Lipid nanoparticle formulations for mRNA delivery

Abstract

The present invention provides methods of encapsulating messenger RNA in lipid nanoparticles without the use of flammable solvents, and compositions produced by these methods, for mRNA delivery in therapeutic use. In particular, the present invention provides a process of encapsulating messenger RNA in lipid nanoparticles comprising a step of mixing (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids, one or more PEG-modified lipids, and a solvent, wherein the solvent comprises diethylene glycol monoethyl ether or tert-amyl alcohol, thereby forming mRNA encapsulated within the LNPs.

Description

[0001] LIPID NANOPARTICLE FORMULATIONS FOR MRNA DELIVERY

[0002] BACKGROUND OF THE INVENTION

[0003] [1] Messenger RNA (mRNA) therapy is becoming an increasingly important approach for the treatment of a variety of diseases. Messenger RNA therapy involves administration of mRNA to a patient in need of therapy for production of a protein encoded by the mRNA within the patient's body. Lipid encapsulated mRNA formulations, such as lipid nanoparticle (LNP) compositions show a high degree of cellular uptake and protein expression.

[0004] [2] Lipid nanoparticle formulations traditionally use ethanol as a solvent for the lipid solution which is then mixed with an mRNA solution. However, the use of flammable solvents such as ethanol poses safety risks and increases production costs, particularly in large-scale applications. In addition, LNP formulations that are more suitable for dosing and reduce downstream processing volumes and costs, are also currently difficult to obtain using ethanol as a solvent. Such formulations are also desirable as they permit bedside mixing to include other routes of administration, for example, subcutaneous or intramuscular.

[0005] [3] While some alternatives to ethanol have been identified, they present some drawbacks such as the dissolution of certain plastics, such as polycarbonate, during the encapsulation process and / or the degradation of some components of LNPs, such as DOPE. Triethylene glycol monomethyl ether (mTEG) has notably been previously identified as a non-flammable alternative to ethanol for the manufacturing of LNPs encapsulating mRNA (mRNA-LNPs). However, the safety profile of this polymer in humans has not yet been determined. This solvent will therefore require further testing before it can be envisaged in pharmaceutical formulations for use in humans. Thus, there remains a need for non-flammable solvents that can be used in place of ethanol in the manufacturing of mRNA-LNPs. In particular, there remains a need for non-flammable solvents that have a known safety profile or that are already actively used in pharmaceutical applications in humans, that are biocompatible and biodegradable, that are cost-effective, and / or that can be used in large-scale manufacturing without negatively impacting the activity or stability profiles of the mRNA-LNP formulations that are obtained. The identification of such solvents would allow for mRNA-LNP manufacturing to be to be used in direct therapeutic applications and could further streamline regulatory approval processes and accelerate development cycles. SUMMARY OF THE INVENTION

[0006] [4] The present invention provides, among other things, a method of encapsulating mRNA in LNPs without the use of flammable solvents that yields LNPs with high encapsulation efficiency for mRNA delivery in therapeutic applications. In one aspect, the present invention provides a safer and more cost-effective method for large-scale manufacturing of mRNA-LNPs. In another aspect, the present invention provides a method for producing LNP formulations that can both reduce downstream processing in manufacturing and be directly suitable for dosing and bedside mixing. The invention is based on the surprising discovery that mixing an mRNA solution and a lipid solution in the presence of particular solvents, i.e., diethylene glycol monoethyl ether or tert-amyl alcohol, generates mRNA encapsulated within LNPs (mRNA-LNPs) without negatively impacting the physical characteristics of the LNPs (e.g. degradation of DOPE) or mRNA translation efficiency. In addition, contrary to mTEG, these solvents advantageously do not dissolve plastics such as polycarbonate during the encapsulation process. Such solvents have also advantageously been previously described in use human pharmaceuticals, and thus have known safety profiles. Finally, such solvents are advantageously biocompatible and biodegradable. The present invention thus provides, among other things, a safe, efficient, and cost-effective process for preparing a composition comprising mRNA-loaded lipid nanoparticles. The present invention further provides compositions comprising mRNA-loaded lipid nanoparticles that can be used in pharmaceutical applications in humans.

[0007] [5] In one aspect, the present invention provides a process of encapsulating mRNA in LNPs comprising a step of mixing (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids, one or more PEG- modified lipids, and a solvent, wherein the solvent comprises diethylene glycol monoethyl ether or tert-amyl alcohol, thereby forming mRNA encapsulated within the LNPs (mRNA-LNPs).

[0008] [6] In some embodiments, the solvent comprises diethylene glycol monoethyl ether. In other embodiments, the solvent comprises tert-amyl alcohol.

[0009] [7] In some embodiments, the solvent further comprises propylene glycol, polyethylene glycol, 1,3-propanediol, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), or a combination of two or more thereof. In some embodiments, the solvent further comprises propylene glycol. In some embodiments, the solvent further comprises polyethylene glycol. In some embodiments, the solvent further comprises 1,3-propanediol. In some embodiments, the solvent further comprises PVP. In some embodiments, the solvent further comprises PVA. [8] In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% v / v. Thus, in some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 30% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 40% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 50% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 60% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 70% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 80% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 90% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 95% v / v. In some embodiments, the solvent consists of diethylene glycol monoethyl ether.

[0010] [9] In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of between 15% and 23% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of 16% to 22% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of 17% to 21% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 20% v / v. In some embodiments, the solvent comprises tertamyl alcohol at a concentration of between 15% and 23% v / v in propylene glycol. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of 16% to 22% v / v in propylene glycol. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of 17% to 21% v / v in propylene glycol. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 20% v / v in propylene glycol.

[0011]

[0010] In some embodiments, the mRNA solution and / or the lipid solution are at about ambient temperature.

[0012]

[0011] In some embodiments, the ambient temperature is less than about 35°C. In some embodiments, the ambient temperature is less than about 30°C. In some embodiments, the ambient temperature is less than about 26°C. In some embodiments, the ambient temperature is less than about 25°C. In some embodiments, the ambient temperature is less than about 24°C. In some embodiments, the ambient temperature is less than about 23°C. In some embodiments, the ambient temperature is less than about 22°C. In some embodiments, the ambient temperature is less than about 21°C. In some embodiments, the ambient temperature is less than about 20°C. In some embodiments, the ambient temperature is less than about 18°C.

[0012] In some embodiments, the ambient temperature ranges from about 18-32°C. In some embodiments, the ambient temperature ranges from about 21-26°C. In some embodiments, the ambient temperature ranges from about 23-25°C.

[0013]

[0013] In some embodiments, the ambient temperature is about 18°C. In some embodiments, the ambient temperature is about 20°C. In some embodiments, the ambient temperature is about 21°C. In some embodiments, the ambient temperature is about 22°C. In some embodiments, the ambient temperature is about 23°C. In some embodiments, the ambient temperature is about 24°C. In some embodiments, the ambient temperature is about 25°C. In some embodiments, the ambient temperature is about 26°C. In some embodiments, the ambient temperature is about 30°C. In some embodiments, the ambient temperature is about 35°C.

[0014]

[0014] In some embodiments, the process does not require a step of heating the mRNA solution and the lipid solution prior to the mixing step.

[0015]

[0015] In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 12 L of the mRNA solution. In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 10 L of the mRNA solution. In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 8 L of the mRNA solution. In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 6 L of the mRNA solution. In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 4 L of the mRNA solution. In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 2 L of the mRNA solution. In some embodiments, the mRNA solution comprises at least about 1 g of mRNA per 1 L of the mRNA solution.

[0016]

[0016] In some embodiments, the concentration of mRNA in the mRNA solution is greater than about 0.125 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than about 0.25 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than about 0.5 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than about 1.0 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than about 1.5 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than about 2.0 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is between about 0.05 mg / mL and about 0.5 mg / mL. In particular embodiments, the concentration of mRNA in the mRNA solution is between about 0.1 mg / mL to about 0.5 mg / mL, for example about 0.1 mg / mL or about 0.35 mg / mL.

[0017]

[0017] In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of between 1:1 and 10:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of between 2:1 and 6:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 2:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 3:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 4:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 5:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 6:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of greater than about 2:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of greater than about 3:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of greater than about 4:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of greater than about 5:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of greater than about 6:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of 1-8:1. In some embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of 1-4:1. In particular embodiments, the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 1:1.

[0018]

[0018] In some embodiments, the mRNA solution is a buffered solution, optionally an acidic buffered solution. In some embodiments, the mRNA solution has a pH between 3.0 and 5.5. In some embodiments, the mRNA solution has a pH between 3.0 and 5.0. In some embodiments, the mRNA solution has a pH between 3.5 and 4.5. In some embodiments, the mRNA solution has a pH of about 3.0. In some embodiments, the mRNA solution has a pH of about 3.5. In some embodiments, the mRNA solution has a pH of about 4.0. In some embodiments, the mRNA solution has a pH of about 4.5. In some embodiments, the mRNA solution has a pH of about 5.0. In some embodiments, the mRNA solution has a pH of about 5.5.

[0019]

[0019] In some embodiments, the mRNA solution comprises about 37.5 mM to about 300 mM sodium chloride (NaCI). In some embodiments, the mRNA solution comprises about 37.5 mM NaCI. In some embodiments, the mRNA solution comprises about 75 mM NaCI. In some embodiments, the mRNA solution comprises about 100 mM NaCI. In some embodiments, the mRNA solution comprises about 150 mM NaCI. In some embodiments, the mRNA solution comprises about 300 mM NaCI.

[0020]

[0020] In some embodiments, the mRNA solution comprises citrate. In some embodiments, the mRNA solution comprises less than 5 mM of citrate. In some embodiments, the mRNA solution comprises 1 mM citrate. In some embodiments, the mRNA solution comprises 1 mM citrate and 150 mM NaCI with a pH of about 4.5.

[0021] In some embodiments, the mRNA solution comprises trehalose. In some embodiments, trehalose is present at a concentration of about 5-20% v / v. In some embodiments, trehalose is present at a concentration of about 10-20% v / v. In some embodiments, the mRNA solution comprises about 20% v / v trehalose. In some embodiments, the mRNA solution comprises about 15% v / v trehalose. In some embodiments, the mRNA solution comprises about 10% v / v trehalose. In some embodiments, the mRNA solution comprises about 5% v / v trehalose.

[0021]

[0022] In some embodiments, the lipid solution comprises four lipid components.

[0022]

[0023] In some embodiments, the lipid solution further comprises one or more cholesterol-based lipids. In some embodiments, the lipid solution further comprises cholesterol.

[0023]

[0024] In some embodiments, the lipid solution comprises a PEG-modified lipid, a cationic lipid (e.g., ML-2, OF-02, cKK-ElO or GL-HEPES-E3-E12-DS-4-E10), a non-cationic lipid (e.g., DSPC or DOPE), and optionally cholesterol. In some embodiments, the molar ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is 35-55:5-35:20-40:1-15, respectively.

[0024]

[0025] In some embodiments, the lipid solution comprises cationic lipid at a molar ratio of 40%, DMG-PEG2K at a molar ratio of 1.5%, DOPE at a molar ratio of 30%, and cholesterol at a molar ratio of 28.5%. In some embodiments, the lipid solution comprises cationic lipid at a molar ratio of 40%, DMG-PEG2K at a molar ratio of 1.5%, DSPC at a molar ratio of 30%, and cholesterol at a molar ratio of 28.5%.

[0025]

[0026] In particular embodiments, the lipid solution and the mRNA solution (e.g., an aqueous solution comprising a citrate buffer) are mixed at a volumetric ratio of 1:1-4 with a final concentration of mRNA of about 0.05-0.5 mg / mL, and the ratio of cationic lipid(s) to noncationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is 35-55:25-35:20-40:1-15 (for example about 40:30:25:5), such that the cationic lipid(s) to mRNA N / P ratio is about 2-6 (e.g. about 4).

[0026]

[0027] In some embodiments, the step of mixing occurs in a total volume of between about 3 and 10 mL. In some embodiments, the step of mixing occurs in a total volume of between about 1 and 10 mL. In some embodiments, the step of mixing occurs in a total volume of between about 1 and 15 mL. In some embodiments, the step of mixing occurs in a total volume of about 1 mL. In some embodiments, the step of mixing occurs in a total volume of about 2 mL. In some embodiments, the step of mixing occurs in a total volume of about 3 mL. In some embodiments, the step of mixing occurs in a total volume of about 4 mL. In some embodiments, the step of mixing occurs in a total volume of about 5 mL. In some embodiments, the step of mixing occurs in a total volume of about 6 mL. In some embodiments, the step of mixing occurs in a total volume of about 7 mL. In some embodiments, the step of mixing occurs in a total volume of about 8 mL. In some embodiments, the step of mixing occurs in a total volume of about 9 mL. In some embodiments, the step of mixing occurs in a total volume of about 10 mL. In some embodiments, the step of mixing occurs in a total volume of about 12 mL. In some embodiments, the step of mixing occurs in a total volume of about 13 mL. In some embodiments, the step of mixing occurs in a total volume of about 14 mL. In some embodiments, the step of mixing occurs in a total volume of about 15 mL.

[0027]

[0028] In some embodiments, the process further comprises a step of incubating the mRNA-LNPs (i.e., post-mixing). In some embodiments, the mRNA-LNPs are incubated at a temperature of between 21°C and 65°C.

[0028]

[0029] In some embodiments, the mRNA-LNPs are incubated at a temperature of about 26°C, about 30°C, or about 65°C.

[0029]

[0030] In some embodiments, the mRNA-LNPs are incubated for greater than about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 120 minutes. In some embodiments, the mRNA-LNPs are incubated for greater than about 20 minutes. In some embodiments, the mRNA-LNPs are incubated for greater than about 30 minutes. In some embodiments, the mRNA-LNPs are incubated for greater than about 60 minutes. In some embodiments, the mRNA-LNPs are incubated for greater than about 90 minutes. In some embodiments, the mRNA-LNPs are incubated for greater than about 120 minutes. In a particular embodiment, the mRNA-LNPs are incubated for about 60 minutes.

[0030]

[0031] In some embodiments, the lipid solution does not comprise volatile organic compounds (VOCs). In some embodiments, the lipid solution does not comprise a flammable solvent. In some embodiments, the lipid solution does not comprise alcohol. In some embodiments, the lipid solution does not comprise ethanol.

[0031]

[0032] In some embodiments, the mRNA-LNPs are purified by tangential flow filtration (TFF).

[0032]

[0033] In some embodiments, at least about 1 g, 5 g, 10 g, 20 g, 50 g, 100 g, or 1 kg of mRNA is encapsulated in LNPs in a single batch.

[0033]

[0034] In some embodiments, the mRNA solution and the lipid solution are mixed by a pulse-less flow pump. In some embodiments, the pump is a gear pump. In some embodiments, the pump is a centrifugal pump.

[0034]

[0035] In some embodiments, the mRNA solution is mixed at a flow rate ranging from about 150-

[0035] 250 mL / min, 250-500 mL / min, 500-1000 mL / min, 1000-2000 mL / min, 2000-3000 mL / min, 3000- 4000 mL / min, 4000-5000 mL / min, 6000-8000 mL / min, 8000-10000 mL / min or 10000-12000 mL / min.

[0036]

[0036] In some embodiments, the mRNA solution is mixed at a flow rate of at least about 100 mL / min, at least about 150 mL / min, at least about 200 mL / min, at least about 250 mL / min, at least about 500 mL / min, at least about 800 mL / min, at least about 1000 mL / min, at least about 1200 mL / min, at least about 2000 mL / min, at least about 3000 mL / min, at least about 4000 mL / min, at least about 5000 mL / min, at least about 6000 mL / min, at least about 8000 mL / min, at least about 10000 mL / min, at least about 12000 mL / min, or at least about 15000 mL / min.

[0037]

[0037] In some embodiments, the mRNA solution is mixed at a flow rate of about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 400 mL / min, about 500 mL / min, about 600 mL / min, about 800 mL / min, about 1000 mL / min, about 1200 mL / min, about 1400 mL / min, about 1600 mL / min, about 1800 mL / min, about 2000 mL / min, about 2400 mL / min, about 3000 mL / min, about 4000 mL / min, about 5000 mL / min, about 6000 mL / min, about 7000 mL / min, about 8000 mL / min, about 9000 mL / min, about 10000 mL / min, about 12000 mL / min, about 15000 mL / min.

[0038]

[0038] In some embodiments, the lipid solution is mixed at a flow rate ranging from about 25-75 mL / min, about 75-200 mL / min, about 200-350 mL / min, about 350-500 mL / min, about 500-650 mL / min, about 650-850 mL / min, or about 850-1000 mL / min.

[0039]

[0039] In some embodiments, the lipid solution is mixed at a flow rate of at least about 25 mL / min, at least about 75 mL / min, at least about 200 mL / min, at least about 350 mL / min, at least about 500 mL / min, at least about 650 mL / min, at least about 850 mL / min, or at least about 1000 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 25 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 75 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 200 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 350 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 500 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 650 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 850 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of at least 1000 mL / min.

[0040]

[0040] In some embodiments, the lipid solution is mixed at a flow rate of about 50 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min, about 800 mL / min, about 850 mL / min, about 900 mL / min, about 950 mL / min, about 1000 mL / min, about 1200 mL / min, or about 1500 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 100 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 150 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 200 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 250 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 300 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 350 mL / min.

[0041]

[0041] In some embodiments, the flow rate of the mRNA solution is same as the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 2 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 3 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 4 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 4.5 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 5 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 5.5 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 6 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 8 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is at least about 10 times greater than the flow rate of the lipid solution.

[0042]

[0042] In some embodiments, the mRNA is encapsulated in a process free of VOCs. In some embodiments, the mRNA is encapsulated in a process free of flammable solvent. In some embodiments, the mRNA is encapsulated in a process free of alcohol. In some embodiments, the mRNA is encapsulated in a process free of ethanol.

[0043]

[0043] In another aspect, the present invention provides a composition comprising one or more lipid nanoparticles encapsulating mRNA (mRNA-LNPs), wherein the lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, and wherein the composition further comprises diethylene glycol monoethyl ether or tert-amyl alcohol.

[0044]

[0044] In some embodiments, the composition further comprises polyethylene glycol, propylene glycol, 1,3-propanediol, PVP, PVA, or a combination of two or more thereof. In some embodiments, the composition further comprises polyethylene glycol. In some embodiments, the composition further comprises propylene glycol. In some embodiments, the composition further comprises 1,3- propanediol. In some embodiments, the composition further comprises PVP. In some embodiments, the composition further comprises PVA.

[0045]

[0045] In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 6%, 8%, 10%, 12%, 14%, 16%, 18%, 19%, or 20% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 6% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 8% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 10% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 12% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 14% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 16% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 18% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of at least about 19% v / v. In some embodiments, the composition comprises diethylene glycol monoethyl ether at a concentration of about 6%, 8%, 10%, 12%, 14%, 16%, 18%, 19%, or 20% v / v. In some embodiments, the diethylene glycol monoethyl ether is present at a concentration of about 20% v / v.

[0046]

[0046] In some embodiments, the composition comprises tert-amyl alcohol at a concentration of 3% to 5% v / v. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 3.2% to 4.6% v / v. In some embodiments, the composition comprises tertamyl alcohol at a concentration of about 3.4% to 4.4% v / v. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 4% to 4.2% v / v. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 4% v / v.

[0047]

[0047] In some embodiments, the composition comprises tert-amyl alcohol at a concentration of 3% to 5% v / v in propylene glycol. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 3.2% to 4.6% v / v in propylene glycol. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 3.4% to 4.4% v / v in propylene glycol. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 4% to 4.2% v / v in propylene glycol. In some embodiments, the composition comprises tert-amyl alcohol at a concentration of about 4% v / v in propylene glycol.

[0048]

[0048] In some embodiments, the composition comprises at least 1 g, 5 g, 10 g, 20 g, 50 g, 100 g, or 1 kg of mRNA. In some embodiments, the composition comprises at least 1 g of mRNA. In some embodiments, the composition comprises at least 5 g of mRNA. In some embodiments, the composition comprises at least 10 g of mRNA. In some embodiments, the composition comprises at least 20 g of mRNA. In some embodiments, the composition comprises at least 50 g of mRNA. In some embodiments, the composition comprises at least 100 g of mRNA. In some embodiments, the composition comprises at least 1 kg of mRNA.

