Stable liquid lipid nanoparticle formulation

Abstract

This invention provides stable lipid nanoparticles that are resistant to freeze / thaw cycles. [Solution] A liquid lipid nanoparticle (LNP) formulation is provided, which encapsulates mRNA encoding a peptide or polypeptide, and comprises cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, and also comprises a sugar or sugar alcohol, pH buffer, and an agent that provides ionic strength, and is resistant to aggregation and mRNA degradation after multiple freezing and thawing cycles at -20°C.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 118,243, filed on 25 November 2020, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Nucleic acid-based technologies are becoming increasingly important for a variety of therapeutic applications, including, but not limited to, messenger RNA therapy. Efforts to deliver nucleic acids involve the creation of compositions prepared to protect nucleic acids from degradation when delivered in vivo. One type of delivery vehicle for nucleic acids is lipid nanoparticles. Key parameters to consider for the successful use of lipid nanoparticles as a delivery vehicle include lipid nanoparticle formation, the physical properties of the lipid components, nucleic acid encapsulation efficiency, in vivo nucleic acid release potential, and lipid nanoparticle toxicity. [Overview of the initiative] [Problems that the invention aims to solve]

[0003] The production of stable lipid nanoparticles that are resistant to freeze / thaw cycles remains a challenge in this field. [Means for solving the problem]

[0004] The present invention provides, in particular, liquid lipid nanoparticle (LNP) formulations for encapsulating mRNA encoding peptides or polypeptides that are resistant to aggregation and / or mRNA degradation after multiple freeze-and-thaw cycles at -20°C. The inventors have surprisingly discovered that LNP formulations with high ionic strength prevent LNP aggregation and / or mRNA degradation after multiple freeze-and-thaw cycles. The inventors have also surprisingly discovered that stable, high-ionic-strength LNP formulations resistant to aggregation and / or mRNA degradation can be achieved by using either higher buffering strength or higher salt concentrations in the LNP formulation.

[0005] In some embodiments, a liquid lipid nanoparticle (LNP) formulation is provided to encapsulate mRNA encoding a peptide or polypeptide that is resistant to aggregation and mRNA degradation, wherein the LNP formulation comprises: a. one or more LNPs having lipid components including or consisting of cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol; b. mRNA encoding a peptide or polypeptide encapsulated within one or more lipid nanoparticles; c. sugar or sugar alcohol; d. pH of the LNP formulation being 6.0 to 8.0; e. pH buffer providing the pH of the LNP formulation at a minimum buffer ion strength; f. .Optionally comprising one or more additional agents that provide ionic strength to the LNP formulation, the total concentration of the pH buffer from (e) and optionally one or more additional agents from (f) provides an ionic strength of the LNP formulation at least twice greater than the minimum buffered ion strength, and after three freeze-and-thaw cycles at -20°C, the LNP formulation exhibits (i) less aggregation, (ii) less degradation of encapsulated mRNA, or (iii) both (i) and (ii) compared to the same LNP formulation having only the minimum buffered ion strength in the LNP formulation, instead of an ionic strength at least twice greater than the minimum buffered ion strength.

[0006] In some embodiments, the LNP formulation comprises one or more cryoprotective agents. The cryoprotective agents may be permeable or non-permeable. For example, in some embodiments, The permeable cryoprotectant comprises glycerin, ethylene glycol, triethylene glycol, propylene glycol, or tetraethylene glycol. Therefore, in some embodiments, the permeable cryoprotectant comprises glycerin. In some embodiments, the permeable cryoprotectant comprises ethylene glycol. In some embodiments, the permeable cryoprotectant comprises triethylene glycol. In some embodiments, the permeable cryoprotectant comprises propylene glycol. In some embodiments, the permeable cryoprotectant comprises tetraethylene glycol.

[0007] In some embodiments, the impermeable cryoprotectant is selected from sugars and / or polymers. For example, in some embodiments, the impermeable cryoprotectant is selected from one or more of the following sugars: dextrose, sorbitol, trehalose, sucrose, raffinose, dextran, or inulin. Thus, in some embodiments, the impermeable cryoprotectant comprises dextrose. In some embodiments, the impermeable cryoprotectant comprises sorbitol. In some embodiments, the impermeable cryoprotectant comprises trehalose. In some embodiments, the impermeable cryoprotectant comprises sucrose. In some embodiments, the impermeable cryoprotectant comprises raffinose. In some embodiments, the impermeable cryoprotectant comprises dextran. In some embodiments, the impermeable cryoprotectant comprises inulin.

[0008] In some embodiments, the non-permeable cryoprotectant is selected from one or more of the following polymers: PVP, PVA, poloxamer, or PEG. Therefore, in some embodiments, the non-permeable cryoprotectant is selected from PVP. In some embodiments, the non-permeable cryoprotectant is selected from poloxamer. In some embodiments, the non-permeable cryoprotectant is selected from PEG.

[0009] In some embodiments, a method is provided for preparing a stable liquid solution of mRNA in LNPs. For example, in some embodiments, the mRNA encapsulated in the LNP is produced by in vitro transcription (IVT). In some embodiments, the mRNA is synthesized using a suitable RNA polymerase such as SP6 RNA polymerase. Therefore, in some embodiments, the mRNA is synthesized using SP6 RNA polymerase. The LNPs include, for example, cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol.

[0010] In some embodiments, the noncationic lipids are 1,2-dielycoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosph Selected from phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, or 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE).

[0011] In some embodiments, the noncationic lipids have a molar ratio higher than 10%. For example, in some embodiments, the noncationic lipids have a lipid molar ratio of 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%, and 44%. The percentages are 45%, 46%, 47%, 48%, 49%, or 50%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 15%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 20%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 25%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 30%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 35%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 40%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 45%. In some embodiments, the noncationic lipid has a lipid molar ratio of about 50%.

[0012] In some embodiments, the noncationic lipid is dioleoylphosphatidylethanolamine (DOPE).

[0013] In some embodiments, DOPE has a lipid molar ratio greater than 10%. For example, in some embodiments, DOPE has a lipid molar ratio of 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%, or 50%. In some embodiments, DOPE has a lipid molar ratio of approximately 15%. In some embodiments, DOPE has a lipid molar ratio of approximately 20%. In some embodiments, DOPE has a lipid molar ratio of approximately 25%. In some embodiments, DOPE has a lipid molar ratio of approximately 30%. In some embodiments, DOPE has a lipid molar ratio of approximately 35%. In some embodiments, DOPE has a lipid molar ratio of approximately 40%. In some embodiments, DOPE has a lipid molar ratio of approximately 45%. In some embodiments, DOPE has a lipid molar ratio of approximately 50%. In some embodiments, DOPE has a lipid molar ratio between approximately 10% and 30%.

[0014] In some embodiments, the cationic lipid is a lipidoid. In some embodiments, the lipidoid has a molar ratio of, for example, about 40% to 60%. In some embodiments, the lipidoid has a molar ratio of about 50% to 60%. In some embodiments, the lipidoid has a molar ratio of about 40%. In some embodiments, the lipidoid has a molar ratio of about 50%. In some embodiments, the lipidoid has a molar ratio of about 60%.

[0015] In some embodiments, the mRNA encodes a protein that is missing in the subject. For example, in some embodiments, the protein missing in the subject is CFTR.

[0016] In some embodiments, the mRNA encodes a vaccine antigen. For example, in some embodiments, the vaccine antigen is the SARS-CoV-2 antigen.

[0017] In some embodiments, the sugar is a disaccharide. In some embodiments, the disaccharide is trehalose.

[0018] In some embodiments, the sugar or sugar alcohol is selected from the group consisting of dextrose, sorbitol, trehalose, sucrose, raffinose, dextran, and inulin. Therefore, in some embodiments, the sugar or sugar alcohol is dextrose. In some embodiments, the sugar or sugar alcohol is sorbitol. In some embodiments, the sugar or sugar alcohol is trehalose. In some embodiments, the sugar or sugar alcohol is sucrose. In some embodiments, the sugar or sugar alcohol is raffinose. In some embodiments, the sugar or sugar alcohol is dextran. In some embodiments, the sugar or sugar alcohol is inulin.

[0019] In some embodiments, the concentration of trehalose is between approximately 1% and 20%. In some embodiments, the concentration of trehalose is between approximately 2.5% and 3.0%. In some embodiments, the concentration of trehalose is between approximately 5.0% and 15%. In some embodiments, the concentration of trehalose is between approximately 10% and 20%.

[0020] In some embodiments, the pH is between approximately 6.0 and approximately 8.0. For example, in some embodiments, the pH is between approximately 6.0 and 7.0, 6.5 and 7.5, or 7.0 and 8.0. Therefore, in some embodiments, the pH is between approximately 6.0 and 7.0. In some embodiments, the pH is between approximately 6.5 and 7.5. In some embodiments, the pH is between approximately 7.0 and 8.0. In some embodiments, the pH is approximately 7.4. In some embodiments, the pH is 7.4.

[0021] In some embodiments, the pH buffer has a pKa between 6.0 and 8.2. Therefore, in some embodiments, the pH buffer has a pKa of approximately 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or 8.2. In some embodiments, the pH buffer has a pKa of approximately 6.2. In some embodiments, the pH buffer has a pKa of approximately 6.4. In some embodiments, the pH buffer has a pKa of approximately 6.6. In some embodiments, the pH buffer has a pKa of approximately 6.8. In some embodiments, the pH buffer has a pKa of approximately 7.0. In some embodiments, the pH buffer has a pKa of approximately 7.2. In some embodiments, the pH buffer has a pKa of approximately 7.4. In some embodiments, the pH buffer has a pKa of approximately 7.6. In some embodiments, the pH buffer has a pKa of approximately 7.8. In some embodiments, the pH buffer has a pKa of approximately 8.0. In some embodiments, the pH buffer has a pKa of approximately 8.2.

[0022] In some embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, imidazole buffer, histidine buffer, and Good's buffer. Therefore, in some embodiments, the buffer is phosphate buffer. In some embodiments, the buffer is citrate buffer. In some embodiments, the buffer is imidazole buffer. In some embodiments, the buffer is histidine buffer. In some embodiments, the buffer is Good's buffer. In some embodiments, Good's buffer is Tris buffer or HEPES buffer.

[0023] In some embodiments, the pH buffer is a phosphate buffer (e.g., citrate-phosphate buffer), a Tris buffer, or an imidazole buffer.

[0024] In some embodiments, the minimum buffer ion strength is at least 75 mM, at least 100 mM, at least 125 mM, at least 150 mM, or at least 200 mM. Therefore, in some embodiments, the minimum buffer ion strength is at least 75 mM. In some embodiments, the minimum buffer ion strength is at least 100 mM. In some embodiments, the minimum buffer ion strength is at least 125 mM. In some embodiments, the minimum buffer ion strength is at least 150 mM. In some embodiments, the minimum buffer ion strength is at least 200 mM.

[0025] In some embodiments, the minimum buffer ion strength is between approximately 75 mM and 200 mM, 75 mM and 150 mM, 75 mM and 100 mM, or 100 mM and 200 mM. Therefore, in some embodiments, the minimum buffer ion strength is between approximately 75 mM and 200 mM. In some embodiments, the minimum buffer ion strength is between approximately 75 mM and 150 mM. In some embodiments, the minimum buffer ion strength is between approximately 75 mM and 100 mM. In some embodiments, the minimum buffer ion strength is between approximately 100 mM and 200 mM.

[0026] In some embodiments, the minimum buffer ion strength is obtained by either increasing the buffer concentration in the formulation and / or increasing the salt concentration in the formulation. Therefore, in some embodiments, the minimum buffer ion strength is obtained by increasing the buffer concentration. In some embodiments, the minimum buffer ion strength is obtained by increasing the salt concentration in the formulation. In some embodiments, the minimum buffer ion strength is obtained by increasing the buffer concentration in the formulation and increasing the salt concentration in the formulation.

[0027] In some embodiments, the disaccharide-to-buffer ratio is between 0.1 and 0.9. In some embodiments, the disaccharide-to-buffer ratio is between 0.1 and 0.7. In some embodiments, the disaccharide-to-buffer ratio is between 0.2 and 0.7. In some embodiments, the disaccharide-to-buffer ratio is between 0.2 and 0.5.

[0028] In some embodiments, one or more agents providing ionic strength include a salt. In some embodiments, the salt is selected from the group consisting of NaCl, KCl, and CaCl2. Thus, in some embodiments, the salt is NaCl. In some embodiments, the salt is KCl. In some embodiments, the salt is CaCl2.

