Methods and compositions for delivering mRNA-encoded antibodies

Abstract

The present invention provides, inter alia, a method of treating an immune disease in a subject, comprising administering to a subject in need thereof one or more mRNAs encoding the heavy and light chains of an antibody that binds to a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4R, IL-5R, IL-6R, IL-9R, IL-13R, IL-25R, or IL-33R, wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).The present invention also provides, inter alia, a composition comprising one or more mRNAs encoding the heavy and light chains of an antibody that binds to a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4R, IL-5R, IL-6R, IL-9R, IL-13R, IL-25R, or IL-33R, wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 137,528, filed January 14, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the text file named "MRT-2210WO_SL.txt", created on January 10, 2022 and having a size of 60614 bytes, are incorporated by reference in their entirety into this specification. [Background technology]

[0003] Antibodies have powerful therapeutic effects and are currently used to treat a range of diseases, including cancer, immune disorders, cardiovascular diseases, and transplant rejection. Traditionally, therapeutic antibodies are produced by recombinant technology, formulated, and then administered to patients in need of antibody therapy. However, the production and formulation of antibodies is very expensive. In addition, many antibodies have a very short half-life in vivo, so they may not reach the target antigen or target tissue before being degraded. To achieve the desired effect, antibody therapy often requires high doses and frequent administration, which can result in undesirable off-target effects. Summary of the Invention [Means for solving the problem]

[0004] The present invention provides, inter alia, a method for treating a disease in a subject. In some aspects, the present invention provides a method for treating an immune disease in a subject. In some embodiments, the method involves administering to the subject a composition comprising an mRNA encoding an antibody that targets a driver of type 2 inflammation, such targets including, for example, IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R). In some embodiments, the method involves administering to a subject a composition comprising an mRNA encoding an antibody that binds and / or inhibits a driver of type 2 inflammation, such targets including, for example, IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R). The inventors have surprisingly discovered a robust and efficient means to deliver mRNA encoding an antibody encapsulated in lipid nanoparticles (LNPs) that targets a specific cytokine involved in immune disease. The methods and compositions disclosed herein can be used to treat a variety of diseases, such as those associated with lung-associated immune diseases. Examples of such lung-related immune diseases include asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic obstructive pulmonary disease (COPD), systemic sclerosis-interstitial lung disease (SSc-ILD), idiopathic pulmonary fibrosis IPF, sarcoidosis, or allergies. The inventors have also surprisingly discovered that administering the compositions described herein by inhalation or nebulization may result in low or no systemic exposure, thus avoiding systemic undesirable side effects.

[0005] In some aspects, a method of treating an immune disease in a subject, the method comprising administering to a subject in need thereof one or more mRNAs encoding heavy and light chains of an antibody that binds to and / or inhibits a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP). In some embodiments, the protein target is IL-4. In some embodiments, the protein target is IL-5. In some embodiments, the protein target is IL-6. In some embodiments, the protein target is IL-9. In some embodiments, the protein target is IL-13. In some embodiments, the protein target is IL-25. In some embodiments, the protein target is IL-33. In some embodiments, the protein target is an IL-4 receptor (IL-4R, e.g., IL-4Rα). In some embodiments, the protein target is an IL-5 receptor (IL-5R). In some embodiments, the protein target is an IL-6 receptor (IL-6R). In some embodiments, the protein target is an IL-9 receptor (IL-9R). In some embodiments, the protein target is an IL-13 receptor (IL-13R). In some embodiments, the protein target is an IL-25 receptor (IL-25R). In some embodiments, the protein target is an IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1).

[0006] In some aspects, provided herein is a method of treating an immune disease in a subject, the method comprising administering to a subject in need thereof one or more mRNAs encoding heavy and light chains of an antibody that binds to and / or inhibits a protein selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP). In some embodiments, the antibody binds to and / or inhibits IL-4. In some embodiments, the antibody binds to and / or inhibits IL-5. In some embodiments, the antibody binds to and / or inhibits IL-6. In some embodiments, the antibody binds to and / or inhibits IL-9. In some embodiments, the antibody binds to and / or inhibits IL-13. In some embodiments, the antibody binds to and / or inhibits IL-25. In some embodiments, the antibody binds to and / or inhibits IL-33. In some embodiments, the antibody binds to and / or inhibits IL-4 receptor (IL-4R, e.g., IL-4Rα). In some embodiments, the antibody binds to and / or inhibits IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1).

[0007] In some embodiments, the antibody is an anti-IL6R antibody or an anti-IL4Rα antibody. In some embodiments, the antibody is an anti-IL6R antibody. In some embodiments, the antibody is an anti-IL4Rα antibody.

[0008] In some embodiments, the immune disorder is associated with an increase in type 2 inflammation-associated cytokines.

[0009] In some embodiments, the immune disease is selected from asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic obstructive pulmonary disease (COPD), systemic sclerosis-interstitial lung disease (SSc-ILD), idiopathic pulmonary fibrosis IPF, sarcoidosis, or allergy. In some embodiments, the immune disease is asthma. In some embodiments, the immune disease is chronic rhinosinusitis with nasal polyps (CRSwNP). In some embodiments, the immune disease is chronic obstructive pulmonary disease (COPD). In some embodiments, the immune disease is systemic sclerosis-interstitial lung disease (SSc-ILD). In some embodiments, the immune disease is idiopathic pulmonary fibrosis IPF. In some embodiments, the immune disease is sarcoidosis. In some embodiments, the immune disease is allergy.

[0010] In some embodiments, administration is by nebulization, intratracheal delivery, or inhalation. In some embodiments, administration is by nebulization. In some embodiments, pulmonary delivery is by nebulization of the compound using a nebulizer, preferably a mesh nebulizer. In some embodiments, the nebulizer delivers the compound to the lung cells in the form of an aerosol. In some embodiments, the lung cells are lung epithelial cells. In some embodiments, compositions having lipid nanoparticles with an average size of about 50-70 nm are particularly suitable for pulmonary delivery by nebulization.

[0011] In some embodiments, administration is by intratracheal delivery, hi some embodiments, administration is by inhalation.

[0012] In some embodiments, the administration results in administration of the mRNA to lung tissue.

[0013] In some embodiments, administration results in antibody expression for at least about 48 hours, 72 hours, 96 hours, or 120 hours. Thus, in some embodiments, administration results in antibody expression for at least about 48 hours. In some embodiments, administration results in antibody expression for at least about 72 hours. In some embodiments, administration results in antibody expression for at least about 96 hours. In some embodiments, administration results in antibody expression for at least about 120 hours. In some embodiments, administration results in antibody expression for about 72-120 hours. In some embodiments, administration results in antibody expression for 96-120 hours.