[0049]

[0049] In some embodiments, the mRNA comprises one or more modified nucleotides.

[0050]

[0050] In some embodiments, mRNA is unmodified.

[0051]

[0051] In some embodiments, the mRNA is at least about 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 8 kb, 10 kb, 20 kb, 30 kb, or 40 kb in length. In some embodiments, the mRNA is at least about 0.5 kb in length. In some embodiments, the mRNA is at least about 1 kb in length. In some embodiments, the mRNA is at least about 2 kb in length. In some embodiments, the mRNA is at least about 3 kb in length. In some embodiments, the mRNA is at least about 4 kb in length. In some embodiments, the mRNA is at least about 5 kb in length. In some embodiments, the mRNA is at least about 8 kb in length. In some embodiments, the mRNA is at least about 10 kb in length. In some embodiments, the mRNA is at least about 20 kb in length. In some embodiments, the mRNA is at least about 30 kb in length. In some embodiments, the mRNA is at least about 40 kb in length.

[0052]

[0052] In some embodiments, the mRNA-LNPs have an average size of less than 150 nm, less than 125 nm, or less than 100 nm. In some embodiments, the mRNA-LNPs have an average size less than 150 nm. In some embodiments, the mRNA-LNPs have an average size of less than 125 nm. In some embodiments, the mRNA-LNPs have an average size less than 100 nm. In some embodiments, the mRNA-LNPs have an average size less than 95 nm. In some embodiments, the mRNA-LNPs have an average size less than 90 nm. In some embodiments, the mRNA-LNPs have an average size less than 85 nm. In some embodiments, the mRNA-LNPs have an average size less than 80 nm. In some embodiments, the mRNA-LNPs have an average size less than 75 nm. In some embodiments, the mRNA-LNPs have an average size less than 70 nm.

[0053]

[0053] In some embodiments, the mRNA-LNPs have an average size ranging from 80-130 nm.

[0054]

[0054] In some embodiments, the LNPs have a PDI of less than about 0.3, about 0.2, or about 0.18. In some embodiments, the LNPs have a PDI of less than about 0.3. In some embodiments, the LNPs have a PDI of less than about 0.2. In some embodiments, the LNPs have a PDI of less than about 0.18. In some embodiments, the LNPs have a PDI of less than about 0.15. In some embodiments, the LNPs have a PDI of less than about 0.12. In some embodiments, the LNPs have a PDI of less than about 0.10.

[0055] In some embodiments, the encapsulation efficiency (EE) of the mRNA in the LNPs is greater than about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 70%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 75%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 80%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 85%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 90%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 91%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 92%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 93%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 94%. In some embodiments, the EE of the mRNA in the LNPs is greater than about 95%.

[0055]

[0056] In some embodiments, the one or more non-cationic lipids in the lipid solution or the mRNA- LNPs is selected from DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-0- dimethyl PE, 18-1-trans PE, SOPE, DOPS, DEPE, DPOC, DLPC, DLPE, or a combination of two or more thereof.

[0056]

[0057] In some embodiments, the non-cationic lipid is DSPC. In some embodiments, the noncationic lipid is DOPC. In some embodiments, the non-cationic lipid is DPPC. In some embodiments, the non-cationic lipid is DOPG. In some embodiments, the non-cationic lipid is DPPG. In some embodiments, the non-cationic lipid is DOPE. In some embodiments, the non-cationic lipid is POPC. In some embodiments, the non-cationic lipid is POPE. In some embodiments, the non-cationic lipid is DOPE-mal. In some embodiments, the non-cationic lipid is DPPE. In some embodiments, the noncationic lipid is DMPE. In some embodiments, the non-cationic lipid is DSPE. In some embodiments, the non-cationic lipid is phosphatidylserine. In some embodiments, the non-cationic lipid is sphingolipid. In some embodiments, the non-cationic lipid is cerebrosides. In some embodiments, the non-cationic lipid is gangliosides. In some embodiments, the non-cationic lipid is 16-O- monomethyl PE. In some embodiments, the non-cationic lipid is 16-O-dimethyl PE. In some embodiments, the non-cationic lipid is 18-1-trans PE. In some embodiments, the non-cationic lipid is SOPE. In some embodiments, the non-cationic lipid is DOPS. In some embodiments, the noncationic lipid is DEPE. In some embodiments, the non-cationic lipid is DPOC. In some embodiments, the non-cationic lipid is DLPC. In some embodiments, the non-cationic lipid is DLPE.

[0057]

[0058] In some embodiments, the one or more cationic lipids is selected from DOTMA, DOTAP, DSDMA, ML-2, cKK-ElO, CKK-E12, OF-02, C12-200, DLinDMA, DLinkC2DMA, ICE (Imidazol-based), HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4004, HGT4005, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DLenDMA, CLinDMA, Octyl-CLinDMA, Octyl- CLinDMA (2R), Octyl-CLinDMA (2S), ALNY-100, NC98-5, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLin-K-DMA, DLin-K-XTC2-DMA, cDD-TE-4-E10, CDD-TE-4-E12, DLin-MC3- DMA, DLin-KC2-DMA, Dlin-DMA, L319, SM-102, ALC-0315, DC-Chol, 3060110, 9A1P9, A2-lso5- 2DC18, BAME-O16B, FTT5, OF-Deg-Lin, TT3, Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5-tricarboxamide, MVL5, Lipid 5, ATX-126, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS- 4-E10, GL-HEPES-E3-E12-DS-3-E14, IM-001, IS-001, or a combination of two or more thereof. In some embodiments, the one or more cationic lipids is ML-2, OF-02, cKK-ElO, GL-HEPES-E3-E12-DS- 4-E10, IM-001, IS-001, or a combination of two or more thereof. In some embodiments, the one or more cationic lipids is ML-2, OF-02, cKK-ElO, GL-HEPES-E3-E12-DS-4-E10 or a combination of two or more thereof.

[0058]

[0059] In some embodiments, the cationic lipid is DOTMA. In some embodiments, the cationic lipid is DOTAP. In some embodiments, the cationic lipid is DSDMA. In some embodiments, the cationic lipid is ML-2. In some embodiments, the cationic lipid is cKK-ElO. In some embodiments, the cationic lipid is CKK-E12. In some embodiments, the cationic lipid is OF-02. In some embodiments, the cationic lipid is C12-200. In some embodiments, the cationic lipid is DLinDMA. In some embodiments the cationic lipid is DLinkC2DMA. In some embodiments, the cationic lipid is ICE (Imidazol-based). In some embodiments, the cationic lipid is HGT5000. In some embodiments, the cationic lipid is HGT5001. In some embodiments, the cationic lipid is HGT5002. In some embodiments, the cationic lipid is HGT4001. In some embodiments, the cationic lipid is HGT4002. In some embodiments, the cationic lipid is HGT4003. In some embodiments, the cationic lipid is DODAC. In some embodiments, the cationic lipid is DDAB. In some embodiments, the cationic lipid is DMRIE. In some embodiments, the cationic lipid is DOSPA. In some embodiments, the cationic lipid is DOGS. In some embodiments, the one or more cationic lipids is DODAP. In some embodiments, the one or more cationic lipids is DODMA. In some embodiments, the cationic lipid is DMDMA. In some embodiments, the cationic lipid is DLenDMA. In some embodiments, the cationic lipid is CLinDMA. In some embodiments, the cationic lipid is Octyl-CLinDMA. In some embodiments, the cationic lipid is Octyl-CLinDMA (2R). In some embodiments, the cationic lipid is Octyl-CLinDMA (2S). In some embodiments, the cationic lipid is ALNY-100. In some embodiments, the cationic lipid is NC98-5. In some embodiments, the cationic lipid is CpLinDMA. In some embodiments, the cationic lipid is DMOBA. In some embodiments, the cationic lipid is DOcarbDAP. In some embodiments, the cationic lipid is DLinDAP. In some embodiments, the cationic lipid is DLincarbDAP. In some embodiments, the cationic lipid is DLinCDAP. In some embodiments, the cationic lipid is DLin-K-DMA. In some embodiments, the cationic lipid is DLin-K-XTC2-DMA. In some embodiments, the cationic lipid is cDD-TE-4-E10. In some embodiments, the cationic lipid is cDD- TE-4-E12. In some embodiments, the cationic lipid is DLin-MC3-DMA. In some embodiments, the cationic lipid is DLin-KC2-DMA. In some embodiments the cationic lipid is Dlin-DMA. In some embodiments, the cationic lipid is L319. In some embodiments, the cationic lipid is SM-102. In some embodiments, the cationic lipid is ALC-0315. In some embodiments, the cationic lipid is DC-Chol. In some embodiments, the cationic lipid is 3060110. In some embodiments, the cationic lipid is 9A1P9. In some embodiments, the cationic lipid is A2-lso5-2DC18. In some embodiments, the cationic lipid is BAME-O16B. In some embodiments, the cationic lipid is FTT5. In some embodiments, the cationic lipid is OF-Deg-Lin. In some embodiments, the cationic lipid is TT3. In some embodiments, the cationic lipid is Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene-l,3,5-tricarboxamide, MVL5, Lipid 5, ATX-126, GL-HEPES-E3-E10-DS-3-E18-1. In some embodiments, the cationic lipid is GL- HEPES-E3-E12-DS-4-E10. In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14. In some embodiments, the cationic lipid is IM-001. In some embodiments, the cationic lipid is IS-001. In some embodiments, the cationic lipid is a combination of two or more of said cationic lipids.

[0059]

[0060] In some embodiments, the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, the PEG-modified lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0060]

[0061] In some embodiments, the LNPs comprise four lipid components.

[0061]

[0062] In some embodiments, the LNPs further comprise one or more cholesterol-based lipids, optionally cholesterol.

[0062]

[0063] In some embodiments, the LNPs comprise a PEG-modified lipid, a cationic lipid (e.g., ML-2, OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10), a non-cationic lipid (e.g., DSPC or DOPE), and optionally cholesterol. In some embodiments, the molar ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is 35-55:5-35:20-40:1-15, respectively.

[0063]

[0064] In some embodiments, the lipid solution comprises cationic lipid at a molar ratio of 40%, DMG-PEG2K at a molar ratio of 1.5%, DOPE at a molar ratio of 30%, and cholesterol at a molar ratio of 28.5%. In some embodiments, the LNPs comprises cationic lipid at a molar ratio of 40%, DMG- PEG2K at a molar ratio of 1.5%, DSPC at a molar ratio of 30%, and cholesterol at a molar ratio of 28.5%.

[0064]

[0065] In some embodiments, the composition further comprises trehalose.

[0066] In some embodiments, the mRNA-LNPs have an N / P ratio of between 1 and 10. In some embodiments, the mRNA-LNPs have an N / P ratio from 2 to 6. In some embodiments, the mRNA- LNPs have an N / P ratio of about 2. In some embodiments, the mRNA-LNPs have an N / P ratio of about 4. In some embodiments, the mRNA-LNPs have an N / P ratio of about 6. In particular embodiments, the mRNA solution and lipid solution are mixed at a N / P ratio of about 4. As shown in the examples, such an N / P ratio yielded LNPs of suitable size and encapsulation efficiencies for therapeutic use.

[0065]

[0067] In some embodiments, the composition is free of VOCs. In some embodiments, the composition does not comprise a flammable solvent. In some embodiments, the composition does not comprise alcohol. In some embodiments, the composition does not comprise ethanol.

[0066]

[0068] In particular embodiments, the four lipid components of the lipid solution or the composition are a PEG-modified lipid, a cationic lipid (e.g., ML-2, OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10), cholesterol, and a helper (e.g., non-cationic) lipid (e.g., DSPC or DOPE). In particular embodiments, the lipid components are a PEG-modified lipid, ML-2, cholesterol, and DSPC. In particular embodiments, the lipid components are a PEG-modified lipid, ML-2, cholesterol, and DOPE. In particular embodiments, the lipid components are a PEG-modified lipid, OF-02, cholesterol, and DSPC. In particular embodiments, the lipid components are a PEG-modified lipid, a cationic lipid OF- 02, cholesterol, and lipid DOPE. In particular embodiments, the four lipid components are a PEG- modified lipid, a cationic lipid cKK-ElO, cholesterol, and DSPC. In particular embodiments, the lipid components are a PEG-modified lipid, cKK-ElO, cholesterol, and DOPE. In particular embodiments, the lipid components are a PEG-modified lipid, GL-HEPES-E3-E12-DS-4-E10, cholesterol, and DSPC. In particular embodiments, the lipid components are a PEG-modified lipid, GL-HEPES-E3-E12-DS-4- E10, cholesterol, and DOPE.

[0067]

[0069] In another aspect, the present invention provides a composition comprising mRNA-LNPs prepared by the process provided herein.

[0068]

[0070] In another aspect, the composition comprising mRNA-LNPs provided herein is used for treating a disease in a subject.

[0069]

[0071] In another aspect, a kit of parts is provided, said kit of parts comprising a first container and a second container, wherein the first container comprises an mRNA solution comprising one or more mRNAs as provided herein and the second container comprises a lipid solution as provided herein.

[0070]

[0072] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072]

[0073] The following figures are for illustration purposes only and not for limitation.

[0073]

[0074] FIG. 1 depicts the level of in vitro protein expression obtained when mRNA coding for human erythropoietin (hEPO) was transfected into HeLa cells in an LNP or lipofectamine formulation. hEPO mRNA was encapsulated in an LNP composed of a cationic lipid, DOPE, cholesterol, and DMG-PEG2K by mixing an aqueous solution of mRNA with lipids dissolved in ethanol (group 1) or 20% v / v TAA- PG (group 2). Group 3 corresponds to mRNA administered with lipofectamine (positive control). Panels A and B show the level of expression obtained with mRNA-LNPs formulated using ML-2 or cKK-ElO, respectively, as the cationic lipid. Individual points correspond to independent in vitro transfections (n=6).

[0074]

[0075] FIG. 2 depicts the level of in vivo expression of hEPO mRNA in mice 24 hours after a single intramuscular injection of hEPO mRNA-LNPs. hEPO mRNA was encapsulated in an LNP composed of a cationic lipid, DOPE, cholesterol, and DMG-PEG2K by mixing an aqueous solution of mRNA with lipids dissolved in ethanol (groups 1, 3, 5, and 7) or 20% v / v TAA-PG (groups 2, 4, 6, and 8). mRNA- LNPs contained ML-2 (groups 1, 2), OF-02 (groups 3, 4), cKK-ElO (groups 5, 6), or GL-HEPES-E3-E12- DS-4-E10 (groups 7, 8) as the cationic lipid. Error bars correspond to the standard deviation (n=3).

[0075]

[0076] FIG. 3 depicts neutralizing antibody titers in mice as measured by the hemagglutination inhibition (HAI) assay and expressed as geometric mean titers (GMTs) following intramuscular injection of hemagglutinin (HA) mRNA-LNPs. HA mRNA-LNPs were obtained by mixing an aqueous solution of mRNA with lipids dissolved in ethanol, 20% v / v TAA in PG ("TAA"), or diethylene glycol monoethyl ether ("TP"). mRNA-LNPs contained either cKK-ElO (panel A) or GL-HEPES-E3-E12-DS- 4-E10 (panel B) as the cationic lipid. Each point on the graph represents an individual mouse titer value. The bars and error bars represent the geometric mean with 95% confidence intervals, respectively.

[0076]

[0077] FIG. 4 depicts injection site reactogenicity in mice following intramuscular injection of hemagglutinin mRNA-LNPs that were obtained in an ethanol-based process ("EtOH") or an ethanol- free process using 20% v / v TAA in PG ("TAA") or diethylene glycol monoethyl ether ("TP") in place of ethanol. The three bars for each condition correspond to reactogenicity measurements taken at days 22, 23, and 24, respectively, immediately following the second injection on day 21. Reactogenicity was indicated as an Edema score on a grading scale of 0 to 5, with a score of 0 indicating of no swelling and a score of 5 indicative of significant severe swelling requiring veterinary intervention. mRNA-LNPs contained either cKK-ElO (panel A) or GL-HEPES-E3-E12-DS-4- E10 (panel B) as the cationic lipid.

[0077]

[0078] FIG. 5 depicts stability of mRNA-LNPs that were obtained using an ethanol-based process ("EtOH") or an ethanol-free process using 20% v / v TAA in PG ("TAA", see panels A, B, C) or diethylene glycol monoethyl ether ("TP", see panels D, E, F) in place of ethanol after one month at -80°C. mRNA-LNP stability was determined by measuring particle size (panels A, D), percent mRNA integrity (panels B, E), and percent encapsulation efficiency ("EE"; panels C, F). mRNA-LNPs contained either cKK-ElO ("CL1") or GL-HEPES-E3-E12-DS-4-E10 ("CL2") as the cationic lipid.

[0078]

[0079] FIG. 6 depicts stability of mRNA-LNPs that were obtained using an ethanol-based process ("EtOH") or an ethanol-free process using 20% v / v TAA in PG ("TAA", see panels A, B, C) or diethylene glycol monoethyl ether ("TP", see panels D, E, F) over the course of five days. Briefly, the formulations were stored at either -80°C or 25°C and subjected to freeze thaw cycles, with one cycle per day over the course of 5 days. mRNA-LNP stability was determined by measuring particle size (panels A, D), percent mRNA integrity (panels B, E), and percent encapsulation efficiency ("EE"; panels C, F). mRNA-LNPs contained either cKK-ElO ("CL1") or GL-HEPES-E3-E12-DS-4-E10 ("CL2") as the cationic lipid.

[0079] DETAILED DESCRIPTION

[0080]

[0080] The present disclosure is directed to, inter alia, methods of encapsulating mRNA in LNPs without the use of ethanol or other flammable solvents, and compositions produced by these methods, for mRNA delivery in therapeutic use. Accordingly, this disclosure provides methods of making and using stable, safe, cost-effective ethanol-free LNP formulations that have a high mRNA encapsulation efficiency for efficient mRNA delivery for therapeutic use.

[0081]

[0081] In order for the present invention to be more readily understood, certain terms are defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0082]

[0082] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, may provide one of skill with a general dictionary of many of the terms used in this disclosure. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0083]

[0083] Throughout this specification and embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It is further understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0084]

[0084] It is to be noted that the terms "a" or "an" refer to one or more of the corresponding entity. For example, "an LNP" is understood to represent one or more LNPs. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0085]

[0085] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In this application, the use of "or" means "and / or" unless stated otherwise.

[0086]

[0086] Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0087] The term "approximately" or "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% or ±0.001%. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "approximately" or "about".

[0087]

[0088] As used herein, the term "batch" refers to a quantity or amount of mRNA-LNPs produced at one time, e.g., produced according to a single manufacturing order during the same cycle of manufacture.

[0088]

[0089] As used herein, the term "mixing" is interchangeably used with combining or blending. Mixing refers to putting together two or more discrete solutions having different compositions or properties, for example, combining an mRNA solution with a lipid solution, to obtain a composition of mRNA encapsulated in LNPs. In some embodiments, the combining of the two solutions is performed at a specific ratio of the components being combined. In some embodiments, the resultant composition obtained from the combining has a property distinct from any one or both of its components.

[0089]

[0090] As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery). In some embodiments, delivery is pulmonary delivery, e.g., comprising nebulization.

[0090]

[0091] As used herein, the term "dsRNA" refers to the production of complementary RNA sequences during an in vitro transcription (IVT) reaction. Complimentary RNA sequences can be produced for a variety of reasons including, for example, short abortive transcripts that can hybridize to complimentary sequences in the nascent RNA strand, short abortive transcripts acting as primers for RNA dependent DNA independent RNA transcription, and possible RNA polymerase template reversal.

[0092] As used herein, the term "efficacy," or grammatical equivalents, refers to an improvement of a biologically relevant endpoint, as related to delivery of mRNA that encodes a relevant protein or peptide.

[0091]

[0093] As used herein, the term "encapsulation," or its grammatical equivalents, refers to the process of confining a nucleic acid molecule within a nanoparticle.

[0092]

[0094] As used herein, "expression" of a nucleic acid sequence refers to the translation of an mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody), and / or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms "expression" and "production," and grammatical equivalent, are used interchangeably when used in an in vivo context.