[0029] In some embodiments, the total concentration of one or more additional agents providing ionic strength is between approximately 50–500 mM, 100–400 mM, or 200–300 mM. Therefore, in some embodiments, the total concentration of one or more agents is between approximately 50–500 mM. In some embodiments, the total concentration of one or more agents is between approximately 100–400 mM. In some embodiments, the total concentration of one or more agents is between approximately 200–300 mM. In some embodiments, the total concentration of one or more agents providing ionic strength is between approximately 50–300 mM, 50–150 mM, or 75–125 mM. In some embodiments, the total concentration of one or more agents providing ionic strength is between approximately 50–300 mM. In some embodiments, the total concentration of one or more agents providing ionic strength is between approximately 50–150 mM. In some embodiments, the total concentration of one or more agents providing ionic strength is between approximately 75 and 125 mM.

[0030] In some embodiments, the total concentration of the pH buffer is between approximately 100–300 mM, 200–300 mM, or 250–300 mM. Therefore, in some embodiments, the total concentration of the pH buffer is between approximately 100–300 mM. In some embodiments, the total concentration of the pH buffer is between 200–300 mM. In some embodiments, the total concentration of the pH buffer is between 250–300 mM. In some embodiments, the total concentration of the pH buffer is between approximately 15–250 mM, 30–150 mM, or 40–50 mM. Therefore, in some embodiments, the total concentration of the pH buffer is between approximately 15–250 mM. In some embodiments, the total concentration of the pH buffer is between approximately 30–150 mM. In some embodiments, the total concentration of the pH buffer is between approximately 40–50 mM.

[0031] In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is selected from approximately 40 mM Tris buffer and approximately 75-200 mM NaCl, approximately 50 mM Tris buffer and approximately 75-200 mM NaCl, approximately 100 mM Tris buffer and approximately 75-200 mM NaCl, approximately 40 mM imidazole and approximately 75-200 mM NaCl, approximately 50 mM imidazole and approximately 75-200 mM NaCl, approximately 100 mM imidazole and approximately 75-200 mM NaCl, approximately 40 mM phosphate and approximately 75-200 mM NaCl, approximately 50 mM phosphate and approximately 75-200 mM NaCl, approximately 100 mM phosphate and 75-200 mM NaCl. Therefore, in some embodiments, pH buffer and ionic strength are provided. The total concentration of one or more additional agents is approximately 40 mM Tris buffer and approximately 75-200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is approximately 50 mM Tris buffer and approximately 75-200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is approximately 100 mM Tris buffer and approximately 75-200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is approximately 40 mM imidazole and approximately 75-200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is 50 mM imidazole and 75-200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is 100 mM imidazole and 75 mM to 200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is about 40 mM imidazole, about 75 mM to 200 mM NaCl, and 2.5 to 10% trehalose. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is 50 mM imidazole, about 75 mM to 200 mM NaCl, and 2.5 to 10% trehalose. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is 100 mM imidazole, about 75 mM to 200 mM NaCl, and 2.5 to 10% trehalose.

[0032] In some embodiments, the ionic strength of the LNP formulation is at least 2.25 times greater, at least 2.5 times greater, at least 2.75 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, and at least 5 times greater than the minimum buffered ion strength. Therefore, in some embodiments, the ionic strength of the LNP formulation is at least 2.25 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 2.5 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 2.75 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 3.0 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 3.5 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 4.0 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 4.5 times greater than the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is at least 5.0 times greater than the minimum buffered ion strength.

[0033] In some embodiments, the ionic strength of the LNP formulation is less than 20 times, less than 19 times, less than 18 times, less than 17 times, less than 16 times, less than 15 times, less than 14 times, less than 13 times, less than 12 times, less than 11 times, less than 10 times, less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, and less than 4 times the minimum buffered ion strength. Therefore, in some embodiments, the ionic strength of the LNP formulation is less than 20 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 19 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 18 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 17 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 16 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 15 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 14 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 13 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 12 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 11 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 10 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 9 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 8 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 7 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 6 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 5 times the minimum buffered ion strength. In some embodiments, the ionic strength of the LNP formulation is less than 4 times the minimum buffered ion strength.

[0034] In some embodiments, the ionic strength of the LNP formulation is at least twice greater than the minimum buffered ion strength and less than 20 times greater, and the ionic strength of the LNP formulation is between approximately 150 mM and 750 mM, 150 mM and 500 mM, 150 mM and 400 mM, 150 mM and 300 mM, 150 mM, and 200 mM. Therefore, in some embodiments, the ionic strength of the LNP formulation is at least twice greater than the minimum buffered ion strength and less than 20 times greater, and the ionic strength of the LNP formulation is between approximately 150 mM and 750 mM. In some embodiments, the ionic strength of the LNP formulation is at least twice greater than the minimum buffered ion strength and less than 20 times greater, and the ionic strength of the LNP formulation is between approximately 150 mM and 500 mM. In some embodiments, the ionic strength of the LNP formulation is at least twice greater than the minimum buffered ion strength and less than 20 times greater, and the ionic strength of the LNP formulation is between approximately 150 mM and 400 mM. In some embodiments, the ionic strength of the LNP formulation is at least twice greater than the minimum buffered ion strength and less than 20 times greater, and the ionic strength of the LNP formulation is between approximately 150 mM and 300 mM.

[0035] In some embodiments, the ionic strength of the LNP formulation is at least twice greater than the minimum buffered ion strength and less than 20 times greater, and the ionic strength of the LNP formulation is 150 mM or greater than 150 mM.

[0036] In some embodiments, less aggregation is determined by turbidity analysis. In some embodiments, less degradation of encapsulated mRNA is determined by turbidity analysis. Various methods can be used to measure turbidity, including, for example, the use of visual analysis and / or spectroscopic analysis.

[0037] In some embodiments, after more than three freeze-and-thaw cycles at -20°C, the LNP formulation exhibits (i) less aggregation, (ii) less degradation of encapsulated mRNA, or (iii) both (i) and (ii) compared to the same LNP formulation having only the minimum buffer ion strength in the LNP formulation, instead of (i) an ionic strength at least twice greater than the minimum buffer ion strength.

[0038] In some embodiments, the LNP has a diameter of less than approximately 100 nm. In some embodiments, the LNP has a diameter between approximately 70 nm and 90 nm. For example, in some embodiments, the LNP has a diameter between approximately 70 nm and 85 nm. In some embodiments, the LNP has a diameter between approximately 70 nm and 80 nm. In some embodiments, the LNP has a diameter between approximately 70 nm and 75 nm. In some embodiments, the LNP has a diameter between approximately 80 nm and 90 nm. In some embodiments, the LNP has a diameter between approximately 85 nm and 90 nm. In some embodiments, the LNP has a diameter between approximately 75 nm and 90 nm. In some embodiments, the LNP has a diameter between approximately 75 nm and 85 nm. In some embodiments, the LNP has a diameter between approximately 75 nm and 80 nm. In some embodiments, the LNP has a diameter of less than approximately 70 nm.

[0039] In some embodiments, the lipid components are DMG-PEG-2000, cKK-E10, and It contains or consists of sterols and DOPE. Therefore, in some embodiments, the lipid component contains DMG-PEG-2000, cKK-E10, cholesterol, and DOPE. In some embodiments, the lipid component consists of DMG-PEG-2000, cKK-E10, cholesterol, and DOPE.

[0040] In some embodiments, the N / P ratio is between approximately 3 and 5. For example, in some embodiments, the N / P ratio is approximately 3. In some embodiments, the N / P ratio is approximately 4. In some embodiments, the N / P ratio is approximately 5.

[0041] In some embodiments, the final concentration of mRNA is between approximately 0.05 mg / mL and 1.0 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.05 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.1 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.1 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.2 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.3 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.4 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.5 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.6 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.7 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.8 mg / mL. In some embodiments, the final concentration of mRNA is approximately 0.9 mg / mL. In some embodiments, the mRNA concentration is approximately 1.0 mg / mL.

[0042] In some embodiments, the mRNA concentration is between approximately 0.2 mg / mL and 0.5 mg / mL.

[0043] In some embodiments, LNP is stable at -20°C for at least 3 months, 6 months, 12 months, or more than 12 months. Therefore, in some embodiments, LNP is stable at -20°C for at least 3 months. In some embodiments, LNP is stable at -20°C for at least 6 months. In some embodiments, LNP is stable at -20°C for at least 12 months. In some embodiments, LNP is stable at -20°C for more than 12 months.

[0044] In some embodiments, the LNP formulation is stable after dilution.

[0045] In some embodiments, subcutaneous or intramuscular delivery of the formulation is associated with reduced pain compared to formulations without a buffer having a concentration of 300 mM or less and a pH between approximately 7.0 and 7.5.

[0046] In some embodiments, pain reduction is assessed by a 10 cm Visual Analog Scale (VAS) or a 6-item Verbal Rating Scale (VRS).

[0047] In some embodiments, methods are provided for reducing the degradation and / or aggregation of LNPs, which include storing LNPs in a formulation as described herein.

[0048] In this application, the use of “or” means “and / or” unless otherwise specified. When used in this disclosure, the terms “comprise,” as well as terms such as “comprising” and “comprises” The modifications are not intended to exclude other additives, components, integers, or processes. Where used in this application, the terms “about” and “approximately” are used interchangeably. Both terms are intended to cover any normal fluctuations recognized by those skilled in the art.

[0049] Other features, 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 not limiting, and are provided only as examples. Various changes and modifications within the scope of the present invention will be evident to those skilled in the art.

[0050] The drawings are illustrative, not limiting. [Brief explanation of the drawing]

[0051] [Figure 1A]This graph shows the stability of LNP at pH 7.5 as a function of the increase in trehalose concentration in the LNP formulation, and as a function of the minimum buffering strength required to maintain LNP stability at pH 7.5. [Figure 1B] This graph shows the stability of an LNP formulation containing a constant percentage (i.e., 2.7%) of trehalose, as a function of pH fluctuations and as a function of the minimum buffering strength required to maintain the stability of the LNP formulation. [Figure 2] This graph shows the lipid pKa-dependent behavior of tested LNP formulations. In these studies, the LNP formulations contained 2.7% trehalose. [Figure 3A] This figure shows the various conditions for the tested LNP formulations. The table shows the molar concentration of lipids and the concentration of Tris buffer at pH 7.5. A checkmark in the table indicates a stable LNP formulation. An "X" indicates an unstable LNP formulation. [Figure 3B] This graph shows the expression of human EPO protein derived from LNPs encapsulating human EPO mRNA in animal models, either 6 or 24 hours after administration. Various LNP constituent lipids are shown. [Figure 4A] A series of tables showing the various compositions of the tested LNP formulations are presented. The tables show the molar concentration of the tested buffer (i.e., Tris or imidazole) and the corresponding salt concentration (i.e., NaCl) tested in the various LNP formulations evaluated. A checkmark in the table indicates a stable LNP formulation. An "X" indicates an unstable LNP formulation. [Figure 4B] A table showing the evaluation of various LNP formulations is presented. The LNP formulations were evaluated with respect to either the Tris or phosphate buffer concentration. The stability of the LNPs after dilution was assessed. Stable LNPs are indicated with a checkmark, while unstable LNP formulations are indicated with an "X". [Figure 5A] The graph shows the encapsulation efficiency % of LNP formulations with various trehalose-to-PBS ratios (e.g., approximately 0.2-0.5) at 4°C. [Figure 5B]The graph shows the encapsulation efficiency % of LNP formulations with various trehalose-to-PBS ratios (e.g., approximately 0.2-0.5) at 25°C. [Figure 6A] The graph shows the LNP size (nanometers) of LNP formulations containing various trehalose-to-PBS ratios (e.g., approximately 0.2-0.5) at 4°C. [Figure 6B] The graph shows the LNP size (nanometers) of LNP formulations containing various trehalose-to-PBS ratios (e.g., approximately 0.2-0.5) at 25°C. [Modes for carrying out the invention]

[0052] 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 present invention and to provide additional details relating to its implementation are incorporated herein by reference.

[0053] 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, unless otherwise stated or evident from the context (except when such a number exceeds 100% of the possible values), the terms “approximately” or “about” refer to a range of the applicable value, in either direction (greater or less) of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% of the reference value mentioned above.