[0014] In some embodiments, low or no systemic exposure of the mRNA occurs following administration of the composition to the lungs of a subject.

[0015] In some embodiments, the immune disease is selected from autoimmune dermatitis or atopic dermatitis. Thus, in some embodiments, the immune disease is autoimmune dermatitis. In some embodiments, the immune disease is atopic dermatitis.

[0016] In some embodiments, administration is intravenous. In some embodiments, administration is intraperitoneal. In some embodiments, the antibody is expressed systemically, for example, when administered intravenously or intraperitoneally.

[0017] In some embodiments, the LNPs comprise one or more of a cationic lipid, a non-cationic lipid, and a PEG-modified lipid.

[0018] In some embodiments, the LNP further comprises cholesterol.

[0019] In some embodiments, the LNPs have a molar ratio of cationic lipids to non-cationic lipids to PEG-modified lipids of about 30-60:25-35:1-15, respectively.

[0020] In some embodiments, the non-cationic lipid is 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-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).

[0021] In some embodiments, the LNPs comprise DMG-PEG-2000, Guan-SS-Chol, and DOPE. In some embodiments, the LNPs comprise DMG-PEG-2000. In some embodiments, the LNPs comprise Guan-SS-Chol. In some embodiments, the LNPs comprise DOPE.

[0022] In some embodiments, DMG-PEG-2000, Guan-SS-Chol, and DOPE are present in a ratio of about 1-15:30-60:25-35. In some embodiments, DMG-PEG-2000, Guan-SS-Chol, and DOPE are present in a ratio of about 5:60:35.

[0023] In some embodiments, the heavy and light chains are encoded by a single mRNA.

[0024] In some embodiments, the heavy and light chains are encoded by separate mRNAs.

[0025] In some embodiments, the LNPs have a size of 150 nm or less.

[0026] In some embodiments, the LNPs have a size of 100 nm or less.

[0027] In some embodiments, the LNPs have a size of 75 nm or less.

[0028] In some embodiments, the LNPs have a size of about 60 nm.

[0029] In some embodiments, one or more mRNAs are modified to enhance stability.

[0030] In some embodiments, one or more mRNAs are modified to include modified nucleotides, a cap structure, a polyA tail, a 5' and / or a 3' untranslated region.

[0031] In some embodiments, one or more mRNAs are unmodified.

[0032] In some aspects, compositions are provided that include one or more mRNAs encoding heavy and light chains of an antibody that binds to and / or inhibits a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP). In some embodiments, the protein target is IL-4. In some embodiments, the protein target is IL-5. In some embodiments, the protein target is IL-6. In some embodiments, the protein target is IL-9. In some embodiments, the protein target is IL-13. In some embodiments, the protein target is IL-25. In some embodiments, the protein target is IL-33. In some embodiments, the protein target is an IL-4 receptor (IL-4R, e.g., IL-4Rα). In some embodiments, the protein target is an IL-5 receptor (IL-5R). In some embodiments, the protein target is an IL-6 receptor (IL-6R). In some embodiments, the protein target is an IL-9 receptor (IL-9R). In some embodiments, the protein target is an IL-13 receptor (IL-13R). In some embodiments, the protein target is an IL-25 receptor (IL-25R). In some embodiments, the protein target is an IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1).

[0033] In some embodiments, the antibody is an anti-IL6R antibody or an anti-IL4Rα antibody. In some embodiments, the antibody is an anti-IL6R antibody. In some embodiments, the antibody is an anti-IL4Rα antibody.

[0034] In some embodiments, the LNPs comprise one or more of a cationic lipid, a non-cationic lipid, and a PEG-modified lipid.

[0035] In some embodiments, the LNP further comprises cholesterol.

[0036] In some embodiments, the LNPs have a molar ratio of cationic lipids to non-cationic lipids to PEG-modified lipids of about 30-60:25-35:1-15, respectively.

[0037] In some embodiments, the non-cationic lipid is 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-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).

[0038] In some embodiments, the LNP comprises DMG-PEG-2000, Guan-SS-Chol, and DOPE.

[0039] In some embodiments, DMG-PEG-2000, Guan-SS-Chol, and DOPE are present in a ratio of about 1-15:30-60:25-35. In some embodiments, DMG-PEG-2000, Guan-SS-Chol, and DOPE are present in a ratio of about 5:60:35.

[0040] In some embodiments, the mRNA is EVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18). In some embodiments, the mRNA encodes an anti-IL6R antibody heavy chain comprising a sequence at least 80% identical to SEQ ID NO: 18. In some embodiments, the mRNA encodes an anti-IL6R antibody heavy chain comprising a sequence 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 18. In some embodiments, the mRNA encodes an anti-IL6R antibody heavy chain comprising a sequence identical to SEQ ID NO: 18.

[0041] In some embodiments, the mRNA encodes an anti-IL6R antibody heavy chain that further comprises a secretory sequence at least 80% identical to MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLS (SEQ ID NO: 26). In some embodiments, the mRNA encodes an anti-IL6R antibody heavy chain that further comprises a secretory sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 26. In some embodiments, the mRNA encodes an anti-IL6R antibody heavy chain that further comprises a secretory sequence of SEQ ID NO: 26.

[0042] In some embodiments, the mRNA encodes an anti-IL6R antibody light chain comprising a sequence at least 80% identical to DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19). In some embodiments, the mRNA encodes an anti-IL6R antibody light chain comprising a sequence 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 19. In some embodiments, the mRNA encodes an anti-IL6R antibody light chain comprising a sequence identical to SEQ ID NO: 19.

[0043] In some embodiments, the mRNA encodes an anti-IL6R antibody light chain that further comprises a secretory sequence at least 80% identical to MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLS (SEQ ID NO: 26). In some embodiments, the mRNA encodes an anti-IL6R antibody light chain that further comprises a secretory sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 26. In some embodiments, the mRNA encodes an anti-IL6R antibody light chain that further comprises a secretory sequence of SEQ ID NO: 26.

[0044] In some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and has the following sequence:

[0045] In some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to one of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and comprises a sequence that is identical to one of SEQ ID NOs: 2, 3, 4, or 5. Thus, in some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 2. In some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 3. In some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the anti-IL6R antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 5.