[0093]

[0095] As used herein, the term "flammable solvent" refers to a liquid solvent having a flash point below 37.8°C. Examples of flammable solvents include, but are not limited to, ethanol, acetone, and benzene.

[0094]

[0096] As used herein, the term "flow rate" refers to the volume of fluid that passes a given point within a given period of time, and may be expressed as (volume) / (time) e.g., mL / min.

[0095]

[0097] As used herein, the terms "improve," "increase" or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0096]

[0098] As used herein, the term "impurities" refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound.

[0097]

[0099] As used herein, the term "in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, etc., rather than within a multi-cellular organism.

[0098]

[0100] As used herein, the term "in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0099]

[0101] The terms "isolated," "isolating," "purified," "purifying," "enriched," and "enriching," when used herein with respect to an mRNA of interest, indicate that the mRNA of interest at some point in time was separated, enriched, sorted, etc., from or with respect to other biological material or chemical components, such as components of an IVT reaction, to yield a higher proportion of the mRNA of interest compared to the other biological material, chemical components, contaminates, or active agents such as enzymes. Isolated mRNA may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which it was initially associated. In some embodiments, isolated mRNA is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. Components from which mRNA may be isolated notably include dsRNA, plasmid DNA, enzymes, and endotoxins. As used herein, calculation of percent purity of isolated substances should not include excipients (e.g., buffer, water, etc.).

[0100]

[0102] As used herein, the term "liposome" or "LNP" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, an LNP as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). In some embodiments, an LNP liposome suitable for the present invention contains one or more cationic lipids and, optionally, one or more noncationic lipids, one or more PEG-modified lipids, and / or one or more cholesterol-based lipid(s).

[0101]

[0103] As used herein, the terms "local distribution," "local delivery," or grammatical equivalents, refer to tissue specific delivery or distribution. Typically, local distribution or delivery requires a peptide or protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulation system. In contrast, the terms "systemic distribution," "systemic delivery," or grammatical equivalents, refer to a delivery or distribution mechanism or approach that affects the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream.

[0102]

[0104] As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. mRNA may contain one or more coding and non-coding regions. A coding region is alternatively referred to as an open reading frame (ORF). mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. mRNA as used herein encompasses both modified and unmodified RNA. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA may contain one or more modifications that typically enhance RNA stability. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C-5 propynyl-cytidine, C-5 propynyl-uridine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-cytidine, C5-methylcytidine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2- thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2' -fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'- / V-phosphoramidite linkages).

[0103]

[0105] As used herein, the term "mRNA integrity" generally refers to the quality of mRNA. In some embodiments, mRNA integrity refers to the percentage of mRNA that is not degraded after a purification process. mRNA integrity may be determined using methods well known in the art, for example, by RNA agarose gel electrophoresis (e.g., Ausubel et al., John Weley & Sons, Inc., 1997, Current Protocols in Molecular Biology) or capillary gel electrophoresis (CGE).

[0104]

[0106] As used herein, the term "N / P ratio" refers to a molar ratio of positively charged molecular units in the cationic lipids in a lipid nanoparticle relative to negatively charged molecular units in the mRNA encapsulated within that lipid nanoparticle. As such, N / P ratio is typically calculated as the ratio of moles of amine groups in cationic lipids in a lipid nanoparticle relative to moles of phosphate groups in mRNA encapsulated within that lipid nanoparticle. For example, a 4-fold molar excess of cationic lipid per mol mRNA is referred to as an "N / P ratio" of about 4.

[0105]

[0107] As used herein, the terms "nucleic acid," "nucleic acid molecule" and the like refer to a polymer of nucleotides of any length. "Nucleic acid" encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences that encode proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs such as those described herein, chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioates and S'-N- phosphoramidite linkages). In some embodiments, the present invention is specifically directed to "unmodified nucleic acids," meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.

[0106]

[0108] The term "pharmaceutically acceptable" as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0107]

[0109] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C4al kyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.

[0110] The term "polydispersity index (PDI)" as used herein is a dimensionless parameter that describes the uniformity of particle size distribution in a sample. A lower PDI, such as less than 0.3, indicates a narrow distribution of particle sizes.

[0108] [Hl] The terms "prematurely aborted RNA sequences", "short abortive RNA species", "shortmers", and "long abortive RNA species" as used herein, refer to incomplete products of an mRNA synthesis reaction (e.g., an in vitro synthesis reaction). For a variety of reasons, RNA polymerases do not always complete transcription of a DNA template; e.g., RNA synthesis terminates prematurely. Possible causes of premature termination of RNA synthesis include quality of the DNA template, polymerase terminator sequences for a particular polymerase present in the template, degraded buffers, temperature, depletion of ribonucleotides, and mRNA secondary structures. Prematurely aborted RNA sequences may be any length that is less than the intended length of the desired transcriptional product. For example, prematurely aborted mRNA sequences may be less than 1000 bases, less than 500 bases, less than 100 bases, less than 50 bases, less than 40 bases, less than 30 bases, less than 20 bases, less than 15 bases, or less than 10 bases in length.

[0109]

[0112] As used herein the term "salt" refers to an ionic compound that does or may result from a neutralization reaction between an acid and a base.

[0110]

[0113] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0111]

[0114] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0112]

[0115] As used herein, the term "substantially free" refers to a state in which relatively little or no amount of a given substance (e.g., shortmers) is present. For example, "substantially free of shortmers" means the shortmers are present at a level less than approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (w / w) of the impurity. Alternatively, "substantially free of shortmers" means the shortmers are present at a level less than about 100 ng, 90 ng, 80 ng, 70 ng, 60 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 1 ng, 500 pg, 100 pg, 50 pg, 10 pg, or less.

[0113]

[0116] As used herein, the term "solvent" refers to a non-aqueous liquid that is capable of dissolving lipids, thereby forming a lipid solution.

[0114]

[0117] As used herein, the term "target tissues" refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease- associated pathology, symptom, or feature.

[0115]

[0118] As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0116]

[0119] The terms "treat," "treatment," or "treating" as used herein, refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0117]

[0120] As used herein, the term "volatile organic compound (VOC)" refers to an organic compound having a composition such that evaporation occurs under normal indoor atmospheric temperature and atmospheric pressure conditions. In particular, a VOC refers to any organic compound having a vapor pressure greater than 2 Torr (0.27 kPa) at 25°C, and may be e.g., a low molecular weight alcohol and / or ether.

[0121] As used herein, the term "yield" refers to the percentage of mRNA recovered after encapsulation as compared to the total mRNA as starting material. In some embodiments, the term "recovery" is used interchangeably with the term "yield".

[0118]

[0122] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention.

[0119] Lipid nanoparticles encapsulating mRNA (mRNA-LNPs)

[0120]

[0123] The method of encapsulating mRNA into LNPs disclosed herein can be applied to various techniques, which are presently known in the art. Various methods are described in U.S. publication nos. US 2011 / 0244026, US 2016 / 0038432, US 2018 / 0153822, US 2018 / 0125989, US 2021 / 0046192, and US 2021 / 353556, and can be used to practice the present invention. One exemplary process entails encapsulating mRNA by mixing it with a lipid solution, without first pre-forming the lipids into LNPs, as described in US 2016 / 0038432. Another exemplary process entails encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in US 2018 / 0153822. In this case, the pre-formed LNPs may be obtained by mixing an aqueous solution that does not comprise mRNA with a lipid solution as provided herein.

[0121]

[0124] For the delivery of nucleic acids, achieving high encapsulation efficiencies is important to protect the drug substance (e.g., mRNA) and reduce loss of activity in vivo. Thus, enhancement of expression of a protein or peptide encoded by the mRNA and its therapeutic effect is highly correlated with mRNA encapsulation efficiency.

[0122]

[0125] To achieve high encapsulation efficiency, the process of preparing mRNA-LNPs typically includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed LNPs, the solution comprising the mRNA, and the mixed solution comprising the LNP-encapsulated mRNA, as provided herein. As described in U.S. publication no. US 2016 / 0038432, heating one or more solutions increases mRNA encapsulation efficiency and recovery rate. The process of encapsulation typically includes 10-100 mM citrate as a buffer in mRNA and / or lipid solutions. Alternatively, a high encapsulation rate can be achieved in a process without heating the mRNA and / or the lipid solutions prior to mixing, by using a low concentration of citrate (i.e., < 5mM) in the mRNA solution. mRNA Solution

[0123]

[0126] Various methods may be used to prepare an mRNA solution suitable for the present invention. In some embodiments, mRNA may be directly dissolved in a buffer solution described herein. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock 1 solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation.

[0124]

[0127] In some embodiments, a suitable mRNA stock solution may contain mRNA in an aqueous solution (e.g. water or a buffer) at a concentration at or greater than about 0.125 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or 1.6 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, or 5.0 mg / mL. In some embodiments, a suitable mRNA stock solution contains the mRNA at a concentration at or greater than about 1 mg / mL, about 10 mg / mL, about 50 mg / mL, or about 100 mg / mL. In some embodiments, the mRNA stock solution contains mRNA in an aqueous solution (e.g., water or a buffer) at a concentration of between about 0.05 mg / mL and about 0.5 mg / mL. In particular embodiments, the mRNA stock solution contains mRNA in an aqueous solution (e.g., water or a buffer) at a concentration of about 0.1 mg / mL to about 0.5 mg / mL, for example about 0.1 mg / mL or about 0.35 mg / mL.

[0125]

[0128] Typically, a suitable mRNA solution may also contain a buffering agent and / or salt. Generally, buffering agents can include HEPES, Tris(hydroxymethyl)aminomethane (Tris), ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, suitable concentration of the buffering agent may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, suitable concentrations of the buffering agent may range from 2.0 mM to 4.0 mM.

[0126]

[0129] In some embodiments, a buffer solution comprises less than about 5 mM of citrate. In some embodiments, a buffer solution comprises less than about 3 mM of citrate. In some embodiments, a buffer solution comprises less than about 1 mM of citrate. In some embodiments, a buffer solution comprises less than about 0.5 mM of citrate. In some embodiments, a buffer solution comprises less than about 0.25 mM of citrate. In some embodiments, a buffer solution comprises less than about 0.1 mM of citrate. In some embodiments, a buffer solution des not comprise citrate.

[0127]

[0130] Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in an mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. Salt concentration in a suitable mRNA solution is or is greater than about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM,

[0128] 90 mM, or 100 mM.

[0129]

[0131] In some embodiments, a buffer solution comprises about 300 mM NaCI. In some embodiments, a buffer solution comprises about 200 mM NaCI. In some embodiments, a buffer solution comprises about 175 mM NaCI. In some embodiments, a buffer solution comprises about 150 mM NaCI. In some embodiments, a buffer solution comprises about 100 mM NaCI. In some embodiments, a buffer solution comprises about 75 mM NaCI. In some embodiments, a buffer solution comprises about 50 mM NaCI. In some embodiments, a buffer solution comprises about 25 mM NaCI.

[0130]

[0132] In some embodiments, a suitable mRNA solution may have a pH ranging from about 3.0-6.5, 3.0-6.0, 3.0-5.5., 3.0-5.0, 3.0-4.5, 3.5-6.5, 3.5-6.0, 3.5-5.5., 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. In some embodiments, a suitable mRNA solution may have a pH of or no greater than about 3.0, 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, or 6.5.

[0131]

[0133] In some embodiments, a buffer solution has a pH of about 5.0. In some embodiments, a buffer solution has a pH of about 4.8. In some embodiments, a buffer solution has a pH of about 4.7. In some embodiments, a buffer solution has a pH of about 4.6. In some embodiments, a buffer solution has a pH of about 4.5. In some embodiments, a buffer solution has a pH of about 4.4. In some embodiments, a buffer solution has a pH of about 4.3. In some embodiments, a buffer solution has a pH of about 4.2. In some embodiments, a buffer solution has a pH of about 4.1. In some embodiments, a buffer solution has a pH of about 4.0. In some embodiments, a buffer solution has a pH of about 3.9. In some embodiments, a buffer solution has a pH of about 3.8. In some embodiments, a buffer solution has a pH of about 3.7. In some embodiments, a buffer solution has a pH of about 3.6. In some embodiments, a buffer solution has a pH of about 3.5. In some embodiments, a buffer solution has a pH of about 3.4. In some embodiments, a buffer solution has a pH of about 3.3. In some embodiments, a buffer solution has a pH of about 3.2. In some embodiments, a buffer solution has a pH of about 3.4. In some embodiments, a buffer solution has a pH of about 3.1. In some embodiments, a buffer solution has a pH of about 3.0.

[0132]

[0134] In some embodiments, a mRNA solution may contain mRNA at a concentration at or greater than about 0.125 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL or 1.0 mg / mL. In some embodiments, the mRNA solution contains mRNA at a concentration of between about 0.05 mg / mL and about 1 mg / mL, e.g., at a concentration of between about 0.05 0.5 mg / mL. In particular embodiments, the mRNA solution contains mRNA at a concentration of about 0.1 mg / mL to about 0.5 mg / mL, for example about 0.1 mg / mL or about 0.35 mg / mL.

[0133]

[0135] In some embodiments, the mRNA solution is obtained by mixing an mRNA stock solution with a buffer solution. In some embodiments, an mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to pulse-less flow pumps, gear pumps, peristaltic pumps and centrifugal pumps.

[0134]

[0136] Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging between about 100-6000 mL / min (e.g., about 100-300 mL / min, 300-600 mL / min, 600-1200 mL / min, 1200-2400 mL / min, 2400-3600 mL / min, 3600-4800 mL / min, 4800-6000 mL / min, or 60-420 mL / min). In some embodiments, a buffer solution is mixed at a flow rate of or greater than about 60 mL / min, 100 mL / min, 140 mL / min, 180 mL / min, 220 mL / min, 260 mL / min, 300 mL / min, 340 mL / min, 380 mL / min, 420 mL / min, 480 mL / min, 540 mL / min, 600 mL / min, 1200 mL / min, 2400 mL / min, 3600 mL / min, 4800 mL / min, or 6000 mL / min.

[0135]

[0137] In some embodiments, an mRNA stock solution is mixed at a flow rate ranging between about 10-600 mL / min (e.g., about 5-50 mL / min, about 10-30 mL / min, about 30-60 mL / min, about 60-120 mL / min, about 120-240 mL / min, about 240-360 mL / min, about 360-480 mL / min, or about 480-600 mL / min). In some embodiments, an mRNA stock solution is mixed at a flow rate of or greater than about 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 60 mL / min, 80 mL / min, 100 mL / min, 200 mL / min, 300 mL / min, 400 mL / min, 500 mL / min, or 600 mL / min.

[0136]

[0138] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging between about 10-30 mL / min, about 30-60 mL / min, about 60-120 mL / min, about 120-240 mL / min, about 240-360 mL / min, about 360-480 mL / min, or about 480-600 mL / min. In some embodiments, the mRNA stock solution is mixed at a flow rate of about 20 mL / min, about 40 mL / min, about 60 mL / min, about 80 mL / min, about 100 mL / min, about 200 mL / min, about 300 mL / min, about 400 mL / min, about 500 mL / min, or about 600 mL / min.

[0137]

[0139] In some embodiments, the mRNA solution is at an ambient temperature. In some embodiments, an mRNA solution is at a temperature of about 20-25 °C. In some embodiments, the mRNA solution is at a temperature of about 18-32°C. In some embodiments, the mRNA solution is at a temperature of about 20-25 °C. In some embodiments, the mRNA solution is at a temperature of about 21-26°C. In some embodiments, the mRNA solution is at a temperature of about 23-25°C. In some embodiments, the mRNA solution is not heated prior mixing with a lipid solution. In some embodiments, the mRNA solution is kept at an ambient temperature. In some embodiments, the mRNA solution is at ambient temperature both prior to mixing and during the step of mixing with a lipid solution.

[0138] Lipid Solution

[0139]

[0140] According to the present invention, a lipid solution contains a mixture of lipids suitable to form lipid nanoparticles for encapsulation of mRNA. According to the present invention, a suitable lipid solution does not contain ethanol (i.e. it is ethanol-free). In some embodiments, the lipid solution does not contain any other flammable organic solvent (e.g., isopropanol).

[0140]

[0141] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of or greater than about 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL, 6.0 mg / mL, 7.0 mg / mL, 8.0 mg / mL, 9.0 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, or 100 mg / mL. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg / mL, 0.5-90 mg / mL, 1-80 mg / mL, 1-70 mg / mL, 1-60 mg / mL, 1-50 mg / mL, 1-40 mg / mL, 1-30 mg / mL, 1-20 mg / mL, 1-15 mg / mL, 1-10 mg / mL, 1-9 mg / mL, 1-8 mg / mL, 1-7 mg / mL, 1-6 mg / mL, or 1-5 mg / mL. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration up to about 100 mg / mL, 90 mg / mL, 80 mg / mL, 70 mg / mL, 60 mg / mL, 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL, or 10 mg / mL.

[0141]

[0142] Any desired lipids may be mixed at any ratios suitable for encapsulating mRNAs. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g., non cationic lipids and / or cholesterol lipids), and / or PEG-modified lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g., non cationic lipids and / or cholesterol lipids), one or more PEG-modified lipids. In some embodiments, the lipid solution comprises three lipid components. In some embodiments, the lipid solution comprises four lipid components. In some embodiments, the lipid solution comprises five lipid components. In particular embodiments, the three or four lipid components of the lipid solution are a PEG-modified lipid, a cationic lipid (e.g ML- 2, OF-02, cKK-ElO or GL-HEPES-E3-E12-DS-4-E10), a helper (e.g., non-cationic) lipid (e.g., DSPC or DOPE), and, optionally, a cholesterol-based lipid (e.g., cholesterol). In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, ML-2, cholesterol, and DSPC. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, ML-2, cholesterol, and DOPE. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, OF-02, cholesterol, and DSPC. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, OF-02, cholesterol, and lipid DOPE. In particular embodiments, the four lipid components of the lipid solution are a PEG- modified lipid, cKK-ElO, cholesterol, and DSPC. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, cKK-ElO, cholesterol, and DOPE. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, GL-HEPES-E3-E12-DS-4-E10, cholesterol, and DSPC. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, GL-HEPES-E3-E12-DS-4-E10, cholesterol, and DOPE. In particular embodiments, the four lipid components of the lipid solution are a PEG- modified lipid, IM-100, cholesterol, and DSPC. In particular embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, IM-100, cholesterol, and DOPE. The lipid components of the lipid solution are dissolved in a solvent. In the context of the present invention, the lipid components are dissolved in a solvent comprising diethylene glycol monoethyl ether or tert-amyl alcohol.

[0142]

[0143] In some embodiments, a lipid solution is at an ambient temperature. In some embodiments, a lipid solution is at a temperature of about 18-32°C. In some embodiments, a lipid solution is at a temperature of about 21-26°C. In some embodiments, a lipid solution is at a temperature of about 23-25 °C. In some embodiments, a lipid solution is not heated prior mixing with a mRNA solution. In some embodiments, a lipid solution is kept at an ambient temperature.

[0143]

[0144] In certain embodiments, provided compositions comprise a LNP wherein the mRNA is associated on both the surface of the LNP and encapsulated within the same LNP. For example, during preparation of the compositions of the present invention, cationic LNPs may associate with the mRNA through electrostatic interactions.

[0144]

[0145] In some embodiments, the compositions and methods of the invention comprise mRNA encapsulated in an LNP (mRNA-LNPs). In some embodiments, the one or more mRNA species may be encapsulated in the same LNP. In some embodiments, the one or more mRNA species may be encapsulated in different LNPs. In some embodiments, the mRNA is encapsulated in one or more LNPs, which differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligands and / or combinations thereof. In some embodiments, the one or more LNP may have a different composition of sterol-based cationic lipids, neutral lipid, PEG-modified lipid and / or combinations thereof. In some embodiments the one or more LNPs may have a different molar ratio of cholesterol-based cationic lipid, neutral lipid, and PEG-modified lipid used to create the LNP. Process of Encapsulation

[0145]

[0146] The process of incorporation of a desired mRNA into an LNP is referred to as "loading." Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The LNP-incorporated nucleic acids may be completely or partially located in the interior space of the LNP, within the bilayer membrane of the LNP, or associated with the exterior surface of the LNP membrane. The incorporation of an mRNA into LNPs is also referred to herein as "encapsulation" wherein the nucleic acid is entirely or substantially contained within the interior space of the LNP.

