Lipid nanoparticle formulations for mRNA delivery

Abstract

This invention provides a method for encapsulating mRNA in lipid nanoparticles without the use of flammable solvents for mRNA delivery in therapeutic applications. [Solution] A method for encapsulating mRNA in LNPs is provided, comprising the steps of (a) mixing an mRNA solution containing one or more mRNAs with (b) a lipid solution containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, wherein the step of mixing the mRNA solution and the lipid solution is to be mixed in the presence of an amphiphilic polymer to form mRNA encapsulated within the LNP in an LNP formulation solution (mRNA-LNP).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests and priority of U.S. Provisional Patent Application No. 63 / 025,355, filed on 15 May 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Messenger RNA (mRNA) therapy is becoming an increasingly important approach to treating a variety of diseases. Messenger RNA therapy involves administering messenger RNA to a patient to produce the mRNA-encoded protein within the patient's body. mRNA preparations encapsulated in lipids, such as lipid nanoparticle (LNP) compositions, exhibit high levels of cellular uptake and protein expression. Lipid nanoparticle preparations traditionally use ethanol as a solvent for the lipid solution, which is subsequently mixed with the mRNA solution.

[0003] However, the use of flammable solvents such as ethanol increases safety risks and manufacturing costs, especially in large-scale applications. Furthermore, low-volume LNP formulations, which are more suitable for administration and reduce downstream processing volume and costs, are currently difficult to obtain using ethanol as a solvent. Low-volume LNP formulations are also desirable because they allow for bedside mixing for other administration routes, such as subcutaneous or intramuscular administration. [Overview of the project] [Means for solving the problem]

[0004] There is a need for stable, safe, and cost-effective ethanol-free LNP formulations with high mRNA encapsulation efficiency for efficient delivery in therapeutic use. The present invention provides a stable, safe, and cost-effective method for encapsulating mRNA in lipid nanoparticles without the use of flammable solvents, resulting in LNPs with high encapsulation efficiency for messenger RNA delivery in therapeutic applications. In one embodiment, the present invention provides a safer and more cost-effective method for large-scale manufacturing processes. In another embodiment, the present invention provides a method for producing low-volume LNP formulations suitable for bedside mixing, which not only reduces downstream processing in manufacturing but also facilitates drug administration and multiple administration routes, including subcutaneous and intramuscular. The present invention is based on the remarkable discovery that mixing an mRNA solution with a lipid solution in the presence of an amphiphilic polymer forms mRNA encapsulated within LNPs (mRNA-LNPs) in an LNP formulation solution. The present invention provides, among other things, a safe, efficient, and cost-effective method for producing compositions containing mRNA-loaded lipid nanoparticles.

[0005] In one embodiment, the present invention provides a method for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), comprising the steps of (a) mixing an mRNA solution containing one or more mRNAs with (b) a lipid solution containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, wherein the step of mixing the mRNA solution and the lipid solution comprises mixing in the presence of an amphiphilic polymer to form mRNA encapsulated within LNPs in an LNP formulation solution (mRNA-LNPs). In some embodiments, the lipid solution contains three lipid components. In some embodiments, the lipid solution contains four lipid components. In certain embodiments, the four lipid components of the lipid solution are a PEG-modified lipid, a cationic lipid (e.g., ML-2 or MC-3), cholesterol, and a helper (e.g., non-cationic) lipid (e.g., DSPC or DOPE).

[0006] In some embodiments, the amphiphilic polymer is pluronic, polyvinylpyrrolidone. This includes polyvinyl alcohol, polyethylene glycol (PEG), or a combination thereof. Therefore, in some embodiments, the amphiphilic polymer contains pluronic. In some embodiments, the amphiphilic polymer contains polyvinylpyrrolidone. In some embodiments, the amphiphilic polymer contains polyethylene glycol.

[0007] In some embodiments, PEG is triethylene glycol monomethyl ether (mTEG). In some embodiments, PEG is methoxypolyethylene glycol (mPEG). In some embodiments, PEG is tetraethylene glycol monomethyl ether. In some embodiments, PEG is pentaethylene glycol monomethyl ether. In some embodiments, PEG is a combination of mTEG, mPEG, tetraethylene glycol monomethyl ether, and / or pentaethylene glycol monomethyl ether.

[0008] In some embodiments, the step of mixing the mRNA solution and the lipid solution results in a PEG concentration of over 25% by volume / volume.

[0009] In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 50% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 45% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 40% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 35% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 30% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 25% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 20% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 15% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 10% volume / volt. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 5% volume / volume. In some embodiments, the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 1% volume / volume. In certain embodiments, PEG is mTEG. Particularly suitable final concentrations of mTEG in mRNA-LNP formulations are approximately 55–65% volume / volume, for example, approximately 50% volume / volume. As shown in the examples, this final concentration of mTEG maintains mRNA solubility and stability, and allows for reduced processing volume and easy production of formulations on a large scale.

[0010] In some embodiments, the mRNA solution contains less than 5 mM citrate and mRNA-LNPs have an inclusion efficiency of more than 60%. In some embodiments, the mRNA solution contains less than 5 mM citrate and mRNA-LNPs have an inclusion efficiency of more than 70%. In some embodiments, the mRNA solution contains less than 5 mM citrate and mRNA-LNPs have an inclusion efficiency of more than 80%. In some embodiments, the mRNA solution contains less than 5 mM citrate and mRNA-LNPs have an inclusion efficiency of more than 90%. In some embodiments, the mRNA solution contains less than 5 mM citrate and mRNA-LNPs have an inclusion efficiency of more than 95%. In some embodiments, the mRNA solution contains less than 5 mM citrate and mRNA-LNPs have an inclusion efficiency of more than 99%.

[0011] In some embodiments, the mRNA solution and / or lipid solution are at approximately ambient temperature.

[0012] In some embodiments, the ambient temperature is less than approximately 35°C. In some embodiments, the ambient temperature is less than approximately 32°C. In some embodiments, the ambient temperature is less than approximately 30°C. In some embodiments, the ambient temperature is less than approximately 28°C. In some embodiments, the ambient temperature is approximately 26°C. It is less than. In some embodiments, the ambient temperature is less than approximately 25°C. In some embodiments, the ambient temperature is less than approximately 24°C. In some embodiments, the ambient temperature is less than approximately 23°C. In some embodiments, the ambient temperature is less than approximately 22°C. In some embodiments, the ambient temperature is less than approximately 21°C. In some embodiments, the ambient temperature is less than approximately 20°C. In some embodiments, the ambient temperature is less than approximately 19°C. In some embodiments, the ambient temperature is less than approximately 18°C. In some embodiments, the ambient temperature is less than approximately 16°C.

[0013] In some embodiments, the ambient temperature is in the range of approximately 15 to 35°C. In some embodiments, the ambient temperature is in the range of approximately 16 to 32°C. In some embodiments, the ambient temperature is in the range of approximately 17 to 30°C. In some embodiments, the ambient temperature is in the range of approximately 18 to 30°C. In some embodiments, the ambient temperature is in the range of approximately 18 to 32°C. In some embodiments, the ambient temperature is in the range of approximately 20 to 28°C. In some embodiments, the ambient temperature is in the range of approximately 20 to 26°C. In some embodiments, the ambient temperature is in the range of approximately 20 to 25°C. In some embodiments, the ambient temperature is in the range of approximately 23 to 25°C. In some embodiments, the ambient temperature is in the range of approximately 21 to 24°C. In some embodiments, the ambient temperature is in the range of approximately 21 to 23°C. In some embodiments, the ambient temperature is in the range of approximately 21 to 26°C.

[0014] In some embodiments, the ambient temperature is approximately 16°C. In some embodiments, the ambient temperature is approximately 18°C. In some embodiments, the ambient temperature is approximately 20°C. In some embodiments, the ambient temperature is approximately 21°C. In some embodiments, the ambient temperature is approximately 22°C. In some embodiments, the ambient temperature is approximately 23°C. In some embodiments, the ambient temperature is approximately 24°C. In some embodiments, the ambient temperature is approximately 25°C. In some embodiments, the ambient temperature is approximately 26°C. In some embodiments, the ambient temperature is approximately 27°C. In some embodiments, the ambient temperature is approximately 28°C. In some embodiments, the ambient temperature is approximately 30°C. In some embodiments, the ambient temperature is approximately 31°C. In some embodiments, the ambient temperature is approximately 32°C. In some embodiments, the ambient temperature is approximately 35°C.

[0015] In some embodiments, one or more noncationic lipids are selected from distearoylphosphatidylcholine (DSPC). In some embodiments, one or more noncationic lipids are dioleoylphosphatidylcholine (DOPC). In some embodiments, one or more noncationic lipids are dipalmitoylphosphatidylcholine (DPPC). In some embodiments, one or more noncationic lipids are dioleoylphosphatidylglycerol (DOPG). In some embodiments, one or more noncationic lipids are dipalmitoylphosphatidylglycerol (DPPG). In some embodiments, one or more noncationic lipids are dioleoylphosphatidylethanolamine (DOPE). In some embodiments, one or more noncationic lipids are palmitoyloleoylphosphatidylcholine (POPC). In some embodiments, one or more noncationic lipids are palmitoyloleoylphosphatidylethanolamine (POPE). In some embodiments, one or more noncationic lipids are dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal). In some embodiments, one or more noncationic lipids are dipalmitoylphosphatidylethanolamine (DPPE). In some embodiments, one or more noncationic lipids are dimyristoylphosphoethanolamine (DMPE). In some embodiments, one or more noncationic lipids are distearoyl-phosphatidyl-ethanolamine (DSPE). In some embodiments, one or more noncationic lipids are phosphatidylserine. In some embodiments, one or more noncationic lipids are sphingolipids. In some embodiments, one or more noncationic lipids are cerebrosides. In some embodiments, one or more noncationic lipids are gangliosides. In some embodiments, one or more noncationic lipids are 16-O-monomethylPE. In some embodiments, one or more noncationic lipids are 16-O-dimethylPE. In some embodiments, one or more noncationic lipids are 18-1-transPE. In some embodiments, one or more noncationic lipids are l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE).

[0016] In some embodiments, the mRNA solution further contains trehalose. In some embodiments, the mRNA solution contains 20% trehalose. In some embodiments, the mRNA solution contains 15% trehalose. In some embodiments, the mRNA solution contains 10% trehalose. In some embodiments, the mRNA solution contains 5% trehalose.

[0017] In some embodiments, this method does not require the step of heating the mRNA solution and lipid solution before the mixing step.

[0018] In some embodiments, the mRNA solution contains more than approximately 1 g of mRNA per 12 L of mRNA solution. In some embodiments, the mRNA solution contains more than approximately 1 g of mRNA per 10 L of mRNA solution. In some embodiments, the mRNA solution contains approximately 1 g of mRNA per 8 L of mRNA solution. In some embodiments, the mRNA solution contains more than approximately 1 g of mRNA per 6 L of mRNA solution. In some embodiments, the mRNA solution contains approximately 1 g of mRNA per 4 L of mRNA solution. In some embodiments, the mRNA solution contains approximately 1 g of mRNA per 2 L of mRNA solution. In some embodiments, the mRNA solution contains more than approximately 1 g of mRNA per 1 L of mRNA solution.

[0019] In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 0.05 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 0.1 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 0.125 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 0.25 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 0.5 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 1.0 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 1.5 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is greater than approximately 2.0 mg / mL. In some embodiments, the concentration of mRNA in the mRNA solution is between approximately 0.05 mg / mL and approximately 0.5 mg / mL. In certain embodiments, the concentration of mRNA in the mRNA solution is between approximately 0.1 mg / mL and approximately 0.5 mg / mL, for example, approximately 0.1 mg / mL or approximately 0.35 mg / mL.

[0020] In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) between 1:1 and 10:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) between 2:1 and 6:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) of approximately 2:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) of approximately 3:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) of approximately 4:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) of approximately 5:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) of approximately 6:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) greater than approximately 2:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio (v / v) greater than approximately 3:1. In some embodiments, the mRNA solution and lipid solution are mixed in a ratio greater than approximately 4:1 (v / v). In some embodiments, the mRNA solution and lipid solution are mixed in a ratio greater than approximately 5:1 (v / v). In some embodiments, the mRNA solution and lipid solution are mixed in a ratio greater than approximately 6:1 (v / v). In some embodiments, the mRNA solution and lipid solution (e.g., approximately 100% mTEG lipid solution) are mixed in a ratio of 1 to 8:1, for example, 1 to 4:1 (v / v). They are mixed together. In certain embodiments, the mRNA solution and the lipid solution (e.g., about 100% mTEG lipid solution) are mixed in a ratio of about 1:1 (v / v). As shown in the examples, this ratio of mRNA solution to lipid solution maintains mRNA solubility and stability, and allows for reduced processing volume and easy production of formulations on a large scale.

[0021] In some embodiments, the mRNA solution has a pH between 2.5 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 approximately 3.0. In some embodiments, the mRNA solution has a pH of approximately 3.5. In some embodiments, the mRNA solution has a pH of approximately 4.0. In some embodiments, the mRNA solution has a pH of approximately 4.5. In some embodiments, the mRNA solution has a pH of approximately 5.0. In some embodiments, the mRNA solution has a pH of approximately 5.5.

[0022] In some embodiments, the mixing process is carried out with a total volume between approximately 3 and 10 mL. In some embodiments, the mixing process is carried out with a total volume between approximately 1 and 10 mL. In some embodiments, the mixing process is carried out with a total volume between approximately 1 and 15 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 1 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 2 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 3 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 4 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 5 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 6 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 7 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 8 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 9 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 10 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 12 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 13 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 14 mL. In some embodiments, the mixing process is carried out with a total volume of approximately 15 mL.

[0023] In some embodiments, this method does not contain alcohol.

[0024] In some embodiments, the method further includes a step of incubating mRNA-LNP. In some embodiments, the method further includes a step of incubating mRNA-LNP after mixing. In some embodiments, mRNA-LNP is incubated at a temperature between 21°C and 65°C. In some embodiments, mRNA-LNP is incubated at a temperature between 25°C and 60°C. In some embodiments, mRNA-LNP is incubated at a temperature between 30°C and 55°C. In some embodiments, mRNA-LNP is incubated at a temperature between 35°C and 50°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 26°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 30°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 31°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 32°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 35°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 36°C. In some embodiments, mRNA-LNP is incubated at a temperature of approximately 38°C. In some embodiments, mRNA-LNP is incubated at approximately 40°C. In some embodiments, mRNA-LNP is incubated at approximately 42°C. In some embodiments, mRNA-LNP is incubated at approximately 45°C. In some embodiments, mRNA-LNP is incubated at approximately 50°C. In some embodiments, mRNA-LNP is incubated at approximately 55°C. In some embodiments, mRNA-LNP is incubated at approximately 60°C. In some embodiments, mRNA-LNP is incubated at approximately 65°C.

[0025] In some embodiments, mRNA-LNP is over-incubated for approximately 20 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 30 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 40 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 50 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 60 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 70 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 80 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 90 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 100 minutes. In some embodiments, mRNA-LNP is over-incubated for approximately 120 minutes. In some embodiments, mRNA-LNP is incubated for approximately 30 minutes. In some embodiments, mRNA-LNP is incubated for approximately 40 minutes. In some embodiments, mRNA-LNP is incubated for approximately 50 minutes. In some embodiments, mRNA-LNP is incubated for approximately 60 minutes. In some embodiments, mRNA-LNP is incubated for approximately 70 minutes. In some embodiments, mRNA-LNP is incubated for approximately 80 minutes. In some embodiments, mRNA-LNP is incubated for approximately 90 minutes. In some embodiments, mRNA-LNP is incubated for approximately 100 minutes. In some embodiments, mRNA-LNP is incubated for approximately 120 minutes. In some embodiments, mRNA-LNP is incubated for approximately 150 minutes. In some embodiments, mRNA-LNP is incubated for approximately 180 minutes.

[0026] In some embodiments, the lipid solution does not contain alcohol.

[0027] In some embodiments, the lipid solution further comprises one or more cholesterol-based lipids.

[0028] In some embodiments, mRNA-LNPs are purified by tangential flow filtration.

[0029] In some embodiments, mRNA-LNPs have an average diameter of less than 200 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 150 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 100 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 95 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 90 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 85 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 80 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 75 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 70 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 65 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 60 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 55 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 50 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 45 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 40 nm. In some embodiments, mRNA-LNPs have an average diameter of less than 35 nm. In some embodiments, mRNA-LNPs have an average diameter in the range of 35 nm to 65 nm. In some embodiments, mRNA-LNPs have an average diameter in the range of 40 nm to 70 nm. In some embodiments, mRNA-LNPs have an average diameter in the range of 40 nm to 60 nm. In some embodiments, mRNA-LNPs have an average diameter in the range of 45 nm to 55 nm.

[0030] In some embodiments, lipid nanoparticles have a PDI of less than approximately 0.3. In some embodiments, lipid nanoparticles have a PDI of less than approximately 0.2. In some embodiments, lipid nanoparticles The molecules have a PDI of less than approximately 0.18. In some embodiments, the lipid nanoparticles have a PDI of less than approximately 0.15. In some embodiments, the lipid nanoparticles have a PDI of less than approximately 0.12. In some embodiments, the lipid nanoparticles have a PDI of less than approximately 0.10.

[0031] In some embodiments, the encapsulation efficiency of mRNA-LNP is over 60%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 65%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 70%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 75%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 80%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 85%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 90%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 95%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 96%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 97%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 98%. In some embodiments, the encapsulation efficiency of mRNA-LNP is over 99%.

[0032] In some embodiments, mRNA-LNPs have an N / P ratio between 1 and 10. In some embodiments, mRNA-LNPs have an N / P ratio between 2 and 6. In some embodiments, mRNA-LNPs have an N / P ratio of about 4. In some embodiments, the mRNA solution and lipid solution are mixed in an N / P ratio between 1 and 10. In some embodiments, the mRNA solution and lipid solution are mixed in an N / P ratio between 2 and 6. In some embodiments, the mRNA solution and lipid solution are mixed in an N / P ratio of about 2. In some embodiments, the mRNA solution and lipid solution are mixed in an N / P ratio of about 4. In some embodiments, the mRNA solution and lipid solution are mixed in an N / P ratio of about 6. In certain embodiments, the mRNA solution and lipid solution are mixed in an N / P ratio of about 4. As shown in the examples, such N / P ratios resulted in LNPs with a diameter and encapsulation efficiency suitable for therapeutic use.