[0046] In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and has the following sequence: (A) ATGGCCACTGGAAGCCGGACAAGCCTGCTGCTGGCCTTTGGCCTGCTGTGTCTGCCTTGGCTGCAGGAGGGAAGCGCATTTCCAACAATTCCTCTGAGCGACATTCAGATGACACAGAGCCCCAGCAGCGTGTCCGCATCAGTGGGAGACAGGGTGACTATCACATGTAGAGCTTCTCAAGGAATTAGCTCTTGGCTGGCCTGGTATCAGCAGAAGCCAGGCAAGGCCCCCAAGCTGCTGATCTATGGAGCTAGCTCTCTGGAGTCTGGGGTGCCATCTAGGTTCAGTGGCTCCGGCAGCGGAACAGACTTCACACTGACTATCAGCAGCCTGCAGCCTGAGGACTTTGCCAGCTACTACTGCCAGCAGGCAAATAGCTTTCCCTATACTTTCGGACAGGGCACCAAGCTGGAGATTAAGCGGACCGTTGCTGCTCCAAGCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGTCTGCTGAACAATTTCTATCCCCGTGAAGCCAAAGTGCAGTGGAAGGTGGATAACGCTCTCCAGTCTGGCAATTCCCAGGAGAGCGTGACAGAGCAGGATTCTAAGGATTCTACCTACTCCCTGTCCAGCACACTGACCCTGAGCAAGGCCGATTACGAAAAACACAAAGTGTACGCCTGCGAAGTCACACACCAGGGGCTGAGCTCCCCAGTGACAAAGAGCTTTAATAGAGGGGAGTGCTGA (SEQ ID NO: 6); (B) ATGGCTACAGGGAGCCGCACTAGCCTGCTGCTGGCTTTTGGCCTGCTGTGCCTGCCATGGCTGCAAGAGGGGTCCGCCTTTCCTACCATCCCCCTGTCCGATATTCAGATGACCCAGTCCCCTAGCAGCGTGTCTGCCAGCGTGGGAGACAGGGTGACTATCACCTGTAGGGCCAGCCAGGGCATTTCTAGCTGGCTGGCTTGGTACCAGCAGAAGCCAGGAAAGGCTCCCAAACTGCTGATCTACGGGGCATCCTCTCTGGAGTCCGGAGTGCCAAGCAGATTCTCTGGGAGCGGCAGCGGGACCGATTTCACACTGACCATTAGCAGCCTGCAGCCAGAAGACTTCGCCAGCTACTATTGTCAGCAGGCAAACTCTTTTCCTTATACCTTCGGGCAGGGGACTAAACTGGAAATCAAGCGGACAGTGGCTGCTCCAAGCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCTTCCGTGGTGTGCCTGCTGAACAACTTTTATCCAAGGGAAGCTAAAGTGCAGTGGAAGGTGGACAACGCTCTGCAGTCTGGAAATAGCCAGGAATCCGTGACTGAGCAGGATAGCAAAGACAGCACATACAGCCTGTCTTCCACTCTGACCCTGAGCAAGGCAGACTACGAGAAACACAAAGTGTATGCCTGTGAGGTGACCCATCAGGGCCTGTCTAGCCCAGTGACCAAGTCCTTTAACAGAGGCGAATGTTGA (SEQ ID NO: 7); (C) ATGGCAACTGGATCCCGGACCTCTCTGCTGCTGGCCTTCGGACTGCTGTGCCTGCCATGGCTGCAGGAGGGGAGCGCTTTTCCTACTATCCCCCTGTCTGACATCCAGATGACTCAGAGCCCAAGCTCTGTGTCCGCAAGCGTGGGGGACAGGGTGACAATCACTTGCAGGGCATCCCAGGGAATCTCCTCTTGGCTGGCATGGTACCAGCAGAAACCTGGAAAAGCCCCAAAACTGCTGATTTATGGCGCTTCCAGCCTCGAATCCGGAGTGCCATCCCGGTTTTCTGGCTCCGGCAGCGGGACAGATTTTACTCTGACCATCTCTAGCCTGCAGCCAGAGGATTTTGCCTCCTATTATTGCCAGCAGGCCAACAGCTTTCCTTATACCTTTGGACAGGGAACTAAGCTGGAGATCAAGAGGACAGTGGCTGCTCCTAGCGTGTTCATCTTCCCACCTTCTGACGAACAGCTGAAGTCTGGAACAGCCTCTGTGGTGTGCCTCCTCAACAACTTCTATCCCCGGGAGGCTAAGGTCCAGTGGAAAGTGGACAACGCCCTGCAGTCCGGAAACTCCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGATAGCACTTATTCCCTGTCCTCCACCCTGACTCTGTCCAAGGCCGACTACGAAAAGCACAAAGTGTACGCCTGCGAAGTCACTCATCAGGGACTGAGCTCCCCCGTGACCAAGAGCTTCAATAGGGGAGAATGTTAG (SEQ ID NO: 8); or It contains a sequence that is at least 80% identical to one of SEQ ID NO:9: (D) ATGGCAACTGGCTCCAGGACTAGCCTGCTGCTGGCATTTGGCCTCCTGTGTCTGCCATGGCTGCAGGAGGGCTCCGCCTTCCCAACAATTCCACTGTCCGACATCCAGATGACACAGTCCCCTAGCAGCGTGAGCGCCTCCGTGGGAGATAGAGTGACAATTACCTGTCGCGCAAGCCAGGGGATCAGCAGCTGGCTGGCCTGGTATCAACAGAAACCTGGAAAAGCCCCCAAGCTCCTGATCTATGGCGCCAGCAGCCTGGAAAGCGGGGTTCCAAGCCGGTTTTCCGGGTCCGGCAGCGGAACTGACTTCACCCTGACAATTTCAAGCCTGCAGCCCGAGGATTTTGCAAGCTACTACTGTCAGCAGGCTAATAGCTTTCCTTACACATTCGGCCAGGGCACCAAGCTCGAAATTAAAAGAACTGTGGCTGCCCCATCCGTGTTTATCTTCCCACCCTCTGACGAACAGCTGAAGTCCGGGACAGCCTCTGTGGTGTGCCTGCTGAACAATTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTCGACAATGCTCTGCAGTCTGGAAACTCCCAGGAGTCTGTGACTGAGCAGGACAGCAAGGACAGCACCTATAGCCTGTCTTCCACCCTGACCCTGAGCAAGGCCGATTACGAAAAGCACAAGGTGTATGCCTGTGAGGTGACCCACCAGGGACTGTCTAGCCCAGTGACTAAATCCTTTAATAGAGGCGAATGCTGA.

[0047] In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to one of the following SEQ ID NOs: 6, 7, 8, or 9. In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and comprises a sequence that is identical to one of the following SEQ ID NOs: 6, 7, 8, or 9. In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 6. In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 7. In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 8. In some embodiments, the mRNA encoding the anti-IL6R antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 9.

[0048] In some embodiments, the mRNA is EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE It encodes an anti-IL4Rα antibody heavy chain comprising a sequence at least 80% identical to FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 20).

