-2S protein of SEQ ID NO: 1 or 7. 168. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein one or more mRNAs are associated with or encapsulated in the LNP. 169. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein the formulation is stored at a temperature above 25°C for a period of at least 12 weeks. 170. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123 or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139 or 141-149, wherein the formulation is stored at a temperature range of about 2-40°C for a period of at least 12 weeks. 171. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein the formulation is characterized in that, compared to the control formulation, the Z-mean of the lipid nanoparticles exhibits a change of less than 20 nm when stored at a temperature of about 2-8°C for at least 12 weeks. 172. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein the formulation is characterized in that, compared to the control formulation, the Z-mean of the lipid nanoparticles exhibits a change of less than 20 nm when stored at a temperature of about 25°C for at least 12 weeks. 173. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein the formulation is characterized in that, compared to the control formulation, the polydispersity index (PDI) of the lipid nanoparticles exhibits a change of less than 0.1 when stored at a temperature of about 2-8°C for at least 12 weeks. 174. The formulation of any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the method of any one of claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139, or 141-149, wherein the formulation is characterized in that, compared to the control formulation, the polydispersity index of the lipid nanoparticles exhibits a change of less than 0.1 when stored at a temperature of about 25°C for at least 12 weeks. 175. The formulation according to any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the formulation according to claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, 64-69. The method of any one of 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139 or 141-149, wherein the formulation is characterized in that, compared with the control formulation, when stored at a temperature of about 2-8°C for a period of at least 12 weeks, the mRNA encapsulation % is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. 176. The formulation according to any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the formulation according to claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59, or 64-69. The method of any one of 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139 or 141-149, wherein the formulation is characterized in that, compared with the control formulation, when stored at a temperature of about 25°C for a period of at least 12 weeks, the mRNA encapsulation % is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. 177. The formulation according to any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the formulation according to claims 4-9, 14-19, 24-29, 34-39, 44-49, 54-59. The method of any one of 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139 or 141-149, wherein the formulation is characterized in that, compared with the control formulation, when stored at a temperature of about 2-8°C for a period of at least 12 weeks, the mRNA expression % is at least 60%, at least 70%, at least 80% or at least 90%. 178. The formulation according to any one of claims 1-3, 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, or 131-133, or the formulation according to claims 4-9, 14-19, 24-29, 34-39, 44-49, or 54-59. The method of any one of 64-69, 74-79, 84-89, 94-99, 104-109, 114-119, 124-129, 134-139 or 141-149, wherein the formulation is characterized in that, compared with the control formulation, when stored at a temperature of about 25°C for a period of at least 12 weeks, the mRNA expression % is at least 60%, at least 70%, at least 80% or at least 90%.