[0054] As used herein, the term “batch” refers to, for example, the content or amount of mRNA synthesized at one time, produced according to a single manufacturing sequence during the same manufacturing cycle. A batch can also refer to the amount of mRNA synthesized in a single reaction occurring via a single aliquot of enzyme and / or a single aliquot of DNA template for sequential synthesis under one set of conditions. In some embodiments, a batch includes mRNA produced from a reaction in which not all reagents and / or components are replenished and / or supplied as the reaction progresses. The term “not a single batch” does not mean mRNA synthesized at different times that are combined to achieve a desired amount.

[0055] Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, mRNA delivery encompasses situations in which mRNA is delivered to a target tissue, the encoded protein is expressed and retained within the 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.

[0056] Encapsulation: As used herein, the term “encapsulation,” or its grammatical equivalent, refers to the process of encapsulating an mRNA molecule within a nanoparticle.

[0057] Engineered or Mutant: As used herein, the terms “engineered” or “mutant,” or their grammatical equivalents, refer to, but are not limited to, a nucleotide or protein sequence that has one or more modifications compared to its naturally occurring sequence, including deletions, insertions, inversions, substitutions, or combinations thereof of heteronucleotides or amino acids.

[0058] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides (e.g., the heavy or light chains of an antibody) into an intact protein (e.g., an antibody), and / or post-translational modification of polypeptides or fully assembled proteins (e.g., antibodies). In this application, the terms “expression” and “production,” as well as their grammatical equivalents, are used interchangeably.

[0059] Functional: As used herein, a “functional” biological molecule is a biological molecule in a form that exhibits the properties and / or activities characterized thereby.

[0060] Half-life: As used herein, the term “half-life” refers to the time required for a content, such as the concentration or activity of a nucleic acid or protein, to decrease to half of its original value, as measured at the beginning of a period.

[0061] To improve, increase, or decrease: When used herein, “to improve” The terms “increase” or “decrease,” or their grammatical equivalents, refer to values ​​relative to baseline measurements, such as measurements in the same individual before the commencement of the treatment described herein, or measurements 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.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] Isolated: As used herein, the term “isolated” means (1) a substance and / or entity that has been separated from at least a portion of the components that were associated with it when it was first produced (whether in nature and / or in an experimental setting), and / or (2) a substance and / or entity that has been produced, manufactured and / or produced by human hands. Isolated substances and / or entities are separated 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 agent is ultrapure of 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 about 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of the percentage purity of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0066] 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. Unless otherwise specified, mRNA sequences are presented in the 5' to 3' direction.

[0067] 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” refers to RNA and single-stranded RNA This includes 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 skeleton. For example, so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in their skeleton, are considered to be within the scope of this 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 that encode proteins contain introns. Nucleic acids 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, nucleic acids may include nucleoside analogs such as analogs having chemically modified bases or sugars, skeleton modifications, etc. Unless otherwise specified, 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. In some embodiments, nucleotides T and U are used interchangeably in sequence descriptions.

[0068] 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., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. Humans include prenatal and postnatal forms.

[0069] 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.

[0070] Stable: As used herein, the term “stable” protein or its grammatical equivalent refers to a protein that retains its physical stability and / or biological activity. In one embodiment, the stability of a protein is determined by a low percentage of degraded (e.g., fragmented) and / or aggregated protein, based on the percentage of monomeric protein in solution. In one embodiment, a stable engineered protein retains or exhibits an increased half-life compared to a wild-type protein. In one embodiment, a stable engineered protein is less susceptible to ubiquitination, which results in proteolysis, compared to a wild-type protein.

[0071] Subject: As used herein, the term "subject" means human or any non-human animal. This refers to an object (e.g., a mouse, rat, rabbit, dog, cat, cow, 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, referring to 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.

[0072] 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.

[0073] Treatment: As used herein, the terms “treatment,” “treatment,” or “to treat” refer to any method used to partially or completely reduce, improve, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce 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.

[0074] Detailed explanation The present invention provides, in particular, improved methods and compositions that result in the production of stable LNP formulations encapsulating mRNA that are resistant to multiple freeze / thaw cycles. Such resistance to multiple freeze / thaw cycles is manifested by at least 1) low aggregation of LNPs after one or more freeze / thaw cycles; and 2) low degradation of the encapsulated mRNA.

[0075] Stable lipid nanoparticle formulations This specification provides formulations for stable liquid lipid nanoparticles (LNPs) that encapsulate mRNA encoding a peptide or polypeptide. Such stable LNPs are resistant to aggregation and mRNA degradation after one or more freeze-thaw cycles. For example, the stable LNPs are resistant to one, two, three, four, five, or more than five freeze-thaw cycles, and the mRNA-encapsulating LNPs are stored at -20°C. In some embodiments, the stable LNPs are resistant to one, two, three, four, five, or more than five freeze-thaw cycles, and the mRNA-encapsulating LNPs are stored at -80°C or below.

[0076] Furthermore, the stable LNP formulations encapsulating mRNA described herein, when administered to subjects requiring them, result in reduced pain. For example, the described LNP formulations reduce pain during administration, such as intramuscular or subcutaneous administration, compared to LNP formulations that do not possess certain ionic strengths as described herein.

[0077] In some embodiments, such a stable LNP formulation comprises: a) one or more LNPs having lipid components including cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol; b) mRNA encapsulated within one or more lipid nanoparticles encoding a peptide or polypeptide; c) sugar or sugar alcohol; d) pH of the LNP formulation between 6.0 and 8.0; e) p providing the pH of the LNP formulation at the minimum buffering ion strength. The solution comprises (e) pH buffer and optionally (f) one or more additional agents that provide ionic strength to the LNP formulation. The stable LNP formulation has the total concentration of the pH buffer from (e) and optionally one or more additional agents from (f) and provides an ionic strength of the LNP formulation that is at least twice greater than the minimum buffered ion strength. After one, two, three or three or more freeze-and-thaw cycles, the described LNP formulation has (i) less aggregation, (ii) less degradation of encapsulated mRNA, or (iii) both (i) and (ii) compared to the same LNP formulation having only the minimum buffered ion strength in the LNP formulation, instead of an ionic strength at least twice greater than the minimum buffered ion strength.

[0078] The one or more additional agents in (f) above may be a salt, a buffer, or a combination of a salt and a buffer. For example, the one or more additional agents in (f) may include, for example, NaCl, KCl, and CaCl2. The buffer may include, for example, a phosphate buffer, a citrate buffer, an imidazole buffer, a histidine buffer, or a Good's buffer. Various types of Good's buffers are known in the art and include, for example, MES, bis-trismethane, ADA, bis-trispropane, PIPES, ACES, POPSO, cholamine chloride, MOPS, BES, AMPB, TES, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, Tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, CAPSO, AMP, CAPS, and CABS. In some embodiments, the Good's buffer is either a Tris buffer or a HEPES buffer.

[0079] In some embodiments, one or more additional agents have concentrations between approximately 50–500 mM, 100–400 mM, or 200–300 mM. The buffer pH of the LNP formulations described herein has concentrations between approximately 100–300 mM, 200–300 mM, or 250–300 mM.

[0080] The minimum buffer ion strength of the stable LNP formulations for encapsulating mRNA described herein is, for example, at least 15 mM, at least 25 mM, at least 50 mM, at least 75 mM, at least 100 mM, at least 125 mM, at least 150 mM, or at least 200 mM. In embodiments, the stable LNP formulations for encapsulating mRNA described herein are, for example, between approximately 15 mM and 200 mM, 50 mM and 200 mM, 75 mM and 200 mM, 15 mM and 150 mM, 50 mM and 150 mM, 75 mM and 150 mM, 15 mM and 100 mM, 50 mM and 100 mM, 75 mM and 100 mM, or 100 mM and 200 mM. The minimum buffer ion strength can be obtained by various methods. For example, in some embodiments, the minimum buffer ion strength is obtained by increasing the buffer concentration. Alternatively, the minimum buffer ion strength can be obtained by increasing the salt concentration. In some embodiments, the minimum buffer ion strength can be obtained by increasing both the buffer concentration and the salt concentration. For example, in some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is selected from approximately 40 mM Tris buffer and approximately 75-200 mM NaCl, approximately 50 mM Tris buffer and approximately 75-200 mM NaCl, approximately 100 mM Tris buffer and approximately 75-200 mM NaCl, approximately 40 mM imidazole and approximately 75-200 mM NaCl, approximately 50 mM imidazole and 75-200 mM NaCl, approximately 100 mM imidazole and 75-200 mM NaCl, approximately 40 mM phosphate and approximately 75-200 mM NaCl, approximately 50 mM phosphate and 75-200 mM NaCl, approximately 100 mM phosphate and 75-200 mM NaCl. In some embodiments, the total concentration of one or more additional agents providing pH buffer and ionic strength is 40 mM Tris buffer, about 75-200 mM NaCl, and about 2.5-10% trehalose, or about 50 mM Tris buffer, about 75-200 mM NaCl, and about 2.5-10% trehalose. % trehalose, approximately 100 mM Tris buffer, approximately 75 mM to 200 mM NaCl and approximately 2.5 to 10% trehalose, approximately 40 mM imidazole, approximately 75 mM to 200 mM NaCl and approximately 2.5 to 10% trehalose, approximately 50 mM imidazole, 75 mM to 200 mM NaCl and approximately 2.5 to 10% trehalose, approximately 100 mM imidazole, 75 mM to The following are selected: 200 mM NaCl and approximately 2.5-10% trehalose, approximately 40 mM phosphate; approximately 75-200 mM NaCl and approximately 2.5-10% trehalose, approximately 50 mM phosphate; 75-200 mM NaCl and approximately 2.5-10% trehalose, approximately 100 mM phosphate; and 75-200 mM NaCl and approximately 2.5-10% trehalose.

[0081] In some embodiments, the buffers are used interchangeably. In some embodiments, Tris buffer is substituted with imidazole buffer or phosphate buffer. In some embodiments, Tris buffer is substituted with imidazole buffer. In some embodiments, Tris buffer is substituted with phosphate buffer. In some embodiments, imidazole buffer is substituted with phosphate buffer or Tris buffer. In some embodiments, imidazole buffer is substituted with phosphate buffer. In some embodiments, imidazole buffer is substituted with Tris buffer. In some embodiments, phosphate buffer is substituted with Tris buffer or imidazole buffer. In some embodiments, phosphate buffer is substituted with Tris buffer. In some embodiments, phosphate buffer is substituted with imidazole buffer.

[0082] In some embodiments, Tris buffer, imidazole buffer, or phosphate buffer has a high buffering strength (e.g., 100 mM or higher). In some embodiments, Tris buffer, phosphate buffer, or imidazole buffer with a low buffering strength (e.g., 15–20 mM) is used with a high salt concentration (e.g., 200 mM or higher NaCl). In some embodiments, Tris buffer, phosphate buffer, or imidazole buffer with a moderate buffering strength (e.g., 40–50 mM) is used with a moderate salt concentration (e.g., 50–100 mM NaCl).

[0083] In some embodiments, Tris buffer, phosphate buffer, or imidazole buffer is used with low trehalose concentrations (e.g., 50–100 mM NaCl). In some embodiments, the stability of the LNP formulation was greater at lower sugar / buffer ratios. In some embodiments, a lower trehalose-to-buffer ratio of the LNP formulation was beneficial in preventing encapsulation degradation. In some embodiments, a lower trehalose-to-buffer ratio prevented an increase in LNP size.

[0084] In some embodiments, the LNP formulation has an ionic strength that is at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, or at least 5 times greater than the minimum buffer ion strength. In some embodiments, the LNP formulation has an ionic strength that is less than 20 times, less than 19 times, less than 18 times, less than 17 times, less than 16 times, less than 15 times, less than 14 times, less than 13 times, less than 12 times, less than 11 times, less than 10 times, less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, or less than 4 times the minimum buffer ion strength. In some embodiments, the ionic strength of the LNP formulation is at least twice the minimum buffered ion strength and less than 20 times, and the ionic strength of the LNP formulation is between approximately 150 mM and 750 mM, 150 mM and 500 mM, 150 mM and 400 mM, 150 mM and 300 mM, 150 mM, and 200 mM. In some embodiments, the ionic strength of the LNP formulation is at least twice the minimum buffered ion strength and less than 20 times, and the ionic strength of the LNP formulation is 150 mM or greater than 150 mM. The minimum buffered ion strength referenced throughout is at least 75 mM, at least 100 mM, at least 125 mM, at least 150 mM, or at least 200 mM.