[0049] In some embodiments, the mRNA encodes an anti-IL4Rα antibody heavy chain that comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 20. In some embodiments, the mRNA encodes an anti-IL4Rα antibody heavy chain that comprises a sequence that is identical to SEQ ID NO: 20.

[0050] In some embodiments, the mRNA encodes an anti-IL4Rα antibody heavy chain that further comprises a secretory sequence at least 80% identical to MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLS (SEQ ID NO: 26). In some embodiments, the mRNA encodes an anti-IL4Rα antibody heavy chain that further comprises a secretory sequence that is 70%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 26. In some embodiments, the mRNA encodes an anti-IL4Rα antibody heavy chain that further comprises a secretory sequence identical to SEQ ID NO: 26.

[0051] In some embodiments, the mRNA is DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21). In some embodiments, the mRNA encodes an anti-IL4Rα antibody light chain comprising a sequence that is at least 80% identical to SEQ ID NO: 21. In some embodiments, the mRNA encodes an anti-IL4Rα antibody light chain comprising a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 21. In some embodiments, the mRNA encodes an anti-IL4Rα antibody light chain comprising a sequence that is identical to SEQ ID NO: 21.

[0052] In some embodiments, the mRNA encodes an anti-IL4Rα antibody light chain that further comprises a secretory sequence that is at least 80% identical to MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLS (SEQ ID NO: 26). In some embodiments, the mRNA encodes an anti-IL4Rα antibody light chain that further comprises a secretory sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 26.

[0053] In some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and has the following sequence:

[0054] In some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to one of SEQ ID NOs: 10, 11, 12, or 13. In some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and comprises a sequence that is identical to one of SEQ ID NOs: 10, 11, 12, or 13. Thus, in some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 10. In some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 11. In some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 12. In some embodiments, the mRNA encoding the anti-IL4Rα antibody heavy chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 13.

[0055] In some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and contains a sequence that is at least 80% identical to one of the following sequences: (A) ATGGCCACAGGCTCTAGGACATCCCTGCTGCTGGCCTTCGGACTGCTGTGTCTGCCTTGGCTGCAGGAGGGATCTGCATTCCCAACTATCCCTCTGTCCGATATTGTGATGACCCAGAGCCCCCTGAGCCTGCCAGTGACTCCTGGGGAGCCCGCATCTATCAGCTGCCGGTCCTCTCAGTCTCTGCTGTATTCTATCGGGTACAACTACCTGGATTGGTACCTGCAGAAAAGTGGGCAGAGCCCCCAGCTGCTCATCTATCTGGGGTCCAACAGGGCTAGTGGCGTGCCAGACCGGTTCTCCGGATCCGGCTCCGGAACAGACTTTACACTGAAAATTAGCCGCGTGGAGGCCGAGGACGTGGGGTTTTATTATTGTATGCAGGCCCTGCAGACCCCATACACATTTGGCCAGGGGACAAAGCTGGAAATTAAGCGCACTGTGGCCGCTCCGTCTGTGTTCATCTTTCCTCCCAGCGATGAACAGCTGAAGTCTGGGACCGCTAGCGTCGTGTGCCTGCTGAACAATTTTTACCCCAGGGAGGCTAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGGAACAGCCAGGAGAGTGTTACTGAGCAGGATTCTAAAGATTCCACCTATTCCCTGTCTTCCACCCTGACTCTGTCTAAGGCCGATTACGAAAAACATAAGGTGTACGCATGCGAGGTGACCCACCAGGGGCTGAGCTCTCCCGTGACTAAGAGCTTCAATCGCGGAGAGTGCTGA(SEQ ID NO: 14); (B) ATGGCTACAGGCAGCAGAACCAGCCTGCTGCTGGCATTTGGCCTGCTGTGCCTGCCTTGGCTGCAGGAGGGGAGCGCTTTTCCCACAATTCCTCTGTCTGATATCGTCATGACCCAATCTCCCCTGTCCCTGCCTGTGACTCCAGGAGAGCCCGCTAGCATTTCTTGCAGGTCTTCCCAGAGCCTGCTGTACAGCATCGGCTATAACTACCTGGATTGGTATCTGCAGAAAAGCGGGCAGTCTCCTCAGCTGCTGATCTACCTGGGCTCTAACAGAGCCTCTGGGGTCCCCGACAGGTTTTCCGGAAGCGGCTCTGGCACCGACTTTACTCTCAAAATCAGCCGCGTGGAGGCAGAGGACGTGGGCTTCTATTACTGCATGCAGGCCCTGCAGACACCATATACATTCGGACAGGGGACCAAGCTGGAGATTAAGAGAACAGTGGCTGCCCCAAGCGTGTTTATCTTTCCTCCCTCCGATGAACAGCTGAAAAGCGGCACTGCTTCCGTGGTGTGCCTGCTGAATAATTTCTACCCTAGAGAGGCCAAAGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAAAGTGTCACCGAGCAGGATTCCAAGGATTCCACATATTCTCTGTCCAGCACTCTGACACTGTCCAAGGCAGACTACGAAAAACACAAGGTCTACGCCTGCGAAGTGACCCACCAGGGACTGTCTAGCCCTGTGACTAAGTCTTTTAATAGGGGGGAGTGTTAG(SEQ ID NO: 15); (C) ATGGCTACTGGCAGCAGAACCAGCCTGCTGCTGGCATTCGGGCTGCTCTGCCTGCCATGGCTGCAGGAGGGATCCGCCTTCCCAACTATCCCCCTGAGCGATATCGTGATGACCCAGTCTCCCCTGAGCCTGCCAGTTACACCCGGCGAACCTGCTAGCATCAGCTGCAGATCCTCCCAGTCTCTCCTGTACTCCATCGGGTACAATTATCTGGATTGGTATCTGCAGAAGTCTGGCCAATCCCCCCAGCTGCTGATCTACCTGGGCTCCAACAGAGCAAGCGGCGTGCCCGATAGATTCAGCGGCAGCGGGAGCGGCACTGATTTTACTCTGAAGATCAGCAGGGTGGAGGCCGAAGATGTGGGATTTTACTACTGCATGCAAGCACTGCAGACTCCTTACACATTCGGCCAGGGAACTAAGCTGGAGATCAAAAGAACCGTGGCAGCTCCAAGCGTCTTCATTTTCCCACCTTCTGACGAGCAGCTGAAGTCCGGCACAGCTTCCGTCGTGTGCCTCCTGAACAACTTCTACCCCAGGGAGGCAAAGGTGCAATGGAAAGTGGACAACGCTCTGCAGAGCGGAAACAGTCAGGAGTCCGTGACCGAGCAGGACAGCAAAGACTCCACTTACAGCCTGAGCTCTACTCTGACCCTGAGCAAAGCTGACTACGAGAAGCATAAGGTGTATGCTTGCGAGGTCACCCACCAGGGCCTCTCTTCTCCCGTGACCAAGAGCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 16); or It contains a sequence that is at least 80% identical to one of the following sequences: ATGGCAACTGGAAGCAGGACCTCCCTGCTCCTGGCTTTCGGCCTGCTCTGTCTGCCATGGCTGCAAGAAGGATCTGCCTTTCCTACAATTCCACTGTCCGACATCGTGATGACACAGTCCCCCCTGTCTCTGCCTGTCACCCCAGGCGAACCAGCCTCTATTTCTTGTCGGTCCTCTCAGTCCCTGCTGTATAGCATCGGATATAATTATCTGGACTGGTACCTGCAAAAATCCGGCCAGTCTCCTCAGCTGCTGATCTATCTGGGCTCCAACCGGGCTAGCGGAGTCCCAGACCGGTTTTCCGGGTCTGGCAGTGGGACAGATTTTACACTGAAAATTTCCCGGGTGGAGGCTGAGGACGTGGGATTTTACTACTGTATGCAGGCCCTGCAAACCCCATATACTTTCGGACAGGGAACAAAGCTGGAGATCAAAAGAACCGTGGCCGCCCCCAGCGTTTTCATCTTCCCACCAAGCGACGAGCAGCTCAAATCTGGGACCGCTAGCGTGGTCTGTCTGCTGAATAACTTCTACCCAAGGGAAGCAAAGGTGCAGTGGAAGGTCGACAACGCACTGCAGAGCGGGAACTCCCAGGAGAGCGTGACTGAACAGGACAGCAAGGACAGCACCTATAGCCTCAGCAGCACTCTGACCCTGTCTAAAGCTGATTACGAAAAACACAAGGTGTATGCTTGTGAAGTGACTCACCAGGGCCTGTCTTCCCCTGTTACAAAGTCCTTCAATAGAGGAGAATGTTAA