[0146]

[0147] As used herein, a process for the formation of mRNA-loaded lipid nanoparticles (mRNA- LNPs) is used interchangeably with the term "mRNA encapsulation" or grammatical variants thereof. Specifically, an mRNA solution and a lipid solution are mixed such that the mRNA becomes encapsulated in lipid nanoparticles (mRNA-LNPs). The solution in which the mRNA-LNPs are present following mixing of the mRNA and lipid solutions may be referred to herein a formulation or encapsulation solution.

[0147]

[0148] In the present invention, the mixing of the mRNA solution and lipid solution results in the formation mRNA encapsulated within the LNPs (mRNA-LNPs) in an LNP formulation solution. Thus, the present invention more particularly provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) comprising a step of mixing (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising: i) one or more cationic lipids; ii) one or more non-cationic lipids; iii) one or more PEG-modified lipids; and iv) a solvent, wherein the solvent comprises diethylene glycol monoethyl ether or tert-amyl alcohol, thereby forming (or obtaining) mRNA encapsulated within the LNPs (mRNA-LNPs) in an LNP formulation solution.

[0148]

[0149] In some embodiments, an LNP formulation without ethanol according to the present invention may be compared to a conventional ethanol LNP formulation or encapsulation solution that includes a solvent such as ethanol. In previous LNP formulations which used ethanol as a solvent, the formulation solution comprised ethanol at about 10%-40% volume. Other previous LNP formulations used isopropyl alcohol as a solvent at about 10%-40% volume. In contrast, the instant invention provides a method of LNP encapsulation that does not require the use of flammable solvents, such as ethanol.

[0150] Accordingly, in some embodiments, a suitable formulation or encapsulation solution of the present invention does not include a flammable solvent. In some embodiments, a suitable formulation or encapsulation solution does not include ethanol (i.e. it is ethanol-free).

[0149]

[0151] The LNP formulation solution comprises diethylene glycol monoethyl ether or tert-amyl alcohol as provided herein. As a particular example, when diethylene glycol monoethyl ether is used as the solvent, the mRNA-LNPs may be provided in an LNP formulation solution comprising about 10-40% v / v diethylene glycol monoethyl ether, e.g., about 20% v / v. As an alternative example, when about 20% v / v tert-amyl alcohol in propylene glycol is used as the solvent, the mRNA-LNPs may be provided in an LNP formulation solution comprising 3-5% v / v tert-amyl alcohol, e.g., about 4% v / v tert-amyl alcohol.

[0150]

[0152] In some embodiments, a suitable formulation or encapsulation solution may also contain a buffering agent or salt. Exemplary buffering agents include HEPES, Tris, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. Exemplary salt may include sodium chloride, magnesium chloride, and potassium chloride.

[0151]

[0153] In some embodiments, ethanol, citrate buffer, and / or other destabilizing agents are absent during the addition of mRNA and hence the formulation does not require any further downstream processing. In some embodiments, the formulation solution comprises trehalose. The lack of destabilizing agents and the stability of trehalose solution increase the ease of scaling up the formulation and production of mRNA-encapsulated lipid nanoparticles.

[0152]

[0154] In some embodiments, the lipid solution contains one or more cationic lipids, one or more non-cationic lipids, and one or more PEG lipids. In some embodiments, the lipid solution further comprises one or more cholesterol-based lipids.

[0153]

[0155] In some embodiments, the lipid and mRNA solutions are mixed using a pump system. In some embodiments, the pump system comprises a pulse-less flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the process using a pump system is performed at large scale. For example, in some embodiments, the process includes using pumps as described herein to mix a solution of at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, 1 g, 10 g, 50 g, or 100 g or more of mRNA with a lipid solution, to produce mRNA encapsulated in lipid nanoparticles. In some embodiments, the process of mixing mRNA and lipid solutions provides a composition according to the present invention that contains at least about 1 mg, 5mg, 10 mg, 50 mg, 100 mg, 500 mg, 1 g, 10 g, 50 g, or 100 g or more of encapsulated mRNA.

[0156] In some embodiments, the step of mixing an mRNA solution with a lipid solution is performed using a pump system. Such mixing may notably be performed using a centrifugal pump or gear pump.

[0154]

[0157] In some embodiments, the mRNA and lipid solutions are mixed are mixed at a flow rate ranging from about 25-75 mL / min, about 75-200 mL / min, about 200-350 mL / min, about 350-500 mL / min, about 500-650 mL / min, about 650-850 mL / min, or about 850-1000 mL / min. In some embodiments, the mRNA solution and lipid solutions are mixed at a flow rate of about 50 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min, about 800 mL / min, about 850 mL / min, about 900 mL / min, about 950 mL / min, or about 1000 mL / min.

[0155]

[0158] In some embodiments, the mRNA solution is mixed at a flow rate of at least 50 mL / min, at least 100 mL / min, at least 150 mL / min, at least 250 mL / min, at least 300 mL / min, at least 350 mL / min, at least 400 mL / min, at least 450 mL / min, at least 500 mL / min, at least 600 mL / min, at least 800 mL / min, at least 1000 mL / min, at least 1200 mL / min, at least 2000 mL / min, at least 3000 mL / min, or at least 4000 mL / min, at least 5000 mL / min, at least 6000 mL / min, at least 8000 mL / min, at least 10000 mL / min, at least 12000 mL / min, or at least 15000 mL / min.

[0156]

[0159] In some embodiments, the mRNA solution is mixed at a flow rate of about 100 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 150 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 250 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 400 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 500 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 600 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 800 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 1000 mL / min. In some embodiments, the mRNA solution is mixed at a flow of about 1200 mL / min. In some embodiments, the mRNA solution is mixed at a flow of about 1400 mL / min. In some embodiments, the mRNA solution is mixed at a flow of about 1600 mL / min. In some embodiments, the mRNA solution is mixed at a flow of about 1800 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 2000 mL / min. In some embodiments, the mRNA solution is mixed at a flow of about 2400 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 3000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 4000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 5000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 6000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 7000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 8000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 9000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 10000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 12000 mL / min. In some embodiments, the mRNA solution is mixed at a flow rate of about 15000 mL / min.

[0157]

[0160] In some embodiments, the lipid solution is mixed at a flow rate of at least 25 mL / min, at least 75 mL / min, at least 200 mL / min, at least 350 mL / min, at least 500 mL / min, at least 650 mL / min, at least 850 mL / min, at least 1000 mL / min, at least 1200 mL / min, at least 1500 mL / min, at least 2000 mL / min, at least 2200 mL / min, at least 2400 mL / min, or at least 3000 mL / min.

[0158]

[0161] In some embodiments, the lipid solution is mixed at a flow rate of about 25 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 50 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 75 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 100 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 200 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 250 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 300 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 350 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 400 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 450 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 500 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 550 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 600 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 650 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 700 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 750 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 800 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 850 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 900 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 950 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 1000 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 1200 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 1500 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 2000 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 2200 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 2400 mL / min. In some embodiments, the lipid solution is mixed at a flow rate of about 3000 mL / min.

[0162] In some embodiments, the mixing of an mRNA solution with a lipid solution is performed in absence of any pump.

[0159]

[0163] In some embodiments, the process according to the present invention includes maintaining one or more of (i) the lipid solution, (ii) the mRNA solution, and (iii) the mixed solution comprising the mRNA-LNPs at ambient temperature (i.e., not applying heat from a heat source to the solution). In some embodiments, the process includes the step of maintaining at ambient temperature one or both of the mRNA solution and the lipid solution, prior to the mixing step. In some embodiments, the process includes maintaining at ambient temperature one or more of the lipid solution and the mRNA solution during the mixing step. In some embodiments, the process includes the step of maintaining the mRNA-LNPs at ambient temperature after the mixing step.

[0160]

[0164] In some embodiments, the ambient temperature ranges from about 15-35°C. In some embodiments, the ambient temperature ranges from about 16-32°C. In some embodiments, the ambient temperature ranges from about 17-30°C. In some embodiments, the ambient temperature ranges from about 18-30°C. In some embodiments, the ambient temperature ranges from about 18-32°C. In some embodiments, the ambient temperature ranges from about 20-28°C. In some embodiments, the ambient temperature ranges from about 20-26°C. In some embodiments, the ambient temperature ranges from about 20-25°C. In some embodiments, the ambient temperature ranges from about 23-25°C. In some embodiments, the ambient temperature ranges from about 21-24°C. In some embodiments, the ambient temperature ranges from about 21-23°C. In some embodiments, the ambient temperature ranges from about 21-26°C.

[0161]

[0165] In some embodiments, the ambient temperature is less than about 35°C. In some embodiments, the ambient temperature is less than about 32°C. In some embodiments, the ambient temperature is less than about 30°C. In some embodiments, the ambient temperature is less than about 28°C. In some embodiments, the ambient temperature is less than about 26°C. In some embodiments, the ambient temperature is less than about 25°C. In some embodiments, the ambient temperature is less than about 24°C. In some embodiments, the ambient temperature is less than about 23°C. In some embodiments, the ambient temperature is less than about 22°C. In some embodiments, the ambient temperature is less than about 21°C. In some embodiments, the ambient temperature is less than about 20°C. In some embodiments, the ambient temperature is less than about 18°C. In some embodiments, the ambient temperature is less than about 16°C.

[0162]

[0166] In some embodiments, the ambient temperature is about 16°C. In some embodiments, the ambient temperature is about 18°C. In some embodiments, the ambient temperature is about 20°C. In some embodiments, the ambient temperature is about 21°C. In some embodiments, the ambient temperature is about 22°C. In some embodiments, the ambient temperature is about 23°C. In some embodiments, the ambient temperature is about 24°C. In some embodiments, the ambient temperature is about 25°C. In some embodiments, the ambient temperature is about 26°C. In some embodiments, the ambient temperature is about 27°C. In some embodiments, the ambient temperature is about 28°C. In some embodiments, the ambient temperature is about 30°C. In some embodiments, the ambient temperature is about 32°C. In some embodiments, the ambient temperature is about 35°C.

[0163]

[0167] In some embodiments, the ambient temperature at which one or more of the solutions is maintained is or is less than about 35°C, 30°C, 26°C, 25°C, 23°C, 21°C, 20°C, 18°C, or 16 °C. In some embodiments, the ambient temperature at which one or more of the solutions is maintained ranges from about 15-35°C, about 15-30°C, about 15-25°C, about 15-20°C, about 18-32°C, about 20-35°C, about 25-35°C, about 30-35°C, about 20-30°C, about 25-30°C or about 20-25°C, about 21-26°C or about 23-25°C. In some embodiments, the ambient temperature at which one or more of the solutions is maintained is 20-25°C. In some embodiments, the process according to the present invention does not require a step of heating the mRNA solution and the lipid solution prior to the mixing step.

[0164]

[0168] In some embodiments, the lipid solution is heated prior to and / or during mixing. In some embodiments, the lipid solution is heated at a temperature of between about 35°C and 70°C, of between about 40°C and 68°C, of between about 50°C and 66°C, or of between about 60°C and 65°C. In some embodiments, the lipid solution is heated at a temperature of at least about 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In a particular embodiment, the lipid solution is heated at a temperature of about 65°C.

[0165]

[0169] In some embodiments, the lipid solution is heated for at least about 5 min, at least about 10 min, at least about 15 min, or at least about 20 min. In some embodiments, the lipid solution is heated for about 5-20 min, e.g., about 10 min.

[0166]

[0170] In some embodiments, lipid solution is heated at about 65 °C for about 10 minutes. Heating the lipid solution may advantageously reduce the time necessary for lipid dissolution in the solvent.

[0167]

[0171] In some embodiments, the step of mixing the mRNA and lipid solutions to form mRNA-LNPs is performed at ambient temperature. Thus, in some embodiments, the lipid solution is allowed to reach ambient temperature prior to mixing with the mRNA solution.

[0168]

[0172] In some embodiments, greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs have a size of less than about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified LNPs have a size less than 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified LNPs have a size of about 50-150 nm. In some embodiments, substantially all of the purified LNPs have a size of about 50-150 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified LNPs have a size ranging from 80-150 nm. In some embodiments, substantially all of the purified LNPs have a size ranging from 80-150 nm.

[0169]

[0173] A variety of methods known in the art are available for sizing of a population of LNPs. A particular method utilizes Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 pl of an LNP sample are mixed with 990 pl of 10% trehalose. This solution is loaded into a cuvette and then put into the Zetasizer machine. The z-average diameter (nm), or cumulants mean, is regarded as the average size for the LNPs in the sample. The Zetasizer machine can also be used to measure the PDI by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. Average LNP diameter may be reduced by sonication of formed LNP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid nanoparticle synthesis.

[0170]

[0174] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in a composition provided herein encapsulate an mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified LNPs in a composition encapsulate an mRNA within each individual particle.

[0171]

[0175] In some embodiments, a process according to the present invention results in an encapsulation of greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the mRNA. In some embodiments, a process according to the present invention results in greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

[0172]

[0176] In some embodiments, the mRNA-LNP encapsulation efficiency in a formulation according to the present invention is at least the same as the mRNA-LNP encapsulation efficiency in an ethanol LNP formulation.

[0177] In some embodiments, a process according to the present invention comprises a step of incubating the mRNA-LNPs post-mixing. A step of incubating the mRNA-LNPs post-mixing is described in U.S. publication no. US 2022 / 0218612 and can be used to practice the present invention. In some embodiments, the mRNA-LNPs are incubated at a temperature of between 21°C and 65 °C. In some embodiments, the mRNA-LNPs are incubated at a temperature of between 25°C and 60°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of between 30°C and 55°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of between 35°C and 50°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 26°C.

[0173] In some embodiments, the mRNA-LNPs are incubated at a temperature of about 30°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 31°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 32°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 35°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 36°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 38°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 40°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 42°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 45°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 50°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 55°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 60°C. In some embodiments, the mRNA-LNPs are incubated at a temperature of about 65°C. In some embodiments, mRNA-LNPs are incubated at a temperature of about 26°C, about 30°C, or about

[0174] 65°C.

[0175]

[0178] In some embodiments, at least 1 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 5 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 10 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 15 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 20 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 25 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 30 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 40 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 50 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 75 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 100 g of mRNA is encapsulated in LNPs in a single batch. In some embodiments, at least 1 kg of mRNA is encapsulated in LNPs in a single batch. Purification

[0176]

[0179] In some embodiments, the mRNA-LNPs are purified and / or concentrated. Various purification methods may be used. In some embodiments, the mRNA-LNPs are purified by tangential flow filtration (TFF). In some embodiments, the mRNA-LNPs are purified by gravitybased normal flow filtration (NFF). In some embodiments, the mRNA-LNPs are purified by any other suitable filtration process. In some embodiments, the mRNA-LNPs are purified by centrifugation. In some embodiments, the mRNA-LNPs are purified by chromatographic methods.

[0177] Delivery Vehicles

[0178]

[0180] According to the present invention, mRNA encoding a protein or a peptide (e.g., a full length, fragment, or portion of a protein or a peptide) as described herein may be delivered to a subject via a delivery vehicle. As used herein, the terms "delivery vehicle," "lipid nanoparticle," and grammatical equivalents thereof, are used interchangeably.

[0179]

[0181] A delivery vehicle can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. For example, mRNA-LNPs can be formed as described above. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid to a target cell.

[0180]

[0182] In some embodiments, mRNAs encoding at least one protein or peptide may be delivered via a single delivery vehicle. In some embodiments, mRNAs encoding at least one protein or peptide may be delivered via one or more delivery vehicles each of a different composition. In some embodiments, the one or more mRNAs and / or are encapsulated within the same LNPs. In some embodiments, the one or more mRNAs are encapsulated within separate LNPs. In some embodiments, at least some of the LNPs are empty.

[0181]

[0183] Liposomal delivery vehicles, i.e., LNPs, are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of LNPs are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the LNPs can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, an LNP typically serves to transport a desired nucleic acid (e.g., mRNA) to a target cell or tissue. In some embodiments, an LNP comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and / or one or more PEG-modified lipids. In some embodiments, an LNP comprises no more than three distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid.

[0182] Ionizable Lipids

[0183]

[0184] An ionizable lipid facilitates mRNA encapsulation and may be a cationic lipid. A cationic lipid affords a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA drug substance.

[0184]

[0185] Suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in U.S. publication no. US 2010 / 324120. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, [(6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino) butanoate (also known as DLin-MC3- DMA or MC3, CAS no. 1224606-06-7) and pharmaceutically acceptable salts thereof.

[0185]

[0186] Other suitable cationic lipids for use in the compositions and methods of the present invention include ionizable cationic lipids as described in U.S. publication no. US 2015 / 166462.

[0186]

[0187] In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-15,18-dien-l- amine ("HGT5000") as described in U.S. publication no. US 2020 / 0237671, and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z, 18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-4,15,18-trien-l-amine ("HGT5001") as described in U.S. publication no. US 2020 / 0237671 and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)- octadeca-9,12-dien-l-yl) tetracosa-5,15,18-trien-l-amine ("HGT5002") as described in U.S. publication no. US 2020 / 0237671 and pharmaceutically acceptable salts thereof.

[0187]

[0188] Other suitable cationic lipids for use in the compositions and methods of the invention include cationic lipids described as aminoalcohol lipidoids in U.S. publication no. US 2010 / 331234.

[0188]

[0189] Other suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in U.S. publication nos. US 2018 / 085474 or US 2018 / 000953.

[0189]

[0190] Other suitable cationic lipids for use in the compositions and methods of the invention include a cationic lipid having the formula of 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0191] Other suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in U.S. publication nos. US 2013 / 158021 and US 2016 / 367686.

[0190]

[0192] Other suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in U.S. publication nos. US 2017 / 0246319, US 2016 / 002178, or US 2023 / 0071228.

[0191]

[0193] Other suitable cationic lipids for use in the compositions and methods of the present invention include the cationic lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277.

[0192]

[0194] Other suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in U.S. publication nos. US 2015 / 376115, US 2016 / 376224, US 2017 / 119904, US 2019 / 022247, or US 2017 / 210697.

[0193]

[0195] Other suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in U.S. publication nos. US 2019 / 136231 and US 2016311759.

[0194]

[0196] Other suitable cationic lipids for use in the compositions and methods of the present invention include cleavable cationic lipids as described in US publication no. US 2014 / 288160. In some embodiments, the cationic lipid is "HGT4001," "HGT4002," "HGT4003," "HGT4004" (also referred to as 5-(((2,3-bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)propyl)disulfanyl)methyl)-lH- imidazole), or "HGT4005" as described in US 2014 / 288160, and pharmaceutically acceptable salts thereof.

[0195]

[0197] Other suitable cationic lipids for use in the compositions and methods of the present invention include cleavable cationic lipids as described in U.S. publication no. US 2022 / 0323355.

[0196]

[0198] In certain embodiments, the compositions and methods of the present invention include a cationic lipid that is any of general formulas or any of structures (la)-(21a) and (lb) - (21b) and (22)-(237) described in U.S. publication no. US 2022 / 0323355.

[0197]

[0199] In certain embodiments, the compositions and methods of the present invention include a cationic lipid that is Compound (139) of U.S. publication no. US 2022 / 0323355.

[0198]

[0200] In some embodiments, the cationic lipid is ML-2 (also referred to as CKK-E12 and having CAS no: 1432494-65-9).

[0199]

[0201] In some embodiments, the cationic lipid is OF-02. OF-02 is a non-degradable structural analog of OF-Deg-Lin (CAS No.: 1883431-67-1). OF-Deg-Lin contains degradable ester linkages to attach the diketopiperazine core and the doubly-unsaturated tails, whereas OF-02 contains non-degradable 1,2-amino-alcohol linkages to attach the same diketopiperazine core and the doubly-unsaturated tails (Fenton et al., Adv Mater. (2016) 28:2939; U.S. Pat. 10,201,618).

[0200]

[0202] In some embodiments, the cationic lipid is cKK-ElO (Dong et al., PNAS (2014) 111(11):3955- 60; U.S. Pat. 9,512,073; Formula (II) in W02022 / 099003).