[0085] In some embodiments, the stable LNP formulations described herein further comprise one or more cryoprotective agents. Cryoprotective agents can be characterized as either "penetrating" or "impermeable" cryoprotective agents. Suitable cryoprotective agents for the LNP formulations described herein can be selected from penetrating and / or impermeable cryoprotective agents. Exemplary impermeable cryoprotective agents include sugars such as dextrose, sorbitol, trehalose, sucrose, raffinose, dextran, and inulin. Another category of impermeable cryoprotective agents includes polymers such as PVP, PVA, poloxamer, and PEG. Examples of penetrating cryoprotective agents include, for example, glycerol, ethylene glycol, triethylene glycol, propylene glycol, and tetraethylene glycol. Any one or more of the described cryoprotective agents are suitable for inclusion in the stable LNP formulations described herein. In some embodiments, the cryoprotective agent in the LNP formulation comprises trehalose at a concentration between 1% and 20%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration between approximately 2.5% and 3.0%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration of approximately 2.5%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration of approximately 2.6%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration of approximately 2.7%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration of approximately 2.8%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration of approximately 2.9%. In some embodiments, the cryoprotective agent in the LNP formulation contains trehalose at a concentration of approximately 3.0%.

[0086] Various noncationic lipids can be used in the LNP formulations described herein. For example, suitable cationic lipids for the LNP formulations described herein include 1,2-dielycoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoyl The following can be selected: 2-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, or 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE). In some embodiments, the noncationic lipid is DOPE.

[0087] Noncationic lipids in LNP formulations may have a lipid molar ratio exceeding 10%. For example, in some embodiments, noncationic lipids have lipid molar ratios of 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%, or 50%.

[0088] In some embodiments, cationic lipids are selected from lipidoids. Various lipidoids are known in the art. For example, lipidoids are described in Goldberg M. (2013) Lipidoids: A Combinatorial Approach to siRNA Delivery. In: Howard K. (ed.) RNA Interference from Biology to Therapeutics. The details are described in Advances in Delivery Science and Technology, Springer, Boston, MA, and are incorporated herein by reference. In some embodiments, the lipidoid is cationic. In some embodiments, the lipidoid includes up to seven tails. The seven tails can be extended, for example, from an amine skeleton. In some embodiments, the lipidoid has an inversion of its ester bond with respect to its aliphatic chain compared to natural lipids such as triglycerides. In some embodiments, the lipidoid does not have an inversion of its ester bond with respect to its aliphatic chain compared to natural lipids such as triglycerides.

[0089] In some embodiments, the lipidoid includes, for example, an amino alcohol lipidoid. In some embodiments, the lipidoid is selected from cKK-E10, OF-02, or C12-200. Therefore, in some embodiments, the lipidoid is cKK-E-10. In some embodiments, the lipidoid is OF-02. In some embodiments, the lipidoid is C12-200.

[0090] The LNP formulation of the present invention can have a pH between approximately 6.0 and 8.0. For example, in some embodiments, the LNP formulation can have a pH between approximately 6.0 and 7.0. In some embodiments, the LNP formulation can have a pH between approximately 6.5 and 7.5. In some embodiments, the LNP formulation can have a pH between approximately 7.0 and 8.0. In some embodiments, the LNP formulation has a pH of approximately 7.4. In some embodiments, the LNP formulation has a pH equivalent to physiological pH.

[0091] The pH buffer of an LNP formulation can have a pKa between approximately 6.0 and 8.2. For example, the pH buffer of an LNP formulation may have a pKa of approximately 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or 8.2. In some embodiments, the pH buffer has a pKa of approximately 6.2. In some embodiments, the pH buffer has a pKa of approximately 6.4. In some embodiments, the pH buffer has a pKa of approximately 6.6. In some embodiments, the pH buffer has a pKa of approximately 6.8. In some embodiments, the pH buffer has a pKa of approximately 7.0. In some embodiments, the pH buffer has a pKa of approximately 7.2. In some embodiments, the pH buffer has a pKa of approximately 7.4. In some embodiments, the pH buffer has a pKa of approximately 7.6. In some embodiments, the pH buffer has a pKa of approximately 7.8. In some embodiments, the pH buffer has a pKa of approximately 8.0. In some embodiments, the pH buffer has a pKa of approximately 8.2.

[0092] As described above, the LNP formulations described herein exhibit less aggregation after one or more freeze-thaw cycles. Various methods exist in the art for determining LNP aggregation, for example, by any one of the following: dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), turbidity analysis, flow microscopy, flow cytometry, FTIR microscopy, resonance mass measurement (RMM), Raman microscopy, filtration, laser diffraction, electron microscopy, atomic force microscopy (AFM), static light scattering (SLS), polygonal static light scattering (MALS), field flow fractionation (FFF), and analytical ultracentrifugation (AUC). LNP aggregation can be evaluated using any one or more of these methods.

[0093] The LNP formulations described herein also exhibit less mRNA degradation after one or more freeze-thaw cycles. Various methods in the art exist to determine mRNA degradation, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), turbidity analysis, flow microscopy, flow cytometry, FTIR microscopy, resonance mass measurement (RMM), Raman microscopy, filtration, laser diffraction, electron microscopy, atomic force microscopy (AFM), static light scattering (SLS), polygonal static light scattering (MALS), field flow fractionation (FFF), and analytical ultracentrifugation (AUC). Any one of these methods... mRNA degradation can be evaluated using one or more of these methods.

[0094] The LNP formulations described herein have a diameter of less than 100 nm. For example, in some embodiments, the LNP has a diameter between 70 nm and 90 nm. In some embodiments, the LNP has a diameter of less than 70 nm.

[0095] As described throughout, various types of lipid components are suitable for the LNPs described herein. In some embodiments, the LNP formulation has a lipid component comprising DMG-PEG-2000, cKK-E10, cholesterol, and DOPE. In some embodiments, the LNP formulation has a lipid component comprising DMG-PEG-2000, cKK-E10, cholesterol, and DOPE.

[0096] LNP formulations can have an N / P ratio in the range of approximately 3 to 5. In some embodiments, the N / P ratio is approximately 3. In some embodiments, the N / P ratio is approximately 4. In some embodiments, the N / P ratio is approximately 5.

[0097] LNP formulations encapsulate mRNA. Any mRNA can be encapsulated by the LNP formulations described herein. The final concentration of mRNA encapsulated within the LNP may be in the range of approximately 0.05 mg / mL to 1.0 mg / mL. In some embodiments, the mRNA encapsulated within the LNP is in the range of approximately 0.2 mg / mL to approximately 0.5 mg / mL.

[0098] The LNP formulations described herein are stable when stored at -20°C, -80°C, or below -80°C. Therefore, in some embodiments, the LNP formulations described herein are stable when stored at -20°C. In some embodiments, the LNP formulations described herein are stable when stored below -80°C. In some embodiments, the LNP formulations described herein are stable when stored below -80°C. For example, the LNP formulations are stable for at least 3 months, 6 months, 12 months, or more than 12 months when stored at -20°C. Furthermore, the LNP formulations are stable after dilution.

[0099] mRNA synthesis The mRNA according to the present invention can be synthesized according to any of the various known methods. 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.

[0100] In some embodiments, a DNA template is transcribed in vitro to prepare the mRNA according to the present invention. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired mRNA and a desired nucleotide sequence for a termination signal.

[0101] mRNA synthesis using SP6 RNA polymerase In some embodiments, mRNA is produced using SP6 RNA polymerase. SP6 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for the SP6 promoter sequence. SP6 polymerase catalyzes the 5'→3' in vitro synthesis of RNA on single-stranded or double-stranded DNA downstream from the promoter, incorporating native ribonucleotides and / or modified ribonucleotides and / or labeled ribonucleotides into the polymerized transcript. Examples of such labeled ribonucleotides include... Examples include biotin-, fluorescein-, digoxigenin-, aminoallyl-, and isotopically labeled nucleotides.

[0102] The initial amino acid sequence of bacteriophage SP6 RNA polymerase is as follows: MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELIAPMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLNTDATLQAIAM SVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYRHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQ TSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFK PLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRF TEGRPVNGVEALKWFCINGANLWGWDKKTFDRVVSNVLDEEFQDMCRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYS AMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQ KAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIV DEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQKLLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFA It was described as having (GenBank:Y00105.1).

[0103] A suitable SP6 RNA polymerase for the present invention is any enzyme having substantially the same polymerase activity as bacteriophage SP6 RNA polymerase. Therefore, in some embodiments, a suitable SP6 RNA polymerase for the present invention is modified from SEQ ID NO: 16. For example, a suitable SP6 RNA polymerase may include one or more amino acid substitutions, deletions, or additions. In some embodiments, a suitable SP6 RNA polymerase has an amino acid sequence that is approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, or 60% identical or homologous to SEQ ID NO: 16. In some embodiments, a suitable SP6 RNA polymerase may be a cleaved protein (from the N-terminus, C-terminus, or internally) but retain polymerase activity. In some embodiments, the appropriate SP6 RNA polymerase is a fusion protein.

[0104] SP6 RNA polymerases suitable for the present invention may be commercially available products from, for example, Aldevron, Ambion, New England Biolabs (NEB), Promega, and Roche. SP6 can be ordered and / or custom-designed from commercial or non-commercial sources according to the amino acid sequence of SEQ ID NO: 16 or variants of SEQ ID NO: 16 described herein. SP6 may be a standard fidelity polymerase or a high fidelity / high efficiency / high volume polymerase modified to promote RNA polymerase activity, for example, mutations in the SP6 RNA polymerase gene or post-translational modifications of the SP6 RNA polymerase itself. Examples of SP6 include Ambion's SP6 RNA polymerase-Plus®, NEB's HiScribe SP6, and Promega's RiboMAX® and Riboprobe® systems.

[0105] In some embodiments, the appropriate SP6 RNA polymerase is a fusion protein. For example, the SP6 RNA polymerase may contain one or more tags to facilitate the isolation, purification, or solubility of the enzyme. The appropriate tag may be located at the N-terminus, C-terminus, and / or internally. Non-limiting examples of appropriate tags include calmodulin-binding protein (CBP); Fasciola liver 8-kDa antigen (Fh8); FLAG tag peptide; glutathione-S-transferase (GST); histidine tag (e.g., hexahistidine tag (His6)); maltose-binding protein (MBP); N-utilizing agent (NusA); small ubiquitin-associated modifier (SUMO) fusion tag; streptavidin-binding peptide (STREP); tandem affinity purification (TAP); and thioredoxin (TrxA). Other tags may be used in the present invention. These and other fusion tags are described, for example, by Costa et al., Frontiers in Microbiology vol. 5 (2014): pp. 63 and PCT / US16 / 57044, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the His tag is located at the N-terminus of SP6.

[0106] DNA template Typically, DNA templates are either fully double-stranded or mostly single-stranded with a double-stranded SP6 promoter sequence.

[0107] Linearized plasmid DNA (linearized via one or more restriction enzymes), linearized genomic DNA fragments (via restriction enzymes and / or physical means), PCR products, and / or synthetic DNA oligonucleotides can be used as templates for in vitro transcription using SP6, provided that they contain a double-stranded SP6 promoter upstream (and forward) of the DNA sequence to which they are transcribed.

[0108] In some embodiments, the linearized DNA template has blunt ends.

[0109] In some embodiments, the transcribed DNA sequence is optimized to facilitate more efficient transcription and / or translation. For example, the DNA sequence may be optimized with respect to cis-regulatory elements (e.g., TATA boxes, stop signals, and protein-binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slip sites, and / or other elements related to transcription; the DNA sequence may be optimized with respect to potential splice sites, mRNA secondary structure, stable free energy of mRNA, repeat sequences, RNA unstable motifs, and / or other elements related to mRNA processing and stability; the DNA sequence may be optimized with respect to codon use bias, codon compatibility, internal chi sites, ribosome-binding sites (e.g., IRES), immature poly-A sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA sequence may be optimized with respect to codon context, codon-anticodon interactions, translation pause sites, and / or other elements related to protein folding. Optimization methods known in the art, such as the GeneOptimizer by ThermoFisher and OptimumGene® described in U.S. Patent Application Publication No. 2011 / 0081708, which is incorporated herein by reference in its entirety, can be used in this invention.