[0056] In some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 14, 15, 16, or 17. In some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 14, 15, 16, or 17. Thus, in some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 14. In some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and comprises a sequence that is identical to SEQ ID NO: 15. In some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and comprises the identical sequence that is SEQ ID NO: 16. In some embodiments, the mRNA encoding the anti-IL4Rα antibody light chain is codon optimized and comprises the identical sequence that is SEQ ID NO: 17.

[0057] In some embodiments, the mRNA comprises a 5'UTR and a 3'UTR sequence. In some embodiments, the 5'UTR sequence comprises a sequence having at least 80% identity to GGACAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCTTGACACG (SEQ ID NO: 27). In some embodiments, the 5'UTR sequence comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 27. In some embodiments, the 5'UTR sequence comprises a sequence that is identical to SEQ ID NO: 27.

[0058] In some embodiments, the 3'UTR sequence comprises a sequence having at least 80% identity to CGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATC (SEQ ID NO: 28). In some embodiments, the 3'UTR sequence comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 28. In some embodiments, the 3'UTR sequence comprises a sequence that is identical to SEQ ID NO: 28.

[0059] In this application, the use of "or" means "and / or" unless otherwise specified. As used in this disclosure, the term "comprise" and variations of the term, such as "comprises" and "comprises," are not intended to exclude other additives, components, integers, or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Both terms are meant to cover any normal variations recognized by those skilled in the relevant art.

[0060] Other features, objects, and advantages of the present invention will be apparent in the following detailed description, drawings, and claims. It should be understood, however, that the detailed description, drawings, and claims, while indicating embodiments of the present invention, are given by way of illustration only, and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0061] The drawings are for illustrative purposes, not for limiting purposes. [Brief description of the drawings]

[0062] [Figure 1] 1 is an exemplary graph comparing human IgG levels in bronchoalveolar lavage fluid (BALF) 72 hours after administration of mRNA encoding anti-IL6R. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference.

[0064] Antibody: As used herein, the term "antibody" encompasses both intact antibodies and active antibody fragments. Typically, an intact "antibody" is an immunoglobulin that specifically binds to a particular antigen. An antibody can be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgE, and IgD. A typical immunoglobulin (antibody) structural unit as understood in the art is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light and heavy chains, respectively. Each variable region is further subdivided into a hypervariable (HV) region and a framework (FR) region. The hypervariable region comprises three hypervariable sequence regions (CDR1, CDR2 and CDR3) called complementarity determining regions, separated by four framework regions (FR1, FR2, FR2 and FR4), which form a beta-sheet structure and function as a scaffold to hold the HV region in place. The C-terminus of each heavy and light chain defines a constant region consisting of one domain (CL) of the light chain and three domains (CH1, CH2 and CH3) of the heavy chain. In some embodiments, the term "intact antibody" or "fully assembled antibody" is used in reference to an antibody, meaning that it contains two heavy chains and two light chains, optionally linked by disulfide bonds, as in naturally produced antibodies. In some embodiments, the antibody according to the invention is an antibody fragment. As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding region or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.For example, antibody fragments include "Fv" fragments, which are isolated fragments consisting of the variable regions of the heavy and light chains, recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are connected by a peptide linker ("ScFv protein"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable region. In many embodiments, the antibody fragment contains sufficient sequence of the parent antibody that the fragment binds to the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs).

[0065] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value referred to. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater or smaller) of the reference value mentioned above, unless otherwise stated or clear from the context (unless such number exceeds 100% of possible values).

[0066] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses the situation where mRNA is delivered to a target tissue, and the encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and the situation where mRNA is delivered to a target tissue, and 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 referred to as "systemic distribution" or "systemic delivery"). In some embodiments, delivery is pulmonary delivery, including, for example, nebulization.

[0067] Encapsulation: As used herein, the term "encapsulation," or grammatical equivalents, refers to the process of confining mRNA molecules within a nanoparticle.

[0068] Engineered or Mutant: As used herein, the terms "engineered" or "mutant," or grammatical equivalents, refer to a nucleotide or protein sequence that contains one or more modifications compared to its naturally occurring sequence, including, but not limited to, a deletion, a heterologous nucleic acid or amino acid insertion, an inversion, a substitution, or a combination thereof.

[0069] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., heavy or light chains of an antibody) into an intact protein (e.g., an antibody), and / or the post-translational modification of a polypeptide or a fully assembled protein (e.g., an antibody). In this application, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0070] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0071] Half-life: As used herein, the term "half-life" is the time required for a content, such as a nucleic acid or protein concentration or activity, to fall to half of its value when measured at the beginning of a period.