[0033] In some embodiments, 5 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 10 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 15 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 20 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 25 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 30 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 40 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 50 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 75 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 100 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 150 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 200 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 250 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 500 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 750 g or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 1 kg or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 5 kg or more of mRNA is encapsulated in lipid nanoparticles in a single batch. In some embodiments, 10 kg or more of mRNA is encapsulated in lipid nanoparticles in a single batch.

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

[0035] In some embodiments, the mRNA solution is mixed at flow rates in the range of approximately 150-250 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] In some embodiments, the mRNA solution is mixed at a flow rate of approximately 100 ml / min, 200 ml / min, 500 ml / min, 800 ml / min, 1000 ml / min, 1200 ml / min, 2000 ml / min, 3000 ml / min, 4000 ml / min, 5000 ml / min, 6000 ml / min, 8000 ml / min, 10000 ml / min, 12000 ml / min, or 15000 ml / min.

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

[0038] In some embodiments, the lipid solution is mixed at a flow rate in the range of approximately 25-75 ml / min, approximately 75-200 ml / min, approximately 200-350 ml / min, approximately 350-500 ml / min, approximately 500-650 ml / min, approximately 650-850 ml / min, or approximately 850-1000 ml / min. In some embodiments, the lipid solution is mixed at a flow rate of approximately 50 ml / min, 100 ml / min, 150 ml / min, 200 ml / min, 250 ml / min, 300 ml / min, 350 ml / min, 400 ml / min, 450 ml / min, 500 ml / min, 550 ml / min, 600 ml / min, 650 ml / min, 700 ml / min, 750 ml / min, 800 ml / min, 850 ml / min, 900 ml / min, 950 ml / min, 1000 ml / min, 1200 ml / min, or 1500 ml / min.

[0039] In some embodiments, the flow rate of the mRNA solution is the same as the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is twice as large as the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is three times as large as the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is four times as large as the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is The flow rate of the mRNA solution is 4.5 times greater than that of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 5 times greater than that of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 5.5 times greater than that of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 6 times greater than that of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 8 times greater than that of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 10 times greater than that of the lipid solution.

[0040] In some embodiments, compositions containing mRNA encapsulated in lipid nanoparticles are produced by this method.

[0041] In some embodiments, the composition contains 1 g or more of mRNA. In some embodiments, the composition contains 5 g or more of mRNA. In some embodiments, the composition contains 10 g or more of mRNA. In some embodiments, the composition contains 15 g or more of mRNA. In some embodiments, the composition contains 20 g or more of mRNA. In some embodiments, the composition contains 25 g or more of mRNA. In some embodiments, the composition contains 50 g or more of mRNA. In some embodiments, the composition contains 75 g or more of mRNA. In some embodiments, the composition contains 100 g or more of mRNA. In some embodiments, the composition contains 125 g or more of mRNA. In some embodiments, the composition contains 150 g or more of mRNA. In some embodiments, the composition contains 250 g or more of mRNA. In some embodiments, the composition contains 500 g or more of mRNA. In some embodiments, the composition contains 1 kg or more of mRNA.

[0042] In some embodiments, the mRNA contains one or more modified nucleotides.

[0043] In some embodiments, the mRNA is unmodified.

[0044] In some embodiments, mRNA is greater than approximately 0.5kb. In some embodiments, mRNA is greater than approximately 1kb. In some embodiments, mRNA is greater than approximately 2kb. In some embodiments, mRNA is greater than approximately 3kb. In some embodiments, mRNA is greater than approximately 4kb. In some embodiments, mRNA is greater than approximately 5kb. In some embodiments, mRNA is greater than approximately 6kb. In some embodiments, mRNA is greater than approximately 8kb. In some embodiments, mRNA is greater than approximately 10kb. In some embodiments, mRNA is greater than approximately 20kb. In some embodiments, mRNA is greater than approximately 30kb. In some embodiments, mRNA is greater than approximately 40kb. In some embodiments, mRNA is greater than approximately 50kb.

[0045] In some embodiments, the lipid solution contains four lipid components. In some embodiments, the lipid solution contains PEG-modified lipids, cationic lipids (e.g., ML-2 or MC-3), helper (e.g., non-cationic) lipids (e.g., DSPC or DOPE), and optionally cholesterol. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids and PEG-modified lipids in the LNP is 35-55:5-35:20-40:1-15. In certain embodiments, a lipid solution containing mTEG (e.g., 100% mTEG) as a solvent and an aqueous solution of mRNA (e.g., citrate buffer) are mixed in a volume ratio of 1:1 to 4 (e.g., about 1:1), the final mRNA concentration is about 0.05 to 0.5 mg / mL, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids to PEG-modified lipids in the LNP is 35 to 55:25 to 35:20 to 40:1 to 15 (e.g., about 40:30:25:5), and as a result the N / P ratio of cationic lipids to mRNA is about 2 to 6 (e.g., about 4). As shown in the examples, these preparations are particularly suitable for use in the formulations of the present invention because they ensure appropriate mRNA-LNP diameter and encapsulation efficiency. Furthermore, such mRNA-LNP formulations with high lipid and mRNA concentrations are advantageous in reducing processing volume and thereby increasing ease of processing in manufacturing.

[0046] In some embodiments, mRNA is purified using or without volatile organic compounds. In some embodiments, mRNA is purified using a method that does not contain volatile organic compounds. In some embodiments, mRNA is purified using an alcohol-free method. In some embodiments, mRNA is purified using an isopropyl alcohol-free method. In some embodiments, mRNA is purified using a benzyl alcohol-free method.

[0047] In some embodiments, mRNA is purified and encapsulated in LNPs using a method that does not contain volatile organic compounds. In some embodiments, mRNA is purified and encapsulated in LNPs using a method that does not contain alcohol. In some embodiments, mRNA is encapsulated in LNPs using a method that does not contain volatile organic compounds. In some embodiments, mRNA is encapsulated in LNPs using a method that does not contain alcohol.

[0048] Other functions, purposes, and advantages of the present invention are evident in the following detailed description, drawings, and claims. However, it should be understood that the detailed description, drawings, and claims illustrate embodiments of the present invention, but are given merely as examples and not as limitations. Various changes and modifications within the scope of the present invention will be evident to those skilled in the art.

[0049] The following drawings are for illustrative purposes only and not for limitation. [Brief explanation of the drawing]

[0050] [Figure 1] This graph shows the mean radiance p / sec / cm2 / sr from mice administered with firefly luciferase (FFL) mRNA-LNP encapsulated in ethanol-free formulations (i.e., mTEGs) or ethanol-containing formulations. Furthermore, the data also shows data obtained from formulations prepared at high volumes (1:4 lipid solution vs. mRNA solution) or low volumes (1:1 lipid solution vs. mRNA solution). [Figure 2] This graph shows the total OTC (ng / mg) of total protein from mice administered ornithine transcarbamylase (OTC) mRNA-LNP encapsulated in an ethanol-free formulation (i.e., mTEG). The data also shows data obtained from formulations prepared at high volume (1:4 lipid solution vs. mRNA solution) or low volume (1:1 lipid solution vs. mRNA solution). [Modes for carrying out the invention]

[0051] definition To make the present invention easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are given throughout this specification. Publications and other reference materials referenced herein to explain the background of the invention and to provide additional details relating to its implementation are incorporated herein by reference.

[0052] Terms like "or more," "at least," and "greater than," for example, "at least one," are not limited to just one, but include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 ,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,10 3, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, It is understood to include 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or any number greater than or equal to the value mentioned. It also includes any larger number or fractions in between.

[0053] Conversely, the term "less than or equal to" includes each value less than the value mentioned. For example, "less than or equal to 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 5 Includes 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fractions in between are also included.

[0054] Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limited to these, but include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 ,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 It is understood to include 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Any larger number or fractions in between are also included.

[0055] Amino Acids: As used herein, the term “amino acid” means in its broadest sense any compound and / or substance incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. “Standard amino acid” refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” refers to any amino acid other than a standard amino acid, whether synthetically produced or obtained from a natural source. As used herein, “synthetic amino acid” includes, but is not limited to, salts, amino acid derivatives (such as amides), and / or substituted, chemically modified amino acids. Amino acids containing carboxy- and / or amino-terminal amino acids in peptides may negatively impact methylation, amidation, acetylation, protecting groups, and / or their activity. Amino acids can be modified by substitution with other chemical groups that can alter the cyclic half-life of the peptide without affecting its overall function. Amino acids can participate in disulfide bonds. Amino acids can undergo one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl, acetate, acetyl, phosphate, formyl, isoprenoid, sulfate, polyethylene glycol, lipid, carbohydrate, biotin, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and refers to free amino acids and / or amino acid residues of peptides. Whether this term refers to free amino acids or peptide residues is clear from the context in which it is used.

[0056] Animals: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any stage of development. In some embodiments, “animal” refers to a non-human animal at any stage of development. In certain embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, an animal includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or bugs. In some embodiments, an animal is a transgenic animal, a genetically modified animal, and / or a clone.

[0057] Approximately or about: As used herein, the terms “approximately” or “about” refer to a value similar to the reference value mentioned, when applied to one or more of the values ​​of interest. In certain embodiments, the terms “approximately” or “about” refer to being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the value mentioned. Unless otherwise clearly indicated by the context, all numerical values ​​provided herein are qualified by the terms “approximately” or “about.”

[0058] Batch: As used herein, the term “batch” refers to, for example, the content or amount of mRNA purified at one time according to a single manufacturing sequence during the same manufacturing cycle. A batch may also refer to the amount of mRNA purified in a single reaction.

[0059] Biologically active: As used herein, the phrase “biologically active” refers to the characteristic of any drug that is active in a biological system, and in particular in living organisms. For example, a drug is considered biologically active if, when administered to an organism, it has a biological effect on that organism.

[0060] Comprising: As used herein, the term "comprising," or variations such as "comprises" or "comprising," is understood to mean the inclusion of the element, integer, or process, or group of elements, integers, or processes, being referred to, but not the exclusion of any other element, integer, or process, or group of elements, integers, or processes.

[0061] Combining: As used herein, the term “combining” is used interchangeably with “mixing” or “blending.” Combining refers to bringing together discrete LNP particles having distinct properties into the same solution, for example, by combining mRNA-LNPs and empty LNPs to obtain an mRNA-LNP composition. In some embodiments, the combining of two LNPs is carried out in a specific ratio of the components being combined. In some embodiments, the resulting composition obtained from combining has properties different from one or both of its components.

[0062] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, mRNA delivery means that mRNA is delivered to a target tissue and encoded… This includes situations in which a protein is expressed and retained within a target tissue (also called "local distribution" or "local delivery"), and situations in which mRNA is delivered to a target tissue, the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), distributed throughout the body, and taken up by other tissues (also called "systemic distribution" or "systemic delivery"). In some embodiments, delivery is pulmonary delivery, including, for example, spraying.

[0063] dsRNA: As used herein, the term “dsRNA” refers to the production of complementary RNA sequences during in vitro transcription (IVT) reactions. Complementary RNA sequences are produced for a variety of reasons, including, for example, short interrupted transcripts that can hybridize to complementary sequences in nascent RNA strands, short interrupted transcripts that act as primers for RNA-dependent DNA-independent RNA transcription, and possible RNA polymerase template inversion.

[0064] Efficacy: As used herein, the term “efficacy,” or its grammatical equivalent, refers to the improvement of a biologically relevant endpoint related to the delivery of mRNA encoding the relevant protein or peptide.

[0065] Encapsulation: As used herein, the term “encapsulation,” or its grammatical equivalent, refers to the process of encapsulating nucleic acid molecules within nanoparticles.

[0066] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide (e.g., the heavy or light chain of an antibody), the assembly of multiple polypeptides (e.g., the heavy or light chain of an antibody) into an intact protein (e.g., an antibody), and / or post-translational modification of a polypeptide or a fully assembled protein (e.g., an antibody). In this application, the terms “expression” and “production,” as well as their grammatical equivalents, are used interchangeably.

[0067] Functional: As used herein, a “functional” biological molecule is a biological molecule in a form that exhibits the properties and / or activities characterized thereby.

[0068] To improve, increase, or decrease: As used herein, the terms “improve,” “increase,” or “decrease,” or their grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual before the commencement of the treatment described herein, or a measurement in a control subject (or more control subjects) in the absence of the treatment described herein. A “control subject” is a subject of approximately the same age as the subject being treated and suffering from the same form of disease as the subject being treated.

[0069] Impurities: As used herein, the term “impurity” refers to a limited amount of substance within a liquid, gas, or solid that differs from the chemical composition of the target material or compound. Impurities are also called “contaminants.”

[0070] In vitro: As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0071] In vivo: As used herein, the term “in vivo” refers to events occurring within multicellular organisms such as humans and non-human animals. In the context of cell systems, the term is used to refer to events occurring within living cells (for example, in contrast to in vitro systems).

[0072] Isolated: As used herein, the term “isolated” means (1) (natural (2) substances and / or entities that have been isolated from at least some of the components that were associated with them when they were first produced (in either the experimental setting or otherwise), and / or (2) produced, manufactured, and / or fabricated by human hands. The isolated substances and / or entities are isolated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, 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 they were first associated. In some embodiments, the isolated agents are 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. As used herein, the calculation of the percent purity of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0073] Liposomes: As used herein, the term “liposome” refers to any layered, multilayered, or solid nanoparticle vesicles. Typically, liposomes are formed by mixing one or more lipids, or by mixing one or more lipids with a polymer, as used herein. In some embodiments, liposomes suitable for the present invention contain cationic lipids and optionally non-cationic lipids, optionally cholesterol-based lipids, and / or optionally PEG-modified lipids.

[0074] Local Distribution or Delivery: As used herein, the terms “local distribution,” “local delivery,” or their grammatical equivalents refer to tissue-specific delivery or distribution. Typically, local distribution or delivery requires that a peptide or protein encoded by mRNA (e.g., an enzyme) be translated and expressed within a cell or in limited secretion, avoiding entry into the patient’s circulatory system.

[0075] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA contains one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or chemically synthesized. Where appropriate, for example, in chemically synthesized molecules, mRNA may contain nucleoside analogs such as chemically modified bases or sugars, or analogs with skeletal modifications. mRNA sequences are presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, mRNA is derived from natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-amino Denosine, 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); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoamidite bonds), or comprising these.

[0076] mRNA integrity: As used herein, the term “mRNA integrity” generally refers to the quality of mRNA. In some embodiments, mRNA integrity is defined as the quality of mRNA after the purification process. This refers to the percentage of undegraded mRNA. mRNA integrity is 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).

[0077] N / P Ratio: As used herein, the term “N / P ratio” refers to the molar ratio of positively charged molecular units in the cationic lipid within a lipid nanoparticle to negatively charged molecular units in the mRNA encapsulated within that lipid nanoparticle. Thus, the N / P ratio is typically calculated as the ratio of moles of amine groups in the cationic lipid within the lipid nanoparticle to moles of phosphate groups in the mRNA encapsulated within that lipid nanoparticle. For example, a 4-molar excess of cationic lipid per mole of mRNA is called an “N / P ratio” of approximately 4.

[0078] Nucleic Acids: As used herein, the term “nucleic acid” means, in its broadest sense, any compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain via phosphodiester bonds. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single-stranded 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 something other than a phosphodiester backbone. For example, so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in their backbone, are considered to be within the scope of the invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and / or encoding the same amino acid sequence. Nucleic acid sequences and / or RNA encoding proteins contain introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and sometimes purified, or chemically synthesized. Where appropriate, for example, in chemically synthesized molecules, nucleic acids may contain nucleoside analogs such as chemically modified bases or sugars, or analogs with skeletal modifications. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.In some embodiments, nucleic acids are natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoamidite bonds), or comprising these. In some embodiments, the present invention specifically relates to “unmodified nucleic acids” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified to facilitate or achieve delivery.

[0079] Patient: As used herein, the terms “patient” or “subject” refer to any organism to which the provided composition can be administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mice, rats, rabbits, etc.) This includes non-human primates and / or mammals such as humans. In some embodiments, the patient is human. Humans include prenatal and postnatal forms.

[0080] Pharmacologically acceptable: As used herein, the term "pharmaceutically acceptable" means a substance that, within the bounds of reliable medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit-to-risk ratio.

[0081] pharmaceutically acceptable salts: pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed by inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or by organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxyethanesulfonate. These include lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, valersates, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N+ (C 1~4 Alkyl) 4 salts are included. Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed by quaternizing amines using appropriate electrophiles, such as alkyl halides, to form quaternized alkylated amino salts.

[0082] Precipitation: As used herein, the term “precipitation” (or any grammatical equivalent) refers to the formation of a solid in solution. As used in relation to mRNA, the term “precipitation” refers to the formation of mRNA in an insoluble or solid form in a liquid.

[0083] Early Termination RNA Sequences: The terms “early termination RNA sequence,” “short termination RNA species,” “shortmer,” and “long termination RNA species,” as used herein, refer to incomplete products of mRNA synthesis reactions (e.g., in vitro synthesis reactions). For various reasons, RNA polymerase does not always complete the transcription of a DNA template; for example, RNA synthesis may terminate prematurely. Possible causes of premature termination of RNA synthesis include the quality of the DNA template, polymerase termination sequences for specific polymerases present in the template, degradation buffers, temperature, ribonucleotide depletion, and mRNA secondary structure. An early termination RNA sequence is any length shorter than the intended length of the desired transcript. For example, an early termination mRNA sequence may be less than 1000 bases, less than 500 bases, or less than 100 bases. Less than 50 bases, less than 40 bases, less than 30 bases, less than 20 bases, less than 15 bases, less than 10 bases, or less than or equal to that.

[0084] Salt: As used herein, the term "salt" refers to an ionic compound that results from, or can result from, a neutralization reaction between an acid and a base.

[0085] Systemic distribution or delivery: As used herein, the terms “systemic distribution,” “systemic delivery,” or their grammatical equivalents refer to a mechanism or approach of delivery or distribution that affects the whole body or organism. Typically, systemic distribution or delivery is achieved through the body’s circulatory system, e.g., blood flow. Compare with the definition of “local distribution or delivery.”

[0086] Subject: As used herein, the term “subject” means a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, meaning a human being who is under the care of a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject may be susceptible to or prone to a disease or disorder, and may or may not exhibit symptoms of the disease or disorder.