[0201]

[0203] In some embodiments, the cationic lipid is a HEPES-based disulfide cationic lipid with a piperazine core as described in WO2022 / 221688. In some embodiments, the cationic lipid is GL- HEPES-E3-E10-DS-3-E18-1 (2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate) (Formula (III) in W02022 / 099003). In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS- 4-E10 (2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate) (Formula (IV) in W02022 / 099003). In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate) (Formula (V) in W02022 / 099003).

[0202]

[0204] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES- E3-E12-DS-3-E14 can be synthesized according to the general synthetic scheme set out in Scheme 1:

[0203] Scheme 1: General Synthetic Scheme

[0204]

[0205] In some embodiments, the cationic lipid is MC3.

[0205]

[0206] In some embodiments, the cationic lipid is SM-102 (CAS No.: 2089251-47-6; 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate).

[0206]

[0207] In some embodiments, the cationic lipid is ALC-0315 (CAS No.: 2036272-55-4; [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)).

[0207]

[0208] In some embodiments, the cationic lipid is cOrn-EEl, having the Formula I:

[0208]

[0209] In certain embodiments, the cationic lipid is IM-001, having the Formula II (provided as compound 3 in W02025 / 003759):

[0209] Formula (II)

[0210]

[0210] IM-001 can be synthesized according to the general procedure set out in Scheme 2, provided in Example 10.

[0211]

[0211] In some embodiments, the cationic lipid is IS-001, having the Formula III (provided as compound 24 in W02025 / 003759):

[0212] Formula (III)

[0213]

[0212] IS-001 can be synthesized according to the general procedure set out in Scheme 3, provided in Example 11.

[0214]

[0213] In certain embodiments, the cationic lipid is N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride ("DOTMA") (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference).

[0215]

[0214] In some embodiments, the cationic lipid may be selected from the group comprising ML-2,

[0216] OF-02, cKK-ElO, CKK-E12, C12-200, DLinkC2DMA, ICE, HGT5000, HGT5001, HGT4003, DMDMA, KLin-K-DMA, cDD-TE-4-E10, CDD-TE-4-E12, DLin-MC3-DMA, Dlin-DMA, L319, SM-102 (9- heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), ALC-0315, DC- Chol, 3060110, 9A1P9, A2-lso5-2DC18, BAME-O16B, hexa(octan-3-yl) 9, 9', 9", 9"', 9"", 9""'- ((((benzene-l,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3, 1-diyl)) tris(azanetriyl))hexanonanoate (FTT5), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, 1- diyl))bis(azanetriyl))tetrakis(ethane-2, 1-diyl) (9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetrakis

[0217] (octadeca-9,12-dienoate) (OF-Deg-Lin), TT3, N1,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5-tricarboxamide, Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3- aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), ATX-126 (CAS No.: 2230647-37-5; 4,4'-[[[[3-(dimethylamino)propyl]thio]carbonyl]imino]b / s-butanoic acid, l,l'-b / s(l- heptyloctyl) ester), GL-HEPES-E3-E10-DS-3-E18, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS- 3-E14, IM-001, IS-001, HGT5002, HGT4001, HGT4002, HGT4004, HGT4005, 5- carboxyspermylglycinedioctadecylamide ("DOGS"), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium ("DOSPA") (Behr et al. Proc. Natl Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761), l,2-Dioleoyl-3-Dimethylammonium- Propane ("DODAP"), or l,2-Dioleoyl-3-Trimethylammonium-Propane ("DOTAP").

[0218]

[0215] Additional exemplary cationic lipids suitable for the compositions and methods of the present invention also include: l,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2- dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); l,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane ("DLinDMA"); l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N- dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy l-(cis,cis-9', l-2'-octadecadienoxy)propane ("CpLinDMA"); N,N-dimethyl-3,4- dioleyloxybenzylamine ("DMOBA"); l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'- Dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"); l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane ("DLinCDAP"); 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propane-l-amine ("Octyl-CLinDMA"); (2R)-2-((8-[(3beta)-cholest-5-en-3- yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-l-amine ("Octyl- CLinDMA (2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyh3-[(9Z, 12Z)- octadeca-9, 12-dien-l-yloxy]propan-l-amine ("Octyl-CLinDMA (2S)"); HGT5002; HGT4004; 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)- octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see U.S. publication no. US 2011 / 256175; Semple et al., Nature Biotech. 28: 172-176 (2010)). (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8): 1003- 1007 (2005); U.S. publication no. US 2006 / 008910); DLin-K-XTC2-DMA; (3aR,5s,6aS)-N,N-dimethyl- 2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [l,3]dioxol-5-amine ("ALNY- 100"); and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-Nl,N16-diundecyl-4,7,10,13- tetraazahexadecane-l,16-diamide ("NC98-5"). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0216] In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the composition, e.g., a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total lipid content in the composition, e.g., a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30- 55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the composition, e.g., a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol %, of the total lipid content in the composition, e.g., a lipid nanoparticle.

[0219]

[0217] In some embodiments, the cationic lipid is biodegradable.

[0220]

[0218] In some embodiments, the cationic lipid is not biodegradable.

[0221]

[0219] In some embodiments, the cationic lipid is cleavable.

[0222]

[0220] In some embodiments, the cationic lipid is not cleavable.

[0223]

[0221] Cationic lipids are described in further detail in Dong et al. (PNAS. lll(ll):3955-60. 2014); Fenton et al. (Adv Mater. 28:2939. 2016); U.S. Pat. No. 9,512,073; and U.S. Pat. No. 10,201,618, each of which is incorporated herein by reference.

[0224] Non-Cationic Lipids

[0225]

[0222] In some embodiments, the LNPs contain one or more non-cationic ("helper") lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. A non-cationic lipid enhances the structural stability of the LNP and helps the LNP in endosome escape. It improves uptake and release of the mRNA drug payload. In some embodiments, the non-cationic lipid is a zwitterionic lipid, which has fusogenic properties for enhancing uptake and release of the drug payload.

[0226]

[0223] The non-cationic lipid may include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3- phospho-L-serine (DOPS), l,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DPOC), l,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), or a combination of two or more thereof.

[0227]

[0224] In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and / or administered.

[0228]

[0225] In some embodiments, such non-cationic lipids may be used alone. However, typically, noncationic lipids are used in combination with other lipids, for example, cationic lipids.

[0229]

[0226] In some embodiments, a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in an LNP may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in an LNP may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of noncationic lipid in an LNP is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in an LNP may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0230]

[0227] In some embodiments, a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in an LNP may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in an LNP may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of noncationic lipid in an LNP is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in an LNP may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

[0231] Cholesterol-Based Lipids

[0232]

[0228] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNPs comprise one or more cholesterol-based lipids. Suitable cholesterol-based lipids include: DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat. 5,744,335), imidazole cholesterol ester ("ICE"; W02011 / 068810), sitosterol (22,23-dihydrostigmasterol), p-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3R-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (A5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); zymosterol (5a-cholesta-8,24-dien-3R-ol); lathosterol (5a-cholest-7-en- 3R-ol); diosgenin ((3P,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3R-ol); campestanol (5a- campestan-3b-ol); 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3R-ol); cholesteryl margarate (cholest-5-en-3R-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol.

[0233]

[0229] In some embodiments, the cholesterol-based lipid used in the LNPs is cholesterol. In some embodiments, the cholesterol-based lipid may comprise a molar ratio (mol %) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in an LNP. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0234]

[0230] In some embodiments, a cholesterol-based lipid may be present in a weight ratio (wt %) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in an LNP. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

[0235] PEG-Modified Lipids

[0236]

[0231] In some embodiments, the LNP comprises one or more PEGylated lipids. The PEGylated lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0237]

[0232] Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20(e.g., C8, C10, C12, C14, C16, or C18) length, such as a derivatized ceramide (PEG-CER), e.g., N-octanoyl-sphingosine-1- [succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide) or N-Octanoyl-Sphingosine-1- [Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14or C18). In some embodiments, the PEGylated lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); l,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); l,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2- distearoyl-rac-glycero-polyethelene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; a PEG-dialkyoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159); and combinations thereof.

[0238]

[0233] In certain embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2K, DSPE-PEG2K, DLPE-PEG2K, DSG-PEG2K, C8 PEG2K, or ALC-0159. In certain embodiments, the PEGylated lipid herein is DMG-PEG2K.

[0234] The PEG-modified phospholipid and derivatized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the LNP delivery vehicle. In some embodiments, one or more PEG-modified lipids constitute about 4% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute about 5% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute about 6% of the total lipids by molar ratio.

[0239]

[0235] In various embodiments, the LNPs comprise (i) a cationic lipid selected from OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, IM-001, or IS-001; (ii) DMG-PEG2K; (iii) cholesterol; and (iv) DOPE. In other embodiments, the LNPs comprise (i) SM-102, (ii) DMG-PEG2K, (iii) cholesterol, and (iv) DSPC. In yet other embodiments, the LNPs comprise (i) ALC-0315, (ii) ALC-0159, (iii) cholesterol, and (iv) DSPC. In yet other embodiments, the LNPs comprise (i) ATX-126, (ii) DMG-PEG2K, (iii) cholesterol, and (iv) DSPC.

[0240] Polymers

[0241]

[0236] In some embodiments, the LNP may further comprise one or more polymers. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactidepolyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

[0242]

[0237] According to various embodiments, the selection of lipids (e.g., cationic lipids, non-cationic lipids, PEG-modified lipids, and / or cholesterol-based lipids) which comprise the LNP, as well as the relative molar ratio of such lipids to each other, may vary depending on the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the nucleic acid to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and tolerability of the selected lipid(s). Thus, the lipid composition itself and / or molar ratios of the selected lipids may be adjusted accordingly.

[0243] Use of non-flammable solvents in LNP formulations

[0244]

[0238] The solvent used in the methods provided herein comprises tert-amyl alcohol or diethylene glycol monoethyl ether.

[0245]

[0239] In some embodiments, the solvent comprises tert-amyl alcohol. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of between 15% and 23% v / v (i.e., 16%, 17%, 18%, 19%, 20%, 21%, or 22% v / v). In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 16% to 22% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 17% to 21% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 17% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 18% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 19% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 20% v / v. In some embodiments, the solvent comprises tert-amyl alcohol at a concentration of about 21% v / v. In some embodiments, the tert-amyl alcohol is present at a concentration of between 15% and 23% v / v in propylene glycol (i.e., 16%, 17%, 18%, 19%, 20%, 21%, or 22% v / v tert-amyl alcohol in propylene glycol). In some embodiments, the tertamyl alcohol is present at a concentration of 16% to 22% v / v in propylene glycol. In some embodiments, the tert-amyl alcohol is present at a concentration of 17% to 21% v / v in propylene glycol. In some embodiments, the tert-amyl alcohol is present at a concentration about 20% v / v in propylene glycol.

[0246]

[0240] In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 30% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 40% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 50% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 60% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 70% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 80% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 90% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 95% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 96% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 97% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 98% v / v. In some embodiments, the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 99% v / v. In some embodiments, the solvent consists of diethylene glycol monomethyl ether.

[0247]

[0241] In some embodiments, the solvent used in the methods provided herein further comprises propylene glycol, polyethylene glycol, 1,3-propanediol, PVP, or PVA, or a combination of two or more thereof.

[0242] In some embodiments, the formulation is alcohol free. In some embodiments, the formulation is produced without the use of any non-aqueous solvent (e.g., without the use of alcohol). In some embodiments, the solvent is free of flammable agents. In some embodiments, a solvent is free of ethanol. In some embodiments, a solvent is free of isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, and combinations thereof. In some embodiments, the solvent is free of a ketone solvent (e.g., acetone, methyl ethyl ketone, or methyl isobutyl ketone). In some embodiments, the mRNA-LNP formulation is aqueous. In some embodiments, the solvent is free of triethylene glycol monomethyl ether (mTEG). In some embodiments, the formulation is produced without the use of mTEG. In some embodiments, the formulation is free of triethylene glycol monomethyl ether.

[0248]

[0243] In some embodiments, the mRNA is encapsulated in the absence of ethanol. In some embodiments, the mRNA is purified in the absence of ethanol. In some embodiments, the mRNA purification, mRNA encapsulation, or both processes, are in the absence of ethanol. In some embodiments, mRNA purification, mRNA encapsulation, or both processes, are free of flammable agents. In some embodiments, mRNA purification, mRNA encapsulation, or both processes, are free of volatile organic compounds.

[0249] Ratio of Distinct Lipid Components

[0250]

[0244] A suitable LNP for the present invention may include one or more of any of the cationic lipids, non-cationic lipids, cholesterol lipids, and / or PEG-modified lipids described herein at various molar ratios. In some embodiments, a LNP comprises five and no more than five distinct components. In some embodiments, a LNP comprises four and no more than four distinct components. In some embodiments, a lipid nanoparticle comprises three and no more than three distinct components.

[0251]

[0245] As a particular example, in cases where the LNP comprises four distinct components (e.g., a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a non-cationic lipid), the molar ratio of the cationic lipid, the PEGylated lipid, the cholesterol-based lipid, and the helper lipid is A: B: C: D, where A + B + C + D = 100%. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is 35-55%, such as 35-50% (e.g., 38-42% such as 40%, or 45- 50%). In some embodiments, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is 0.25-2.75% (e.g., 1-2% such as 1.5%). In some embodiments, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is 20-50% (e.g., 27-30% such as 28.5%, or 38-43%). In some embodiments, the molar ratio of the non-cationic lipid relative to the total lipids (i.e., D) is 5-35% (e.g., 28-32% such as 30%, or 8-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the non-cationic lipid. In some embodiments, the LNPs contain a molar ratio of the cationic lipid to the non-cationic lipid that is more than 1.

[0252]

[0246] In various embodiments, cationic lipids constitute about 30-60 % (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the LNP by molar ratio. In some embodiments, the percentage of cationic lipids is or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the LNP by molar ratio.

[0253]

[0247] In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol- based lipid(s) to PEG-modified lipid(s) may be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:28.5:1.5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 50:10:35:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 60:35:0:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5.

[0254]

[0248] In certain embodiments, the LNP comprises: a cationic lipid at a molar ratio of 35% to 55% or 40% to 50% (e.g., a cationic lipid at a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41% 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%); a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a PEGylated lipid at a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); a cholesterol-based lipid at a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., a cholesterol-based lipid at a molar ratio of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% 31% 32% 33% 34% 35% 36% 37% 38% 39% 40% 41% 42%, 43%, 44%, 45%, 46%, 47%,

[0255] 48%, 49%, or 50%); and a non-cationic lipid at a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., a helper lipid at a molar ratio of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%), wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0256]

[0249] An exemplary mixture of lipids for use with the invention is composed of four lipid components: a cationic lipid (e.g., ML-2, OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10), a noncationic lipid (e.g., DSPC, DPPC, DOPE or DEPE), a cholesterol-based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K). In some embodiments, the molar ratio of cationic lipid(s) (e.g. CKK-ElO or GL-HEPES-E3-E12-DS-4-E10) to non-cationic lipid(s) (e.g. DSPC or DOPE) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs may be between about 35-55:5- 35:20-40:1-15, respectively. In some embodiments, the molar ratio of cationic lipid(s) (e.g. CKK-ElO or GL-HEPES-E3-E12-DS-4-E10) to noncationic lipid(s) (e.g. DSPC or DOPE) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is 35-45:25-35:20-30:1-10. In particular embodiments, the molar ratio of cationic lipid(s) (e.g. CKK-ElO or GL-HEPES-E3-E12-DS-4-E10) to noncationic lipid(s) (e.g. DSPC or DOPE) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is about 40:30:25:5. In some embodiments, the molar ratio of cationic lipid(s) (e.g. MC-3) to noncationic lipid(s) (e.g. DSPC or DOPE) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is 45- 55:5-15:30-40:1-10. In some embodiments, the molar ratio of cationic lipid(s) (e.g. MC-3) to noncationic lipid(s) (e.g. DSPC or DOPE) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is about 50:10:35:5. In some embodiments, the molar ratio of cationic lipid(s) (e.g. CKK-ElO or GL-HEPES-E3-E12-DS-4-E10) to noncationic lipid(s) (e.g. DSPC or DOPE) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the LNPs is about 40:30:28.5:1.5. As shown in the examples, such preparations are suitable for use in the formulations of the invention as they ensure suitable mRNA- LNP size and encapsulation efficacy.

[0257]

[0250] In some embodiments, a mixture of lipids for use with the invention may comprise no more than three distinct lipid components. In some embodiments, one distinct lipid component in such a mixture is a cholesterol-based or imidazol-based cationic lipid. An exemplary mixture of lipids may be composed of three lipid components: a cationic lipid (e.g., a cholesterol-based or imidazol- based cationic lipid), a non-cationic lipid (e.g., DSPC, DPPC, DOPE, or DEPE) and a PEG-modified lipid (e.g., DMG-PEG2K). In some embodiments, the molar ratio of cationic lipid to non-cationic lipid to PEG-modified lipid may be between about 55-65:30-40:1-15, respectively. In some embodiments, the molar ratio of cationic lipid (e.g., ICE) to non-cationic lipid (e.g., DSPC) to PEG-modified lipid in the LNPs is 55-65:30-40:1-15. In particular embodiments, the molar ratio of cationic lipid (e.g., ICE) to non-cationic lipid (e.g., DSPC or DOPE) to PEG-modified lipid in the LNPs is 60:35:5.

[0258]

[0251] In some embodiments, the concentration of the lipids and mRNA in the mRNA-LNP is such that the cationic lipid(s) (e.g., CKK-E10 or GL-HEPES-E3-E12-DS-4-E10) to mRNA N / P ratio from 2 to 6. In some embodiments, the concentration of the lipids and mRNA in the mRNA-LNP is such that the cationic lipid(s) (e.g., CKK-E10 or GL-HEPES-E3-E12-DS-4-E10) to mRNA N / P ratio is about 2, 3, 4, 5, or 6. A particularly suitable N / P ratio is about 4, which allows efficient LNP formation and mRNA encapsulation efficiency.

[0259]

[0252] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 35% to 55%; DMG- PEG2K at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0260]

[0253] In certain embodiments, the LNP comprises: cKK-ElO at a molar ratio of 35% to 55%; DMG- PEG2K at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0261]

[0254] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 35% to 55%; DMG-PEG2K at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0262]

[0255] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2K at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0263]

[0256] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 35% to 55%; DMG-PEG2K at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0264]

[0257] In certain embodiments, the LNP comprises: SM-102 at a molar ratio of 35% to 55%; DMG- PEG2K at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0265]

[0258] In certain embodiments, the LNP comprises: ALC-0315 at a molar ratio of 35% to 55%; ALC- 0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0266]

[0259] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0260] In certain embodiments, the LNP comprises: cKK-ElO at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0267]

[0261] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0268]

[0262] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0269]

[0263] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0270]

[0264] In certain embodiments, the LNP comprises DLin-MC3-DMA (MC3) at a molar ratio of 50%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 38.5%; and DSPC at a molar ratio of 10%.

[0271]

[0265] In certain embodiments, the LNP comprises: IM-001 at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0272]

[0266] In certain embodiments, the LNP comprises: IS-001 at a molar ratio of 40%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0273]

[0267] In certain embodiments, the LNP comprises: SM-102 at a molar ratio of 50%; DSPC at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and DMG-PEG2K at a molar ratio of 1.5%.

[0274]

[0268] In certain embodiments, the LNP comprises: ALC-0315 at a molar ratio of 46.3%; DSPC at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and ALC-0159 at a molar ratio of 1.6%.

[0275]

[0269] In certain embodiments, the LNP comprises: ALC-0315 at a molar ratio of 47.4%; DSPC at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and ALC-0159 at a molar ratio of 1.7%.

[0276]

[0270] In certain embodiments, the LNP comprises ATX-126 at a molar ratio of 50%; DMG-PEG2K at a molar ratio of 1.5%; cholesterol at a molar ratio of 38.5%; and DSPC at a molar ratio of 10%.

[0277]

[0271] To calculate the actual amount of each lipid to be put into an LNP formulation, the molar amount of the cationic lipid is first determined based on a desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.

[0278] Nucleic acids encapsulated in LNPs

[0279]

[0272] The LNP compositions described herein may comprise a nucleic acid (e.g., a mRNA) as provided herein.

[0280]

[0273] mRNAs provided herein may be synthesized according to any of a variety of known methods. Various methods are described in U.S. publication no. US 2018 / 0258423 and can be used to prepare mRNAs of the present invention. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary according to the specific application. A suitable DNA template typically has a promoter, for example, a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.