[0110] In some embodiments, the DNA template includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region is one or that affects mRNA stability or translation. This includes additional elements, such as iron-responsive elements. In some embodiments, the 5' untranslated region may be between approximately 50 and 500 nucleotides in length.

[0111] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, a protein binding site that affects the stability of mRNA at its intracellular location, or one or more miRNA binding sites. In some embodiments, the 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0112] Exemplary 3' and / or 5'UTR sequences can be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzymes) to increase the stability of the sense mRNA molecule. For example, the 5'UTR sequence may contain a portion or fragment of the CMV pre-early 1 (IE1) gene to improve nuclease resistance and / or improve the half-life of the polynucleotide. Alternatively, to further stabilize the polynucleotide, a sequence or fragment encoding human growth hormone (hGH) may be included in the 3' end or untranslated region of the polynucleotide (e.g., mRNA). In general, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to its unmodified counterpart, and include modifications made to improve, for example, the resistance of such polynucleotides to in vivonuclease digestion.

[0113] Large-scale mRNA synthesis In some embodiments, the present invention can be used for the large-scale production of mRNA encapsulated in stable LNPs. In some embodiments, methods according to the present invention synthesize at least 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 25 g, 500 g, 75 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg or more of mRNA in a single batch. As used herein, the term “batch” refers to the content or amount of mRNA synthesized at one time, for example, the content or amount of mRNA produced according to a single production setting. A batch can also refer to the amount of mRNA synthesized in a single reaction occurring via a single aliquot of enzyme and / or a single aliquot of DNA template for sequential synthesis under one set of conditions. mRNA synthesized in a single batch does not contain mRNA synthesized at different times that would be combined to achieve the desired amount. Generally, the reaction mixture includes SP6 RNA polymerase, a linear DNA template, and RNA polymerase reaction buffer (which may contain ribonucleotides or may require the addition of ribonucleotides).

[0114] According to the present invention, 1 to 100 mg of SP6 polymerase is typically used per gram (g) of the generated mRNA. In some embodiments, approximately 1 to 90 mg, 1 to 80 mg, 1 to 60 mg, 1 to 50 mg, 1 to 40 mg, 10 to 100 mg, 10 to 80 mg, 10 to 60 mg, or 10 to 50 mg of SP6 polymerase is used per gram of the generated mRNA. In some embodiments, approximately 5 to 20 mg of SP6 polymerase is used to produce approximately 1 gram of mRNA. In some embodiments, approximately 0.5 to 2 grams of SP6 polymerase is used to produce approximately 100 grams of mRNA. In some embodiments, approximately 5 to 20 grams of SP6 polymerase is used to produce approximately 1 kilogram of mRNA. In some embodiments, at least 5 mg of SP6 polymerase is used to produce at least 1 gram of mRNA. In some embodiments, at least 500 mg of SP6 polymerase is used to produce at least 100 grams of mRNA. In some embodiments, at least 5 grams of SP6 polymerase are used to produce at least 1 kilogram of mRNA. In some embodiments, approximately 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg per gram of produced mRNA. mg or 100 mg of plasmid DNA is used. In some embodiments, about 10–30 mg of plasmid DNA is used to produce about 1 gram of mRNA. In some embodiments, about 1–3 grams of plasmid DNA is used to produce about 100 grams of mRNA. In some embodiments, about 10–30 grams of plasmid DNA is used to produce about 1 kilogram of mRNA. In some embodiments, at least 10 mg of plasmid DNA is used to produce at least 1 gram of mRNA. In some embodiments, at least 1 gram of plasmid DNA is used to produce at least 100 grams of mRNA. In some embodiments, at least 10 grams of plasmid DNA is used to produce at least 1 kilogram of mRNA.

[0115] In some embodiments, the concentration of SP6 RNA polymerase in the reaction mixture may be about 1–100 nM, 1–90 nM, 1–80 nM, 1–70 nM, 1–60 nM, 1–50 nM, 1–40 nM, 1–30 nM, 1–20 nM, or about 1–10 nM. In certain embodiments, the concentration of SP6 RNA polymerase is about 10–50 nM, 20–50 nM, or 30–50 nM. SP6 RNA polymerase can be used in concentrations of 100 to 10,000 units / ml, for example, 100 to 9,000 units / ml, 100 to 8,000 units / ml, 100 to 7,000 units / ml, 100 to 6,000 units / ml, 100 to 5,000 units / ml, 100 to 1,000 units / ml, 200 to 2,000 units / ml, 500 to 1,000 units / ml, 500 to 2,000 units / ml, 500 to 3,000 units / ml, 500 to 4,000 units / ml, 500 to 5,000 units / ml, 500 to 6,000 units / ml, 1,000 to 7,500 units / ml, and 2,500 to 5,000 units / ml.

[0116] The concentrations of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture range from about 0.1 mM to about 10 mM, for example, between about 1 mM and about 10 mM, between about 2 mM and about 10 mM, between about 3 mM and about 10 mM, between about 1 mM and about 8 mM, between about 1 mM and about 6 mM, between about 3 mM and about 10 mM, between about 3 mM and about 8 mM, between about 3 mM and about 6 mM, and between about 4 mM and about 5 mM. In some embodiments, each ribonucleotide is present in a concentration of about 5 mM in the reaction mixture. In some embodiments, the total concentration of rNTPs (e.g., combinations of ATP, GTP, CTP, and UTP) used in the reaction ranges from 1 mM to 40 mM. In some embodiments, the total concentration of rNTPs (e.g., combinations of ATP, GTP, CTP, and UTP) used in the reaction is in the range of 1 mM to 30 mM, or 1 mM to 28 mM, or 1 mM to 25 mM, or 1 mM to 20 mM. In some embodiments, the total rNTP concentration is less than 30 mM. In some embodiments, the total rNTP concentration is less than 25 mM. In some embodiments, the total rNTP concentration is less than 20 mM. In some embodiments, the total rNTP concentration is less than 15 mM. In some embodiments, the total rNTP concentration is less than 10 mM.

[0117] RNA polymerase reaction buffers typically contain salts / buffers, such as Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, sodium chloride, and magnesium chloride.

[0118] The pH of the reaction mixture can be about 6–8.5, about 6.5–8.0, or about 7.0–7.5, and in some embodiments, the pH is 7.5.

[0119] Combine a linear or linearized DNA template (e.g., in sufficient quantity / concentration to provide the desired amount of RNA, as described above), RNA polymerase reaction buffer, and SP6 RNA polymerase to form the reaction mixture. Incubate the reaction mixture at approximately 37°C to 42°C for 30 minutes to 6 hours, for example, approximately 60 to 90 minutes.

[0120] In some embodiments, approximately 5 mM NTP, approximately 0.05 mg / mL SP6 polymerase, and approximately 0.1 mg / mL DNA template are incubated in a suitable RNA polymerase reaction buffer (the pH of the final reaction mixture is approximately 7.5) at approximately 37°C to approximately 42°C for 60 to 90 minutes.

[0121] In some embodiments, the reaction mixture contains an SP6 polymerase-specific promoter, SP6 The reaction mixture contains RNA polymerase, an RNase inhibitor, pyrophosphatase, 29 mM NTP, 10 mM DTT, and a reaction buffer (800 mM HEPES, 20 mM spermidine, 250 mM MgCl2, pH 7.7 for 10×) and a linearized double-stranded DNA template with sufficient content (QS) to reach the desired reaction volume with RNase-free water. The reaction mixture is then incubated at 37°C for 60 minutes. The polymerase reaction is then quenched by adding DNase I and DNase I buffer (100 mM Tris-HCl, 5 mM MgCl2, and 25 mM CaCl2, pH 7.6 for 10×) to facilitate the digestion of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[0122] In some embodiments, the reaction mixture comprises NTP at a concentration in the range of 1 to 10 mM, DNA template at a concentration in the range of 0.01 to 0.5 mg / ml, and SP6 RNA polymerase at a concentration in the range of 0.01 to 0.1 mg / ml. For example, the reaction mixture may contain NTP at a concentration of 5 mM, DNA template at a concentration of 0.1 mg / ml, and SP6 at a concentration of 0.05 mg / ml. Contains RNA polymerase.

[0123] nucleotide mRNA according to the present invention can be generated using various naturally occurring or modified nucleosides. In some embodiments, the mRNA is a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); a nucleoside analog (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-deazag Anosine, 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); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite bonds) or comprising them.

[0124] In some embodiments, mRNA contains one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methylcytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thiouridine ("2sU"). For consideration of such residues and their incorporation into mRNA, see, for example, U.S. Patent No. 8,278,036 or International Publication No. 2011 / 012316. mRNA can 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 for 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 reference in their entirety. The presence of non-standard nucleotide residues makes mRNA different from mRNA that has the same sequence but contains only standard residues. The mRNA can be made more stable and / or less immunogenic than the control mRNA. 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 nucleotide—for example, individually or in combination—at 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, over 95%, or 100% of the constituent nucleotide.

[0125] 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.

[0126] The 5' cap is typically added as follows: firstly, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; secondly, guanosine triphosphate (GTP) is added to the terminal phosphate via guanytransferase to form a 5'5'5 triphosphate bond; and thirdly, the 7-nitrogen of guanine is methylated by methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Further cap structures are described in U.S. Patent Application Publication No. 2016 / 0032356 and U.S. Provisional Patent Application No. 62 / 464,327, filed on 27 February 2017, which are incorporated herein by reference.

[0127] 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, and at least 500 adenosine or cytosine nucleotides, respectively. The material 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 10-8 kb each. 00 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 nucleotides can be 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.

[0128] 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.

[0129] mRNA synthesized according to the present invention can be used without further purification. In particular, mRNA synthesized according to the present invention can be used without the step of removing the shorter. In some embodiments, mRNA synthesized according to the present invention can be further purified. mRNA synthesized according to the present invention can be purified using various methods. For example, mRNA purification can be carried out 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, mRNA is purified by HPLC. In some embodiments, mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, which is well known to those skilled in the art. In some embodiments, mRNA is purified using tangential flow filtration. Appropriate purification methods include U.S. Patent Application Publication No. 2016 / 0040154, U.S. Patent Application Publication No. 2015 / 0376220, and International Patent Application No. PCT / US18 / 19954, both filed on February 27, 2018, and also filed on February 27, 2018, both titled "METHODS FOR PURIFICATION OF MESSENGER RNA". This includes, but is not limited to, the provisions described in International Patent Application No. PCT / US18 / 19978, which are all incorporated herein by reference and can be used to carry out the present invention.

[0130] 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.

[0131] In some embodiments, mRNA is purified by centrifugation either before or after capping and tailing, or both before and after.

[0132] In some embodiments, mRNA is purified by filtration either before or after capping and tailing, or both before and after.

[0133] In some embodiments, mRNA is purified by tangential flow filtration (TFF) either before or after capping and tailing, or both before and after.

[0134] In some embodiments, mRNA is purified by chromatography either before or after capping and tailing, or both before and after.

[0135] mRNA characterization Full-length or interrupted mRNA transcripts can be detected and quantified using any method available in the art. In some embodiments, synthesized mRNA molecules are detected using blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. Other detection methods known in the art are included in the present invention. In some embodiments, synthesized mRNA molecules are detected using UV absorption spectroscopy with separation by capillary electrophoresis. In some embodiments, mRNA is first denatured with a glyoxal dye before gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, synthesized mRNA is characterized before capping or tailing. In some embodiments, synthesized mRNA is characterized after capping and tailing.

[0136] In some embodiments, the mRNA produced by the methods disclosed herein contains impurities other than full-length mRNA in amounts of 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 less than 0.1%. These impurities include IVT contaminants, such as proteins, enzymes, free nucleotides, and / or shortmers.

[0137] In some embodiments, mRNA produced according to the present invention is substantially free of shortmers and interrupted transcripts. In particular, mRNA produced according to the present invention contains shortmers or interrupted transcripts at levels undetectable by capillary electrophoresis or glyoxal gel electrophoresis. As used herein, the terms “shortmer” or “interrupted transcript” refer to any transcript that is less than full length. In some embodiments, “shortmer” or “interrupted transcript” are nucleotides with a length of less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides. In some embodiments, shortmers are detected or quantified after the addition of a 5'-cap and / or 3'-poly-A tail.