[0087] Substantially: As used herein, the term “substantially” refers to a quantitative state that indicates all or nearly all of the characteristics or properties of the subject. Those skilled in the biological art will understand that biological and chemical phenomena rarely complete and / or progress to completion, or achieve or avoid absolute results. Therefore, the term “substantially” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0088] Substantially Free: As used herein, the term “substantially free” means that the removed substance (e.g., prematurely interrupted RNA sequences) is present in relatively small amounts or none at all. For example, “substantially free of prematurely interrupted RNA sequences” means that prematurely interrupted RNA sequences are present at impurity levels of 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). Alternatively, “substantially free of prematurely interrupted RNA sequences” means that prematurely interrupted RNA sequences are present at levels of approximately 100ng, 90ng, 80ng, 70ng, 60ng, 50ng, 40ng, 30ng, 20ng, 10ng, 1ng, 500pg, 100pg, 50pg, 10pg or less.

[0089] Target tissue: As used herein, the term “target tissue” refers to any tissue affected by the disease being treated. In some embodiments, target tissue includes tissue exhibiting a disease-related condition, symptom, or function.

[0090] Therapeutic dose: As used herein, the term “therapeutic dose” of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, disorder, and / or condition when administered to a subject who is afflicted with or susceptible to the disease, disorder, and / or condition. It will be recognized by those skilled in the art that a therapeutic dose is typically administered via a dosing regimen containing at least one unit dose.

[0091] To treat: When used herein, “to treat,” “treatment,” or “treatment” The term "to reduce, improve, alleviate, inhibit, prevent, delay the onset, reduce the severity, and / or decrease the occurrence of one or more symptoms or functions of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who are not showing signs of the disease and / or who are showing only early signs of the disease, for the purpose of reducing the risk of developing a disease-related condition.

[0092] Yield: As used herein, the term “yield” refers to the percentage of mRNA recovered after encapsulation compared to the total mRNA as starting material. In some embodiments, the term “recovery rate” is used interchangeably with the term “yield.”

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood and used by those skilled in the art to which this application belongs; such art as is incorporated by reference. In case of any conflict, this specification, including its definitions, shall prevail.

[0094] Detailed explanation The present invention provides, in particular, methods and compositions for formulations comprising mRNA encapsulated in lipid nanoparticles, without the use of ethanol or other flammable solvents in the formulation. Accordingly, the present disclosure provides methods for producing and using stable, safe, and cost-effective ethanol-free LNP formulations having high mRNA encapsulation efficiency for efficient mRNA delivery for therapeutic use.

[0095] Various aspects of the present invention are described in detail in the following sections. The use of these sections is not intended to limit the present invention. Each section can be applied to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise specified.

[0096] Liposomes containing mRNA (mRNA-LNP) The methods for encapsulating mRNA in lipid nanoparticles disclosed herein can be applied to various techniques currently known in the art. Various methods are described in U.S. Patent Publication No. 2011 / 0244026, U.S. Patent Publication No. 2016 / 0038432, U.S. Patent Publication No. 2018 / 0153822, U.S. Patent Publication No. 2018 / 0125989 and U.S. Provisional Patent Application No. 62 / 877,597 filed on 23 July 2019, which can be used to carry out the present invention, and all of these are incorporated herein by reference. A conventional method for encapsulating mRNA, also known as Method A, described in U.S. Patent Publication No. 2016 / 0038432, includes the step of mixing mRNA with a mixture of lipids without first pre-forming the lipids into lipid nanoparticles. Alternatively, another method for encapsulating messenger RNA (mRNA) by mixing it with pre-formed lipid nanoparticles, as described in U.S. Patent Application Publication No. 2018 / 0153822, is known as Method B.

[0097] Achieving high encapsulation efficiency is crucial for nucleic acid delivery, protecting the active pharmaceutical ingredient (e.g., mRNA) and reducing in vivo activity loss. Therefore, the expression of the mRNA-encoded protein or peptide and the enhancement of its therapeutic effect are highly correlated with mRNA encapsulation efficiency.

[0098] To achieve high sealing efficiency using Method A, the Method typically involves heating or adding heat to one or more solutions in 10 mM citrate buffer to achieve or maintain a temperature higher than ambient temperature. As described in U.S. Patent Application Publication No. 2016 / 0038432, heating one or more solutions... This increases mRNA encapsulation efficiency and recovery rate. Furthermore, Method A typically includes 10-100 mM citrate as a buffer in the mRNA and / or lipid solution. Alternatively, high encapsulation rates can be achieved without heating the mRNA and / or lipid solution before mixing by using a low concentration of citrate (i.e., 5 mM or less) in the mRNA solution.

[0099] mRNA solution Various methods can be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA can be directly dissolved in the buffer described herein. In some embodiments, the mRNA solution can be prepared by mixing the mRNA stock solution with the buffer before mixing it with the mounting lipid solution. In some embodiments, the mRNA solution can be prepared by mixing the mRNA stock solution with the buffer immediately before mixing it with the mounting lipid solution. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at concentrations of approximately 0.2 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 or higher. In some embodiments, a suitable mRNA stock solution contains mRNA at a concentration of about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml or higher. In some embodiments, the mRNA stock solution contains mRNA in water at a concentration between about 0.05 mg / mL and about 0.5 mg / mL. In specific embodiments, the mRNA stock solution contains mRNA in water at a concentration between about 0.1 mg / mL and about 0.5 mg / mL, for example, about 0.1 mg / mL or about 0.35 mg / mL.

[0100] Typically, a suitable mRNA solution may also contain a buffer and / or salt. Generally, buffers can include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, suitable concentrations of buffers can 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 buffers can range from 2.0 mM to 4.0 mM.

[0101] In some embodiments, the buffer contains less than approximately 5 mM of citrate. In some embodiments, the buffer contains less than approximately 3 mM of citrate. In some embodiments, the buffer contains less than approximately 1 mM of citrate. In some embodiments, the buffer contains less than approximately 0.5 mM of citrate. In some embodiments, the buffer contains less than approximately 0.25 mM of citrate. In some embodiments, the buffer contains less than approximately 0.1 mM of citrate. In some embodiments, the buffer does not contain citrate.

[0102] Exemplary salts include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable salt concentrations in mRNA solution can 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. Suitable salt concentrations in mRNA solution are about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, or greater.

[0103] In some embodiments, the buffer contains approximately 300 mM NaCl. In some embodiments, the buffer contains approximately 200 mM NaCl. In some embodiments, the buffer contains approximately 175 mM NaCl. In some embodiments, the buffer contains approximately 150 mM NaCl. In some embodiments, the buffer contains approximately 100 mM NaCl. In some embodiments, the buffer contains approximately 75 mM NaCl. In some embodiments, the buffer contains approximately 50 mM NaCl. In some embodiments, the buffer contains approximately 25 mM NaCl.

[0104] In some embodiments, a suitable mRNA solution may have a pH in the range of approximately 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 approximately 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, and 6.5 or lower.

[0105] In some embodiments, the buffer has a pH of approximately 5.0. In some embodiments, the buffer has a pH of approximately 4.8. In some embodiments, the buffer has a pH of approximately 4.7. In some embodiments, the buffer has a pH of approximately 4.6. In some embodiments, the buffer has a pH of approximately 4.5. In some embodiments, the buffer has a pH of approximately 4.4. In some embodiments, the buffer has a pH of approximately 4.3. In some embodiments, the buffer has a pH of approximately 4.2. In some embodiments, the buffer has a pH of approximately 4.1. In some embodiments, the buffer has a pH of approximately 4.0. In some embodiments, the buffer has a pH of approximately 3.9. In some embodiments, the buffer has a pH of approximately 3.8. In some embodiments, the buffer has a pH of approximately 3.7. In some embodiments, the buffer has a pH of approximately 3.6. In some embodiments, the buffer has a pH of approximately 3.5. In some embodiments, the buffer has a pH of approximately 3.4.

[0106] In some embodiments, the mRNA stock solution is mixed with a buffer using a pump. Exemplary pumps include, but are not limited to, pulseless flow pumps, gear pumps, peristaltic pumps, and centrifugal pumps.

[0107] Typically, the buffer is mixed at a flow rate exceeding that of the mRNA stock solution. For example, the buffer is mixed at a flow rate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times greater than that of the mRNA stock solution. In some embodiments, the buffer is mixed at a flow rate in the range of approximately 100 to 6000 ml / min (e.g., approximately 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, 4800 to 6000 ml / min, or 60 to 420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of approximately 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 or more.

[0108] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10 to 600 ml / min (e.g., approximately 5 to 50 ml / min, approximately 10 to 30 ml / min, approximately 30 to 60 ml / min, approximately 60 to 120 ml / min, approximately 120 to 240 ml / min, approximately 240 to 360 ml / min, approximately 360 to 480 ml / min, or approximately 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 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 The mixture is mixed at a flow rate of 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 or more.

[0109] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10–30 ml / min, 30–60 ml / min, 60–120 ml / min, 120–240 ml / min, 240–360 ml / min, 360–480 ml / min, or 480–600 ml / min. In some embodiments, the mRNA stock solution is mixed at a flow rate of approximately 20 ml / min, 40 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.

[0110] In some embodiments, the mRNA solution is at ambient temperature. In some embodiments, the mRNA solution is at a temperature of approximately 20–25°C. In some embodiments, the mRNA solution is at a temperature of approximately 21–23°C. In some embodiments, the mRNA solution is not heated before being mixed with the lipid solution. In some embodiments, the mRNA solution is maintained at ambient temperature.

[0111] Lipid solution According to the present invention, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. According to the present invention, in some embodiments, the suitable lipid solution does not contain ethanol, isopropanol, or any other flammable organic solvent.

[0112] A suitable lipid solution can contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution can contain a mixture of desired lipids at a total concentration of approximately 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 or more. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration in the range of approximately 0.1–100 mg / ml, 0.5–90 mg / ml, 1.0–80 mg / ml, 1.0–70 mg / ml, 1.0–60 mg / ml, 1.0–50 mg / ml, 1.0–40 mg / ml, 1.0–30 mg / ml, 1.0–20 mg / ml, 1.0–15 mg / ml, 1.0–10 mg / ml, 1.0–9 mg / ml, 1.0–8 mg / ml, 1.0–7 mg / ml, 1.0–6 mg / ml, or 1.0–5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of up to approximately 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.

[0113] Any desired lipids can be mixed in any ratio suitable for encapsulating mRNA. 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), amphiphilic block copolymers (e.g., poloxamers) and / or PEGylated 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) and one or more PEGylated lipids. In some embodiments, the lipid solution contains three lipid components. In some embodiments, the lipid solution contains four lipid components. In certain embodiments, the three or four lipid components of the lipid solution include PEGylated lipids, cationic lipids (e.g., ML-2 or MC-3), helper (e.g., non-cationic) lipids (e.g., DSPC or DOPE), and optionally PEGylated lipids. It is sterol.

[0114] In some embodiments, the lipid solution is at ambient temperature. In some embodiments, the lipid solution is at a temperature of approximately 20–25°C. In some embodiments, the lipid solution is at a temperature of approximately 21–23°C. In some embodiments, the lipid solution is not heated before mixing with the lipid solution. In some embodiments, the lipid solution is maintained at ambient temperature.

[0115] In certain embodiments, the provided composition comprises liposomes in which mRNA is associated on both surfaces of the liposomes and encapsulated within the liposomes. For example, during the preparation of the composition of the present invention, cationic liposomes can associate with mRNA through electrostatic interactions.

[0116] In some embodiments, the compositions and methods of the present invention include mRNA encapsulated in liposomes. In some embodiments, one or more mRNA species are encapsulated in the same liposome. In some embodiments, one or more mRNA species are encapsulated in different liposomes. In some embodiments, mRNA is encapsulated in one or more liposomes that differ in their lipid composition, molar ratio of lipid components, diameter, charge (zeta potential), targeted ligand, and / or combination thereof. In some embodiments, one or more liposomes may have different compositions of sterol-based cationic lipids, neutral lipids, PEG-modified lipids, and / or combinations thereof. In some embodiments, one or more liposomes may have different molar ratios of cholesterol-based cationic lipids, neutral lipids, and PEG-modified lipids used to construct the liposomes.

[0117] Enclosure method As used herein, the method of forming mRNA-borne lipid nanoparticles (mRNA-LNPs) is used interchangeably with the term "mRNA encapsulation" or its grammatical equivalent. In some embodiments, mRNA-LNPs are formed by mixing an mRNA solution with a lipid solution, and the mRNA solution and / or lipid solution are maintained at ambient temperature before mixing.

[0118] In some embodiments, the mRNA solution and the lipid solution are mixed in the solution so that the mRNA is encapsulated in lipid nanoparticles. Such a solution is also called a formulation or encapsulation solution.

[0119] In some embodiments, for example, the ethanol-free LNP formulation according to the present invention can be compared with conventional ethanol-based LNP formulations or encapsulation solutions containing a solvent such as ethanol. In previous LNP formulations using ethanol as a solvent, the formulation contained approximately 10% to 40% ethanol by volume. Other previous LNP formulations used isopropyl alcohol as a solvent at approximately 10% to 40% by volume. In contrast, in some embodiments, the present invention provides an LNP encapsulation method that does not contain a flammable solvent.

[0120] Therefore, in some embodiments, the suitable formulation or encapsulation solution of the present invention does not contain a flammable solvent. In some embodiments, the suitable formulation or encapsulation solution does not contain ethanol.

[0121] In some embodiments, the appropriate formulation or encapsulation solution may also contain buffers or salts. Exemplary buffers include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts include sodium chloride, magnesium chloride, and potassium chloride.

[0122] In some embodiments, ethanol, citrate buffer, and other destabilizers are not present during mRNA addition, and therefore the formulation does not require any further downstream processing. In the embodiment, the formulation solution contains trehalose. The absence of destabilizers and the stability of the trehalose solution increase the scalability of the formulation and the ease of producing mRNA-encapsulated lipid nanoparticles.

[0123] 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 lipids also contain one or more cholesterol lipids.

[0124] In some embodiments, the lipid solution and the mRNA solution are mixed using a pump system. In some embodiments, the pump system includes a pulseless flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a spindle pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the method using a pump system is carried out on a large scale. For example, in some embodiments, the method includes using a pump described herein to mix a solution containing 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 method of mixing the mRNA solution and the lipid solution provides a composition according to the present invention containing 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 encapsulated mRNA.

[0125] In some embodiments, the step of combining the mRNA encapsulated in lipid nanoparticles with the lipid solution is carried out using a pump system. In some embodiments, the mRNA solution and the lipid solution are mixed at flow rates in the range of approximately 25–75 ml / min, approximately 75–200 ml / min, approximately 200–350 ml / min, approximately 350–500 ml / min, approximately 500–650 ml / min, approximately 650–850 ml / min, or approximately 850–1000 ml / min. In some embodiments, the mRNA solution and lipid solution are mixed at a flow rate of approximately 50 ml / min, 100 ml / min, 150 ml / min, 200 ml / min, 250 ml / min, 300 ml / min, 350 ml / min, 400 ml / min, 450 ml / min, 500 ml / min, 550 ml / min, 600 ml / min, 650 ml / min, 700 ml / min, 750 ml / min, 800 ml / min, 850 ml / min, 900 ml / min, 950 ml / min, or 1000 ml / min.

[0126] In some embodiments, the step of mixing the mRNA solution with the lipid solution is carried out in the absence of a pump.

[0127] In some embodiments, the method according to the present invention includes maintaining one or more of the lipid-containing solution, the mRNA-containing solution, and the mixed solution containing mRNA encapsulated in lipid nanoparticles at ambient temperature (i.e., without adding heat from a heat source to the solution). In some embodiments, the method includes maintaining one or both of the mRNA solution and the lipid solution at ambient temperature before the mixing step. In some embodiments, the method includes maintaining one or more of the lipid-containing solution and the mRNA-containing solution at ambient temperature during the mixing step. In some embodiments, the method includes maintaining the mRNA encapsulated in lipid nanoparticles at ambient temperature after the mixing step. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is about 35°C, 30°C, 25°C, 20°C, or 16°C or lower. In some embodiments, the ambient temperature at which one or more solutions are maintained is in the range of about 15–35°C, about 15–30°C, about 15–25°C, about 15–20°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. In some embodiments, the ambient temperature at which one or more solutions are maintained is 20–25°C.

[0128] In some embodiments, the method according to the present invention includes a step of mixing an mRNA solution and a lipid solution to form mRNA-encapsulated lipid nanoparticles, performed at ambient temperature.

[0129] In some embodiments, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a diameter of less than about 150 nm (for example, 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 less than about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a diameter of less than 150 nm (e.g., 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 less than about 50 nm). In some embodiments, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have a diameter in the range of 50 to 150 nm. In some embodiments, substantially all of the purified nanoparticles have a diameter in the range of 50 to 150 nm. In some embodiments, over 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have a diameter in the range of 80–150 nm. In some embodiments, substantially all of the purified nanoparticles have a diameter in the range of 80–150 nm.

[0130] In some embodiments, the method according to the present invention yields inclusion rates of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%. In some embodiments, the method according to the present invention yields mRNA recovery rates of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%.

[0131] In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is the same as the mRNA-LNP encapsulation efficiency in the ethanol-LNP formulation.

[0132] In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 2% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 4% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 5% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 8% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 10% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 12% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 15% higher than that of the ethanol-LNP formulation. In some embodiments, the mRNA-LNP encapsulation efficiency in the formulation according to the present invention is at least 20% higher than that of the ethanol-LNP formulation.

[0133] In some embodiments, the method according to the present invention includes a step of incubating mRNA-LNP after mixing. The step of incubating mRNA-LNP after mixing is described in U.S. Provisional Patent Application No. 62 / 847,837, filed on 14 May 2019, which can be used to carry out the present invention and is incorporated herein by reference in its entirety.

[0134] purification In some embodiments, mRNA-LNPs are purified and / or concentrated. Various purification methods can be used. In some embodiments, mRNA-LNPs are purified by tangential flow filtration (TFF). In some embodiments, mRNA-LNPs are purified by gravity-normal flow filtration (NFF). In some embodiments, mRNA-LNPs are purified by any other suitable filtration method. In some embodiments, mRNA-LNPs are purified by centrifugation. In some embodiments, mRNA-LNPs are purified by chromatography.

[0135] Delivery vehicle According to the present invention, mRNA encoding a protein or peptide described herein (e.g., the full length, fragment, or portion of a protein or peptide) is delivered as naked RNA (unpackaged) or via a delivery vehicle. Where used herein, the terms “delivery vehicle,” “introduction vehicle,” and “nanoparticles” or their grammatical equivalents are interchangeable.

[0136] The delivery vehicle is formulated into a pharmacological composition that is combined with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or mixed with appropriate excipients. For example, liposomes encapsulating mRNA are formed as described above. Techniques for formulating and administering drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pennsylvania. Specific delivery vehicles are selected based on their ability to facilitate the transfection of nucleic acids into target cells.