[0281]

[0274] In some embodiments, a suitable mRNA sequence is an mRNA sequence encoding a protein or a peptide. In some embodiments, a suitable mRNA sequence is codon optimized for efficient expression in human cells. In some embodiments, a suitable mRNA sequence is naturally-occurring or a wild-type sequence. In some embodiments, a suitable mRNA sequence encodes a protein or a peptide that contains one or mutations in amino acid sequence.

[0282]

[0275] The present invention may be used to deliver mRNAs of a variety of lengths. In some embodiments, the present invention may be used to deliver in vitro synthesized mRNA of or greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 20 kb, 30 kb, 40 kb, or 50 kb in length. In some embodiments, the present invention may be used to deliver in vitro synthesized mRNA ranging from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-50 kb in length.

[0283]

[0276] In some embodiments, mRNA is purified prior to use in the process of the invention. In some embodiments, the process further comprises a step of obtaining a purified mRNA in an mRNA solution before the step of mixing the mRNA solution with the lipid solution.

[0284]

[0277] Where desired, the LNP may be multi-valent. In some embodiments, the LNP may carry nucleic acids, such as mRNAs, that encode more than one polypeptide, such as two, three, four, five, six, seven, or eight polypeptides. For example, the LNP may carry multiple nucleic acids (e.g., mRNA), each encoding a different polypeptide; or carry a polycistronic mRNA that can be translated into more than one polypeptide (e.g., each antigen-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). An LNP carrying different nucleic acids (e.g., mRNA) typically comprises (encapsulate) multiple copies of each nucleic acid. For example, an LNP carrying or encapsulating two different nucleic acids typically carries multiple copies of each of the two different nucleic acids.

[0285] Nucleotides

[0286]

[0278] Various naturally-occurring or modified nucleosides may be used to produce mRNA of the present invention. In some embodiments, an mRNA is or comprises naturally-occurring nucleosides (or unmodified nucleotides; e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-l-methyl-pseudouridine), 2- thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2' -fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'- / V-phosphoramidite linkages).

[0287]

[0279] In some embodiments, a suitable mRNA may contain backbone modifications, sugar modifications and / or base modifications. For example, modified nucleotides may include, but not be limited to, modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl- adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl- cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7- methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4- thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro- uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl- uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5- methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5- oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, beta-D- mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine. The preparation of such analogues is known to a person skilled in the art e.g., from U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530 and U.S. Pat. No. 5,700,642.

[0288]

[0280] In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine ("5mC"), pseudouridine ("\| / U"), Nl-methyl-pseudouridine ("ml\| / U"), and / or 2-thio-uridine ("2sU"). The mRNA may be mRNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. See, e.g., U.S. Patent No. US 8,278,036 or U.S. publication no. US 2012 / 0195936 or US 2016 / 0177295 for a discussion of such residues and their incorporation into mRNA. The presence of nonstandard nucleotide residues may render an mRNA more stable and / or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions {e.g., one or more of a 2'-O-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNA). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modification may include, but are not limited to a 2'-deoxy-2'-fluoro modification, a 2'-O-methyl modification, a 2'-O-methoxyethyl modification and a 2'-deoxy modification.

[0289]

[0281] In some embodiments, mRNAs may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-O-(l-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0290]

[0282] In some embodiments, mRNAs may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2' -fluoro-oligoribonucleotide (2'- fluoro-2'-deoxycytidine 5' -triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'- deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'- deoxyuridine 5' -triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy-2'-C- alkyloligoribonucleotide (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5' -triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5' -triphosphate, 2'-arauridine 5'- triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5' -triphosphate, 2'-azido-2'- deoxyuridine 5'-triphosphate).

[0291]

[0283] In some embodiments, any of the modifications described herein may be present in 0-100% of the nucleotides of the mRNA. For example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0292]

[0284] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0293] Post-synthesis processing

[0294]

[0285] Typically, a 5' cap and / or a 3' tail may be added after mRNA synthesis. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a tail serves to protect the mRNA from exonuclease degradation.

[0295]

[0286] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in U.S. publication nos. US 2016 / 0032356 and US 2018 / 0125989, which are incorporated herein by reference.

[0296]

[0287] Typically, a tail structure includes a poly(A) and / or poly(C) tail. A poly-A or poly-C tail on the 3' terminus of mRNA typically includes at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 adenosine or cytosine nucleotides, respectively. In some embodiments, a poly(A) or poly(C) tail may be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200, about 10 to 300, about 10 to 400, about 10 to 500, about 10 to 550, about 10 to 600, about 50 to 600, about 100 to 600, about 150 to 600, about 200 to 600, about 250 to 600, about 300 to 600, about 350 to 600, about 400 to 600, about 450 to 600, about 500 to 600, about 10 to 150, about 10 to 100, about 20 to 70, or about 20 to 60 adenosine or cytosine nucleotides respectively. In some embodiments, a tail structure includes is a combination of poly(A) and poly(C) tails with various lengths described herein. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0297]

[0288] In some embodiments, the 5' cap and / or the 3' tail are added to the synthesized mRNA before the mRNA is purified as described herein. In other embodiments, the 5' cap and / or the 3' tail are added to the synthesized mRNA after the mRNA is purified as described herein.

[0298]

[0289] Synthesized mRNA may be used in the present invention without further purification. In some embodiments, synthesized mRNA may be further purified for use according to the present invention. Various methods may be used to purify synthesized mRNA. For example, purification of mRNA can be performed using centrifugation, filtration and / or chromatographic methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation, filtration, chromatography, gel purification, or any other suitable means or combinations thereof. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, well known to one of skill in the art. In some embodiments, the mRNA is purified using TFF. Suitable purification methods include those described in U.S. publication nos. US 2016 / 0040154, US 2015 / 0376220, US 2018 / 0251755, US 2018 / 0251754, US 2021 / 0388338, US 2023 / 0062449, and US 2024 / 0043826, any of which may be used to purify mRNA.

[0299]

[0290] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.

[0300]

[0291] In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by centrifugation. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by filtration. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by TFF.

[0301]

[0292] In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing by chromatography.

[0302]

[0293] In some embodiments, the mRNA is purified without the use of ethanol or any other flammable solvent. Characterization of purified mRNA

[0303]

[0294] The mRNA described herein (e.g., in the mRNA solution) is substantially free of impurities comprising short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual free nucleotides, residual solvent and / or residual salt.

[0304]

[0295] The mRNA described herein has a purity of between about 60% and about 100%. Accordingly, in some embodiments, the purified mRNA has a purity of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 90%, or 100%.

[0305]

[0296] In some embodiments, the mRNA described herein has less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, and / or less than 0.1% impurities other than full-length mRNA. The impurities include those from IVT, e.g., proteins, enzymes, DNA templates, free nucleotides, residual solvent, residual salt, dsRNA, short abortive RNA species, and / or long abortive RNA species. In some embodiments, the purified mRNA is substantially free of process enzymes.

[0306]

[0297] In some embodiments, the residual plasmid DNA in the purified mRNA of the present invention is less than about 1 pg / mg, less than about 2 pg / mg, less than about 3 pg / mg, less than about 4 pg / mg, less than about 5 pg / mg, less than about 6 pg / mg, less than about 7 pg / mg, less than about 8 pg / mg, less than about 9 pg / mg, less than about 10 pg / mg, less than about 11 pg / mg, or less than about 12 pg / mg.

[0307]

[0298] In some embodiments, more than about 90%, 95%, 96%, 97%, 98%, 99% or substantially all prematurely aborted RNA sequences have been removed from the mRNA provided herein (e.g., in the mRNA solution). In some embodiments, mRNA is substantially free of prematurely aborted RNA sequences. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of prematurely aborted RNA sequences. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of prematurely aborted RNA sequences. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) undetectable prematurely aborted RNA sequences as determined by, e.g., high-performance liquid chromatography (HPLC) (e.g., shoulders or separate peaks), ethidium bromide, Coomassie staining, capillary electrophoresis or Glyoxal gel electrophoresis (e.g., presence of separate lower band). In some embodiments, shortmers are detected or quantified after adding a 5' -cap, and / or a 3'-poly A tail. In some embodiments, shortmers are less than 100 nucleotides in length, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length. In some embodiments, shortmers comprise less than 15 bases (e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 bases). In some embodiments, the shortmers contain about 8-15, 8-14, 8-13, 8-12, 8-11, or 8-10 bases.

[0308]

[0299] In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) is substantially free of enzyme reagents used in in vitro synthesis including, but not limited to, RNA polymerase, DNase I, pyrophosphatase, and / or RNase inhibitor. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of enzyme reagents used in in vitro synthesis. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of enzyme reagents used in in vitro synthesis including. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) contains undetectable enzyme reagents used in in vitro synthesis including as determined by, e.g., silver stain, gel electrophoresis, HPLC, ultra-performance liquid chromatography (UPLC), and / or capillary electrophoresis, ethidium bromide and / or Coomassie staining.

[0309]

[0300] In various embodiments, mRNA provided herein (e.g., in the mRNA solution) maintains high degree of integrity. As used herein, the term "mRNA integrity" generally refers to the quality of mRNA after purification. mRNA integrity may be determined using methods well known in the art, for example, by RNA agarose gel electrophoresis. In some embodiments, mRNA integrity may be determined by banding patterns of RNA agarose gel electrophoresis. In some embodiments, mRNA provided herein (e.g., in the mRNA solution) shows little or no banding compared to reference band of RNA agarose gel electrophoresis. In some embodiments, a purified mRNA of the present invention has an integrity greater than about 95% (e.g., greater than about 96%, 97%, 98%, 99% or more). In some embodiments, mRNA provided herein (e.g., in the mRNA solution) has an integrity greater than 98%. In some embodiments provided herein (e.g., in the mRNA solution) has an integrity greater than 99%. In some embodiments, provided herein (e.g., in the mRNA solution) has an integrity of approximately 100%.

[0310]

[0301] In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) is assessed for one or more of the following characteristics: appearance, identity, quantity, concentration, presence of impurities, microbiological assessment, pH level and activity. In some embodiments, acceptable appearance includes a clear, colorless solution, essentially free of visible particulates. In some embodiments, the identity of the mRNA is assessed by sequencing methods. In some embodiments, the concentration is assessed by a suitable method, such as UV spectrophotometry. In some embodiments, a suitable concentration is between about 90% and 110% nominal (0.9-1.1 mg / mL).

[0302] In some embodiments, assessing the purity of the mRNA includes assessment of mRNA integrity, assessment of residual plasmid DNA, and assessment of residual solvent. In some embodiments, acceptable levels of mRNA integrity are assessed by agarose gel electrophoresis. The gels are analyzed to determine whether the banding pattern and apparent nucleotide length is consistent with an analytical reference standard. Additional methods to assess RNA integrity include, for example, assessment of the purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, acceptable purity of the purified mRNA as determined by CGE is that the purified mRNA composition has no greater than about 55% long abortive / degraded species. In some embodiments, residual plasmid DNA is assessed by methods in the art, for example by the use of qPCR. In some embodiments, less than 10 pg / mg (e.g., less than 10 pg / mg, less than 9 pg / mg, less than 8 pg / mg, less than 7 pg / mg, less than 6 pg / mg, less than 5 pg / mg, less than 4 pg / mg, less than 3 pg / mg, less than 2 pg / mg, or less than 1 pg / mg) is an acceptable level of residual plasmid DNA. In some embodiments, acceptable residual solvent levels are not more than 10,000 ppm, 9,000 ppm, 8,000 ppm, 7,000 ppm, 6,000 ppm, 5,000 ppm, 4,000 ppm, 3,000 ppm, 2,000 ppm, 1,000 ppm.

[0311]

[0303] In some embodiments, microbiological tests are performed on the mRNA provided herein (e.g. in the mRNA solution), which include, for example, assessment of bacterial endotoxins. In some embodiments, bacterial endotoxins are < 0.5 EU / mL, <0.4 EU / mL, <0.3 EU / mL, <0.2 EU / mL or <0.1 EU / mL. In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) has no more than 1 CFU / lOmL, 1 CFU / 25mL, lCFU / 50mL, lCFU / 75mL, or no more than 1 CFU / lOOmL.

[0312]

[0304] In some embodiments, the pH of the mRNA provided herein (e.g., in the mRNA solution) is assessed. In some embodiments, acceptable pH of the mRNA provided herein (e.g., in the mRNA solution) is between 5 and 8. Accordingly, in some embodiments, the mRNA provided herein (e.g., in the mRNA solution) has a pH of about 5. In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) has a pH of about 6. In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) has a pH of about 7. In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) has a pH of about 7.5. In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) has a pH of about 8.

[0313]

[0305] In some embodiments, the translational fidelity of mRNA provided herein (e.g., in the mRNA solution) is assessed. The translational fidelity can be assessed by various methods and include, for example, transfection and Western blot analysis. Acceptable characteristics of the purified mRNA includes banding pattern on a Western blot that migrates at a similar molecular weight as a reference standard.

[0306] In some embodiments, the mRNA provided herein (e.g., in the mRNA solution) is assessed for conductance. In some embodiments, acceptable characteristics of the mRNA provided herein (e.g., in the mRNA solution) include a conductance of between about 50% and 150% of a reference standard.

[0314]

[0307] The mRNA provided herein (e.g., in the mRNA solution) is also assessed for Cap percentage and for PolyA tail length. In some embodiments, an acceptable Cap percentage includes Capl, % Area: NLT90. In some embodiments, an acceptable poly(A) tail length is about 100 -1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides).

[0315]

[0308] Various methods of detecting and quantifying mRNA purity are known in the art. For example, such methods include, blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver stain, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. In some embodiments, mRNA is first denatured by a Glyoxal dye before gel electrophoresis ("Glyoxal gel electrophoresis"). In some embodiments, synthesized mRNA is characterized before capping or tailing. In some embodiments, synthesized mRNA is characterized after capping and tailing.

[0316] Compositions

[0317]

[0309] The present invention further provides a composition comprising one or more lipid nanoparticles encapsulating mRNA (mRNA-LNPs), wherein the lipid nanoparticle (LNP) comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, and wherein the composition further comprises diethylene glycol monoethyl ether or tert-amyl alcohol. The mRNA encapsulated in lipid nanoparticles (mRNA-LNPs) may be prepared by the process provided herein. Thus, a composition comprising mRNA-LNPs prepared by the process described herein is further provided.

[0318]

[0310] The LNP encapsulating the mRNA may comprise the lipid components provided herein in the appropriate molar ratios. In particular, the LNP comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

[0319]

[0311] The amount of diethylene glycol monoethyl ether or tert-amyl alcohol present in the composition may depend on the initial concentration of said solvent in the lipid solution and the respective volumes of the mRNA solution and lipid solutions that are mixed together. Typically, the solvent is diluted about 5-fold as a result of encapsulation. In some embodiments, diethylene glycol monoethyl ether is present in the composition at a concentration of at least about 6%, 8%, 10%, 12%, 14%, 16%, 18%, 19%, or 20% v / v.

[0312] In some embodiments, the composition further comprises polyethylene glycol, propylene glycol, 1,3-propanediol, PVP, PVA, or a combination of two or more thereof.

[0320]

[0313] In some embodiments, tert-amyl alcohol is present in the composition at a concentration of 3% to 5% v / v. In some embodiments, tert-amyl alcohol is present in the composition at a concentration of 3.2% to 4.6% v / v. In some embodiments, tert-amyl alcohol is present in the composition at a concentration of 3.4% to 4.4% v / v. In some embodiments, tert-amyl alcohol is present in the composition at a concentration of 4% to 4.2% v / v. In some embodiments, tert-amyl alcohol is present in the composition at a concentration of about 4% v / v. In some embodiments, when residual tert-amyl alcohol is present in the composition, residual propylene glycol is also present. For example, when tert-amyl alcohol is present in the composition at a concentration of about 4% v / v, propylene glycol may further be present in the composition at a concentration of about 16% v / v.

[0321]

[0314] The present invention further relates to a composition as provided herein for use as a medicament. The present invention further relates to the use of a composition as provided herein for treating a disease in a subject is provided. The present invention further relates to a composition as provided herein for the manufacture of a medicament for the treatment of a disease in a subject.

[0322]

[0315] A kit of parts is also provided herein, wherein said kit of parts comprises a first container and a second container, wherein the first container comprises an mRNA solution as provided herein and the second container comprises a lipid solution as provided herein.

[0323] Therapeutic Use of Compositions

[0324]

[0316] To facilitate expression of mRNA in vivo, mRNA-LNPs can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0325]

[0317] mRNA-LNPs provided herein and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The "effective amount" for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical, and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement, or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression, or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

[0326]

[0318] The present invention provides methods of delivering mRNA for in vivo protein production, comprising administering mRNA to a subject in need of delivery. In some embodiments, mRNA is administered via a route of delivery selected from the group consisting of intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, intratracheal injection, pulmonary delivery (e.g., nebulization or instillation), intramuscular delivery, intrathecal delivery, or intraarticular delivery. Accordingly, in some embodiments, the present invention provides methods of delivering mRNA for in vivo protein production comprising intravenous delivery. In some embodiments, the present invention provides methods of delivering mRNA for in vivo protein production comprising intramuscular delivery. In some embodiments, the present invention provides methods of delivering mRNA for in vivo protein production comprising intratracheal injection. In some embodiments, the present invention provides methods of delivering mRNA for in vivo protein production comprising pulmonary delivery (e.g., nebulization or instillation).

[0327]

[0319] The development of ethanol-free LNP formulations, using solvents with established safety profiles in humans, greatly reduces and / or eliminates fire safety concerns and also allows for bedside mixing. Accordingly, in some embodiments, the mRNA-LNP formulations and compositions comprising mRNA-LNPs provided herein are suitable for preparation and administration in various settings, including for example bedside mixing, hospital on-site mixing, and pharmacy on-site mixing.

[0328]

[0320] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In some embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments, the administration results in delivery of the mRNA to a muscle cell. In some embodiments, the administration results in delivery of the mRNA to a hepatocyte (i.e., liver cell). In a particular embodiment, the intramuscular administration results in delivery of the mRNA to a muscle cell.

[0329]

[0321] Additional teaching of pulmonary delivery and nebulization are described in U.S. publication nos. US 2018 / 0125989 and US 2018 / 0333457, each of which is incorporated by reference in its entirety.

[0322] Alternatively or additionally, mRNA-LNPs and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, e.g. in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection. Formulations containing provided compositions complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.

[0330]

[0323] Provided methods of the present invention contemplate single as well as multiple administrations of a therapeutically effective amount of the therapeutic agents (e.g., compositions comprising mRNA-LNPs) described herein. Therapeutic agents can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition. In some embodiments, a therapeutically effective amount of the therapeutic agent or composition of the present invention is administered intrathecally periodically at regular intervals (e.g., once every year, once every six months, once every three months, monthly (once every month), twice a month, once every 30 days, once every 28 days, once every 14 days, weekly, twice a week, daily, every other day, or continuously).

[0331]

[0324] In some embodiments, provided mRNA-LNPs and / or compositions are formulated such that they are suitable for extended-release of the mRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice a day, daily, or every other day. In another embodiment, the mRNA-LNPs and / or compositions are administered to a subject twice a week, once a week, once every 14-days, once every 28-days, once every 30-days, once-a-month, twice-a-month, once every six-weeks, once every other month, once every three-months, once every six-months, or annually. Also contemplated are compositions and mRNA-LNPs that are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release therapeutic agent (e.g., mRNA) over extended periods of time. In one aspect, the extended-release means employed are combined with modifications made to the mRNA to enhance stability.

[0332]

[0325] As used herein, the "therapeutically effective amount" is largely determined based on the total amount of the therapeutic agent contained in the compositions provided herein. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and / or ameliorating a disease or disorder). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., mRNA) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other related factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

[0333]

[0326] A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0334]

[0327] Also contemplated herein are lyophilized compositions comprising one or more of the LNPs disclosed herein and related methods for the use of such compositions as disclosed for example, in U.S. publication no. US 2019 / 0314284. For example, lyophilized compositions may be reconstituted prior to administration or can be reconstituted in vivo. For example, a lyophilized composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.