[0138] mRNA solution In some embodiments, mRNA can be provided in a solution to be mixed with a lipid solution so that it can be encapsulated in lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations. For example, a suitable mRNA solution may contain mRNA at concentrations of about 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml or greater. In some embodiments, a suitable mRNA solution is approximately 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml, 0.0 It may contain mRNA at concentrations in the range of 5-0.8 mg / ml, 0.05-0.7 mg / ml, 0.05-0.6 mg / ml, 0.05-0.5 mg / ml, 0.05-0.4 mg / ml, 0.05-0.3 mg / ml, 0.05-0.2 mg / ml, 0.05-0.1 mg / ml, 0.1-1.0 mg / ml, 0.2-0.9 mg / ml, 0.3-0.8 mg / ml, 0.4-0.7 mg / ml, or 0.5-0.6 mg / ml. In some embodiments, a suitable mRNA solution may contain mRNA at concentrations of up to approximately 5.0 mg / ml, 4.0 mg / ml, 3.0 mg / ml, 2.0 mg / ml, 1.0 mg / ml, 0.09 mg / ml, 0.08 mg / ml, 0.07 mg / ml, 0.06 mg / ml, or 0.05 mg / ml.

[0139] 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 the buffer are approximately 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or greater.

[0140] 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.

[0141] 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 less.

[0142] 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.

[0143] In some embodiments, the mRNA stock solution is mixed with a buffer using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0144] 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.

[0145] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10 to 600 ml / min (for example, 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 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, 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.

[0146] Delivery vehicle The stable lipid nanoparticle formulations described herein are suitable as delivery vehicles for mRNA.

[0147] As used herein, the terms “delivery vehicle,” “introduction vehicle,” and “nanoparticles” or their grammatical equivalents are used interchangeably.

[0148] The delivery vehicle is formulated into a pharmacological composition that is combined with one or more additional nucleic acids, carriers, targeted ligands, or stabilizing reagents, or mixed with appropriate excipients. The technology for formulating and administering the drug is described in "Remington's Pharmacology." This can be found in the latest edition of "Maceutical 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.

[0149] 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 an outer medium by one or more bilayer membranes. The bilayer membrane of a liposome is typically formed by amphiphilic molecules such as synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of a liposome 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 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, and 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.

[0150] 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.

[0151] 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.

[0152] 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 is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention are of 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 Each is independently selected from the group consisting of acyls; L1 and L2 are hydrogen, and optionally substituted C1-C 30 Alkyl, optionally substituted, variably unsaturated C1-C 30 Alkenyls, and optionally substituted C1-C 30Each 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.

[0153] 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.

[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. 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.

[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. 2016 / 118724, 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.

[0156] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids having the formula 14,25-ditridecyl15,18,21,24-tetraaza-octatriacontane and pharmaceutically acceptable salts thereof.

[0157] Other cationic lipids suitable for use in the compositions and methods of the present invention include those described in International Publication Nos. 2013 / 063468 and International Publication Nos. 2016 / 205691, each of which is 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 R LEach example is independent, and depending on the case, C6~C 40 It contains (which is an alkenyl). 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.

[0158] 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 is 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 Ais independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; each R B is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optiona lly substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen). In certain specific embodiments, the compositions and methods of the present invention comprise

Chemical Formula

[0159] 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 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure:

Chemical Formula

Chemical Formula

Chemical Formula

[0160] 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 10 It 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.

[0161] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described by reference herein by 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.

[0162] 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 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 comprises cationic lipids having the same and pharmaceutically acceptable salts thereof.

[0163] 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] comprises a cationic lipid having the structure and a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a compound having the structure:

化

化

化

化

化

化

化

化

[0164] 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 based on 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 2The 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 Alkyl; R 1 and R 2 Each of these is independent, C6~C 24 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 Alkyl; R 5 (x is H or C1-C6 alkyl; x is 0, 1 or 2).

[0165] 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.

[0166] 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] comprises a cationic lipid having the compound structure and a pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the invention comprise

化

化

化

[0167] Other cationic lipids suitable for use in the compositions and methods of the present invention include those cationic lipids described in International Publication No. WO 2017 / 173054 and International Publication No. WO 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention comprise

化

化

化

[0168] 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 are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention are of the following formula: [ka] cationic lipids (wherein R1 is imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylaminos (e.g., alkylaminos such as dimethylamino) and pyri Selected from the group consisting of Zilles; R2 is given by the following two equations: [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 "HGT4002" (also referred to herein as "Guan-SS-Chol") having the compound structure of the present invention 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 "HGT4003" 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 "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.

[0169] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids described in U.S. Provisional Patent Application No. 62 / 672,194, filed May 16, 2018, 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 U.S. Provisional Patent Application No. 62 / 672,194. 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 are each independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L -; each L 4A and L 5A are independently -C(O)-, -C(O)O-, or -C(O)NR L -; each L 4B and L 5B are independently C1~C 20 alkylene; C2~C 20 alkenylene; or C2~C 20 alkynylene; each B and B' is NR 4 R 5 or 5- to 10-membered nitrogen-containing heteroaryl; each R 1 , R 2 , and R 3 are independently C6~C 30 alkyl, C6~C 30 alkenyl, or C6~C 30 alkynyl; each R 4 and R 5 are 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.

[0170] In certain specific embodiments, the compositions and methods of the present invention

Chemical Structure

[0171] 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-1-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").

[0172] 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-hydroxyl Diethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholesta-5-ene-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-dioleyl oxy Sibenzylamine ("DMOBA"); 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-Dilinoleyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"); 1,2-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminometh Lu-[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 [n-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholesta-5-en-3-yloxy]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 include 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethaneamine ("DLin-KC2-DMA") (incorporated herein by reference, see International Publication 2010 / 042877; see Semple et al., Nature Biotech. 28:172~176 (2010)) (Heyes, J. et al., J Controlled Release 107:276~287 (2005); Morrissey, DV. et al., Nat. Biotechnol. 23(8):1003~1007 (2005); International Publication 2005 / 121348). In some embodiments, one or more cationic lipids include at least one imidazole, dialkylamino, or guanidinium moiety. 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").

[0173] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid, TL1-04D-DMA, which has the compound structure of the above.

[0174] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid, GL-TES-SA-DME-E18-2, which has the compound structure shown.

[0175] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid, SY-3-E14-DMAPr, which has the compound structure shown.

[0176] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid, TL1-01D-DMA, which has the compound structure of the above.

[0177] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid, TL1-10D-DMA, which has the compound structure of the above.

[0178] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid, GL-TES-SA-DMP-E18-2, which has the compound structure shown.

[0179] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid that is HEP-E4-E10 having the compound structure shown.

[0180] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are [ka] It contains a cationic lipid that is HEP-E3-E10 having the compound structure shown.

[0181] 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.

[0182] Noncationic / Helper Lipids In some embodiments, the provided liposomes contain one or more noncationic ("helper") lipids. As used herein, the phrase “noncationic lipid” refers to any neutral, zwitterionic, or anionic lipid. 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 are not limited to these, but include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, and gangliosides. This includes sid, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof.

[0183] In some embodiments, such noncationic lipids are used alone, but are preferably used in combination with other lipids, such as cationic lipids. In some embodiments, noncationic lipids may constitute about 5% to about 90%, or about 10% to about 70%, of the total lipids present in the liposomes in molar ratio. In some embodiments, the noncationic lipids are neutral lipids, i.e., lipids that have no net charge under the conditions in which the composition is formulated and / or administered. In some embodiments, the percentage of noncationic lipids in the liposomes can be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0184] Cholesterol-based lipids In some embodiments, the provided 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, p. 280 (1991); Wolf et al., BioTechniques Vol. 23, p. 139 (1997); U.S. Patent No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipids may constitute about 2% to about 30% or about 5% to about 20% of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles can be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0185] PEG modified lipid For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER) containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), 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). The intended PEG-modified lipids are not limited to C6-C6.20 comprises a polyethylene glycol chain of up to SkDa in length covalently bound to a lipid having a long alkyl chain. The addition of such components prevents complex aggregation, increases circulation half-life, and can also provide a means for increasing the delivery of lipid-nucleic acid compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or these are selected to be rapidly exchanged from the preparation in vivo (see U.S. Patent No. 5,885,613). Particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 ) are PEG-ceramides. The PEG-modified phospholipids and derivatized lipids of the present invention can constitute a molar ratio of from about 0% to about 20%, from about 0.5% to about 20%, from about 1% to about 15%, from about 4% to about 10%, or about 2% of the total lipids present in a liposomally introduced vehicle.

[0186] According to various embodiments, the selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids that constitute the lipid nanoparticles, and the relative molar ratios of such lipids to each other, are based on the characteristics of the selected lipids, the nature of the intended target cells, and the characteristics of the MCNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, membrane fusogenicity and toxicity of the selected lipids. Accordingly, the molar ratios can be adjusted accordingly.

[0187] Polymers In some embodiments, a suitable delivery vehicle is a polymer as a carrier, alone or These are formulated 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, dextran, albumin, gelatin, alginates, collagen, chitosan, 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).

[0188] Liposomes suitable for use according to the present invention Liposomes suitable for the present invention may contain one or more of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids and / or polymers described herein in various proportions. As non-limiting examples, suitable liposome formulations may include cKK-E12, 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.

[0189] 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.

[0190] 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 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids, cholesterol lipids, and PEG-modified lipids is approximately 50:25:20:5.

[0191] In certain embodiments, liposomes for use according to the present invention comprise lipid components consisting of cationic lipids, non-cationic lipids (e.g., DOPE or DEPE), PEG-modified lipids (e.g., DMG-PEG2K), and optionally cholesterol. Cationic lipids particularly suitable for inclusion in such liposomes include GL-TES-SA-DME-E18-2, TL1-01D-DMA, SY-3-E14-DMAPr, TL1-10D-DMA, HGT4002 (also referred to herein as Guan-SS-Chol), GL-TES-SA-DMP-E18-2, HEP-E4-E10, HEP-E3-E10, and TL1-04D-DMA. These cationic lipids have been found to be particularly suitable for use in liposomes administered via pulmonary delivery through spraying. Among these, HEP-E4-E10, HEP-E3-E10, GL-TES-SA-DME-E18-2, GL-TES-SA-DMP-E18-2, TL1-01D-DMA, and TL1-04D-DMA performed particularly well.

[0192] Examples of liposomes include GL-TES-SA-DME-E18-2, TL1-01D-DMA, SY-3-E14-DMAPr, TL1-10D-DMA, GL-TES-SA-DMP-E18-2, HEP-E4-E10, HEP-E3-E10, and TL1-04D-DMA as cationic lipid components, DOPE as a non-cationic lipid component, cholesterol as a helper lipid component, and one of DMG-PEG2K as a PEG-modified lipid component. In some embodiments, the molar ratios of cationic lipids, non-cationic lipids, cholesterol, and PEG-modified lipids may be between approximately 30-60:25-35:20-30:1-15, respectively. In some embodiments, the molar ratios of cationic lipids, non-cationic lipids, cholesterol, and PEG-modified lipids are approximately 40:30:20:10, respectively. In some embodiments, the molar ratios of cationic lipids, non-cationic lipids, cholesterol, and PEG-modified lipids are approximately 40:30:25:5, respectively. In some embodiments, the molar ratios of cationic lipids, non-cationic lipids, cholesterol, and PEG-modified lipids are approximately 40:32:25:3, respectively. In some embodiments, the molar ratios of cationic lipids, non-cationic lipids, cholesterol, and PEG-modified lipids are approximately 50:25:20:5.

[0193] In some embodiments, the lipid components of liposomes particularly suitable for pulmonary delivery consist of HGT4002 (also referred to herein as Guan-SS-Chol), DOPE, and DMG-PEG2K. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to PEG-modified lipids is approximately 60:35:5.

[0194] Ratio of separate lipid components 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.

[0195] 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.

[0196] 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.

[0197] In some embodiments, the lipid component (1), represented by the variable "x", is a sterol-based cationic lipid.

[0198] In some embodiments, the lipid component (2), represented by the variable "y", is a helper lipid.

[0199] In some embodiments, the lipid component (3), represented by the variable "z", is a PEG lipid.

[0200] 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%.

[0201] In some embodiments, the molars of lipid component (1) (e.g., sterol-based cationic lipids) The variable "x" representing the percentage is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, or less than or equal to 10%. In the embodiment, the variable "x" is approximately 65%, 60%, 55%, 50%, or less than or equal to 40%.

[0202] 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%.

[0203] 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%.

[0204] 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%.

[0205] 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%.

[0206] 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%.