[0137] In some embodiments, mRNA encoding at least one protein or peptide is delivered via a single delivery vehicle. In some embodiments, mRNA encoding at least one protein or peptide is delivered via one or more delivery vehicles of different compositions. In some embodiments, one or more mRNAs are encapsulated within the same lipid nanoparticle. In some embodiments, one or more mRNAs are encapsulated within separate lipid nanoparticles. In some embodiments, the lipid nanoparticles are empty.

[0138] According to various embodiments, suitable delivery vehicles include, but are not limited to, polymer carriers such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, exosomes of both natural and synthetic origin, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, calcium silicate phosphor nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, nanocrystalline particles, semiconductor nanoparticles, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other directional tags. The use of bionanopapellet and other viral capsid protein assemblies as suitable delivery vehicles is also envisioned (Hum. Gene Ther. 2008 Sept;19(9):887-95).

[0139] Liposome delivery vehicle In some embodiments, a suitable delivery vehicle is a liposome delivery vehicle, e.g., lipid nanoparticles. As used herein, a liposome delivery vehicle, e.g., lipid nanoparticles, is typically characterized as a microscopic vesicle having an internal water space isolated from the outer medium by one or more bilayer membranes. The bilayer membrane of a liposome is typically space Liposomes are formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, that contain hydrophilic and hydrophobic domains that are separated (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, liposome delivery vehicles typically serve to transport desired nucleic acids (e.g., mRNA) to target cells or tissues. In some embodiments, the nanoparticle delivery vehicle is a liposome. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids. In some embodiments, the liposome comprises three or fewer distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid.

[0140] Cationic lipids As used herein, the phrase “cationic lipid” refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH.

[0141] Cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2010 / 144740, which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention are [ka] The compound comprises a cationic lipid having the following structure, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate, and pharmaceutically acceptable salts thereof.

[0142] Other cationic lipids suitable for use in the compositions and methods of the present invention include ionizable cationic lipids described in International Publication No. 2013 / 149140, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention are based on the following formula: [ka] One cationic lipid or a pharmaceutically acceptable salt thereof (wherein R1 and R2 are hydrogen, optionally substituted, variable saturated or unsaturated C1-C) 20 Alkyl and optionally substituted, variably saturated or unsaturated C6-C 20 From the group consisting of acyl Each is selected independently; L1 and L2 are hydrogen, and possibly substituted C1-C 30 Alkyl, optionally substituted, variably unsaturated C1-C 30 Alkenyls, and optionally substituted C1-C 30 Each is independently selected from the group consisting of alkynnyls; m and o are independently selected from the group consisting of 0 and any positive integer (for example, m is 3); and n is 0 or any positive integer (for example, n is 1). In certain embodiments, the compositions and methods of the present invention are: [ka] The present invention comprises a cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the compound structure thereof and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention are [ka] The present invention comprises a cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") having the compound structure thereof and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention are [ka] The compound comprises cationic lipids having the following compound structure and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002") and pharmaceutically acceptable salts thereof.

[0143] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids described as amino alcohol lipidoids in International Publication No. 2010 / 053572, which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention are [ka] This includes cationic lipids having the compound structure and pharmaceutically acceptable salts thereof.

[0144] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2016 / 118725, which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention are [ka] This includes cationic lipids having the compound structure and pharmaceutically acceptable salts thereof.

[0145] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof.

[0146] Other cationic lipids suitable for use in the compositions and methods of the present invention include a cationic lipid having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and a pharmaceutically acceptable salt thereof.

[0147] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. WO 2013 / 063468 and International Publication No. WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention have the following formula: [Chemical formula] a cationic lipid or a pharmaceutically acceptable salt thereof (where each example of R L is independently, optionally substituted C6-C 40 alkenyl). In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention [ka] The present invention comprises cationic lipids having the compound structure thereof and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention are [ka] This includes cationic lipids having the compound structure and pharmaceutically acceptable salts thereof.

[0148] Other cationic lipids suitable for use in the compositions and methods of the present invention include those described in International Publication No. 2015 / 184256, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention are based on the following formula: [ka] Cationic lipids or their pharmaceutically acceptable salts (wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R A R is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyryl, optionally substituted 3-14 member heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 member heteroaryl, or halogen; each R B These are independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyryl, and optionally The composition and method of the present invention include a substituted 3- to 14-membered heterocycline, optionally a substituted C6- to 14-membered aryl, optionally a substituted 5- to 14-membered heteroaryl or halogen. In certain embodiments, the composition and method of the present invention are [ka] The compound comprises a cationic lipid having the compound structure of "Target 23" and a pharmaceutically acceptable salt thereof.

[0149] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2016 / 004202, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention have the following compound structures: [ka] It comprises a cationic lipid having the compound structure or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] It comprises a cationic lipid having the compound structure or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] It contains cationic lipids having the same properties or pharmaceutically acceptable salts thereof.

[0150] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in U.S. Provisional Patent Application No. 62 / 758,179, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention are based on the following formula: [ka] Cationic lipids or their pharmaceutically acceptable salts (wherein each R) 1 and R 2 Each is independently H or C1-C6 aliphatic; each m is independently an integer with a value between 1 and 4; each A is independently covalent or arrine; each L 1 These are independently ester, thioester, disulfide, or anhydride groups; each L 2 These are independent, C2~C 10It is aliphatic; each X 1 Each R is independently either H or OH; 3 These are independent, C6~C 20 (Aliphatic) is included. In some embodiments, the compositions and methods of the present invention are of the following formula: [ka] It comprises a cationic lipid or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention are of the following formula: [ka] It comprises a cationic lipid or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention are of the following formula: [ka] It contains cationic lipids or pharmaceutically acceptable salts thereof.

[0151] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described by reference herein, J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277. In certain embodiments, the cationic lipids of the compositions and methods of the present invention are [ka] This includes cationic lipids having the compound structure and pharmaceutically acceptable salts thereof.

[0152] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2015 / 199952, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention have the following compound structures: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the same and pharmaceutically acceptable salts thereof. In some embodiments, The compositions and methods of the present invention have a compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] It comprises cationic lipids having the same and pharmaceutically acceptable salts thereof.

[0153] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2017 / 004143, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention have the following compound structures: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] It includes a cationic lipid having and its pharmaceutically acceptable salts. In some embodiments, the compositions and methods of the present invention have the compound structure:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0154] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2017 / 075531, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention are of the following formula: [ka] Cationic lipids or their pharmaceutically acceptable salts (wherein L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O- and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or direct bond; G 1 and G 2 Each of these is independently of the unsubstituted C1~C 12 Alkylene or C1-C 12 It is an alkenylene; G 3 is C1~C 24 Alkylene, C1~C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1~C 12 It is alkyl; R 1 Oh biR 2 Each of these is independent, C6~C24 Alkyl or C6-C 24 It is an alkenil; R 3 H, OR 5 , CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 And; R 4 is C1~C 12 It is alkyl; R 5 (x is H or C1-C6 alkyl; x is 0, 1 or 2).

[0155] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2017 / 117528, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention have the following compound structures: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] The present invention comprises cationic lipids having the compound structure and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention have the compound structure: [ka] It comprises cationic lipids having the same and pharmaceutically acceptable salts thereof.

[0156] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2017 / 049245, which are incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the present invention is of the following formula: [ka] It comprises one compound and its pharmaceutically acceptable salts. For any one of these four formulas, R4 is -(CH2) n Q and -(CH2) n Selected independently from CHQR; Q is -OR, -OH, -O(CH2) n Selected from the group consisting of N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), and heterocycles; n is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention are [ka] The present invention comprises cationic lipids having the compound structure thereof and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention are [ka] The present invention comprises cationic lipids having the compound structure thereof and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention are [ka] The present invention comprises cationic lipids having the compound structure thereof and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention are [ka] This includes cationic lipids having the compound structure and pharmaceutically acceptable salts thereof.

[0157] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Publication No. 2017 / 173054 and International Publication No. 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention [Chemical formula] include a cationic lipid having the compound structure of and a pharmaceutically acceptable salt thereof.

[0158] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids described in International Publication No. 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention have the following formula: [Chemical formula] a cationic lipid of (wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (such as alkylamino like dimethylamino) and pyr dyl; R2 has the following two formulas: [ka] Selected from a group consisting of one of the following, R3 and R4 are variably saturated or unsaturated C6~C, which may be substituted. 20 Alkyl and optionally substituted, variably saturated or unsaturated C6-C 20 Each acyl is independently selected from the group consisting of acyls; n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In certain embodiments, the compositions and methods of the present invention are: [ka] The present invention comprises a cationic lipid having the compound structure of "HGT4001" and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention are [ka] The present invention comprises a cationic lipid having the compound structure of "HGT4002" and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention are [ka] A cationic lipid having the compound structure of "HGT4003" and its pharmaceutically acceptable Contains a salt. In certain embodiments, the compositions and methods of the present invention are [ka] The present invention comprises a cationic lipid having the compound structure of "HGT4004" and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention are [ka] This product contains the cationic lipid "HGT4005" having the compound structure described above, and a pharmaceutically acceptable salt thereof.

[0159] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids described in International Application No. PCT / US2019 / 032522, which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having any of the general formulas or structures (1a)-(21a) and (1b)-(21b) and (22)-(237) described in International Application No. PCT / US2019 / 032522. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the structure of formula (I'). [ka] (In the formula, R X -H and -L are independent of each other. 1 -R 1 , or -L 5A -L 5B -B' is; L 1 , L 2 , and L 3 Each of these can be independently covalent, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L -and; Each L 4A and L 5A These are independently -C(O)-, -C(O)O-, or -C(O)NR L -and; Each L 4B and L 5B These are independent of C1~C 20 Alkylene; C2~C 20 Alkenylene; or C2~C 20 It is alkynylene; Each B and B' is NR 4 R 5 or a 5-10 member nitrogen-containing heteroaryl; Each R 1 , R 2 , and R3 is independently C6-C 30 alkyl, C6-C 30 alkenyl, or C6-C 30 alkynyl; Each R 4 and R 5 is independently hydrogen, C1-C 10 alkyl; C2-C 10 alkenyl; or C2-C 10 alkynyl; Each R L is independently hydrogen, C1-C 20 alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl) comprises.

[0160] In certain embodiments, the compositions and methods of the present invention

Chemical formula

[0161] In some embodiments, the compositions and methods of the present invention include a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA") (incorporated herein by reference, Feigner et al., (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Patent No. 4,897,355). Other cationic lipids suitable for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2-(spermine-carboxamide)ethyl]-N,N-dimethyl--propaneaminium ("DOSPA") (Behr et al., Proc. Nat'l) Acad.Sci.86, 6982 (1989), U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761); contains 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP") and 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0162] Additional exemplary cationic lipids suitable for the compositions and methods of the present invention include: 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholesta-5-en-3-beta-oxybutane- 4-Oxy)-1-(cis,cis-9,12-octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleyl Railcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"); 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminopropane Nomethyl-[1,3]-dioxolane("DLin-K-DMA");2-((8-[(3P)-cholesta-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine("Octyl-CLinDMA");(2R)-2-((8-[(3beta)-cholesta-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine("Octyl-CLinDMA(2R)");(2S)-2-((8-[(3P)-cholesta-5-en- 3-Iloxy]octyl)oxy)-N,fsl-dimethyh3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2S)"); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethaneamine ("DLin-KC2-DMA") (incorporated herein by reference, International Publication No. 2010 / 042877; Semple et al., Nature) This includes (see 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); International Publication No. 2005 / 121348). In some embodiments, one or more cationic lipids include at least one imidazole, dialkylamino, or guanidinium moiety.

[0163] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100"); and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0164] In some embodiments, the compositions of the present invention include one or more cationic lipids constituting at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content of the composition, for example, lipid nanoparticles, as measured by weight. In some embodiments, the composition of the present invention comprises one or more cationic lipids constituting 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%) of the total lipid content of the composition, for example, lipid nanoparticles, as measured by weight.

[0165] Noncationic / Helper Lipids In some embodiments, liposomes contain one or more noncationic ("helper") lipids. As used herein, the phrase "noncationic lipids" is optional. This refers to neutral, zwitterionic, or anionic lipids. As used herein, the phrase “anionic lipid” refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH. Noncationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine. This includes mine-4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof.

[0166] In some embodiments, the noncationic lipid is a neutral lipid, i.e., a lipid that has no net charge under the conditions under which the composition is formulated and / or administered.

[0167] In some embodiments, such noncationic lipids are used alone, but are preferably used in combination with other lipids, such as cationic lipids.

[0168] In some embodiments, noncationic lipids may be present in the composition at 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 some embodiments, all noncationic lipids may be present in the composition at 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 some embodiments, the percentage of non-cationic lipids in liposomes can 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 total non-cationic lipids in liposomes can 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 non-cationic lipids in liposomes is about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less. In some embodiments, the percentage of total non-cationic lipids in liposomes can be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0169] In some embodiments, noncationic lipids may be present in the composition at a weight ratio (weight %) 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 some embodiments, all noncationic lipids may be present in the composition at a weight ratio (weight %) 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 some embodiments, the percentage of non-cationic lipids in liposomes can be more than about 5% by weight, more than about 10% by weight, more than about 20% by weight, more than about 30% by weight, or more than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in liposomes can be more than about 5% by weight, more than about 10% by weight. The percentage of noncationic lipids in liposomes can be about 20% by weight or more, about 30% by weight or more, or about 40% by weight or more. In some embodiments, the percentage of noncationic lipids in liposomes can be about 5% by weight or less, about 10% by weight or less, about 20% by weight or less, about 30% by weight or less, or about 40% by weight or less. In some embodiments, the percentage of total noncationic lipids in liposomes can be about 5% by weight or less, about 10% by weight or less, about 20% by weight or less, about 30% by weight or less, or about 40% by weight or less.

[0170] Cholesterol-based lipids In some embodiments, liposomes contain one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), or the following structures [ka] It contains imidazole cholesterol ester (ICE) which has [a specific characteristic].

[0171] In some embodiments, the cholesterol-based lipid is cholesterol.

[0172] In some embodiments, cholesterol-based lipids can constitute approximately 1% to 30% or 5% to 20% (mol%) of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles can be greater than approximately 5 mol%, greater than approximately 10 mol%, greater than approximately 20 mol%, greater than approximately 30 mol%, or greater than approximately 40 mol%. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles can be less than or equal to approximately 5 mol%, less than or equal to approximately 10 mol%, less than or equal to approximately 20 mol%, less than or equal to approximately 30 mol%, or less than or equal to approximately 40 mol%.

[0173] In some embodiments, cholesterol-based lipids may be present in the liposome at a weight ratio (wt%) of about 1% to about 30% or about 5% to about 20% of the total lipids present. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles may be less than or equal to about 5% by weight, less than or equal to about 10% by weight, less than or equal to about 20% by weight, less than or equal to about 30% by weight, or less than or equal to about 40% by weight.

[0174] PEG modified lipid In some embodiments, the liposome contains one or more PEGylated lipids.

[0175] For example, a derivatized ceramide containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide) The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as mid (PEG-CER), is also intended by the present invention, either alone or preferably in combination with other lipid formulations containing an introduction vehicle (e.g., lipid nanoparticles).

[0176] The PEG-modified lipids intended are not limited to these, but include C6-C 20 The lipids include polyethylene glycol chains up to 5 kDa in length, covalently bonded to lipids having long alkyl chains. In some embodiments, the PEG-modified or PEGylated lipids are PEGylated cholesterol or PEG-2K. The addition of such components can also provide a means to prevent complex aggregation, increase circulating lifespan, and increase the delivery of the lipid-nucleic acid composition to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they are selected to be rapidly replaced from the formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful replaceable lipids are those with shorter acyl chains (e.g., C 14 or C 18 It is a PEG-ceramide that has )

[0177] The PEG-modified phospholipids and derivatized lipids of the present invention can constitute approximately 0% to approximately 20%, approximately 0.5% to approximately 20%, approximately 1% to approximately 15%, approximately 4% to approximately 10%, or approximately 2% of the total lipids present in the liposome introduction vehicle. In some embodiments, one or more PEG-modified lipids constitute approximately 4% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute approximately 5% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute approximately 6% of the total lipids by molar ratio.

[0178] Amphiphilic block copolymers In some embodiments, a suitable delivery vehicle contains an amphiphilic block copolymer (e.g., poloxamer). The present invention can be carried out using a variety of amphiphilic block copolymers. In some embodiments, the amphiphilic block copolymer is also called a surfactant or nonionic surfactant. In some embodiments, amphiphilic polymers suitable for the present invention are selected from poloxamer (Pluronic®), poloxamine (Tetronic®), polyoxyethylene glycol sorbitan alkyl ester (polysorbate), and polyvinylpyrrolidone (PVP).

[0179] Poloxamer In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer has the following structure: [ka] (In the formula, a is an integer between 10 and 150, and b is an integer between 20 and 60.) For example, a is approximately 12 and b is approximately 20, or a is approximately 80 and b is approximately 27, or a is approximately 64 and b is approximately 37, or a is approximately 141 and b is approximately 44, or a is approximately 101 and b is approximately 56.

[0180] In some embodiments, the poloxamer suitable for the present invention has about 10 to about 150 ethylene oxide units. In some embodiments, the poloxamer has about 10 to about 100 ethylene oxide units.

[0181] In some embodiments, the appropriate poloxamer is poloxamer 84. In some embodiments, the appropriate poloxamer is poloxamer 101. In some embodiments, the appropriate poloxamer is poloxamer 105. In some embodiments, the appropriate poloxamer is poloxamer 108. In some embodiments, the appropriate poloxamer is poloxamer 122. In some embodiments, the appropriate poloxamer is poloxamer 123. In some embodiments, the appropriate poloxamer is poloxamer 124. In some embodiments, the appropriate poloxamer is poloxamer 181. In some embodiments, the appropriate poloxamer is poloxamer 182. In some embodiments, the appropriate poloxamer is poloxamer 183. In some embodiments, the appropriate poloxamer is poloxamer 184. In some embodiments, the appropriate poloxamer is poloxamer 185. In some embodiments, the appropriate poloxamer is poloxamer 188. In some embodiments, the appropriate poloxamer is poloxamer 212. In some embodiments, the appropriate poloxamer is poloxamer 215. In some embodiments, the appropriate poloxamer is poloxamer 217. In some embodiments, the appropriate poloxamer is poloxamer 231. In some embodiments, the appropriate poloxamer is poloxamer 234. In some embodiments, the appropriate poloxamer is poloxamer 235. In some embodiments, the appropriate poloxamer is poloxamer 237. In some embodiments, the appropriate poloxamer is poloxamer 238. In some embodiments, the appropriate poloxamer is poloxamer 282. In some embodiments, the appropriate poloxamer is poloxamer 284. In some embodiments, the appropriate poloxamer is poloxamer 288. In some embodiments, the appropriate poloxamer is poloxamer 304. In some embodiments, the appropriate poloxamer is poloxamer 331. In some embodiments, the appropriate poloxamer is poloxamer 333. In some embodiments, the appropriate poloxamer is poloxamer 334. In some embodiments, the appropriate poloxamer is poloxamer 335. In some embodiments, the appropriate poloxamer is poloxamer 338.In some embodiments, the suitable poloxamer is poloxamer 401. In some embodiments, the suitable poloxamer is poloxamer 402. In some embodiments, the suitable poloxamer is poloxamer 403. In some embodiments, the suitable poloxamer is poloxamer 407. In some embodiments, the suitable poloxamer is a combination thereof.