[0335]

[0328] Provided mRNA-LNPs and compositions may be administered to any desired tissue. In some embodiments, the mRNA delivered by provided LNPs or compositions is expressed in the tissue in which the LNPs and / or compositions were administered. In some embodiments, the mRNA delivered is expressed in a tissue different from the tissue in which the LNPs and / or compositions were administered. Exemplary tissues in which delivered mRNA may be delivered and / or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[0336]

[0329] In some embodiments, administering the provided composition results in an increased mRNA expression level in a biological sample from a subject as compared to a baseline expression level before treatment. Typically, the baseline level is measured immediately before treatment. Biological samples include, for example, whole blood, serum, plasma, urine and tissue samples (e.g., muscle, liver, skin fibroblasts). In some embodiments, administering the provided composition results in an increased mRNA expression level by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline level immediately before treatment. In some embodiments, administering the provided composition results in an increased mRNA expression level as compared to an mRNA expression level in subjects who are not treated.

[0337]

[0330] According to various embodiments, the timing of expression of delivered mRNA can be tuned to suit a particular medical need. In some embodiments, the expression of the protein encoded by delivered mRNA is detectable 1, 2, 3, 6, 12, 24, 48, 72, and / or 96 hours after administration of provided LNPs and / or compositions. In some embodiments, the expression of the protein encoded by delivered mRNA is detectable one-week, two-weeks, and / or one-month after administration.

[0338]

[0331] The present invention also provides delivering a composition having mRNA molecules encoding a peptide or polypeptide of interest for use in the treatment of a subject, e.g., a human subject or a cell of a human subject or a cell that is treated and delivered to a human subject.

[0339] EMBODIMENTS

[0340]

[0332] The present invention includes at least the following numbered embodiments:

[0341] 1. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) comprising a step of mixing (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising i) one or more cationic lipids; ii) one or more non-cationic lipids; iii) one or more PEG-modified lipids; and iv) a solvent, wherein the solvent comprises diethylene glycol monoethyl ether or tert-amyl alcohol, thereby forming mRNA encapsulated within the LNPs (mRNA-LNPs).

[0342] 2. The process of embodiment 1, wherein the solvent further comprises propylene glycol, polyethylene glycol, 1,3-propanediol, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), or a combination of two or more thereof.

[0343] 3. The process of embodiment 1 or 2, wherein the solvent comprises diethylene glycol monoethyl ether at a concentration of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% v / v.

[0344] 4. The process of embodiment 1, wherein the solvent consists of diethylene glycol monoethyl ether.

[0345] 5. The process of embodiment 1 or 2, wherein the solvent comprises tert-amyl alcohol at a concentration of between 15% and 23% v / v.

[0346] 6. The process of embodiment 5, wherein the solvent comprises tert-amyl alcohol at a concentration of 16% to 22% v / v, optionally at a concentration of 17% to 21% v / v, e.g., at a concentration of about 20% v / v.

[0347] 7. The process of embodiment 5 or 6, wherein the tert-amyl alcohol is in propylene glycol.

[0348] 8. The process of any one of embodiments 1-2 and 5-7, wherein the solvent comprises tertamyl alcohol at a concentration of about 20% v / v in propylene glycol.

[0349] 9. The process of any one of the preceding embodiments, wherein the mRNA solution and / or the lipid solution are at about ambient temperature.

[0350] 10. The process of embodiment 9, wherein the ambient temperature is less than about 35°C, less than about 30°C, less than about 26°C, less than about 23°C, less than about 21°C, less than about 20°C, or less than about 18°C.

[0351] 11. The process of embodiment 10, wherein the ambient temperature ranges from about 18- 32°C, about 21-26°C, or about 23-25°C.

[0352] 12. The process of any one of the preceding embodiments, wherein the process does not require a step of heating the mRNA solution and the lipid solution prior to the mixing step.

[0353] 13. The process of any one of the preceding embodiments, wherein the mRNA solution comprises at least about 1 g of mRNA per 12 L of the mRNA solution.

[0354] 14. The process of embodiment 13, wherein the mRNA solution comprises about 1 g of mRNA per 8 L of the mRNA solution.

[0355] 15. The process of embodiment 13, wherein the mRNA solution comprises about 1 g of mRNA per 4 L of the mRNA solution. 16. The process of embodiment 13, wherein the mRNA solution comprises about 1 g of mRNA per 2 L of the mRNA solution.

[0356] 17. The process of any one of the preceding embodiments, wherein the concentration of mRNA in the mRNA solution is greater than about 0.125 mg / mL, greater than about 0.25 mg / mL, greater than about 0.5 mg / mL, or greater than about 1.0 mg / mL.

[0357] 18. The process of any one of the preceding embodiments, wherein the mRNA solution and the lipid solution are mixed at a ratio (v / v) of between 2:1 and 6:1.

[0358] 19. The process of embodiment 18, wherein the mRNA solution and the lipid solution are mixed at a ratio (v / v) of about 4:1.

[0359] 20. The process of any one of the preceding embodiments, wherein the mRNA solution is a buffered solution, optionally an acidic buffered solution.

[0360] 21. The process of any one of the preceding embodiments, wherein the mRNA solution has a pH of between 3.0 and 5.0.

[0361] 22. The process of embodiment 21, wherein the mRNA solution has a pH of about 3.5, 4.0, or 4.5.

[0362] 23. The process of any one of the preceding embodiments, wherein the mRNA solution comprises from about 37.5 mM to about 300 mM NaCI.

[0363] 24. The process of embodiment 23, wherein the mRNA solution comprises about 37.5 mM, about 75 mM, about 100 mM, about 150 mM, or about 300 mM NaCI.

[0364] 25. The process of any one of the preceding embodiments, wherein the mRNA solution comprises less than 5 mM of citrate, optionally wherein the mRNA solution comprises 1 mM citrate and 150 mM NaCI with a pH of about 4.5.

[0365] 26. The process of any one of the preceding embodiments, wherein the mRNA solution further comprises trehalose, optionally about 10-20% % v / v trehalose, e.g., about 10% v / v trehalose.

[0366] 27. The process of any one of the preceding embodiments, wherein the lipid solution further comprises one or more cholesterol-based lipids, optionally cholesterol.

[0367] 28. The process of embodiment 27, wherein the lipid solution comprises cationic lipid at a molar ratio of 40%, DMG-PEG2K at a molar ratio of 1.5%, DOPE at a molar ratio of 30%, and cholesterol at a molar ratio of 28.5%.

[0368] 29. The process of any one of the preceding embodiments, wherein the step of mixing occurs in a total volume of between about 3 and 10 mL.

[0369] 30. The process of embodiment 29, wherein the step of mixing occurs in a total volume of about 3 mL.

[0370] 31. The process of any one of the preceding embodiments, wherein the process further comprises a step of incubating the mRNA-LNPs. 32. The process of embodiment 31, wherein the mRNA-LNPs are incubated at a temperature of between 21°C and 65°C.

[0371] 33. The process of embodiment 32, wherein the mRNA-LNPs are incubated at a temperature of about 26°C, about 30°C, or about 65°C.

[0372] 34. The process of any one of embodiments 31-33, wherein the mRNA-LNPs are incubated for greater than about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes.

[0373] 35. The process of embodiment 34, wherein the mRNA-LNPs are incubated for about 60 minutes.

[0374] 36. The process of any one of the preceding embodiments, wherein the lipid solution does not comprise a flammable solvent, e.g., ethanol.

[0375] 37. The process of any one of the preceding embodiments, wherein the mRNA-LNPs are purified by tangential flow filtration.

[0376] 38. The process of any one of the preceding embodiments, wherein at least 1 g, 5 g, 10 g, 20 g, 50 g, 100 g, or 1 kg of mRNA is encapsulated in lipid nanoparticles in a single batch.

[0377] 39. The process of any one of the preceding embodiments, wherein the mRNA solution and the lipid solution are mixed by a pulse-less flow pump.

[0378] 40. The process of embodiment 39, wherein the pump is a gear pump.

[0379] 41. The process of embodiment 39, wherein the pump is a centrifugal pump.

[0380] 42. The process of any one of the preceding embodiments, wherein the mRNA solution is mixed at a flow rate of at least 150 mL / min, at least 250 mL / min, at least 500 mL / min, at least 1000 mL / min, at least 2000 mL / min, at least 3000 mL / min, or at least 4000 mL / min.

[0381] 43. The process of embodiment 42, wherein the mRNA solution is mixed at a flow rate of about 800 mL / min, about 1000 mL / min, or about 12000 mL / min.

[0382] 44. The process of any one of the preceding embodiments, wherein the lipid solution is mixed at a flow rate of at least about 25 mL / min, at least 75 mL / min, at least 200 mL / min, at least 350 mL / min, at least 500 mL / min, at least 650 mL / min, at least 850 mL / min, or at least 1000 mL / min.

[0383] 45. The process of embodiment 44, wherein the lipid solution is mixed at a flow rate of about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min.

[0384] 46. The process of any one of embodiments 42 to 45, wherein the flow rate of the mRNA solution is at least about 2 times, 4 times, or 6 times greater than the flow rate of the lipid solution.

[0385] 47. The process of any one of the preceding embodiments, wherein the mRNA is encapsulated in a process free of volatile organic compounds. 48. The process of any one of the preceding embodiments, wherein the mRNA is encapsulated in a process free of flammable solvent, e.g., ethanol.

[0386] 49. A composition comprising one or more lipid nanoparticles encapsulating mRNA (mRNA- LNPs), wherein the lipid nanoparticle (LNP) comprises one or more cationic lipids, one or more noncationic lipids, and one or more PEG-modified lipids, and wherein the composition further comprises diethylene glycol monoethyl ether or tert-amyl alcohol.

[0387] 50. The composition of embodiment 49, further comprising polyethylene glycol, propylene glycol, 1,3-propanediol, polyvinyl pyrrolidone (PVP), or polyvinyl alcohol (PVA), or a combination of two or more thereof.

[0388] 51. The composition of embodiment 49 or 50, wherein diethylene glycol monoethyl ether is present in the composition at a concentration of at least about 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 19% v / v.

[0389] 52. The composition of any one of embodiments 49 to 51, wherein diethylene glycol monoethyl ether is present in the composition at a concentration of about 20% v / v.

[0390] 53. The composition of embodiment 49 or 50, wherein tert-amyl alcohol is present in the composition at a concentration of 3% to 5% v / v.

[0391] 54. The composition of any one of embodiments 49, 50, and 53, wherein tert-amyl alcohol is present in the composition at a concentration of 3.2% to 4.6% v / v, optionally at a concentration of 3.4% to 4.4% v / v or 4% to 4.2% v / v.

[0392] 55. The composition of embodiment 49, 53 or 54, wherein tert-amyl alcohol is present in propylene glycol.

[0393] 56. The composition of any one of embodiments 49-50 and 53-55, wherein tert-amyl alcohol is present in the composition at a concentration of about 4% v / v in propylene glycol.

[0394] 57. The composition of any one of embodiments 49 to 56, wherein the composition comprises 1 g or more, 5 g or more, 10 g or more, 20 g or more, 50 g or more, 100 g or more, or 1 kg or more of mRNA.

[0395] 58. The process of any one of embodiments 1 to 48, or the composition of any one of embodiments 49 to 57, wherein the mRNA comprises one or more modified nucleotides.

[0396] 59. The process of any one of embodiments 1 to 48, or the composition of any one of embodiments 49 to 57, wherein the mRNA is unmodified.

[0397] 60. The process of any one of embodiments 1 to 48 and 58 to 59, or the composition of any one of embodiments 49 to 59, wherein the mRNA is at least about 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 8 kb, 10 kb, 20 kb, 30 kb, or 40 kb in length. 61. The process of any one of embodiments 1 to 48 and 58 to 60, or the composition of any one of embodiments 49 to 60, wherein the mRNA-LNPs have an average size of less than 150 nm, less than 125 nm, or less than 100 nm.

[0398] 62. The process of any one of embodiments 1 to 48 and 58 to 61, or the composition of any one of embodiment 49 to 61, wherein the mRNA-LNPs have an average size ranging from 80-130 nm.

[0399] 63. The process of any one of embodiments 1 to 48 and 58 to 62, or the composition of any one of embodiments 49 to 62, wherein the lipid nanoparticles have a PDI of less than about 0.3, less than about 0.2, or less than about 0.18.

[0400] 64. The process of any one of embodiments 1 to 48 and 58 to 63, or the composition of any one of embodiments 49 to 63, wherein the encapsulation efficiency of the mRNA in the LNPs is greater than about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95%.

[0401] 65. The process of any one of embodiments 1 to 48 and 58 to 64, or the composition of any one of embodiments 49 to 64, wherein the one or more non-cationic lipids is selected from DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE, DOPS, DEPE, DPOC, DLPC, DLPE, or a combination of two or more thereof.

[0402] 66. The process or the composition of embodiment 65, wherein the one or more non-cationic lipids is DOPE or DSPC, optionally DOPE.

[0403] 67. The process of any one of embodiments 1 to 48 and 58 to 66, or the composition of any one of embodiments 49 to 66, wherein the one or more cationic lipids is selected from DOTMA, DOTAP, DSDMA, ML-2, cKK-ElO, CKK-E12, OF-02, C12-200, DLinDMA, DLinkC2DMA, ICE (Imidazol- based), HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4004, HGT4005, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DLenDMA, CLinDMA, Octyl-CLinDMA, Octyl-CLinDMA (2R), Octyl-CLinDMA (2S), ALNY-100, NC98-5, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLin-K-DMA, DLin-K-XTC2-DMA, cDD-TE-4-E10, CDD-TE-4-E12, DLin-MC3-DMA, DLin-KC2-DMA, Dlin-DMA, L319, SM-102, ALC-0315, DC-Chol, 3060110, 9A1P9, A2- Iso5-2DC18, BAME-O16B, FTT5, OF-Deg-Lin, TT3, Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide, MVL5, Lipid 5, ATX-126, GL-HEPES-E3-E10- DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, IM-001, or a combination of two or more thereof, optionally ML-2, OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10.

[0404] 68. The process of any one of embodiments 1 to 48 and 58 to 67, or the composition of any one of embodiments 49 to 67, wherein the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. 69. The process or the composition of embodiment 68, wherein the PEG-modified lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0405] 70. The composition of any one of embodiments 49 to 69, wherein the LNPs further comprise one or more cholesterol-based lipids, optionally cholesterol.

[0406] 71. The composition of embodiment 70, wherein the LNPs comprise cationic lipid at a molar ratio of 40%, DMG-PEG2K at a molar ratio of 1.5%, DOPE at a molar ratio of 30%, and cholesterol at a molar ratio of 28.5%.

[0407] 72. The composition of any one of embodiments 49 to 71, wherein the composition further comprises trehalose.

[0408] 73. The process of any one of embodiments 1 to 48 and 58 to 69, or the composition of any one of embodiments 49 to 72, wherein the mRNA-LNPs have an N / P ratio between 1 and 10.

[0409] 74. The process or the composition of embodiment 73, wherein the mRNA-LNPs have an N / P ratio from 2 to 6.

[0410] 75. The process or the composition of embodiment 74, wherein the mRNA-LNPs have an N / P ratio of about 4.

[0411] 76. The composition of any one of embodiments 49 to 75, wherein the composition does not comprise a flammable solvent, e.g., ethanol.

[0412] 77. A composition comprising mRNA encapsulated in lipid nanoparticles (mRNA-LNPs) prepared by the process of any one of embodiments 1-48, 58-69, and 73-75.

[0413] 78. The composition of any one of embodiments 49 to 77, for use in treating a disease in a subject.

[0414] 79. A kit of parts, comprising a first container and a second container, wherein the first container comprises an mRNA solution comprising one or more mRNAs and the second container comprises a lipid solution as described in any one of embodiments 1 to 8, 27-28, and 65-69.

[0415] EXAMPLES

[0416]

[0333] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0417] Example 1. Solvent screening and selection

[0418]

[0334] While ethanol is typically used to dissolve lipids prior to LNP formation, development of ethanol-free LNP formulations would greatly reduce or even eliminate fire safety concerns and also allow for bedside mixing leading to the production of low-volume formulations with 1:1 ratios of mRNA to lipid solutions that would be more suitable for dosing. Such low-volume formulations are currently difficult to obtain using ethanol as a solvent.

[0335] This example illustrates the initial solvent screening process. Briefly, 71 solvents from different classes, including triglycerides, fatty alcohols, ionic liquids, and various mixtures, were screened according to specific characteristics, including appearance, miscibility in water, solubility of various lipids in the solvent, and absence of phase separation. Triethylene glycol monomethyl ether (mTEG), a previously identified solvent, was included as a control. Solvents of interest were liquid in appearance, dissolved all lipids, were miscible in water, and did not undergo phase separation. Solvents not meeting these criteria were excluded from further analysis.

[0419]

[0336] A summary is provided in Table 1. In the case of solvent mixtures, amounts are provided in % volume / volume (v / v). Table 1: Solvent screening

[0420] Choi.: Cholesterol; CL: cationic lipid; Phase sep: Phase separation; misc: miscibility; M: Miscible,

[0421] NM: Not miscible; S: Soluble; NS: Not soluble

[0422]

[0337] The following solvents were selected for further evaluation: heptanol in propylene glycol (PG), tert amyl alcohol (TAA) in PG, diethylene glycol monoethyl ether, l-ethyl-3- methylimidazolium acetate, l-ethyl-3-methylimidazolium propionate, and l-ethyl-3- methylimidazolium diethylphosphate.

[0423] Example 2. Screening of selected solvents based on mRNA-LNP characteristics

[0424]

[0338] Solvents selected in the initial screening were further evaluated either alone or at various concentrations in propylene glycol to determine their impact on mRNA-LNP formation.

[0339] To this end, exemplary formulations were prepared by mixing mRNA in an aqueous solution with lipids dissolved in solvent to form mRNA encapsulated within LNPs (mRNA-LNPs) at a 1 mg batch size. Human erythropoietin (hEPO) mRNA that was produced by in vitro transcription, capped, tailed, and purified was used for encapsulation. Purity, integrity, and capping of mRNA preparations were verified before storage at -20°C. The aqueous mRNA solution was prepared by adding the mRNA to a citrate buffer (1 mM citrate, 150 mM NaCI, pH 4.5), resulting in mRNA at a concentration of 0.0833 mg / mL in the buffer. The lipid solution was prepared by dissolving lipids in solvent. The solvent was either ethanol or an ethanol-free solvent selected from the screening of Example 1. Lipids in the lipid solution were provided at the concentration necessary to provide a cationic lipid to mRNA N / P ratio of 4. mRNA and lipid solutions were heated to 65°C prior to mixing. The mRNA solution was mixed with the lipid solution at volumetric ratio of 4 to 1. mRNA was encapsulated in LNPs to form mRNA-LNPs by mixing the lipid solution and the mRNA solution in a mixer using a pump system. The resultant solution was then subjected to buffer exchange using TFF / dialysis tubes, concentrated, and stored at -80°C. LNPs were composed of a mixture of four lipids: cationic lipid, PEGylated lipid DMG-PEG-2K, cholesterol, and helper lipid, in this order, at a molar ratio of 40:1.5:28.5:30. ML-2 was used as a representative cationic lipid.

[0425]

[0340] Particle size and polydispersity index (PDI) were determined for mRNA-LNPs produced using ethanol free solvents and compared to those obtained when using ethanol. Results are provided in Table 2. LNP particle sizes (DLS) and PDI were determined using a Malvern Zetasizer instrument. Samples were analyzed by diluting the formulation in 10% v / v trehalose to an mRNA concentration of about 0.1 mg / mL and then measuring the size and PDI in an optical grade polystyrene cuvette.

[0426] Table 2: Particle size and PDI of mRNA-LNPs obtained with selected solvents

[0427] ND: not done. PG: propylene glycol

[0428]

[0341] Solvents yielding particle sizes of greater than 150 nm and a PDI above 0.2 were not further evaluated. mRNA-LNPs obtained using lipids dissolved in 20% v / v TAA in PG or in diethylene glycol monoethyl ether showed particle sizes of less than 150 nm and PDIs below 0.2 and were further evaluated. Example 3. Solvent flammability

[0429]

[0342] To verify that selected solvents were indeed non-flammable, they were subjected to a flashpoint analysis using the Cleveland Open Cup assay, as necessary. It was confirmed that the flashpoint of 20% v / v TAA in PG was superior to 37.8°C (41°C, data not shown). Thus, this solvent mixture was non-flammable. Mixtures comprising up to 23% v / v TAA in PG were similarly nonflammable. Information for diethylene glycol monoethyl ether available from the manufacturer confirmed that it was also not flammable.