[0207] 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 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%, or 10%. In embodiments, the variable "z" representing the weight percentage of lipid component (3) (e.g., PEG lipids) is approximately 1% to approximately 10%, approximately 2% to approximately The percentages are 10%, approximately 3% to 10%, approximately 4% to 10%, approximately 1% to 7.5%, approximately 2.5% to 10%, approximately 2.5% to 7.5%, approximately 2.5% to 5%, approximately 5% to 7.5%, or approximately 5% to 10%.

[0208] 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.

[0209] Formation of liposomes that encapsulate mRNA Liposome introduction vehicles for use in the compositions of the present invention can be prepared by various techniques currently known in the art. Liposomes for use in the provided compositions can be prepared by various techniques currently known in the art. For example, multilayer vesicles (MLVs) can be prepared according to a prior art technique in which a selected lipid is deposited on the inner wall of a suitable container or vessel by dissolving it in a suitable solvent, and then the solvent is evaporated to leave a thin film in the container or spray-dried. Next, an aqueous phase can be added to the container by a vortex motion that results in the formation of MLVs. Then, single-layer vesicles (ULVs) are formed by homogenization, sonication, or extrusion of the multilayer vesicles. Furthermore, single-layer vesicles can be formed by surfactant removal techniques.

[0210] In certain embodiments, the provided composition comprises liposomes in which mRNA is associated on both surfaces of the liposome and encapsulated within the liposome. For example, during the preparation of the composition of the present invention, cationic liposomes can associate with mRNA through electrostatic interactions.

[0211] 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.

[0212] Methods for incorporating desired mRNA into liposomes are often referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett., vol. 312: pp. 255–258, 1992, which are incorporated herein by reference. Nucleic acids incorporated into liposomes can be located entirely or partially within the internal space of the liposome, within the bilayer membrane of the liposome, or fully or partially associated with the outer surface of the liposome membrane. The incorporation of nucleic acids into liposomes is also referred herein to as “encapsulation,” in which the nucleic acid is completely contained within the internal space of the liposome. The purpose of incorporating mRNA into a delivery vehicle such as a liposome is often to protect the nucleic acid from environments that may contain enzymes or chemicals that degrade the nucleic acid and / or system or receptor, causing rapid efflux of the nucleic acid. Thus, in some embodiments, a suitable delivery vehicle can enhance the stability of the mRNA contained therein and / or facilitate the delivery of mRNA to target cells or tissues.

[0213] Suitable liposomes according to the present invention can be produced in various sizes. In some embodiments, the provided liposomes can be smaller than known mRNA-encapsulating liposomes. In some embodiments, the reduction in liposome size is associated with more efficient mRNA delivery. The selection of an appropriate liposome size can take into account the site of the target cell or tissue, and to some extent, the intended use for which the liposomes are produced.

[0214] In some embodiments, liposomes of an appropriate size are selected to facilitate the systemic distribution of mRNA-encoded antibodies. In some embodiments, it may be desirable to restrict mRNA transfection to specific cells or tissues. For example, to target hepatocytes, the liposomes may be sized such that their dimensions are smaller than the fenestrated areas of the endothelial layer lining the hepatic sinuses within the liver. In such cases, the liposomes can easily penetrate these endothelial fenestrated areas to reach the target hepatocytes.

[0215] Alternatively, liposomes may be sized such that their dimensions have a diameter sufficient to restrict or explicitly avoid distribution to certain cells or tissues.

[0216] Various alternative methods known in the art are available for sizing liposome populations. One such sizing method is described in U.S. Patent No. 4,737,323, incorporated herein by reference. Sonication of the liposome suspension by either bath or probe sonication results in a gradual size reduction to small ULVs with a diameter of less than approximately 0.05 microns. Homogenization is another method that relies on shear energy to fragment larger liposomes into smaller liposomes. In a typical homogenization procedure, the MLVs are recirculated through a standard emulsion homogenizer until a selected liposome size, typically between approximately 0.1 and 0.5 microns, is observed. Liposome size can be determined by quasi-electric scattering (QELS), as described in Bloomfield, Ann. Rev. Biophys. Bioeng., Vol. 10: pp. 421–150 (1981), incorporated herein by reference. Sonication of the formed liposomes can reduce the mean liposome diameter. To facilitate efficient liposome synthesis, intermittent sonication cycles can be performed alternately with QELS evaluation.

[0217] Therapeutic use of the composition In one embodiment, the present invention provides LNP formulations that encapsulate mRNA useful for therapeutic purposes. For example, in some embodiments, the mRNA encapsulated in the LNP encodes a protein that is deficient in the subject. For example, the mRNA may encode CFTR for treating cystitis fibrosis. Suitable mRNAs encoding CFTR are described, for example, in International Publication 2020 / 106946 and International Application PCT / US20 / 44158, each of which is incorporated herein by reference in its entirety. As another example, the mRNA may encode OTC for treating ornithine transcarbamylase deficiency, as described, for example, in International Publication 2017 / 218524, the contents of which are incorporated herein by reference in their entirety.

[0218] In some embodiments, the mRNA encapsulated in the LNP encodes a protein encoding a vaccine antigen, such as the SARS-CoV-2 antigen. Such a SARS-CoV-2 antigen is described in U.S. Patent Application No. 63 / 021,319, which is incorporated herein by reference.

[0219] In some embodiments, mRNA is codon-optimized. Various codon optimization methods are used in this technology. It is publicly known in the field of technology.

[0220] Gene therapy In some embodiments, the LNP formulations described herein are suitable for pharmaceutical compositions comprising codon-optimized nucleic acids encoding proteins used to treat subjects requiring them. In some embodiments, pharmaceutical compositions comprising the rAAV vectors described herein are used to treat subjects requiring them. Pharmaceutical compositions containing the rAAV vectors or particles of the present invention contain pharmaceutically acceptable excipients, diluents, or carriers. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. The pharmaceutical compositions may be in lyophilized form. Such carriers can be formulated by conventional methods and administered to subjects in therapeutically effective amounts.

[0221] The rAAV vector is administered to the target requiring it via an appropriate route. In some embodiments, the rAAV vector is administered intravenously, intraperitoneally, subcutaneously, or intradermally. In one embodiment, the rAAV vector is administered intravenously. In embodiments, intradermal administration includes administration using a "gene gun" or bioristic particle delivery system. In some embodiments, the rAAV vector is administered via nonviral lipid nanoparticles. For example, a composition containing an rAAV vector may include one or more diluents, buffers, liposomes, lipids, or lipid complexes. In some embodiments, the rAAV vector is contained within microspheres or nanoparticles, such as lipid nanoparticles or inorganic nanoparticles.

[0222] In some embodiments, rAAV is pseudotyped. Pseudotyped rAAV is an infectious virus containing any combination of AAV capsid protein and rAAV genome. Pseudotyped rAAV is useful for altering the tissue or cell specificity of rAAV and can be used alone or in combination with unpseudotyped rAAV to transfer one or more genes into cells, such as mammalian cells. For example, in mammals that have developed an immune response to unpseudotyped rAAV, pseudotyped rAAV can be used after administration with unpseudotyped rAAV. Capsid proteins derived from any AAV serotype can be used with rAAV genomes derived from or obtained from wild-type AAV genomes of different serotypes, or with rAAV genomes that are chimeric genomes, i.e., two or more different serotypes, for example, a chimeric genome with two ITRs, or AAV DNA formed from each ITR derived from different serotypes or chimeric ITRs. The use of chimeric genomes, such as those containing ITRs derived from two AAV serotypes or chimeric ITRs, can result in directed recombination and further enhance the production of transcriptionally active intermolecular concatemers. Therefore, the 5' and 3' ITRs in the rAAV vector of the present invention may be homologous, i.e., from the same serotype; heterogeneous, i.e., from different serotypes; or chimeric, i.e., having ITR sequences derived from more than one AAV serotype.

[0223] In some embodiments, the rAAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the rAAV vector is AAV1. In some embodiments, the rAAV vector is AAV2. In some embodiments, the rAAV vector is AAV3. In some embodiments, the rAAV vector is AAV4. In some embodiments, the rAAV vector is AAV5. In some embodiments, the rAAV vector is AAV6. In some embodiments, the rAAV vector is AAV7. In some embodiments, the rAAV vector is AAV8. In some embodiments, the rAAV vector is AAV9. In some embodiments, the rAAV vector is AAV10. In some embodiments, the rAAV vector is AAV11. In some embodiments, the rAAV vector is sequence-optimized. In some embodiments, the rAAV capsid is modified. For example, in some embodiments, the rAAV8 capsid is modified. [Examples]

[0224] While certain compounds, compositions, and methods of the present invention have been described in a specific manner according to certain embodiments, the following examples are for illustrative purposes only and are not intended to limit the compounds of the present invention. [Examples]

[0225] Effects of sugar, buffering ratio, and pH on LNP stability Analysis was performed to evaluate the stability of LNPs in the presence of various amounts of sugar, in this case trehalose, various buffering strengths, and / or various pH levels. In summary, the data from these studies indicate that lower pH levels require higher minimum buffering strength to maintain stability. Furthermore, the results showed that when sugar, trehalose, is maintained at a constant percentage within the formulation, the minimum buffering strength required to maintain LNP stability decreases as the pH of the formulation increases.

[0226] Figure 1A is a graph showing that at pH 7.5, increasing the percentage of sugar and trehalose in the LNP formulation simultaneously increases the minimum buffering strength required in the LNP formulation. Figure 1B is a graph showing that when the trehalose is maintained at a constant percentage (i.e., 2.7%), the minimum buffering strength decreases as the pH level increases.

[0227] These data indicate that a lower sugar / buffer ratio is required at certain pH levels. Furthermore, the data shows that the lower the pH in the LNP formulation, the higher the buffering strength required to stabilize the LNP at a given sugar concentration. For example, when maintaining a constant sugar concentration, the lower the pH level, the higher the buffering strength required to maintain LNP stability. [Examples]

[0228] Lowering the buffering strength allows for high stability below the pKa of lipids. Studies were conducted to evaluate the lipid pKa-dependent behavior. These studies analyzed LNP formulations, which were formulated with 2.7% trehalose and citrate buffer to pH 4.5. These analyses showed that lowering the buffer strength resulted in higher LNP stability below the lipid pKa. Specifically, LNP stability was observed to decrease with increasing buffer strength, i.e., from 1, 10, 20, 50, 75 to 100 mM. This is illustrated in the graph in Figure 2.

[0229] These data indicate that buffering strength is better for stabilizing LNP formulations after sample dilution. For example, stability was visually observed in the following scenarios: 1) 2.7% trehalose + 100 mM Tris pH 7.5 (solution observation - clear); 2) 2.7% trehalose + 20 mM Tris pH 7.5 + 100 mM NaCl (solution observation - crushed / cloudy); 3) 2.7% trehalose + 16 mM Tris pH 7.5 + 220 mM NaCl (solution observation - clear).

[0230] In summary, these data led to the conclusion that maintaining higher ionic strength is desirable for preventing LNP aggregation and resulting mRNA stability. This is achieved, for example, by 1) having high buffering strength (e.g., 100 mM or higher); or 2) having low buffering strength (e.g., 15-20 mM) and high salt concentration (e.g., 200 mM or higher). It was estimated that this could be achieved in various ways by combining these factors; or by combining a moderate buffer strength (e.g., 40–50 mM) with a moderate salt concentration (e.g., 50–100 mM). [Examples]

[0231] Potency vs. Stability It has been previously observed that highly potent LNPs are associated with higher levels of LNP aggregation and subsequent mRNA degradation. The LNP formulations described herein were investigated to determine whether these formulations have any effect on the ability to obtain mRNA encapsulated in LNPs that are resistant to aggregation and subsequent mRNA degradation.

[0232] Various LNP formulations encapsulating human erythropoietin (EPO) mRNA were tested for stability at 6 and 25 hours. The tested LNP formulations had previously been found to be prone to aggregation. As shown in Figures 3A and 3B, the use of the LNP formulations described herein enabled the successful formulation of desired, highly potent LNPs that are resistant to aggregation.

[0233] The different LNP formulations tested are shown in Figures 3A and 3B. The data from Figure 3B were from in vivo studies in which the described LNP formulations were analyzed at either 6 or 24 hours after administration to mice. The data show human EPO protein expression at both 6 and 24 hours, for example, when using very potent lipids, such as lipidoids with high concentrations of DOPE.