[0182] In some embodiments, suitable poloxamers have an average molecular weight of approximately 4,000 g / mol to approximately 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 1,000 g / mol to approximately 50,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 1,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 2,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 3,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 4,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 5,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 6,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 7,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 8,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 9,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 10,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 25,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 30,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 40,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of approximately 50,000 g / mol.

[0183] Other amphiphilic polymers In some embodiments, the amphiphilic polymer is poloxamine, for example, tetronic 304 or tetronic 904.

[0184] In some embodiments, the amphiphilic polymer is polyvinylpyrrolidone (PVP), for example, PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.

[0185] In some embodiments, the amphiphilic polymer is polyethylene glycol ether (Brij), polysorbate, sorbitan, and derivatives thereof. In some embodiments, the amphiphilic polymer is polysorbate, for example, PS20.

[0186] In some embodiments, the amphiphilic polymer is polyethylene glycol ether (Brij), poloxamer, polysorbate, sorbitan, or a derivative thereof.

[0187] In some embodiments, the amphiphilic polymer is polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of formula (SI): [ka] or its salt or isomer (In the formula, t is an integer between 1 and 100: R 1BRIJ Independently, C 10~40 Alkyl, C 10~40 Alkenyl, or C 10~40 It is alkynyl; in some cases R 5PEG One or more methylene groups are C 3~10 Carbocyclylene, 4-10 member heterocyclylene, C 6~10 Arylene, 4-10 member heteroarylene, -N(R) N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR NC(O)-, -NRC(O)N(R)-, -C(O)O- -OC(O)-, -OC(O)O- -OC(O)N(R N )-, -NR N C(O)O- -C(O)S- -SC(O)-, -C(=NR N )-, -C(=NR)N(R)-, -NRNC(=NR N )- -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O- -OS(O)O- -OS(O)2- -S(O)2O- -OS(O)2O- -N(R N )S(O)-, -S(O)N(R N )- -N(R N )S(O)N(R N )- -OS(O)N(R N )- -N(R N )S(O)O- -S(O)2- -N(R N )S(O)2- -S(O)2N(R N )-,-N(R N )S(O)2N(R N )- -OS(O)2N(R N )- or -N(R N ) is independently replaced by S(O)2O-; R N Each example is independent of hydrogen, C 1~6 (An alkyl or nitrogen protecting group) That is the case.

[0188] In some embodiments, R 1BRIJ C is alkyl. For example, polyethylene glycol ether is a compound of formula (S-Ia): [ka] or its salt or isomer (where s is an integer between 1 and 100).

[0189] In some embodiments, R 1BRIJ C is an alkenyl. For example, a suitable polyethylene glycol ether is a compound of formula (S-Ib): [ka] or its salt or isomer (where s is an integer between 1 and 100).

[0190] Typically, amphiphilic polymers (e.g., poloxamers) are present in the formulation in amounts less than their critical micelle concentration (CMC). In some embodiments, amphiphilic polymers (e.g., poloxamers) are present in the mixture in amounts about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than their CMC. In some embodiments, amphiphilic polymers (e.g., poloxamers) are present in the mixture in amounts about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% lower than their CMC. In some embodiments, an amphiphilic polymer (e.g., poloxamer) is present in the mixture in amounts approximately 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% lower than its CMC.

[0191] In some embodiments, approximately 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or less than 0.01% of the original amount of amphiphilic polymer (e.g., poloxamer) present in the formulation remains after removal. In some embodiments, the residual amount of amphiphilic polymer (e.g., poloxamer) remains in the formulation after removal. As used herein, residual amount means the amount remaining after substantially all of the substance (amphiphilic polymer, e.g., poloxamer) in the composition has been removed. Residual amounts can be detected qualitatively or quantitatively using known techniques. Residual amounts may not be detectable using known techniques.

[0192] In some embodiments, a suitable delivery vehicle includes less than 5% of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle includes less than 3% of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle includes less than 2.5% of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle includes less than 2% of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle includes less than 1.5% of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle includes less than 1% of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle includes less than 0.5% (e.g., less than 0.4%, 0.3%, 0.2%, 0.1%) of the amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 0.01% of an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains a residual amount of the amphiphilic polymer (e.g., poloxamer). As used herein, residual amount means the amount remaining after substantially all of the substance (amphiphilic polymer as described herein, e.g., poloxamer) in the composition has been removed. Residual amount is determined using known techniques. It can be detected qualitatively or quantitatively. Residual levels may not be detectable using known techniques.

[0193] polymer In some embodiments, a suitable delivery vehicle is formulated using the polymer alone or in combination with other carriers containing various lipids as described herein. Thus, in some embodiments, the liposome delivery vehicle also includes polymer-containing nanoparticles, as used herein. Suitable polymers include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumins, gelatins, alginates, collagens, chitosans, cyclodextrins, protamines, PEGylated protamines, PLLs, PEGylated PLLs, and polyethyleneimines (PEIs). If a PEI is present, it may be a branched PEI with a molecular weight in the range of 10 to 40 kDa, for example, a 25 kDa branched PEI (Sigma No. 408727).

[0194] According to various embodiments, the selection of cationic lipids, non-cationic lipids, PEG-modified lipids, cholesterol-based lipids, and / or amphiphilic block copolymers constituting lipid nanoparticles, as well as the relative molar ratios of such components (lipids) to each other, are based on the characteristics of the selected lipids, the properties of the intended target cells, and the characteristics of the nucleic acids to be delivered. Additional considerations include, for example, alkyl chain saturation, as well as the size, charge, pH, pKa, membrane fusionability, and tolerability of the selected lipids. Thus, the molar ratios can be adjusted accordingly.

[0195] Use of amphiphilic polymers in ethanol-free LNP formulations In some embodiments, the amphiphilic polymer used in the methods herein includes one or more pluronics, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or a combination thereof. In some embodiments, the amphiphilic polymer is selected from one or more of the following: PEG triethylene glycol, tetraethylene glycol, PEG200, PEG300, PEG400, PEG600, PEG1,000, PEG1,500, PEG2,000, PEG3,000, PEG3,350, PEG4,000, PEG6,000, PEG8,000, PEG10,000, PEG20,000, PEG35,000, and PEG40,000, or a combination thereof. In some embodiments, the amphiphilic polymer is triethylene glycol. In some embodiments, the amphiphilic polymer is tetraethylene glycol. In some embodiments, the amphiphilic polymer is PEG200. In some embodiments, the amphiphilic polymer is PEG300. In some embodiments, the amphiphilic polymer is PEG400. In some embodiments, the amphiphilic polymer is PEG600. In some embodiments, the amphiphilic polymer is PEG1,000. In some embodiments, the amphiphilic polymer is PEG1,500. In some embodiments, the amphiphilic polymer is PEG2,000. In some embodiments, the amphiphilic polymer is PEG3,000. In some embodiments, the amphiphilic polymer is PEG3,350. In some embodiments, the amphiphilic polymer is PEG4,000. In some embodiments, the amphiphilic polymer is PEG6,000. In some embodiments, the amphiphilic polymer is PEG8,000. In some embodiments, the amphiphilic polymer is PEG10,000. In some embodiments, the amphiphilic polymer is PEG20,000. In some embodiments, the amphiphilic polymer is PEG35,000. In some embodiments, the amphiphilic polymer is PEG40,000.

[0196] In some embodiments, the amphiphilic polymer comprises a mixture of two or more types of PEG polymers with different molecular weights. For example, in some embodiments, 2, 3, 4, 5, 6, 7, 8 PEG polymers with molecular weights of 9, 10, 11, or 12 constitute the amphiphilic polymer. Therefore, in some embodiments, the PEG solution contains a mixture of one or more PEG polymers. In some embodiments, the mixture of PEG polymers contains polymers having distinct molecular weights.

[0197] In some embodiments, the lipid solution comprises one or more amphiphilic polymers. In some embodiments, the solvent in the lipid solution comprises a PEG polymer. Various types of PEG polymers are recognized in the art, some of which have distinct geometric configurations. PEG polymers suitable for the methods herein include, for example, PEG polymers having linear, branched, Y-shaped, or multi-arm configurations. In some embodiments, the PEG is in a suspension comprising one or more PEGs of distinct geometric configurations. In some embodiments, the lipid solution is achieved using PEG-6000 as the solvent. In some embodiments, the lipid solution is achieved using PEG-400 as the solvent. In some embodiments, the lipid solution is achieved using triethylene glycol (TEG) as the solvent. In some embodiments, the lipid solution is achieved using triethylene glycol monomethyl ether (mTEG) as the solvent. In some embodiments, the lipid solution is achieved using tert-butyl-TEG-O-propionate as the solvent. In some embodiments, the lipid solution is achieved using TEG-dimethacrylate as the solvent. In some embodiments, the lipid solution is achieved using TEG-dimethyl ether as the solvent. In some embodiments, the lipid solution is achieved using TEG-divinyl ether as the solvent. In some embodiments, the lipid solution is achieved using TEG-monobutyl ether as the solvent. In some embodiments, the lipid solution is achieved using TEG-methyl ether methacrylate as the solvent. In some embodiments, the lipid solution is achieved using TEG-monodecyl ether as the solvent. In some embodiments, the lipid solution is achieved using TEG-dibenzoate as the solvent. Any one of these PEG or TEG-based reagents can be used as the solvent for the lipid solution mixed with the mRNA solution in the LNP formulation. The structures of each of these reagents are shown below in Table 1.

[0198] [Table 1]

[0199] In some embodiments, the lipid solution comprises a PEG polymer solvent, and the PEG polymer comprises a PEG-modified lipid. In some embodiments, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG-2K). In some embodiments, the PEG-modified lipid is a DOPA-PEG conjugate. In some embodiments, the PEG-modified lipid is a poloxamer-PEG conjugate. In some embodiments, the PEG-modified lipid comprises DOTAP. In some embodiments, the PEG-modified lipid comprises cholesterol.

[0200] In some embodiments, the lipid solution contains an amphiphilic polymer. In some embodiments, the lipid solution contains any of the above PEG reagents. In some embodiments, PEG is present in the suspension at a concentration of about 10% to about 100% by weight / volume. For example, in some embodiments, PEG is present in the suspension at concentrations of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% by weight / volume, and any value in between. In some embodiments, PEG is present in the suspension at a concentration of about 5% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of about 6% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of about 7% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of about 8% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of about 9% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 10% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 12% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 15% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 18% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 20% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 25% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 30% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 35% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 40% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 45% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 50% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 55% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 60% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 65% ​​by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 70% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 75% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 80% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 85% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 90% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 95% by weight / volume. In some embodiments, PEG is present in the suspension at a concentration of approximately 100% by weight / volume.

[0201] In some embodiments, the formulation contains a volume:volume ratio of PEG to the total mRNA suspension volume of about 0.1 to about 5.0. For example, in some embodiments, PEG is present in the formulation in volume:volume ratios of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, and 5.0. Therefore, in some embodiments, PEG is present in the formulation in a volume:volume ratio of about 0.1. In some embodiments, PEG is present in the formulation in a volume:volume ratio of about 0.2. In some embodiments, PEG is present in the formulation in a volume:volume ratio of about 0.3. In some embodiments, PEG is present in the formulation in a volume:volume ratio of about 0.4. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 0.5. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 0.6. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 0.7. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 0.8. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 0.9. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 1.0. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 1.25. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 1.5. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 1.75. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 2.0. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 2.25. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 2.5. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 2.75. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 3.0. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 3.25. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 3.5. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 3.75. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 4.0.In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 4.25. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 4.50. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 4.75. In some embodiments, PEG is present in the formulation at a volume:volt ratio of approximately 5.0.

[0202] In certain embodiments, PEG is mTEG (e.g., about 100% or pure mTEG). In certain embodiments, the lipid solution is about 100% mTEG-lipid. Particularly suitable final concentrations of mTEG in mRNA-LNP formulations are about 55–65% by weight / volume, e.g., about 50% by weight / volume. As shown in the examples, this concentration maintains mRNA solubility and stability, and allows for reduced processing volume and easy production of formulations on a large scale.

[0203] In some embodiments, the mRNA solution and the lipid solution (e.g., about 100% mTEG-lipid solution) are mixed in a ratio of 1 to 8:1, for example, 1 to 4:1 (v / v). In certain embodiments, the mRNA solution and the lipid solution (e.g., about 100% mTEG-lipid solution) are mixed in a ratio of about 1:1 (v / v). As shown in the examples, this ratio of mRNA solution to lipid solution maintains mRNA solubility and stability, and allows for reduced processing volume and easy production of formulations on a large scale.

[0204] In some embodiments, the formulation is alcohol-free. In some embodiments, the formulation is manufactured without the use of any non-aqueous solvent (e.g., alcohol). In some embodiments, the solvent does not contain flammable agents. In some embodiments, the solvent does not contain ethanol. In some embodiments, the solvent does not contain isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, or combinations thereof. In some embodiments, the solvent does not contain alcohol solvents (e.g., methanol, ethanol, or isopropanol). In some embodiments, the solvent does not contain ketone solvents (e.g., acetone, methyl ethyl ketone, or methyl isobutyl ketone). In some embodiments, the formulation is aqueous.

[0205] In some embodiments, mRNA is encapsulated in the absence of ethanol. In some embodiments, mRNA is purified in the absence of ethanol. In some embodiments, mRNA purification, mRNA encapsulation, or both processes are performed in the absence of ethanol. In some embodiments, mRNA purification, mRNA encapsulation, or both processes do not contain flammable agents. In some embodiments, mRNA purification, mRNA encapsulation, or both processes do not contain non-aqueous solvents.

[0206] Ratio of separate lipid components Suitable liposomes of the present invention may contain one or more of cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids, amphiphilic block copolymers, and / or polymers described herein in various ratios. In some embodiments, the lipid nanoparticles include separate components of five and five or fewer nanoparticles. In some embodiments, the lipid nanoparticles include separate components of four or four or fewer nanoparticles. In some embodiments, the lipid nanoparticles include separate components of three or three or fewer nanoparticles. As a non-limiting example, suitable liposome formulations may include cKK-E12 (also known as ML2), DOPE, cholesterol, and DMG-PEG2K; C12-200, DOPE, cholesterol, and DMG-PEG2K; HGT4003, DOPE, cholesterol, and DMG-PEG2K; ICE, DOPE, cholesterol, and DMG-PEG2K; or combinations selected from ICE, DOPE, and DMG-PEG2K.

[0207] In various embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) 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 liposomes in molar ratio. In some embodiments, the percentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% or more of the liposomes in molar ratio.

[0208] In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 50:10:35:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 60:35:0:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 50:25:20:5.

[0209] An exemplary lipid mixture for use in the present invention consists of four lipid components: cationic lipids (e.g., ML-2 or MC-3), non-cationic lipids (e.g., DSPC, DPPC, DOPE, or DEPE), cholesterol-based lipids (e.g., cholesterol), and PEG-modified lipids (e.g., DMG-PEG2K). In some embodiments, the molar ratios of cationic lipids (e.g., ML-2 or MC-3), non-cationic lipids (e.g., DSPC or DOPE), cholesterol-based lipids, and PEG-modified lipids in the LNP can be between approximately 35-55:5, 35:20, 40:1, and 15, respectively. In some embodiments, the molar ratios of cationic lipids (e.g., ML-2), non-cationic lipids (e.g., DSPC or DOPE), cholesterol-based lipids, and PEG-modified lipids in the LNP are 35-45:25, 35:20, 30:1, and 10. In certain embodiments, the molar ratio of cationic lipids (e.g., ML-2), non-cationic lipids (e.g., DSPC or DOPE), cholesterol lipids, and PEG-modified lipids in the LNP is approximately 40:30:25:5. In some embodiments, the molar ratio of cationic lipids (e.g., MC-3), non-cationic lipids (e.g., DSPC or DOPE), cholesterol lipids, and PEG-modified lipids in the LNP is 45-55:5-15:30-40:1-10. In some embodiments, the molar ratio of cationic lipids (e.g., MC-3), non-cationic lipids (e.g., DSPC or DOPE), cholesterol lipids, and PEG-modified lipids in the LNP is approximately 50:10:35:5. As shown in the examples, these preparations ensure appropriate mRNA-LNP diameter and encapsulation efficiency, making them particularly suitable for use in the formulations of the present invention.

[0210] In some embodiments, the lipid mixture for use in the present invention may contain three or fewer distinct lipid components. In some embodiments, one distinct lipid component in such a mixture is a cholesterol-based or imidazole-based cationic lipid. An exemplary lipid mixture consists of three lipid components: a cationic lipid (e.g., a cholesterol-based or imidazole-based cationic lipid, e.g., ICE, HGT4001, or HGT4002), 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 lipids to non-cationic lipids and PEG-modified lipids is between approximately 55-65:30-40:1-15, respectively. In some embodiments, the molar ratio of cationic lipids (e.g., ICE), non-cationic lipids (e.g., DSPC), and PEG-modified lipids in the LNP is 55-65:30-40:1-15. In certain embodiments, the molar ratio of cationic lipids (e.g., ICE), non-cationic lipids (e.g., DSPC or DOPE), and PEG-modified lipids in the LNP is 60:35:5. As shown in the examples, these preparations ensure appropriate mRNA-LNP diameter and encapsulation efficiency, making them particularly suitable for use in the formulations of the present invention.

[0211] In some embodiments, the concentrations of lipids and mRNA in the mRNA-LNP are such that the N / P ratio of cationic lipids (e.g., ML-2 or MC-3) to mRNA is approximately 2, 3, 4, 5, or 6. As shown in the examples, a particularly suitable N / P ratio is approximately 4, which allows for efficient LNP formation and mRNA inclusion.