[0430] Example 4. Characterization of lipid nanoparticle (LNP) formulations obtained using nonflammable solvent

[0431]

[0343] This example illustrates the encapsulation efficiency achieved by using TAA in PG or diethylene glycol monoethyl ether as the solvent present in the lipid solution in the production of ethanol-free LNP formulations.

[0432]

[0344] LNP formulations were prepared as described in Example 2. In this example, the lipids were dissolved in an ethanol-free solution (i.e., 20% v / v TAA in PG or 100% diethylene glycol monoethyl ether) or in ethanol. Four different cationic lipids were assessed: ML-2, GL-HEPES-E3-E12-DS-4-E10, cKK-ElO, and OF-02. Particle size, PDI, zeta potential, encapsulation efficiency (EE), and mRNA integrity of the mRNA-LNPs obtained when using ethanol-free TAA in PG or diethylene glycol monoethyl ether solvents were assessed. Particle size and PDI were measured as described in Example 2. Zeta potential was measured by photon correlation spectroscopy using available equipment systems, for example, Zetasizer Nano (Malvern Instruments). mRNA EE was measured using a Ribogreen™ fluorescence plate-based assay, mRNA integrity was measured by extracting the mRNA from the LNP and analyzing on a fragment analyzer using capillary electrophoresis. Ethanol solvent was used to provide a baseline control. Results are shown in Table 3 below.

[0433] Table 3. Characteristics of mRNA-LNPs prepared using TAA in PG or diethylene glycol monoethyl ether solvent versus ethanol for various cationic lipids.

[0434]

[0345] As seen in Table 3, mRNA-LNP formulations prepared with TAA in PG or diethylene glycol monoethyl ether yielded LNPs of comparable size, polydispersity, and zeta potential to ethanol- prepared LNP formulations, regardless of the LNP composition, as similar results were observed in presence of any of four different cationic lipids. Similarly, no differences were observed in mRNA integrity. Encapsulation efficiencies were found to be equivalent or slightly lower in the TAA in PG and diethylene glycol monoethyl ether formulations relative to ethanol formulations. Nevertheless, all formulations were acceptable as they met a 70% encapsulation efficiency threshold. Furthermore, encapsulation efficiency was superior or equal to 80% for all mRNA-LNPs except the mRNA-LNP having the ML-2 cationic lipid and obtained using TAA in PG (EE of 78%).

[0435]

[0346] Both TAA in PG and diethylene glycol monoethyl ether could be successfully used to produce mRNA-LNPs, indicating that alternative solvents to ethanol can be used to dissolve a variety of lipid compositions.

[0436] Example 5. Scaled up production of mRNA-LNPs using a non-flammable solvent

[0437]

[0347] This example illustrates that production of mRNA-LNPs using a non-flammable solvent can be successfully scaled up.

[0438]

[0348] Exemplary LNP formulations were prepared as described in Examples 2 and 3, with production scaled up 10-fold to produce a lOmg batch of mRNA-LNPs. Lipids were dissolved in 20% v / vTAA in PG and two different cationic lipids were assessed: ML-2 and cKK-ElO. As shown in Table 4, characteristics of mRNA-LNPs produced at a 10 mg scale were similar to those obtained at the 1 mg scale. Encapsulation efficiency was slightly lower when using TAA in PG as solvent as compared to ethanol, but was nevertheless higher than 80%, regardless of the cationic lipid tested. Table 4. Characteristics of mRNA-LNPs prepared using an ethanol-free TAA in PG solvent mixture versus an ethanol mixture at a 10 mg scale.

[0439]

[0349] The results confirm that TAA in PG can be used to dissolve lipids allowing for safe manufacture of ethanol-free LNP formulations, and that formulation can be successfully scaled up to obtain high quality mRNA-LNPs.

[0440] Example 6. In vitro expression of hEPO mRNA after administration of mRNA-LNPs prepared using ethanol based or ethanol free methods

[0441]

[0350] This example illustrates in vitro production of protein following in vitro administration of mRNA-LNPs produced using an ethanol-free solvent.

[0442]

[0351] In this study, Hela cells were transfected with human erythropoietin (hEPO) mRNA-LNPs diluted in 10% v / v trehalose at a quantity equivalent to 1.5 pg mRNA per 106cells in a 96-well plate. mRNA-LNPs were prepared using one of two different cationic lipids (ML-2 or cKK-ElO) according to the process described in Example 2, with the solvent being either ethanol or 20% v / v TAA in PG. Cells were incubated for about 20h at 37°C with 5% CO2, and supernatants were collected and stored at 80°C until use. HeLa cells were transfected with 1.5 pg mRNA in Lipofectamine MessengerMax® as a positive control.

[0443]

[0352] hEPO expression was determined via an ELISA using R and D Systems, Quantikine® IVD® ELISA, Human Erythropoietin Immunoassay kit as per the manufacturer's instructions, and reported as final values of ng / mL. Levels of hEPO expression were comparable for mRNA-LNPs generated with ethanol-based and ethanol-free processes, regardless of the cationic lipid used (see Fig. 1).

[0444] Example 7. In vivo expression of hEPO mRNA after intramuscular delivery of mRNA-LNPs prepared using ethanol based or ethanol free methods

[0445]

[0353] This example illustrates that the production of mRNA-LNPs with a non-flammable solvent such as TAA in PG can successfully replace ethanol.

[0446]

[0354] In this study, mRNA-LNPs were prepared according to the process described in Example 2, with the solvent being either ethanol or 20% v / v TAA in PG. The cationic lipid used was either cKK- E10 or GL-HEPES-E3-E12-DS-4-E10. Female BALB / c mice (n=4 except for groups 2 and 6 where n=8) at 6-8 weeks old were given a single intramuscular injection into the right gastrocnemius muscle at a hEPO mRNA dose of 0.1 pg in a volume of 30 pL. Serum was collected at 6 hours post administration and the amount of hEPO protein in the blood was detected using ELISA as described in Example 6.

[0447]

[0355] As can be seen from Fig. 2, the control mRNA-LNPs prepared with ethanol yielded serum levels of hEPO protein of between about 10 and 25 ng / mL hEPO. Levels varied according to the cationic lipid used. When mRNA-LNPs were prepared under ethanol-free conditions (here with 20% v / v TAA in PG solvent), hEPO serum levels were comparable to those seen for the formulation obtained with the ethanol-based process.

[0448] Example 8: In vivo expression of hemagglutinin (HA) mRNA and reactogenicity after intramuscular delivery of mRNA-LNPs prepared using ethanol based or ethanol free methods

[0449]

[0356] The ability of influenza hemagglutinin (HA) mRNA-LNPs obtained with ethanol-free and ethanol-based processes to induce an immune response was determined in mice. Reactogenicity was also evaluated.

[0450]

[0357] mRNA-LNPs were prepared according to the process described in Example 2, with the solvent being either ethanol, 20% v / v TAA in PG, or diethylene glycol monoethyl ether. The cationic lipid used was either cKK-ElO or GL-HEPES-E3-E12-DS-4-E10. Female BALB / c mice at 6-8 weeks of age were immunized under isoflurane anesthesia with a dose of 0.04 pg of HA mRNA per mouse in 0.05 mL of mRNA-LNPs via the IM route in the quadriceps on day 0 in one hind leg and day 21 in the contralateral leg. Reactogenicity was determined at days 22, 23, and 24 post first administration by scoring on a scale of 0-5 (0: no swelling, 1: Minimal, 2: Mild, 3: Moderate, 4: Severe, 5: Significant severe swelling requiring veterinary intervention). Any mice that lost more than 20% of their initial body weight and displayed severe clinical signs were euthanized after the veterinarian's assessment of the animal's health prior to the study termination by administration of 5 mg / kg of meloxicam by subcutaneous injection.

[0451]

[0358] Serum was collected at day 35 to measure functional antibody titers by the hemagglutination inhibition (HAI) assay. Briefly, blood samples were collected into SST tubes and allowed to clot for 30 minutes to 1 hour at room temperature. The samples were then centrifuged 1000-1300 x g for 5-10 minutes with brakes off. Serum was divided into two 0.5 mL cryovials and stored at -20°C.

[0452]

[0359] HAI assays were performed using the A / Tasmania / 503 / 2020 (H3N2) virus stocks from Microbiologies. Sera were treated with receptor-destroying enzyme (RDE) by diluting one part serum with three parts enzyme and incubated overnight in a 37°C water bath. Enzyme was inactivated by a 30-minute incubation period at 56°C followed by addition of six parts phosphate buffered saline (PBS) for a final dilution of 1 / 10. HAI assays were performed in V-bottom 96-well plates using four hemagglutinating units (HAU) of virus and 0.5% turkey red blood cells (RBC). The reference serum for each strain was included as a positive control on every assay plate. Each plate also included a back-titration to confirm the antigen dose (4 HAU / 25pl) as well as a negative control sample (PBS or naive control serum). The HAI titer was determined as the highest dilution of serum resulting in complete inhibition of hemagglutination. Results were only valid for plates with the appropriate back-titration result (verifying 4 HAU / 25 pl added) and a reference serum titer within 2-fold of the expected titer.

[0453]

[0360] Data analysis was performed using GraphPad Prism, and p values were determined via unpaired two-tailed t-test and 1-way ANOVA, as applicable. P-values of <0.05 were considered to be significant.

[0454] No significant differences could be seen in HAI titers (Fig. 3) or reactogenicity (Fig. 4) between mRNA-LNPs obtained using an ethanol-free process and the ethanol-based process, indicating that these alternative solvents could successfully replace ethanol.

[0455] Example 9: Evaluation of stability of LNP formulations

[0456]

[0361] The stability of mRNA-LNPs obtained with ethanol-free and ethanol-based processes was evaluated via an accelerated stability study.

[0457]

[0362] mRNA-LNPs were prepared according to the process described in Example 2, with the solvent being ethanol, 20% v / v TAA in PG, or diethylene glycol monoethyl ether. The cationic lipid used was either cKK-ElO or GL-HEPES-E3-E12-DS-4-E10. mRNA-LNPs were subjected to biophysical characterizations (particle size, encapsulation efficiency, and mRNA integrity) prior to storage (day 0) and then either following storage for one month at -80°C (Fig. 5) or following daily freeze-thaw cycles from -80°C or 25°C for 5 days (Fig. 6). Particle size was measured using DLS, mRNA encapsulation efficiency was measured using a fluorescence plate-based assay, and mRNA integrity was measured by extracting the mRNA from the LNP and analyzing on a fragment analyzer using capillary electrophoresis.

[0458]

[0363] Stability of LNP formulations were similar between formulations obtained using an ethanol- free process and the ethanol-based process (see Fig. 5 and Fig. 6). Example 10: Synthesis of IM-001 according to Scheme 2

[0459]

[0364] Scheme 2: General Synthetic Scheme for Lipid of Formula (II)

[0460] IM-001 Abbreviations: DCM: Dichloromethane, DIPEA: N,N-Diisopropylethylamine, DMAP: 4- Dimethylaminopyridine, EDC: l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, EtOAc: Ethyl acetate, NaHCO3: Sodium hydrogencarbonate, Py: Pyridine, Na2SO4: Sodium Sulfate, TEA: Triethylamine, TFA: Trifluoroacetic Acid, MS: Mass spectrometry, ESI-MS: Electrospray ionization mass spectrometry, TLC: Thin Layer Chromatography

[0365] Step 1: Synthesis of Intermediate (3)

[0461]

[0366] As depicted in Scheme 2: To a solution of acid (2) (4.58 g, 6.55 mmol) and isomannide (1) (0.38 g, 2.62 mmol) in dichloromethane (40 mL) were added DIPEA (3.65 mL, 20.96 mmol), DMAP (0.32 g, 2.62 mmol) and EDC (1.5 g, 7.86 mmol). The resulting mixture was stirred at room temperature for overnight. After 16 h, MS and TLC (30% EtOAc in hexanes) analysis indicated completion of the reaction. The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4and concentrated. The crude residue was purified, and the desired product was eluted at 6% EtOAc in hexanes. The product containing fractions were concentrated to obtain 2.58 g (65%) of pure product.

[0367] Results: ESI-MS: Calculated C86H177N2O10Si4, [M + H+] = 1510.25, Observed = 1510.3

[0462]

[0368] Step 2: Synthesis of IM-001

[0463]

[0369] As depicted in Scheme 2: To a solution of Intermediate (3) (2.58 g, 1.70 mmol) in tetrahydrofuran (14 mL) was added hydrogen fluoride (70% HF.py complex, 7 mL, 51.23 mmol) at 0 °C and stirred at the same temperature for 5 minutes. Then reaction mixture was warmed to room temperature and stirred for 16 h. MS analysis indicated completion of the reaction. The reaction mixture was diluted with ethyl acetate, quenched by slow addition of solid NaHCO3at 0 °C, followed by saturated NaHCO3solution. The organic layer was washed with sat. NaHCO3solution, water and brine. Then dried over anhydrous Na2SO4and concentrated. The crude residue was purified, and the desired product was eluted at 67% EtOAc in hexanes. The purest fractions were concentrated to obtain 1.1 g (61%) of pure product.

[0464]

[0370] Results:TH NMR (400 MHz, CDCI3) 6 5.13 - 5.03 (m, 2H), 4.73 - 4.65 (m, 2H), 4.29 - 3.83 (m, 8H), 3.52 - 2.98 (m, 12H), 2.69 - 2.49 (m, 4H), 2.32 - 2.09 (m, 4H), 1.73 - 1.12 (m, 72H), 0.88 (t, J = 6.6 Hz, 12H).

[0371] ESI-MS: Calculated C62H121N2O10, [M + H+] = 1053.90, Observed = 1053.2 and 527.2 [M / 2 +

[0465] H+].

[0466] Example 11: Synthesis of IS-001 according to Scheme 3

[0467]

[0372] Scheme 3: General Synthetic Scheme for Lipid of Formula (III)

[0373] Abbreviations are as indicated in Example 10.

[0468]

[0374] Step 1: Synthesis of Intermediate (3)

[0469]

[0375] As depicted in Scheme 3: To a solution of acid (2) (1.2 g, 1.71 mmol) and isosorbide (1) (0.100 g, 0.68 mmol) in dichloromethane (10 mL) were added DIPEA (0.95 mL, 5.47 mmol), DMAP (0.084 g, 0.68 mmol) and EDC (0.393 g, 2.05 mmol). The resulting mixture was stirred at room temperature for overnight. After 16 h, MS and TLC (30% EtOAc in hexanes) analysis indicated completion of the reaction. The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified, and the desired product was eluted at 6% EtOAc in hexanes. The product containing fractions were concentrated to obtain 0.72 g (69%) of pure product.

[0470]

[0376] Results: ESI-MS: Calculated C86H177N2O10Si4, [M + H+] = 1510.25, Observed = 1510.3 and 755.4 [M / 2 + H+]

[0471]

[0377] Step 2: Synthesis of IS-001

[0472]

[0378] As depicted in Scheme 3: To a solution of Intermediate (3) (0.72 g, 0.476 mmol) in tetrahydrofuran (4 mL) was added hydrogen fluoride (70% HF.py complex, 2 mL, 14.298 mmol) at 0 °C and stirred at the same temperature for 5 minutes. Then reaction mixture was warmed to room temperature and stirred for 16 h. MS analysis indicated completion of the reaction. The reaction mixture was diluted with ethyl acetate, quenched by slow addition of solid NaHCO3 at 0 °C, followed by saturated NaHCO3solution. The organic layer was washed with sat. NaHCO3solution, water and brine. Then dried over anhydrous Na2SO4 and concentrated. The crude residue was purified, and the desired product was eluted at 65% EtOAc in hexanes. The purest fractions were concentrated to obtain 0.120 g (24%) of pure product.

[0473]

[0379] Results:1H NMR (400 MHz, CDCI3) 6 5.30 - 5.00 (m, 2H), 4.97 - 4.68 (m, 2H), 4.55 - 3.71 (m, 8H), 3.57 - 2.92 (m, 8H), 2.84 - 2.04 (m, 8H), 1.99 - 1.01 (m, 76H), 0.88 (t, J = 6.8 Hz, 12H). ESI-MS: Calculated C62H121N2O10, [M + H+] = 1053.90, Observed = 1053.2 and 527.3 [M / 2 + H+]

Claims

CLAIMS1. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) comprising a step of mixing (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising i) one or more cationic lipids; ii) one or more non-cationic lipids; iii) one or more PEG-modified lipids; and iv) a solvent, wherein the solvent comprises diethylene glycol monoethyl ether or tert-amyl alcohol, thereby forming mRNA encapsulated within the LNPs (mRNA-LNPs).

2. The process of claim 1, wherein the solvent further comprises propylene glycol, polyethylene glycol, 1,3-propanediol, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), or a combination of two or more thereof.

3. The process of claim 1 or 2, wherein the solvent comprises diethylene glycol monoethyl ether at a concentration of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% v / v, optionally wherein the solvent consists of diethylene glycol monoethyl ether.

4. The process of claim 1 or 2, wherein the solvent comprises tert-amyl alcohol at a concentration of between 15% and 23% v / v e.g., about 20% v / v.

5. The process of any one of claims 1-2 and 4, wherein the solvent comprises tert-amyl alcohol at a concentration of about 20% v / v in propylene glycol.

6. The process of any one of the preceding claims, wherein the mRNA solution and the lipid solution are mixed at a ratio (v / v) of between 2:1 and 6:1, optionally a ratio (v / v) of about 4:1.

7. The process of any one of the preceding claims, wherein at least about 1 g, 5 g, 10 g, 20 g, 50 g, 100 g, or 1 kg of mRNA is encapsulated in the lipid nanoparticles in a single batch.

8. The process of any one of the preceding claims, wherein the mRNA is encapsulated in a process free of alcohol e.g., ethanol.

9. A composition comprising one or more lipid nanoparticles (LNPs) encapsulating mRNA (mRNA-LNPs), wherein the LNP comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, and wherein the composition further comprises diethylene glycol monoethyl ether or tert-amyl alcohol.

10. The composition of claim 9, further comprising propylene glycol, polyethylene glycol, 1,3- propanediol, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), or a combination of two or more thereof.

11. The composition of claim 9 or 10, wherein the diethylene glycol monoethyl ether is present at a concentration of at least about 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 19% v / v, optionally wherein the diethylene glycol monoethyl ether is present at a concentration of about 20% v / v.

12. The composition of claim 9 or 10, wherein tert-amyl alcohol is present at a concentration of 3% to 5% v / v e.g., about 4% v / v, optionally wherein the composition further comprises propylene glycol.

13. The process of any one of claims 1-8 or the composition of any one of claims 9-12, wherein: a. the mRNA-LNPs have an average size of less than 150 nm, less than 125 nm, or less than 100 nm, optionally an average size ranging from 80-130 nm; and / or b. the lipid nanoparticles have a PDI of less than about 0.3, less than about 0.2, or less than about 0.18; and / or c. the encapsulation efficiency of the mRNA in the LNPs is greater than about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95%; and / or d. the mRNA-LNPs have an N / P ratio of between 1 and 10, optionally from 2 to 6, e.g., about 4.

14. The process of any one of claims 1-8 or 13 or the composition of any one of claims 9-13, wherein: a. the one or more non-cationic lipids is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-0- monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2- dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), l,2-dioleoyl-sn-glycero-3- phosphocholine (DPOC), l,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dilauroyl- sn-glycero-3-phosphoethanolamine (DLPE), optionally wherein the one or more non-cationic lipids is DOPE or DSPC; and / or b. the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length, optionally wherein the PEG-modified lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K); and / orc. the one or more cationic lipids is selected from ML-2, OF-02, cKK-ElO, OF-Deg-Lin, GL-HEPES- E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, ALC- 0315, ATX-126, IM-001, IS-001, or a combination of two or more thereof, optionally ML-2, OF- 02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10.

15. A composition comprising mRNA encapsulated in lipid nanoparticles (mRNA-LNPs) prepared by the process of any one of claims 1-7, 13, and 14.

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