[0234] Figure 4A shows various combinations of buffer and salt concentrations tested with LNP formulations, as well as the dilution stability obtained for each LNP formulation. The data are consistent with the results presented in Example 2, namely, that higher ionic strength is desirable for preventing LNP aggregation and resulting mRNA stability. In particular, these data confirm that combining moderate buffer strength (e.g., 40–50 mM) with moderate salt concentration (e.g., 50–125 mM) yields stable LNP formulations after dilution.

[0235] Figure 4B shows a table summarizing the stability of the diluted LNP formulations. In these assays, LNPs changed only with respect to Tris or phosphate buffer concentrations. All LNPs in this study were formulated in Tris or phosphate buffer and 2.7% trehalose. As the data show, these LNP formulations were not stable when the formulation pH was achieved with a buffer strength of 20 mM. When the buffer strength reached 100 mM or higher, the LNP formulations were stable. These data are consistent with the results presented in Example 2, namely that higher ionic strength is desirable for preventing LNP aggregation and resulting mRNA stability. [Examples]

[0236] Effect of sugar-to-buffer ratio on lipid nanoparticle encapsulation efficiency and size Studies were conducted to evaluate the effect of the sugar-to-buffer ratio on the stability of formulations at -20°C. These studies analyzed LNP formulations with exemplary trehalose-to-PBS ratios between 0.19 and 0.47 at start mRNA concentrations between 0.9 mg / ml and 1.6 mg / ml (Table 1). Inclusion efficiency (Figures 5A and 5B) and lipid nanoparticle size (Figures 6A and 6B) were evaluated at 4°C and 25°C with varying trehalose-to-PBS ratios for the LNP formulations. These analyses showed that lower trehalose-to-PBS ratios for LNP formulations were beneficial in preventing decreased encapsulation and increased LNP size, thereby resulting in higher stability of the LNP formulations. Overall, the stability of LNP formulations was greater at lower sugar-to-buffer ratios. This was reflected in the encapsulation efficiency (Figures 5A and 5B) and LNP size. The effect of the sugar-to-buffer ratio in the diagrams (Figures 6A and 6B) is shown in graph form.

[0237] [Table 1]

[0238] Inclusion efficiency was evaluated at various exemplary time points (0 hours, 1 hour, 3 hours, 6 hours, and 24 hours), and the observed inclusion efficiency percentages were graphed at 4°C (Figure 5A) and 25°C (Figure 5B). The results showed that LNP formulations with increasing trehalose-to-PBS ratios exhibited decreased inclusion and reduced stability. The results were particularly pronounced at 4°C, but a similar trend was observed at 25°C.

[0239] LNP size was measured at various exemplary time points (0, 1, 3, 6, and 24 hours), and the observed LNP size (nanometers) was graphed at 4°C (Figure 6A) and 25°C (Figure 6B). The results showed that LNP formulations with increasing trehalose-to-PBS ratio exhibited decreased encapsulation and reduced stability. The results were more pronounced at 25°C, but a similar trend was observed at 4°C.

[0240] Overall, the results of these studies showed that a low trehalose-to-PBS ratio is favorable for increased inclusion and reduced LNP size, corresponding to higher LNP stability.

[0241] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In addition, materials, methods, and examples are illustrative and not intended to limit. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Similar or equivalent methods and materials may be used in the practice or testing of the invention, but suitable methods and materials are described herein.

[0242] Equivalents Those skilled in the art can use only routine experiments to perform the experiments described herein. Many equivalents of the specific embodiments described above will be recognizable or verifiable. The scope of the present invention is not intended to be limited to the above specification, but rather as set forth in the following claims.

Claims

1. A liquid lipid nanoparticle (LNP) formulation that encapsulates mRNA encoding a peptide or polypeptide, which is resistant to aggregation and mRNA degradation, a. One or more LNPs having lipid components including or consisting of cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol; b. mRNA encapsulated within one or more lipid nanoparticles and encoding a peptide or polypeptide; c. Sugars or sugar alcohols; d. pH of the LNP preparation, which is between 6.0 and 8.0; e. A pH buffer that provides the pH of the LNP formulation at the minimum buffer ion strength; f. An LNP formulation comprising, optionally, one or more additional agents that provide ionic strength to the LNP formulation, wherein the total concentration of the pH buffer from (e) and optionally one or more additional agents from (f) provides an ionic strength of the LNP formulation that is at least twice greater than the minimum buffered ionic strength.

2. The LNP formulation according to claim 1, which, after three freezing and thawing cycles at -20°C, exhibits (i) less aggregation, (ii) less degradation of encapsulated mRNA, or (iii) both (i) and (ii), compared to the same LNP formulation having only the minimum buffer ion strength in the LNP formulation, instead of an ion strength at least twice greater than the minimum buffer ion strength.

3. Noncationic lipids include 1,2-dielcyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine. An LNP formulation according to claim 1, selected from (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, or 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE).

4. The LNP formulation according to claim 3, wherein the noncationic lipid is dioleoylphosphatidylethanolamine (DOPE).

5. The LNP formulation according to claim 4, wherein DOPE is 10% or more, for example, a lipid molar ratio of 10% to 30%.

6. An LNP preparation according to any one of claims 1 to 5, wherein the cationic lipid is a lipidoid, and in some cases the lipid molar ratio is 40% to 60%, for example, 40% to 50%.

7. An LNP preparation according to any one of claims 1 to 6, wherein the mRNA encodes a protein that is deficient in the target.

8. An LNP preparation according to any one of claims 1 to 6, wherein the mRNA encodes a vaccine antigen.

9. An LNP preparation according to any one of claims 1 to 8, wherein the sugar or sugar alcohol is selected from the group consisting of dextrose, sorbitol, trehalose, sucrose, raffinose, dextran, and inulin.

10. An LNP preparation according to any one of claims 1 to 9, wherein the sugar is a disaccharide.

11. The LNP preparation according to claim 10, wherein the disaccharide is present in a concentration of approximately 1% to 20%.

12. The LNP preparation according to claim 11, wherein the disaccharide is present at a concentration of approximately 2.5 to 3.0%.

13. The LNP formulation according to claim 12, wherein the ratio of disaccharide to buffer solution is between 0.2 and 0.

5.

14. An LNP preparation according to any one of claims 10 to 13, wherein the disaccharide is trehalose.

15. An LNP formulation according to any one of claims 1 to 14, wherein the pH is between approximately 6.0 and approximately 8.0, for example, 6.0 to 7.0, 6.5 to 7.5, or 7.0 to 8.

0.

16. The LNP formulation according to claim 1, wherein the pH is 7.

4.

17. An LNP formulation according to any one of claims 1 to 16, wherein the pH buffer has a pKa between 6.0 and 8.

2.

18. The LNP formulation according to claim 17, wherein the buffer is selected from the group consisting of phosphate buffer, citrate buffer, imidazole buffer, histidine buffer, and Good's buffer.

19. The LNP formulation according to claim 18, wherein the Good buffer is Tris buffer or HEPES buffer.

20. The LNP formulation according to claim 18 or 19, wherein the pH buffer is a phosphate buffer (e.g., citrate-phosphate buffer), a Tris buffer, or an imidazole buffer.

21. An LNP formulation according to any one of claims 1 to 20, wherein the minimum buffer ion strength is at least 75 mM, at least 100 mM, at least 125 mM, at least 150 mM, or at least 200 mM.

22. The LNP formulation according to claim 21, wherein the minimum buffer ion strength is approximately 75 mM to 200 mM, 75 mM to 150 mM, 75 mM to 100 mM, or 100 mM to 200 mM.

23. The LNP formulation according to claim 22, wherein the minimum buffer ion strength is between 100 mM and 200 mM.

24. An LNP formulation according to any one of claims 1 to 23, wherein one or more agents that provide ionic strength include a salt or a sugar.

25. The salts are NaCl, KCl, and CaCl ２ An LNP preparation according to claim 24, wherein the sugar is selected from the group consisting of the following, and the sugar is trehalose.

26. Any of claims 1 to 22, wherein the total concentration of one or more additional agents providing ionic strength is between approximately 50–300 mM, 50–150 mM, or 75–125 mM. The LNP preparation described in item 1.

27. The LNP formulation according to claim 26, wherein the total concentration of the pH buffer is between approximately 15 and 250 mM, 30 and 150 mM, or 40 and 50 mM.

28. The LNP formulation according to claim 26, wherein the total concentration of the pH buffer providing ionic strength and one or more additional agents is selected from about 40 mM Tris buffer and about 50–200 mM NaCl, about 50 mM Tris buffer and about 50–200 mM NaCl, about 100 mM Tris buffer and about 50–200 mM NaCl, about 40 mM imidazole and about 50–200 mM NaCl, about 50 mM imidazole and about 50–200 mM NaCl, about 100 mM imidazole and 50–200 mM NaCl, about 40 mM phosphate and 50–200 mM NaCl, about 50 mM phosphate and 50–200 mM NaCl, about 100 mM phosphate and 50–200 mM NaCl.

29. The LNP formulation according to any one of claims 1 to 28, wherein the ionic strength of the LNP formulation is at least 2.25 times greater, at least 2.5 times greater, at least 2.75 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, or at least 5 times greater than the minimum buffer ion strength.

30. The LNP preparation according to any one of claims 1 to 28, wherein the ionic strength of the LNP preparation is less than 20 times, less than 19 times, less than 18 times, less than 17 times, less than 16 times, less than 15 times, less than 14 times, less than 13 times, less than 12 times, less than 11 times, less than 10 times, less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, or less than 4 times the minimum buffer ion strength.

31. An LNP formulation according to any one of claims 1 to 30, wherein the ionic strength of the LNP formulation is at least twice greater than the minimum buffer ion strength and less than 20 times, and the ionic strength of the LNP formulation is between approximately 150 mM and 750 mM, 150 mM and 500 mM, 150 mM and 400 mM, 150 mM and 300 mM, 150 mM, and 200 mM.

32. The LNP preparation according to any one of claims 1 to 31, wherein the ionic strength of the LNP preparation is at least twice greater than the minimum buffer ion strength and less than 20 times, and the ionic strength of the LNP preparation is 150 mM or more.

33. An LNP formulation according to any one of claims 1 to 32, wherein less aggregation is determined by turbidity analysis.

34. An LNP preparation according to any one of claims 1 to 33, wherein the degradation of encapsulated mRNA is determined by turbidity analysis.

35. The LNP formulation according to any one of claims 1 to 34, wherein, after more than three freeze-and-thaw cycles at -20°C, the LNP formulation exhibits (i) less aggregation, (ii) less degradation of encapsulated mRNA, or (iii) both (i) and (ii), compared to the same LNP formulation having only the minimum buffer ion strength in the LNP formulation, instead of an ion strength at least twice greater than the minimum buffer ion strength.

36. An LNP formulation according to any one of claims 1 to 35, wherein the LNP has a diameter of less than approximately 100 nm.

37. The LNP formulation according to claim 32, wherein the LNP has a diameter between approximately 70 nm and 90 nm.

38. An LNP preparation according to any one of claims 1 to 37, wherein the lipid component comprises or consists of DMG-PEG-2000, cKK-E10, cholesterol, and DOPE.

39. An LNP formulation according to any one of claims 1 to 38, wherein the N / P ratio is between approximately 3 and 5.

40. The LNP formulation according to claim 39, wherein the N / P ratio is approximately 4.

41. An LNP preparation according to any one of claims 1 to 40, wherein the mRNA concentration is between approximately 0.05 mg / mL and 1.0 mg / mL.

42. The LNP preparation according to claim 41, wherein the mRNA concentration is between approximately 0.2 mg / mL and 0.5 mg / mL.

43. An LNP formulation according to any one of claims 1 to 42, wherein the LNP is stable at -20°C for at least 3 months, 6 months, 12 months, or more than 12 months.

44. An LNP preparation according to any one of claims 1 to 43, which is stable after dilution.

45. The LNP formulation according to any one of claims 1 to 44, wherein subcutaneous or intramuscular delivery of the formulation results in reduced pain compared to a formulation that does not contain a buffer having a concentration of 300 mM or less and a pH between approximately 7.0 and 7.

5.

46. The LNP preparation according to claim 45, wherein pain reduction is assessed by a 10 cm visual analog scale (VAS) or a 6-item verbal rating scale (VRS).

47. A method for reducing the degradation and / or aggregation of LNPs, comprising storing the LNPs in a formulation according to any one of claims 1 to 46.

Images