[0212] In embodiments in which lipid nanoparticles contain three or fewer distinct lipid components, the ratio of the total lipid content (i.e., the ratio of lipid component (1): lipid component (2): lipid component (3)) can be expressed as x:y:z, where, (y+z)=100-x That is the case.

[0213] In some embodiments, "x", "y", and "z" each represent the molar percentage of three distinct components of the lipid, and the ratio is the molar ratio.

[0214] In some embodiments, "x", "y", and "z" each represent the weight percentage of three distinct components of the lipid, and the ratios are weight ratios.

[0215] In some embodiments, the lipid component (1), represented by the variable "x", is a sterol-based cationic lipid.

[0216] In some embodiments, the lipid component (2), represented by the variable "y", is a helper lipid.

[0217] In some embodiments, the lipid component (3), represented by the variable "z", is a PEG lipid.

[0218] In some embodiments, the variable "x" representing the molar percentage of the lipid component (1) (e.g., sterol-based cationic lipids) is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[0219] In some embodiments, the variable "x" representing the molar percentage of lipid component (1) (e.g., sterol-based cationic lipids) is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, or less than or equal to 10%. In some embodiments, the variable "x" is approximately 65%, 60%, 55%, 50%, or less than or equal to 40%.

[0220] In some embodiments, the variable "x" representing the molar percentage of lipid component (1) (e.g., sterol-based cationic lipids) is at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%. In embodiments, the variable "x" is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.

[0221] In some embodiments, the variable "x" representing the weight percentage of the lipid component (1) (e.g., sterol-based cationic lipids) is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[0222] In some embodiments, the variable "x" representing the weight percentage of lipid component (1) (e.g., sterol-based cationic lipids) is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, or less than or equal to 10%. In some embodiments, the variable "x" is approximately 65%, 60%, 55%, 50%, or less than or equal to 40%.

[0223] In some embodiments, the variable "x" representing the weight percentage of lipid component (1) (e.g., sterol-based cationic lipids) is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%. In embodiments, the variable "x" is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.

[0224] In some embodiments, the variable "z" representing the molar percentage of lipid component (3) (e.g., PEG lipids) is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% or less. In some embodiments, the variable "z" representing the molar percentage of lipid component (3) (e.g., PEG lipids) is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In the embodiment, the variable "z" representing the molar percentage of the lipid component (3) (e.g., PEG lipid) is approximately 1% to 10%, 2% to 10%, 3% to 10%, 4% to 10%, 1% to 7.5%, 2.5% to 10%, 2.5% to 7.5%, 2.5% to 5%, 5% to 7.5%, or 5% to 10%.

[0225] In some embodiments, the variable "z" representing the weight percentage of lipid component (3) (e.g., PEG lipids) is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% or less. In the embodiment, the variable "z" representing the weight percentage of the lipid component (3) (e.g., PEG lipid) is approximately 1% to 10%, 2% to 10%, 3% to 10%, 4% to 10%, 1% to 7.5%, 2.5% to 10%, 2.5% to 7.5%, 2.5% to 5%, 5% to 7.5%, or 5% to 10%.

[0226] For compositions having three and three or more distinct lipid components, the variables "x", "y", and "z" can be any combination, as long as the sum of the three variables equals 100% of the total lipid content.

[0227] mRNA synthesis The mRNA according to the present invention can be synthesized according to any of the various known methods. Various methods are described in U.S. Patent Application Publication No. 2018 / 0258423, and the present invention can be carried out using these methods, all of which are incorporated herein by reference. For example, the mRNA according to the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions vary depending on the specific application. ru.

[0228] In some embodiments, the appropriate mRNA sequence is an mRNA sequence encoding a protein or peptide. In some embodiments, the appropriate mRNA sequence is codon-optimized for efficient expression in human cells. In some embodiments, the appropriate mRNA sequence is a naturally occurring sequence or a wild-type sequence. In some embodiments, the appropriate mRNA sequence encodes a protein or peptide containing one or more mutations in its amino acid sequence.

[0229] The present invention can be used to deliver mRNA of various lengths. In some embodiments, the present invention can be used to deliver in vitro synthetic mRNA of about 0.5kb, 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, 15kb, 20kb, 30kb, 40kb, or 50kb length or longer. In some embodiments, the present invention can be used to deliver in vitro synthetic mRNA in the range of about 1-20kb, about 1-15kb, about 1-10kb, about 5-20kb, about 5-15kb, about 5-12kb, about 5-10kb, about 8-20kb, or about 8-50kb length.

[0230] In some embodiments, a DNA template is transcribed in vitro to produce mRNA according to the present invention. A suitable DNA template typically has a promoter for in vitro transcription, e.g., a T3, T7, or SP6 promoter, followed by a desired nucleotide sequence for the desired RNA and a termination signal.

[0231] nucleotide mRNA according to the present invention can be produced using a variety of naturally occurring or modified nucleotides. In some embodiments, the mRNA is a naturally occurring nucleoside (or unmodified nucleotide; e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine Syn, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methylpseudridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoamidite bonds), or comprising these.

[0232] In some embodiments, suitable mRNA may contain skeletal modifications, glycosylation, and / or base modifications. For example, modified nucleotides may include, but are not limited to, modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 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-ino Syn, 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-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil This includes au-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-pseuduracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, and phosphoramidites, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine. The manufacture of such analogues is known to those skilled in the art from U.S. Patents 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, which incorporate by reference, for example, the entire disclosure.

[0233] In some embodiments, the mRNA contains one or more non-standard nucleotide residues. Non-standard nucleotide residues include, for example, 5-methylcytidine ("5mC"), pseudouridine ("ΨU"), and / or 2-thiouridine ("2sU"). For a discussion of such residues and their incorporation into mRNA, see, for example, U.S. Patent No. 8,278,036 or International Publication No. 2011 / 012316. The mRNA may be RNA defined as RNA in which 25% of the U residues are 2-thiouridine and 25% of the C residues are 5-methylcytidine. Teachings regarding the use of RNA are disclosed in U.S. Patent Application Publication No. 2012 / 0195936 and International Publication No. 2011 / 012316, both of which are incorporated herein by whole reference. The presence of non-standard nucleotide residues can make the mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only standard residues. In further embodiments, the mRNA may contain isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurinecytosine, as well as one or more non-standard nucleotide residues selected from combinations of these modifications and other nucleic acid base modifications. Some embodiments may further include additional modifications to the furanose ring or nucleic acid bases. Additional modifications include, for example, sugar modifications or substitutions (e.g., 2'-O-alkyl modifications, one or more loc nucleic acids (LNAs)). In some embodiments, the RNA is complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications include, but are not limited to, 2'-deoxy-2'-fluoro modifications, 2'-O-methyl modifications, 2'-O-methoxyethyl modifications, and 2'-deoxy modifications.In some embodiments, any of these modifications can be present in 0-100% of the nucleotides—for example, individually or in combination—in 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, over 95%, or 100% of the constituent nucleotides.

[0234] In some embodiments, mRNA may contain RNA backbone modifications. Typically, backbone modifications are modifications in which the phosphate esters of the nucleotide backbone contained in the RNA are chemically modified. Exemplary backbone modifications typically include, but are not limited to, methylphosphonates, methylphosphoramidites, phosphoramidites, phosphorothioates (for example) This includes modifications from the group consisting of cytidine 5'-O-(1-thiophosphate), boranophosphate, and positively charged guanidinium groups (meaning the phosphodiester bond is replaced by other anionic, cationic, or neutral groups).

[0235] In some embodiments, mRNA may contain sugar modifications. Typical sugar modifications are, but are not limited to, 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides, and 2'-deoxy-2'-C-alkyloligoribonucleotides. This refers to a chemical modification of sugars in nucleotides contained in mRNA, including a sugar modification selected from the group consisting of goribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracithidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0236] Post-synthesis processing Typically, a 5' cap and / or 3' tail can be added after synthesis. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0237] The 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 phosphate esters; then, guanosine triphosphate (GTP) is added to the terminal phosphate ester via guanylyltransferase to produce a 5'5'5 triphosphate bond; and then, the 7-nitrogen of guanine is methylated by methyltransferase. Examples of cap structures, though not limited to these, include m7G(5')ppp(5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication 2016 / 0032356 and U.S. Patent Application Publication 2018 / 0125989, which are incorporated herein by reference.

[0238] Typically, the tail structure includes a poly(A) and / or poly(C) tail. The polyA or polyC tail on the 3' end of the mRNA typically contains at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, and a small number of adenosine or cytosine nucleotides. It contains at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotides. In some embodiments, the poly-A or poly-C tail contains about 10 to 800 each. adenosine or cytosine nucleotides (for example, approximately 10-200 adenosine or cytosine nucleotides, approximately 10-300 adenosine or cytosine nucleotides, approximately 10-400 adenosine or cytosine nucleotides, approximately 10-500 adenosine or cytosine nucleotides, approximately 10-550 adenosine or cytosine nucleotides, approximately 10-600 adenosine or cytosine nucleotides, approximately 50-600 adenosine or cytosine nucleotides, approximately 100-600 adenosine or cytosine nucleotides, approximately 150-600 adenosine or cytosine nucleotides, approximately 200-600 adenosine or cytosine nucleotides) The creotide can be a combination of approximately 250-600 adenosine or cytosine nucleotides, approximately 300-600 adenosine or cytosine nucleotides, approximately 350-600 adenosine or cytosine nucleotides, approximately 400-600 adenosine or cytosine nucleotides, approximately 450-600 adenosine or cytosine nucleotides, approximately 500-600 adenosine or cytosine nucleotides, approximately 10-150 adenosine or cytosine nucleotides, approximately 10-100 adenosine or cytosine nucleotides, approximately 20-70 adenosine or cytosine nucleotides, or approximately 20-60 adenosine or cytosine nucleotides. In some embodiments, the tail structure is a combination of poly(A) tails and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure contains at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, the tail structure contains at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0239] As described herein, without capping and / or tailing, the size of prematurely interrupted mRNA transcripts is too small to detect; therefore, the addition of a 5' cap and / or 3' tail facilitates the detection of interrupted transcripts produced during in vitro synthesis. Thus, in some embodiments, the 5' cap and / or 3' tail are added to the synthesized mRNA before testing the mRNA for purity (the level of interrupted transcripts present in the mRNA). In some embodiments, the 5' cap and / or 3' tail are added to the synthesized mRNA before purifying the mRNA as described herein. In other embodiments, the 5' cap and / or 3' tail are added to the synthesized mRNA after purifying the mRNA as described herein.

[0240] The synthesized mRNA can be used in the present invention without further purification. In particular, the synthesized mRNA can be used in the present invention without the step of removing the shorter. In some embodiments, the synthesized mRNA can be further purified for use in the present invention. The synthesized mRNA can be purified using a variety of methods. For example, the mRNA can be purified using centrifugation, filtration and / or chromatography. In some embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or by gel purification or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, which is well known to those skilled in the art. In some embodiments, the mRNA is purified using tangential flow filtration. Suitable purification methods are all incorporated herein by reference and can be used to carry out the present invention, including U.S. Patent Application Publication 2016 / 0040154, U.S. Patent Application Publication 2015 / 0376220, U.S. Patent Application Publication 2018 / 0251755, U.S. Patent Application Publication 2018 / 0251754, U.S. Provisional Patent Application 62 / 757,612 filed November 8, 2018, and U.S. Provisional Patent Application 62 filed August 26, 2019. This includes items listed in issues 891 and 781.

[0241] In some embodiments, mRNA is purified before capping and tailing. In some embodiments, mRNA is purified after capping and tailing. In some embodiments, mRNA is purified both before and after capping and tailing.

[0242] In some embodiments, mRNA is purified by centrifugation either before or after capping and tailing, or both before and after.

[0243] In some embodiments, mRNA is purified by filtration either before or after capping and tailing, or both before and after.

[0244] In some embodiments, mRNA is purified by tangential flow filtration (TFF) either before or after capping and tailing, or both before and after.

[0245] In some embodiments, mRNA is purified by chromatography either before or after capping and tailing, or both before and after.

[0246] In some embodiments, mRNA is purified without the use of ethanol or any other flammable solvent.

[0247] Characterization of purified mRNA The mRNA compositions described herein are substantially free of contaminants including short interrupted RNA species, long interrupted RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvents, and / or residual salts.

[0248] The mRNA compositions described herein have a purity between approximately 60% and approximately 100%. Therefore, in some embodiments, the purified mRNA has a purity of approximately 60%. In some embodiments, the purified mRNA has a purity of approximately 65%. In some embodiments, the purified mRNA has a purity of approximately 70%. In some embodiments, the purified mRNA has a purity of approximately 75%. In some embodiments, the purified mRNA has a purity of approximately 80%. In some embodiments, the purified mRNA has a purity of approximately 85%. In some embodiments, the purified mRNA has a purity of approximately 90%. In some embodiments, the purified mRNA has a purity of approximately 91%. In some embodiments, the purified mRNA has a purity of approximately 92%. In some embodiments, the purified mRNA has a purity of approximately 93%. In some embodiments, the purified mRNA has a purity of approximately 94%. In some embodiments, the purified mRNA has a purity of approximately 95%. In some embodiments, the purified mRNA has a purity of approximately 96%. In some embodiments, the purified mRNA has a purity of approximately 97%. In some embodiments, the purified mRNA has a purity of approximately 98%. In some embodiments, the purified mRNA has a purity of approximately 99%. In some embodiments, the purified mRNA has a purity of approximately 100%.

[0249] In some embodiments, the mRNA compositions described herein contain impurities other than full-length mRNA in amounts 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%. These impurities include IVT contaminants, such as proteins, enzymes, DNA templates, free nucleotides, residual solvents, residual salts, double-stranded RNA (dsRNA), prematurely interrupted RNA sequences ("short marshals" or "short interrupted RNA species"), and / or long interrupted RNA species. In this embodiment, the purified mRNA is substantially free of process enzymes.

[0250] In some embodiments, the residual plasmid DNA in the purified mRNA of the present invention is less than approximately 1 pg / mg, less than approximately 2 pg / mg, less than approximately 3 pg / mg, less than approximately 4 pg / mg, less than approximately 5 pg / mg, less than approximately 6 pg / mg, less than approximately 7 pg / mg, less than approximately 8 pg / mg, less than approximately 9 pg / mg, less than approximately 10 pg / mg, less than approximately 11 pg / mg, or less than approximately 12 pg / mg. Therefore, the residual plasmid DNA in the purified mRNA is less than approximately 1 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 2 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 3 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 4 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 5 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 6 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 7 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 8 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 9 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 10 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 11 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than approximately 12 pg / mg.

[0251] In some embodiments, the method according to the present invention removes approximately 90%, 95%, 96%, 97%, 98%, more than 99%, or substantially all of the early interrupted RNA sequences (also known as "shortmers"). In some embodiments, the mRNA composition is substantially free of early interrupted RNA sequences. In some embodiments, the mRNA composition contains less than approximately 5% (e.g., less than approximately 4%, 3%, 2%, or 1%) of early interrupted RNA sequences. In some embodiments, the mRNA composition contains less than approximately 1% (e.g., less than approximately 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of early interrupted RNA sequences. In some embodiments, the mRNA composition contains undetectable early interrupted RNA sequences (e.g., the presence of separate low bands) as determined, for example, by high-performance liquid chromatography (HPLC) (e.g., shoulder peaks or separate peaks), ethidium bromide, Coomassie staining, capillary electrophoresis, or glyoxal gel electrophoresis. As used herein, the terms “shortmer,” “short interrupted RNA species,” “early interrupted RNA sequence,” or “long interrupted RNA species” refer to any transcript shorter than its full length. In some embodiments, a “shortmer,” “short interrupted RNA species,” or “early interrupted RNA sequence” is less than 100 nucleotides, less than 90 nucleotides, less than 80 nucleotides, less than 70 nucleotides, less than 60 nucleotides, less than 50 nucleotides, less than 40 nucleotides, less than 30 nucleotides, less than 20 nucleotides, or less than 10 nucleotides. In some embodiments, the shortmer is detected or quantified after the addition of a 5' cap and / or a 3' poly-A tail. In some embodiments, the early interrupted RNA transcript contains fewer than 15 bases (e.g., fewer than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 bases). In some embodiments, the early-disrupted RNA transcript contains approximately 8–15, 8–14, 8–13, 8–12, 8–11, or 8–10 bases.

[0252] In some embodiments, the purified mRNA of the present invention substantially does not contain enzyme reagents used for in vitro synthesis, including, but not limited to, T7 RNA polymerase, DNAse I, pyrophosphatase, and / or RNAse inhibitors. In some embodiments, the purified mRNA of the present invention contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of enzyme reagents used for in vitro synthesis. The purified mRNA contains less than approximately 1% (e.g., less than approximately 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the enzyme reagents used for in vitro synthesis. In some embodiments, the purified mRNA contains undetectable enzyme reagents used for in vitro synthesis, including those determined by silver staining, gel electrophoresis, high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), and / or capillary electrophoresis, ethidium bromide, and / or Coomassi staining.

[0253] In various embodiments, the purified mRNA of the present invention maintains a high degree of integrity. As used herein, the term “mRNA integrity” generally refers to the quality of the purified mRNA. mRNA integrity can be determined using methods well known in the art, for example, by RNA agarose gel electrophoresis. In some embodiments, mRNA integrity can be determined by the band pattern of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present invention shows little to no banding compared to the reference band of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present invention has integrity of about 95% or more (e.g., about 96%, 97%, 98%, 99%, or more). In some embodiments, the purified mRNA of the present invention has integrity of over 98%. In some embodiments, the purified mRNA of the present invention has integrity of over 99%. In some embodiments, the purified mRNA of the present invention has integrity of about 100%.

[0254] In some embodiments, purified mRNA is evaluated for one or more of the following characteristics: appearance, identity, quantity, concentration, presence of impurities, microbiological evaluation, pH level, and activity. In some embodiments, an acceptable appearance includes a colorless, clear solution that is essentially free of visible particles. In some embodiments, mRNA identity is evaluated by sequencing. In some embodiments, concentration is evaluated by a suitable method such as UV spectrophotometry. In some embodiments, a suitable concentration is between approximately 90% and 110% nominal (0.9 to 1.1 mg / mL).