[0072] Improve, increase, or decrease: As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a 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 the disease as the subject being treated.

[0073] Impurity: As used herein, the term "impurity" refers to a limited amount of a substance within a liquid, gas, or solid that differs from the chemical composition of a target material or compound. Impurities are also called contaminants.

[0074] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0075] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cellular systems, the term is used to refer to events that occur within living cells (e.g., in contrast to in vitro systems).

[0076] Isolated: As used herein, the term "isolated" refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) produced, manufactured, and / or created by the hand of man. 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 greater than about 99% of other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of the percent purity of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0077] 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, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' direction.

[0078] "Nebulization" refers to the delivery of a pharmaceutical composition in a fine spray or dispersed suspension that is inhaled into the lungs, typically with a nebulizer.

[0079] A "nebulizer" is a device that uses a propellant or other suitable energy source, such as oxygen, compressed air, or ultrasound, to convert liquids or particles into a fine spray or mist or dispersed suspension, typically in the form of an aerosol that can be directly inhaled. In some embodiments, the nebulizer used with the present invention contains a piezoelectric element to generate vibrations of a mesh. The vibrations pump the liquid pharmaceutical composition through the mesh. The liquid is released from the mesh in droplets that create an aerosol. Such nebulizers are commonly referred to as (vibrating) mesh nebulizers. In some embodiments, the nebulizer is used to aerosolize pharmaceutical compositions for pulmonary delivery. Inhalation from the nebulizer is via a mouthpiece used by the subject.

[0080] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. 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 that includes individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered to be within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or that encode the same amino acid sequence. Protein-encoding nucleotide sequences and / or RNAs include introns. In some embodiments, the nucleic acids are purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, the nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, the nucleic acid is selected from 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 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, The nucleic acid may be or include C5-propynyl-cytidine, 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); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). 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, the nucleotides T and U are used interchangeably in sequence descriptions.

[0081] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition can be administered, for example, for experimental, diagnostic, prophylactic, 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 a human. Human includes prenatal and postnatal forms.

[0082] Pharmaceutically acceptable: The term "pharmacologically acceptable," as used herein, refers to a material that is suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0083] "Pulmonary delivery" refers to administering the pharmaceutical compositions described herein to lung cells in vivo by delivering the pharmaceutical composition to the lungs. Non-limiting methods of pulmonary delivery include nebulization and intratracheal administration / instillation.

[0084] Stable: As used herein, the term "stable" protein or grammatical equivalents thereof refers to a protein that retains its physical stability and / or biological activity. In one embodiment, protein stability 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 leads to protein degradation, compared to a wild-type protein.

[0085] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, which refers to a human who is presenting to a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject can be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0086] Substantially: As used herein, the term "substantially" refers to a quantitative state that exhibits a degree or degree of all or nearly all of a desired feature or characteristic. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0087] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, 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 can be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease, for the purpose of reducing the risk of developing pathologies associated with the disease.

[0088] Detailed Description The present invention provides, inter alia, improved methods and compositions for delivery of mRNA encapsulated in lipid nanoparticles, the mRNA encoding an antibody targeting a driver of type II inflammation. For example, suitable protein targets associated with type II inflammation include IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1). For example, the antibodies of the invention bind to and / or inhibit IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R) or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1). Various aspects of the invention are further described below.

[0089] mRNA-encoded antibodies The present disclosure provides mRNA-encoded antibodies that can be used to treat various diseases, including, for example, immune-related diseases. Various immune-related diseases are known in the art and can generally be divided into lung immune-related diseases and non-lung-related immune-related diseases. The mRNA-encoded antibody methods and compositions provided herein can be used in the treatment of either lung-related or non-lung-related immune diseases. Examples of lung-related immune diseases include, for example, asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic obstructive pulmonary disease (COPD), systemic sclerosis-interstitial lung disease (SSc-ILD), idiopathic pulmonary fibrosis IPF, sarcoidosis, or allergy. Examples of non-lung-related diseases include autoimmune hepatitis and atopic dermatitis.

[0090] Methods of treating an immune disease in a subject are provided, comprising administering to a subject in need thereof one or more mRNAs encoding heavy and light chains of an antibody that binds to and / or inhibits a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R) or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

[0091] A method of treating an immune disease in a subject is provided, comprising administering to a subject in need thereof one or more mRNAs encoding heavy and light chains of an antibody that binds to a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

[0092] Methods of treating an immune disease in a subject are provided, comprising administering to a subject in need thereof one or more mRNAs encoding the heavy and light chains of an antibody that binds to a protein target selected from IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R) or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

[0093] A method of treating an immune disease in a subject is provided, comprising administering to a subject in need thereof one or more mRNAs encoding heavy and light chains of an antibody that inhibits a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

[0094] Methods of treating an immune disease in a subject are provided, comprising administering to a subject in need thereof one or more mRNAs encoding heavy and light chains of an antibody that inhibits a protein target selected from IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R) or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

[0095] In some aspects, the mRNA-encoded antibody encapsulated in LNPs is used to treat lung-related immune diseases. In this scenario, the mRNA that encodes the antibody and is encapsulated in LNPs is delivered to lung tissue by inhalation, nebulization, or intratracheal instillation. One advantage of this administration method is that it allows low or no systemic exposure to lung-related diseases, thus avoiding undesirable systemic side effects. Thus, in some embodiments, administration of the mRNA that encodes the antibody by inhalation or nebulization does not result in systemic exposure. In some embodiments, administration of the mRNA that encodes the antibody by inhalation or nebulization has low systemic exposure. In some embodiments, administration of the mRNA that encodes the antibody by inhalation or nebulization results in delivery of the mRNA encapsulated in LNPs to lung tissue.

[0096] In some embodiments, the antibody encoded by the mRNA is an anti-IL6R antibody.A variety of anti-IL6R antibodies are known in the art, including sarilumab and tocilizumab.Anti-IL6R antibodies have been used to treat various diseases, including rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, systemic juvenile arthritis, and to treat cytokine storm in cases of severe coronavirus infection or Covid-19 disease.

[0097] In some embodiments, the antibody encoded by the mRNA provided herein is used to treat rheumatoid arthritis. In some embodiments, the antibody encoded by the anti-IL6R mRNA provided herein is used to treat rheumatoid arthritis. In some embodiments, the antibody encoded by the anti-IL6R mRNA provided herein is used to treat polyarticular juvenile idiopathic arthritis. In some embodiments, the antibody encoded by the anti-IL6R mRNA provided herein is used to treat systemic juvenile arthritis. In these scenarios, the antibody encoded by the anti-IL6R mRNA is delivered intravenously or intraperitoneally.