[0255] In some embodiments, the assessment of mRNA purity includes assessment of mRNA integrity, assessment of residual plasmid DNA, and assessment of residual solvent. In some embodiments, an acceptable level of mRNA integrity is assessed by agarose gel electrophoresis. The gel is analyzed to determine whether the binding pattern and apparent nucleotide length are consistent with the analytical reference standard. Additional methods for assessing RNA integrity include, for example, assessment of purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, the acceptable purity of purified mRNA as determined by CGE is that the purified mRNA composition has approximately 55% or less of long-broken / degraded species. In some embodiments, residual plasmid DNA is assessed by methods of the Art, for example, by the use of qPCR. In some embodiments, the acceptable level of residual plasmid DNA is 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). In some embodiments, the acceptable residual solvent levels are 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, and 1,000 ppm or less. Therefore, in some embodiments, the acceptable residual solvent level is 10,000 ppm or less. In some embodiments, the acceptable residual solvent level is 9,000 ppm or less. In some embodiments, the acceptable residual solvent level is 8,000 ppm or less. In some embodiments, the acceptable residual solvent level is 7,000 ppm or less. In some embodiments, the acceptable residual solvent level is 6,000 ppm or less. In some embodiments, the acceptable residual solvent level The allowable residual solvent level is 5,000 ppm or less. In some embodiments, the allowable residual solvent level is 4,000 ppm or less. In some embodiments, the allowable residual solvent level is 3,000 ppm or less. In some embodiments, the allowable residual solvent level is 2,000 ppm or less. In some embodiments, the allowable residual solvent level is 1,000 ppm or less.

[0256] In some embodiments, microbiological tests, including, for example, the evaluation of bacterial endotoxins, are performed on the purified mRNA. In some embodiments, the bacterial endotoxin is less than 0.5 EU / mL, less than 0.4 EU / mL, less than 0.3 EU / mL, less than 0.2 EU / mL, or less than 0.1 EU / mL. Therefore, in some embodiments, the bacterial endotoxin in the purified mRNA is less than 0.5 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is less than 0.4 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is less than 0.3 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is less than 0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is less than 0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is less than 0.1 EU / mL. In some embodiments, the purified mRNA has a concentration of 1 CFU / 10 mL, 1 CFU / 25 mL, 1 CFU / 50 mL, 1 CFU / 75 mL or less, or 1 CFU / 100 mL or less. Therefore, in some embodiments, the purified mRNA has a concentration of 1 CFU / 10 mL or less. In some embodiments, the purified mRNA has a concentration of 1 CFU / 25 mL or less. In some embodiments, the purified mRNA has a concentration of 1 CFU / 50 mL or less. In some embodiments, the purified mRNA has a concentration of 1 CFU / 75 mL or less. In some embodiments, the purified mRNA has a concentration of 1 CFU / 100 mL.

[0257] In some embodiments, the pH of the purified mRNA is evaluated. In some embodiments, the acceptable pH of the purified mRNA is between 5 and 8. Therefore, in some embodiments, the purified mRNA has a pH of approximately 5. In some embodiments, the purified mRNA has a pH of approximately 6. In some embodiments, the purified mRNA has a pH of approximately 7. In some embodiments, the purified mRNA has a pH of approximately 7.5. In some embodiments, the purified mRNA has a pH of approximately 8.

[0258] In some embodiments, the translational fidelity of the purified mRNA is evaluated. Translational fidelity can be evaluated by various methods, including, for example, transfection and Western blot analysis. Acceptable features of the purified mRNA include a band pattern in Western blot that moves at a similar molecular weight to the reference standard.

[0259] In some embodiments, purified mRNA is evaluated for conductance. In some embodiments, acceptable characteristics of purified mRNA include conductances between approximately 50% and 150% of the reference standard.

[0260] The purified mRNA is also evaluated for cap percentage and poly-A tail length. In some embodiments, acceptable cap percentages include cap 1, % area:NLT90. In some embodiments, acceptable poly-A tail lengths are approximately 100 to 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).

[0261] In some embodiments, the purified mRNA is also evaluated for residual PEG. In some embodiments, the purified mRNA is evaluated for 10 ng PEG / mg of purified mRNA. The PEG concentration is less than 000 ng PEG / mg of purified mRNA. Therefore, in some embodiments, the purified mRNA concentration is less than approximately 10 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA concentration is less than approximately 100 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA concentration is less than approximately 250 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA concentration is less than approximately 500 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA concentration is less than approximately 750 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA concentration is less than approximately 1000 ng PEG / mg of purified mRNA.

[0262] Various methods for detecting and quantifying mRNA purity are known in the art. For example, such methods include blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. In some embodiments, mRNA is first denatured with a glyoxal dye before gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, the synthesized mRNA is characterized before capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing.

[0263] Therapeutic use of the composition To facilitate in vivo mRNA expression, delivery vehicles such as liposomes can be formulated into pharmacological compositions that combine with one or more additional nucleic acids, carriers, targeted ligands, or stabilizing reagents, or mixed with appropriate excipients. Techniques for formulating and administering drugs can be found in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania.

[0264] In some embodiments, the composition comprises mRNA encapsulated by or complexed with a delivery vehicle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, lipid nanoparticles, solid-lipid nanoparticles, polymers, viruses, sol-gels, and nanogels.

[0265] The mRNA-loaded nanoparticles provided, and compositions containing such nanoparticles, may be administered and doped in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the schedule of administration, the subject's age, sex, weight, and other factors relevant to a physician skilled in the art. The “effective dose” for the purposes herein may be determined by relevant considerations known to those skilled in the art of experimental clinical research, pharmacology, clinical and medical fields. In some embodiments, the dose administered is effective in achieving stabilization, improvement, or elimination of at least some of the symptoms and other indicators that may be selected by those skilled in the art as appropriate measures of disease progression, regression, or improvement. For example, an appropriate dose and dosing regimen will induce at least transient protein (e.g., enzyme) production.

[0266] The present invention provides a method for delivering mRNA for in vivo protein production, comprising the step of administering the mRNA to a target requiring delivery. In some embodiments, the mRNA is administered via a delivery route selected from the group consisting of intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, intratracheal injection / lung delivery (e.g., spray or infusion), intramuscular delivery, intrathecal delivery, or intra-articular delivery. Accordingly, in some embodiments, the present invention provides a method for delivering mRNA for in vivo protein production, including intravenous delivery. In some embodiments, the present invention provides a method for delivering mRNA for in vivo protein production, including intramuscular delivery. In some embodiments, the present invention provides an intratracheal injection This invention provides a method for delivering mRNA for in vivo protein production, including lung delivery.

[0267] The development of ethanol-free LNP formulations greatly reduces and / or eliminates fire safety concerns and enables bedside mixing, resulting in the production of low-volume formulations with a 1:1 citrate-mRNA to solvent-lipid ratio, which would be more suitable for administration. Therefore, in some embodiments, mRNA LNP formulations are suitable for production and administration in a variety of settings, including, for example, bedside mixing, hospital mixing, and pharmacy mixing.

[0268] Appropriate routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, or intestinal administration including intratracheal or inhalation; intradermal, transdermal (topical), intramuscular, subcutaneous, intrathecal injection, and parenteral delivery including intrathecal, direct intraventricular, intravenous, intraperitoneal, or nasal delivery. In some embodiments, intramuscular administration is to muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, the mRNA is delivered to muscle cells by administration. In some embodiments, the mRNA is delivered to hepatocytes (i.e., liver cells) by administration. In certain embodiments, the mRNA is delivered to muscle cells by intramuscular administration.

[0269] Additional teachings for lung delivery and spraying are described in U.S. Patent Application Publication No. 2018 / 0125989 and U.S. Patent Application Publication No. 2018 / 0333457, which are incorporated by reference in their entirety.

[0270] Alternatively, the mRNA-loaded nanoparticles and compositions of the present invention may be administered topically rather than systemically, for example, by direct injection of the pharmaceutical composition into the target tissue, preferably in a sustained-release formulation. Topical delivery can be carried out in a variety of ways depending on the tissue being targeted. For example, an aerosol containing the composition of the present invention may be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present invention may be injected, for example, into the site of injury, disease manifestation, or pain; the composition may be provided in lozenges for oral, tracheal, or esophageal administration; it may be supplied in liquid, tablet, or capsule form for gastric or intestinal administration; it may be supplied in suppository form for rectal or vaginal administration; or it may even be delivered to the eye by use of cream, droplet, or even injection. Formulations containing the provided composition complexed with a therapeutic molecule or ligand may even be administered surgically, for example, in combination with a polymer or other structure or substance that can allow the composition to diffuse from the implantation site into surrounding cells. Alternatively, these may be administered surgically without the use of a polymer or support.

[0271] The methods provided by the present invention are intended for single and multiple administrations of therapeutically effective amounts of the therapeutic agent described herein (e.g., mRNA). The therapeutic agent may be administered at regular intervals depending on the nature, severity, and degree of the condition in question. In some embodiments, therapeutically effective amounts of the therapeutic agent of the present invention (e.g., mRNA) may be administered intrathecally at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, every other month (once every two months), once a month (once every month), every other week (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily, or continuously).

[0272] In some embodiments, the provided liposomes and / or compositions are formulated to be suitable for extended release of the mRNA contained therein. Such extended-release compositions are conveniently administered to subjects at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to subjects twice daily, daily, or every other day. In preferred embodiments, the compositions of the present invention are administered twice a week, once a week, once every 7 days, once every 10 days, once every 14 days, once every 28 days, once every 30 days, once every 2 weeks, once every 3 weeks, or more preferably. The drug is administered to the subject every four weeks, once a month, twice a month, once every six weeks, once every eight weeks, once every two months, once every three months, once every four months, once every six months, once every eight months, once every nine months, or annually. Compositions and liposomes formulated for depot administration (e.g., intramuscular, subcutaneous, intravitreous) to deliver or release the therapeutic agent (e.g., mRNA) over a long period are also intended. Preferably, the extended release means used is combined with modifications made to the mRNA to enhance stability.

[0273] As used herein, the term “therapeutic dose” is determined primarily based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. Generally, a therapeutic dose is sufficient to achieve a meaningful benefit to a subject (e.g., to treat, regulate, cure, prevent, and / or improve a disease or disorder). For example, a therapeutic dose is sufficient to achieve the desired therapeutic and / or preventive effect. Generally, the amount of therapeutic agent (e.g., mRNA) administered to a subject in need depends on the subject’s characteristics. Such characteristics include the subject’s condition, disease severity, overall health, age, sex, and weight. Those skilled in the art will be able to easily determine the appropriate dosage according to these and other relevant factors. Furthermore, both objective and subjective assays may be used to identify the optimal dosage range.

[0274] Therapeutically effective doses are typically administered in drug regimens that may include multiple unit doses. For a particular therapeutic protein, the therapeutically effective dose (and / or appropriate unit dose within an effective drug regimen) varies depending, for example, on the route of administration and in combination with other medications. Furthermore, the specific therapeutically effective dose (and / or unit dose) for a particular patient depends on a variety of factors, including the disorder being treated and its severity; the activity of the specific drug used; the specific composition used; the patient's age, weight, overall health, sex, and diet; the timing of administration, the route of administration, and / or the excretion or metabolic rate of the specific protein used; the duration of treatment; and similar factors well known in the medical field.

[0275] In some embodiments, the therapeutically effective dose is approximately 0.005 mg / kg body weight to 500 mg / kg body weight, for example, approximately 0.005 mg / kg body weight to 400 mg / kg body weight, approximately 0.005 mg / kg body weight to 300 mg / kg body weight, approximately 0.005 mg / kg body weight to 200 mg / kg body weight, approximately 0.005 mg / kg body weight to approximately 100 mg / kg body weight, approximately 0.005 mg / kg body weight to 90 mg / kg body weight, approximately 0.005 mg / kg body weight to 80 mg / kg body weight, and approximately 0.005 mg / kg body weight. The ranges are approximately 70 mg / kg body weight, 0.005 mg / kg body weight to 60 mg / kg body weight, 0.005 mg / kg body weight to 50 mg / kg body weight, 0.005 mg / kg body weight to 40 mg / kg body weight, 0.005 mg / kg body weight to 30 mg / kg body weight, 0.005 mg / kg body weight to 25 mg / kg body weight, 0.005 mg / kg body weight to 20 mg / kg body weight, 0.005 mg / kg body weight to 15 mg / kg body weight, and 0.005 mg / kg body weight to 10 mg / kg body weight.

[0276] In some embodiments, therapeutically effective doses are approximately 0.1 mg / kg body weight, approximately 0.5 mg / kg body weight, approximately 1.0 mg / kg body weight, approximately 3 mg / kg body weight, approximately 5 mg / kg body weight, approximately 10 mg / kg body weight, approximately 15 mg / kg body weight, approximately 20 mg / kg body weight, approximately 30 mg / kg body weight, approximately 40 mg / kg body weight, approximately 50 mg / kg body weight, approximately 60 mg / kg body weight, approximately 70 mg / kg body weight, approximately 80 mg / kg body weight, approximately 90 mg / kg body weight, approximately 100 mg / kg body weight, approximately 150 mg / kg body weight, approximately 200 mg / kg body weight, approximately 250 mg / kg body weight, approximately 300 mg / kg body weight, approximately 350 mg / kg body weight, approximately 400 mg / kg body weight, approximately 450 mg / kg body weight, and approximately 500 mg / kg body weight. In certain embodiments, the therapeutically effective dose is 1.0 mg / kg. In some embodiments, the therapeutically effective dose of 1.0 mg / kg is administered intramuscularly or intravenously.

[0277] This specification also considers lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein, and related methods for using such compositions, as disclosed in U.S. Provisional Patent Application No. 61 / 494,882, filed June 8, 2011, which incorporates its entire teaching herein by reference. For example, the lyophilized pharmaceutical compositions according to the present invention can be reconstituted before administration or in vivo. For example, the lyophilized pharmaceutical composition may be formulated into a suitable dosage form (e.g., an intradermal dosage form such as a disc, rod, or membrane) and administered so that the dosage form is rehydrated in vivo over time by the body fluids of a solid.

[0278] The provided liposomes and compositions are administered to any desired tissue. In some embodiments, the mRNA delivered by the provided liposomes or compositions is expressed in the tissue to which the liposomes and / or compositions are administered. In some embodiments, the delivered mRNA is expressed in a tissue different from the tissue to which the liposomes and / or compositions are administered. Exemplary tissues to which the delivered mRNA is delivered and / or expressed include, but are not limited to, the liver, kidneys, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[0279] In some embodiments, administration of the provided composition results in elevated mRNA expression levels in a biological sample derived from the subject compared to baseline expression levels before treatment. Typically, baseline levels are 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, administration of the provided composition results in elevated mRNA expression levels of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline levels immediately before treatment. In some embodiments, administration of the provided composition results in elevated mRNA expression levels compared to the mRNA expression levels of an untreated subject.

[0280] According to various embodiments, the timing of the expression of delivered mRNA can be adjusted to suit specific medical needs. In some embodiments, the expression of the protein encoded by the delivered mRNA is detectable 1, 2, 3, 6, 12, 24, 48, 72, and / or 96 hours after administration of the provided liposomes and / or composition. In some embodiments, the expression of the protein encoded by the delivered mRNA is detectable 1 week, 2 weeks, and / or 1 month after administration.

[0281] The present invention also provides the delivery of a composition having an mRNA molecule encoding a target peptide or polypeptide for use in treating a target, such as a human target or cells of a human target or cells that have been treated and delivered to a human target. [Examples]

[0282] While certain compounds, compositions, and methods of the present invention have been specifically described by certain embodiments, the following examples are merely illustrative and not intended to limit the present invention. [Examples]

[0283] Encapsulation efficiency of ethanol-free lipid nanoparticle (LNP) formulations using polymers as solvents instead of ethanol. This example illustrates the encapsulation efficiency achieved by using triethylene glycol monomethyl ether (mTEG) as an exemplary solvent for dissolving various cationic lipids, including ML-2 and ICE, in the production of ethanol-free LNP formulations. The development of ethanol-free LNP formulations greatly reduces fire safety concerns and / or This would allow for bedside mixing, leading to the production of low-volume formulations with a 1:1 citrate-mRNA to solvent-lipid ratio that is more suitable for drug administration. Currently, obtaining such low-volume formulations using ethanol as a solvent is difficult.

[0284] Exemplary LNP formulations were prepared by mixing mRNA in aqueous solution with lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids) dissolved in an amphiphilic polymer solution to form mRNA encapsulated within LNPs (mRNA-LNPs). In this example, lipids were prepared in ethanol-free mTEG solution or ethanol-based solution. Two different cationic lipids, ML-2 and ICE, were evaluated, and the same ratio of PEG:cationic lipid:cholesterol:non-cationic lipid was used for each cationic lipid in both the ethanol-free polymer mixture and the ethanol-containing mixture (see Table 2). The particle size, polydispersity index, and encapsulation efficiency of LNPs from the ethanol-free polymer mixture and the ethanol-containing mixture were evaluated.

[0285] [Table 2]

[0286] As shown in Table 2, the mTEG-manufactured formulation yielded LNPs of comparable diameter with comparable or improved encapsulation efficiency compared to the ethanol-manufactured LNP formulation. The polydispersity index was observed to be slightly higher in the mTEG formulation compared to the ethanol formulation.

[0287] The results demonstrated that using mTEG to dissolve lipids can enable the safe production of ethanol-free LNP formulations with equivalent or improved encapsulation efficiency. [Examples]

[0288] Encapsulation efficiency of mRNA-LNPs produced using a lipid polymer solvent compared to using an ethanol solvent for lipids. This example illustrates the average particle size, polydispersity index ("PDI"), and encapsulation efficiency obtained from LNP formulations formulated using a polymer solvent for lipids compared with LNP formulations formulated using an ethanol solvent for lipids. The LNPs formulated using the polymer solvent (mTEG) or ethanol consisted of PEG-modified lipids, cationic lipids of either ML-2 or MC3, cholesterol, and helper lipids (DSPC).

[0289] Exemplary mRNA-LNP formulations were prepared by dissolving lipids for LNPs in 100% mTEG solution or 100% ethanol solution. The lipids included either ML-2 or MC-3 as cationic lipids, PEG-modified lipids, cholesterol, and helper lipids (DSPC). The mTEG-lipid or ethanol-lipid solution was mixed with an aqueous solution of mRNA (in citrate buffer) in a volume ratio of 1:4 (mTEG-lipid solution to mRNA solution or ethanol-lipid solution to mRNA solution). Before mixing, in the aqueous solution... The mRNA concentration was 0.08 mg / mL, and the lipid concentration in the lipid solution was sufficient to provide the necessary N / P ratio for the four cationic lipids (ML-2 or MC-3) and mRNA. PEG-modified lipids, cholesterol, and helper lipids (DSPCs) were prepared according to the target ratios (relative to cationic lipids) provided in Table 3. The particle size, polydispersity index, and encapsulation efficiency of the obtained mRNA-LNPs were analyzed (Table 3).

[0290] [Table 3]

[0291] As shown in Table 3, mRNA-LNPs of comparable diameter were obtained in mTEG-produced and ethanol-produced formulations where MC-3 was a cationic lipid. For formulations containing ML-2 as the cationic lipid, mRNA-LNPs were larger when obtained from mTEG-produced lipids compared to ethanol-produced lipids. Compared to the 90% encapsulation efficiency obtained with the corresponding ethanol-produced MC-3 mRNA-LNP formulation, a high encapsulation efficiency of 97% was obtained with the mTEG-produced MC-3 mRNA-LNP formulation. For formulations containing ML-2 as the cationic lipid, the encapsulation efficiency was comparable for mRNA-LNPs obtained from mTEG-produced lipids compared to ethanol-produced lipids. The polydispersity index increased in the mTEG-produced MC-3 mRNA-LNP formulation compared to the ethanol-produced mRNA-LNP formulation.

[0292] These results demonstrate that polymer-based LNP formulations, particularly mTEG-based LNP formulations, offer comparable LNP diameters and potentially higher encapsulation efficiency compared to ethanol-based LNP formulations. Ethanol-free mTEG formulations can be manufactured safely on a large scale compared to ethanol-based formulations and require less post-processing to remove the solvent used in the lipid composition, making it advantageous to prepare mRNA-encapsulated LNPs using mTEG instead of ethanol. [Examples]

[0293] Ethanol-free formulations require low-volume mixing. This example demonstrates the significant advantage of using mTEG solvent over ethanol solvent for lipids in the production of mRNA-LNPs, particularly in reducing the volume required in the production of mRNA-LNP formulations, for example, for drug administration. In particular, when a 100% ethanol solution containing lipids in a 1:1 (v / v) ratio is mixed with an aqueous solution containing mRNA, the high concentration (50% vol / vol) of ethanol in the resulting mixture leads to unstable mRNA solubility and, in some cases, mRNA precipitation. Therefore, prior to the present invention, an approach to address this problem is to dilute the ethanol component in the ethanol-lipid solution to result in a lower ethanol concentration in the resulting mixture, thereby avoiding mRNA instability and the possibility of mRNA precipitation. This involved increasing the volume of the mRNA aqueous solution (which could affect lipid solubility) and / or adding a third aqueous solution stream. For example, by mixing an ethanol-lipid solution (containing 100% ethanol) with the mRNA aqueous solution in a 1:4 (v / v) ratio, the amount of ethanol in the resulting mixture is reduced (20% v / v), thereby helping to avoid mRNA instability and precipitation caused by ethanol. Unfortunately, all these approaches require mixing in larger volumes than necessary (e.g., as required by the lowest solubility concentration and desired N / P ratio), typically using substantially diluted amounts of lipid and mRNA in each solution, which significantly increases time and cost at large-scale processing levels, and also requires a subsequent concentration step in addition to the ethanol removal step, which further increases time and cost.

[0294] However, when mRNA-LNPs are prepared by mixing a 100% mTEG solution containing lipids with an aqueous solution containing mRNA in a 1:1 (v / v) ratio, the high concentration of mTEG (50% v / v) in the resulting mixture does not appear to cause mRNA instability or precipitation. Such mRNA-LNP preparations, produced on a large scale using optimized low-volume solutions, i.e., lipid-containing mTEG solutions and mRNA-containing aqueous solutions with high lipid and mRNA concentrations respectively, are advantageous in reducing processing volume and thereby increasing ease of processing in production.

[0295] To evaluate the ability of mTEG solvent for lipids to optimize the volume used for mRNA-LNP formulations compared to ethanol solvent for lipids, mRNA-LNP formulations were prepared in low-volume ratios (1:1 lipid volume to mRNA volume) and high-volume ratios (1:4 lipid volume to mRNA volume) by dissolving lipid-containing volumes in either 100% mTEG or 100% ethanol. The dissolved lipids contained PEG-modified lipids, cationic lipids of either ML-2 or MC3, cholesterol, and helper lipids (DSPC). To ensure that the same total amount of mRNA was mixed in each process, the mRNA aqueous solution for the low-volume mixing had an mRNA concentration of 0.33 mg / mL before mixing, which is four times the mRNA concentration of the mRNA aqueous solution for the high-volume mixing (0.08 mg / mL). In the lipid solution, the lipid (either 100% mTEG or 100% ethanol solution) was at a concentration necessary to provide the cationic lipid (ML-2 or MC-3) to mRNA N / P ratio of 4, based on the concentrations of PEG-modified lipids, cholesterol, and helper lipids (DSPC) prepared according to the target ratios (relative to cationic lipids) provided in Table 4. Each preparation was mixed in low volume (1:1 lipid solution to mRNA solution) or high volume (1:4 lipid solution to mRNA solution), and the resulting mRNA-LNPs were evaluated for diameter, polydispersity (PDI), and mRNA inclusion percentage (%EE).

[0296] A low-volume (1:1) mRNA-LNP formulation prepared using ML-2 as a cationic lipid dissolved in mTEG achieved an encapsulation efficiency of 69%. In contrast, a low-volume (1:1) mRNA-LNP formulation prepared using ML-2 as a cationic lipid dissolved in ethanol could not be stably produced as a low-volume formulation and showed precipitation after mixing.

[0297] A low-volume (1:1) mRNA-LNP preparation containing MC-3 as a cationic lipid and prepared using a lipid dissolved in mTEG achieved an encapsulation efficiency of 99%, while a low-volume (1:1) mRNA-LNP preparation containing MC-3 as a cationic lipid and prepared using a lipid dissolved in ethanol showed an encapsulation efficiency of 95%.

[0298] [Table 4]

[0299] The results showed that mTEG is suitable for use in low-volume ethanol-free mRNA-LNP formulations, providing improved mRNA stability after mixing and potentially improved encapsulation efficiency compared to ethanol-produced LNP formulations. [Examples]

[0300] Testing of ethanol-free formulations using various polymers and lipids This example tests ethanol-free LNP formulations using various polymers and lipids.

[0301] While not limited to these, LNP formulations are manufactured using a variety of amphiphilic polymers, including polyethylene glycol (PEG), mPEG, tetraethylene glycol monomethyl ether, and pentaethylene glycol monomethyl ether.

[0302] Dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipal LNP formulations are manufactured using one or more noncationic lipids, including mitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, and 16-O-dimethylPE, 18-1-transPE, and 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE).

[0303] LNP formulations using components that do not show degradation are further analyzed for encapsulation efficiency, LNP diameter, and polydispersity index, as described in Example 2.

[0304] LNP formulations exhibiting favorable encapsulation efficiency are tested in low-volume formulations as described in Example 3.

[0305] By following the above process, a safe, cost-effective, low-volume, ethanol-free LNP formulation can be manufactured for mRNA delivery. [Examples]

[0306] In vivo testing of mRNA delivery in ethanol-free LNP formulations This example illustrates the measurement of the in vivo efficacy of an ethanol-free LNP formulation.

[0307] Ethanol-free LNP formulations and ethanol-based LNP formulations were prepared for mRNA delivery as described in Example 1.

[0308] To test the in vivo efficacy of ethanol-free formulations, ethanol-free LNP formulations and ethanol-containing LNP formulations containing mRNA-encapsulated LNPs were delivered intravenously (IV) or intratracheally to mice via tail vein injection at various doses ranging from 0.1 to 1.0 mg / kg, e.g., 0.5 mg / kg mouse body weight.

[0309] The in vivo distribution of LNPs in various organs was evaluated by bioluminescence testing and quantitative measurement of mRNA and protein expression. The results of biodistribution, mRNA, and protein expression obtained with ethanol-free LNP formulations were compared with those obtained with ethanol-based LNP formulations.

[0310] pulmonary delivery Mice were administered firefly luciferase (FFL) mRNA encapsulated in LNPs produced using either an ethanol-free or ethanol-containing encapsulation process. For these in vivo studies, mice were administered mRNA-containing LNPs intratracheally via catheter, and FFL protein expression was evaluated approximately 24 hours after administration.

[0311] The characteristics of FFL mRNA LNP administered to mice are shown in Table 5 below. As summarized in Table 5, low-volume (1:1 lipid solution to mRNA solution) or high-volume (1:4 lipid solution to mRNA solution) formulations, either encapsulated under ethanol-free conditions (e.g., using mTEG instead of ethanol) or encapsulated under ethanol-containing conditions, were used in this study.

[0312] [Table 5]

[0313] Table 5 shows that encapsulation of mRNA LNP preparations using ethanol-free mRNA conditions resulted in encapsulation and diameter parameters similar to, or better than, those of, ethanol-containing mRNA-LNP preparations (1:4 lipid solution to mRNA solution; high volume conditions).

[0314] The results of the in vivo lung delivery study are summarized in Figure 1. Figure 1 shows that mice administered mRNA LNPs encapsulated using high-volume conditions (1:4 lipid solution vs. mRNA solution) with an ethanol-free encapsulation process (e.g., mTEG) expressed higher levels of protein in the animals compared to animals that received mRNA LNPs encapsulated using a high-volume (1:4 lipid solution vs. mRNA solution) ethanol-containing encapsulation process.

[0315] The results demonstrated the efficacy and feasibility of ethanol-free LNP formulations in in vivo mRNA delivery.

[0316] Intravenous delivery Mice were administered ornithine transcarbamylase (OTC) mRNA encapsulated in LNPs produced using an ethanol-free encapsulation process. For these in vivo studies, the mRNA-containing LNPs were administered intravenously to the mice via tail vein injection, and OTC protein expression in serum and liver was subsequently evaluated 24 hours after administration.

[0317] The characteristics of OTC mRNA LNP administered to mice are shown in Table 6 below. As summarized in Table 6, low-volume (1:1 lipid solution vs. mRNA solution) or high-volume (1:4 lipid solution vs. mRNA solution) formulations encapsulated under ethanol-free conditions were used in this study. OTC mRNA LNP formulated using MC-3 and DOPE were used as controls in this study.

[0318] [Table 6]

[0319] Data from these studies are presented in Figure 2. The data show that OTC expression was present in serum and liver with the use of the following mRNA-LNP formulations: (MC-3)1:1 mTEG; (ML-2)1:4 mTEG; and (ML-2)1:1 mTEG. The results demonstrate the efficacy and feasibility of ethanol-free LNP formulations in in vivo mRNA delivery.

[0320] Further stability testing of mRNA and protein expression will be compared between ethanol-free and ethanol-based LNP formulations by performing measurements over several hours and several days.

[0321] Equivalents and range Those skilled in the art will be able to recognize or confirm many equivalents of the specific embodiments of the invention described herein using only routine experiments. The scope of the invention is not intended to be limited to the above specification, but rather as set forth in the following claims.

Claims

1. A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), comprising the steps of (a) mixing an mRNA solution containing one or more mRNAs with (b) a lipid solution containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, wherein the step of mixing the mRNA solution and the lipid solution is performed in the presence of an amphiphilic polymer to form mRNA encapsulated within LNPs in an LNP formulation solution (mRNA-LNPs).

2. The method according to claim 1, wherein the amphiphilic polymer includes pluronic, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or a combination thereof.

3. The method according to claim 2, wherein PEG is selected from triethylene glycol monomethyl ether (mTEG), methoxypolyethylene glycol mPEG, tetraethylene glycol monomethyl ether, pentaethylene glycol monomethyl ether, or a combination thereof.

4. The method according to claim 3, wherein PEG is triethylene glycol monomethyl ether (mTEG).

5. The method according to claim 1, wherein the step of mixing the mRNA solution and the lipid solution results in a PEG concentration greater than 25% by volume / volume.

6. The method according to claim 1, wherein the step of mixing the mRNA solution and the lipid solution includes PEG at a concentration of approximately 50% volume / volume.

7. The method according to any one of claims 1 to 6, wherein the mRNA solution contains less than 5 mM citrate, and the mRNA-LNPs have an inclusion efficiency of more than 60%.

8. The method according to any one of claims 1 to 7, wherein the mRNA solution and / or lipid solution are at approximately ambient temperature.

9. The method according to claim 8, wherein the ambient temperature is less than approximately 35°C, less than approximately 30°C, less than approximately 26°C, less than approximately 23°C, less than approximately 21°C, less than approximately 20°C, or less than approximately 18°C.

10. The method according to claim 9, wherein the ambient temperature is in the range of approximately 18 to 32°C, approximately 21 to 26°C, or approximately 23 to 25°C.

11. One or more noncationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidyl Ethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethyl The method according to any one of claims 1 to 10, selected from tyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a mixture thereof.

12. The method according to any one of claims 1 to 11, wherein one or more noncationic lipids are distearoylphosphatidylcholine (DSPC).

13. The method according to any one of claims 1 to 12, wherein the mRNA solution further comprises trehalose.

14. The method according to any one of claims 1 to 13, which does not require a step of heating the mRNA solution and the lipid solution before the mixing step.

15. The method according to any one of claims 1 to 14, wherein the mRNA solution contains more than approximately 1 g of mRNA per 12 L of mRNA solution.

16. The method according to claim 15, wherein the mRNA solution contains approximately 1 g of mRNA per 8 L of mRNA solution.

17. The method according to claim 15, wherein the mRNA solution contains approximately 1 g of mRNA per 4 L of mRNA solution.

18. The method according to claim 15, wherein the mRNA solution contains approximately 1 g of mRNA per 2 L of mRNA solution.

19. The method according to claim 15, wherein the concentration of mRNA in the mRNA solution is greater than approximately 0.125 mg / mL, greater than approximately 0.25 mg / mL, greater than approximately 0.5 mg / mL, or greater than approximately 1.0 mg / mL.

20. The method according to any one of claims 1 to 19, wherein the mRNA solution and the lipid solution are mixed in a ratio (v / v) between 2:1 and 6:

1.

21. The method according to claim 20, wherein the mRNA solution and the lipid solution are mixed in a ratio of approximately 4:1 (v / v).

22. The method according to any one of claims 1 to 21, wherein the mRNA solution has a pH between 3.0 and 5.

0.

23. The method according to claim 22, wherein the mRNA solution has a pH of approximately 3.5, 4.0, or 4.

5.

24. The method according to any one of claims 1 to 23, wherein the mixing step is carried out with a total volume between approximately 3 and 10 mL.

25. The method according to claim 24, wherein the mixing step is carried out with a total volume of approximately 3 mL.

26. The method according to any one of claims 1 to 25, wherein no alcohol is included.

27. The method according to any one of claims 1 to 26, further comprising the step of incubating mRNA-LNPs.

28. The method according to claim 27, wherein the mRNA-LNP is incubated at a temperature between 21°C and 65°C.

29. The method according to claim 28, wherein the mRNA-LNP is incubated at a temperature of approximately 26°C, approximately 30°C, or approximately 65°C.

30. The method according to any one of claims 27 to 29, wherein the mRNA-LNP is super-incubated for approximately 20 minutes, approximately 30 minutes, approximately 60 minutes, approximately 90 minutes, or approximately 120 minutes.

31. The method according to claim 30, wherein the mRNA-LNP is incubated for approximately 60 minutes.

32. The method according to any one of claims 1 to 31, wherein the lipid solution does not contain alcohol.

33. The method according to any one of claims 1 to 32, wherein the lipid solution further comprises one or more cholesterol-based lipids.

34. The method according to any one of claims 1 to 33, wherein mRNA-LNPs are purified by tangential flow filtration.

35. The method according to any one of claims 1 to 34, wherein the mRNA-LNP has an average diameter of less than 150 nm, less than 100 nm, less than 80 nm, less than 60 nm, or less than 40 nm.

36. The method according to claim 35, wherein the mRNA-LNP has an average diameter in the range of 40 to 70 nm.

37. The method according to any one of claims 1 to 36, wherein the lipid nanoparticles have a PDI of less than approximately 0.3, less than approximately 0.2, less than approximately 0.18, less than approximately 0.15, or less than approximately 0.

1.

38. The method according to any one of claims 1 to 37, wherein the mRNA-LNP encapsulation efficiency is approximately 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%.

39. The method according to any one of claims 1 to 38, wherein the mRNA-LNP has an N / P ratio between 1 and 10.

40. The method according to claim 39, wherein the mRNA-LNP has an N / P ratio between 2 and 6.

41. The method according to claim 40, wherein the mRNA-LNP has an N / P ratio of approximately 4.

42. The method according to any one of claims 1 to 41, wherein 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 is encapsulated in lipid nanoparticles in a single batch.

43. The method according to any one of claims 1 to 42, wherein the mRNA solution and the lipid solution are mixed by a pulseless flow pump.

44. The method according to claim 43, wherein the pump is a gear pump.

45. The method according to claim 44, wherein the pump is a centrifugal pump.

46. The method according to any one of claims 1 to 45, wherein the mRNA solution is mixed at a flow rate in the range of approximately 150-250 ml / min, 250-500 ml / min, 500-1000 ml / min, 1000-2000 ml / min, 2000-3000 ml / min, 3000-4000 ml / min, or 4000-5000 ml / min.

47. The method according to claim 46, wherein the mRNA solution is mixed at a flow rate of approximately 800 ml / min, approximately 1000 ml / min, or approximately 12000 ml / min.

48. The method according to any one of claims 1 to 47, wherein the lipid solution is mixed at a flow rate in the range of approximately 25 to 75 ml / min, approximately 75 to 200 ml / min, approximately 200 to 350 ml / min, approximately 350 to 500 ml / min, approximately 500 to 650 ml / min, approximately 650 to 850 ml / min, or approximately 850 to 1000 ml / min.

49. The method according to claim 48, wherein the lipid solution is mixed at a flow rate of approximately 100 ml / min, approximately 150 ml / min, approximately 200 ml / min, approximately 250 ml / min, approximately 300 ml / min, and approximately 350 ml / min.

50. The method according to any one of claims 46 to 49, wherein the flow rate of the mRNA solution is 2 times, 4 times, or 6 times greater than the flow rate of the lipid solution.

51. The method according to any one of claims 1 to 50, wherein mRNA is purified by a method that does not contain volatile organic compounds.

52. The method according to claim 51, wherein the mRNA is purified by an alcohol-free method.

53. A composition comprising mRNA encapsulated in lipid nanoparticles produced by the method described in any one of claims 1 to 52.

54. The composition according to claim 53, comprising 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.

55. The composition according to claim 53 or 54, wherein the mRNA comprises one or more modified nucleotides.

56. The composition according to claim 51 or 52, wherein the mRNA is unmodified.

57. The composition according to any one of claims 53 to 56, wherein the mRNA is approximately 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 8 kb, 10 kb, 20 kb, 30 kb, or greater than 40 kb.

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