[0098] In some embodiments, the mRNA-encoded antibodies provided herein are used to reduce or treat cytokine storm in subjects with coronavirus infection or Covid-19 disease. In some embodiments, the mRNA-encoded antibodies provided herein can be used to treat subjects with coronavirus infection or Covid-19 disease. In this scenario, the mRNA-encoded antibodies are delivered by inhalation or nebulization.

[0099] In some embodiments, the antibody encoded by the mRNA is an anti-IL4α antibody.Various anti-IL4α antibodies are known in the art, including, for example, dupilumab.Anti-IL4α antibodies have been used to treat various diseases, including, for example, the treatment of atopic dermatitis, asthma with eosinophilic phenotype or oral corticosteroid-dependent asthma, chronic sinusitis with nasal polyposis, and have been evaluated for diseases caused by type 2 inflammation, such as pediatric atopic dermatitis, pediatric asthma, eosinophilic esophagitis, COPD, nodular pruritus, chronic spontaneous urticaria and bullous pemphigoid.Anti-IL4α antibodies have also been used to treat grass pollen allergy and peanut allergy.

[0100] In some embodiments, the antibodies encoded by the mRNAs provided herein can be used to treat one or more of atopic dermatitis with an eosinophilic phenotype, asthma with an eosinophilic phenotype or oral corticosteroid-dependent asthma, chronic rhinosinusitis with nasal polyposis, pediatric atopic dermatitis, pediatric asthma, eosinophilic esophagitis, COPD, pruritus nodularis, chronic spontaneous urticaria and bullous pemphigoid, grass pollen allergy, and peanut allergy.

[0101] Thus, in some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat atopic dermatitis. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat moderate atopic dermatitis. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat severe atopic dermatitis. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat asthma. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat severe asthma. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat moderate asthma. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat chronic sinusitis with nasal polyposis. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat pruritus nodularis. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat eosinophilic esophagitis. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat bullous pemphigoid. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat chronic spontaneous urticaria. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat COPD. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat grass pollen allergy. In some embodiments, the antibodies encoded by the anti-IL4α mRNA provided herein are used to treat peanut allergy.

[0102] Exemplary sequences of mRNA encoding the antibodies described herein are provided in the table below. In some embodiments, the nucleotide sequence is a codon optimized sequence.

[0103] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0104] Exemplary heavy and light chain antibody sequences are provided in the table below.

[0105] [Table 2-1] [Table 2-2] [Table 2-3]

[0106] As used herein, the term "antibody" encompasses both intact antibodies and antibody fragments. Typically, an intact "antibody" is an immunoglobulin that specifically binds to a particular antigen. An antibody can be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgE, IgA, and IgD. Typically, an intact antibody is a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these corresponding regions on the light and heavy chains, respectively. Each variable region can be further subdivided into a hypervariable (HV) region and a framework (FR) region. The hypervariable region consists of three hypervariable sequence regions (CDR1, CDR2 and CDR3), called complementarity determining regions, separated by four framework regions (FR1, FR2, FR2 and FR4), which form a beta sheet structure and act as a scaffold to hold the HV region in position. The C-terminus of each heavy and light chain defines a constant region consisting of one domain (CL) in the light chain and three domains (CH1, CH2 and CH3) in the heavy chain. The light chain of immunoglobulins can be further differentiated into isotypes kappa and lambda.

[0107] In some embodiments, the term "intact antibody" or "fully assembled antibody" is used in reference to an antibody that comprises two heavy chains and two light chains, optionally joined by disulfide bonds, as in a naturally produced antibody. In some embodiments, an antibody according to the invention is an antibody fragment.

[0108] In some embodiments, the present invention can be used to deliver "antibody fragments." As used herein, an "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments consisting of the variable regions of the heavy and light chains, "Fv" fragments, recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are linked by a peptide linker ("ScFv protein"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable region. In many embodiments, an antibody fragment includes sufficient sequence of a parent antibody that the fragment binds to the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs).

[0109] The present invention can be used to deliver any antibody known in the art and that can be raised against a desired antigen using standard methods. The present invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0110] Synthesis of mRNA The mRNA of the present invention can be synthesized according to any of a variety of known methods.For example, the mRNA of the present invention can be synthesized through in vitro transcription (IVT).In brief, IVT is typically 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.

[0111] In some embodiments, to prepare the mRNA according to the present invention, the DNA template is transcribed in vitro.Suitable DNA template typically has a promoter for in vitro transcription, such as T3, T7 or SP6 promoter, followed by the desired nucleotide sequence for the desired mRNA and termination signal.

[0112] Synthesis of mRNA 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 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 natural and / or modified and / or labeled ribonucleotides into the polymerized transcript. Examples of such labeled ribonucleotides include biotin-, fluorescein-, digoxigenin-, aminoallyl-, and isotope-labeled nucleotides.

[0113] The sequence of bacteriophage SP6 RNA polymerase initially consisted of the following amino acid sequence: (SEQ ID NO:1) It was described as having the following structure (GenBank: Y00105.1).

[0114] The SP6 RNA polymerase suitable for the present invention is any enzyme that has substantially the same polymerase activity as bacteriophage SP6 RNA polymerase. Thus, in some embodiments, the SP6 RNA polymerase suitable for the present invention is modified from SEQ ID NO: 1. 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 about 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: 1. In some embodiments, a suitable SP6 RNA polymerase may be a truncated protein (from the N-terminus, C-terminus, or internally) but retains polymerase activity. In some embodiments, a suitable SP6 RNA polymerase is a fusion protein.

[0115] SP6 RNA polymerase suitable for the present invention may be, for example, a commercially available product from Aldevron, Ambion, New England Biolabs (NEB), Promega and Roche. SP6 may be ordered and / or custom-designed from commercial or non-commercial sources according to the amino acid sequence of SEQ ID NO: 1 or the variant of SEQ ID NO: 1 described herein. SP6 may be a standard fidelity polymerase, or may be a high fidelity / high efficiency / high capacity modified to promote RNA polymerase activity, for example, a mutation in the SP6 RNA polymerase gene or a post-translational modification of the SP6 RNA polymerase itself. Examples of such modified SP6 include Ambion's SP6 RNA Polymerase-Plus™, NEB's HiScribe SP6, and Promega's RiboMAX™ and Riboprobe™ systems.

[0116] In some embodiments, the suitable SP6 RNA polymerase is a fusion protein. For example, the SP6 RNA polymerase may include one or more tags to facilitate the isolation, purification, or solubility of the enzyme. Suitable tags can be located at the N-terminus, C-terminus, and / or internally. Non-limiting examples of suitable 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-utilization substance (NusA); small ubiquitin-related modifier (SUMO) fusion tag; streptavidin-binding peptide (STREP); tandem affinity purification (TAP); and thioredoxin (TrxA). Other tags can be used in the present invention. These and other fusion tags are described, for example, in Costa et al., Frontiers in Microbiology 5 (2014):63 and PCT / US16 / 57044, the contents of which are incorporated by reference herein in their entireties. In certain embodiments, the His tag is located at the N-terminus of SP6.

[0117] DNA template Typically, the DNA template is either fully double-stranded or mostly single-stranded with the double-stranded SP6 promoter sequence.

[0118] 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 with SP6, provided that they contain a double-stranded SP6 promoter upstream (and in the forward orientation) of the DNA sequence to be transcribed.

[0119] In some embodiments, the linearized DNA template has blunt ends.

[0120] In some embodiments, the transcribed DNA sequence is optimized to promote more efficient transcription and / or translation. For example, the DNA sequence is optimized for cis-regulatory elements (e.g., TATA boxes, termination signals and protein binding sites), artificial recombination sites, Chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements related to transcription; the DNA sequence is optimized for potential splice sites, mRNA secondary structures, stable free energy of mRNA, repetitive sequences, RNA instability motifs, and / or other elements related to mRNA processing and stability; the DNA sequence is optimized for codon usage bias, codon compatibility, internal Chi sites, ribosome binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA sequence is optimized for codon context, codon-anticodon interactions, translation pause sites, and / or other elements related to protein folding. Optimization methods known in the art can be used in the present invention, such as GeneOptimizer by ThermoFisher and OptimumGene™, described in U.S. Patent Application Publication No. 2011 / 0081708, which is incorporated by reference in its entirety.

[0121] In some embodiments, the DNA template comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length.

[0122] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA in its location within a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be between 50-500 nucleotides in length or more.

[0123] Exemplary 3' and / or 5' UTR sequences can be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, the 5' UTR sequence can include a subsequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the polynucleotide. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. In general, these modifications include modifications made to improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to its unmodified counterpart, e.g., to improve the resistance of such polynucleotides to in vivo nuclease digestion.

[0124] Large-scale mRNA synthesis In some embodiments, the present invention can be used for large-scale production of stable LNP-encapsulated mRNA. In some embodiments, the method according to the present invention synthesizes 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, 250 g, 500 g, 750 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, e.g., the content or amount of mRNA produced according to a single manufacturing setup. A batch can refer to the amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and / or a single aliquot of DNA template for continuous synthesis under one set of conditions. mRNA synthesized in a single batch does not include mRNAs synthesized at different times that are combined to achieve a desired amount. In general, the reaction mixture includes SP6 RNA polymerase, a linear DNA template, and an RNA polymerase reaction buffer (which may contain ribonucleotides or may require the addition of ribonucleotides).

[0125] According to the present invention, 1-100 mg of SP6 polymerase is typically used per gram (g) of mRNA produced. In some embodiments, about 1-90 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 10-100 mg, 10-80 mg, 10-60 mg, 10-50 mg of SP6 polymerase is used per gram of mRNA produced. In some embodiments, about 5-20 mg of SP6 polymerase is used to produce about 1 gram of mRNA. In some embodiments, about 0.5-2 grams of SP6 polymerase is used to produce about 100 grams of mRNA. In some embodiments, about 5-20 grams of SP6 polymerase is used to produce about 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, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg of plasmid DNA are used per gram of mRNA produced. In some embodiments, about 10-30 mg of plasmid DNA are used to produce about 1 gram of mRNA. In some embodiments, about 1-3 grams of plasmid DNA are used to produce about 100 grams of mRNA. In some embodiments, about 10-30 grams of plasmid DNA are 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.

[0126] In some embodiments, the concentration of SP6 RNA polymerase in the reaction mixture can 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. Concentrations of SP6 RNA polymerase in the range 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 can be used.

[0127] The concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture is about 0.1 mM to about 10 mM, e.g., between about 1 mM to about 10 mM, between about 2 mM to about 10 mM, between about 3 mM to about 10 mM, between about 1 mM to about 8 mM, between about 1 mM to about 6 mM, between about 3 mM to about 10 mM, between about 3 mM to about 8 mM, between about 3 mM to about 6 mM, between about 4 mM to about 5 mM. In some embodiments, each ribonucleotide is about 5 mM in the reaction mixture. In some embodiments, the total concentration of rNTPs (e.g., a combination of ATP, GTP, CTP, and UTP) used in the reaction ranges between 1 mM and 40 mM. In some embodiments, the total concentration of rNTPs (e.g., a combination of ATP, GTP, CTP, and UTP) used in the reaction ranges between 1 mM and 30 mM, or between 1 mM and 28 mM, or between 1 mM and 25 mM, or between 1 mM and 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.

[0128] 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.

[0129] The pH of the reaction mixture can be about 6-8.5, 6.5-8.0, 7.0-7.5, and in some embodiments, the pH is 7.5.

[0130] Combine the linearized or straightened DNA template (e.g., as described above, in a sufficient amount / concentration to provide the desired amount of RNA), RNA polymerase reaction buffer, and SP6 RNA polymerase to form a reaction mixture. Incubate the reaction mixture at about 37°C to about 42°C for 30 minutes to 6 hours, e.g., about 60 to about 90 minutes.

[0131] In some embodiments, about 5 mM NTPs, about 0.05 mg / mL SP6 polymerase, and about 0.1 mg / ml DNA template in a suitable RNA polymerase reaction buffer (pH of the final reaction mixture is about 7.5) are incubated at about 37° C. to about 42° C. for 60 to 90 minutes.

[0132] In some embodiments, the reaction mixture contains an SP6 polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs, 10 mM DTT, and reaction buffer (800 mM HEPES, 20 mM spermidine, 250 mM MgCl for 10×). 2 The reaction mixture is then incubated at 37° C. for 60 minutes. The polymerase reaction is then incubated with DNase I and DNase I buffer (100 mM Tris-HCl, 5 mM MgCl for 10×). 2 and 25 mM CaCl 2 , pH 7.6) to facilitate digestion of double-stranded DNA templates in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[0133] In some embodiments, the reaction mixture comprises NTPs at a concentration ranging from 1 to 10 mM, DNA template at a concentration ranging from 0.01 to 0.5 mg / ml, and SP6 RNA polymerase at a concentration ranging from 0.01 to 0.1 mg / ml, for example, the reaction mixture comprises NTPs at a concentration of 5 mM, DNA template at a concentration of 0.1 mg / ml, and SP6 RNA polymerase at a concentration of 0.05 mg / ml.

[0134] nucleotide A variety of naturally occurring or modified nucleosides can be used to generate the mRNA according to the present invention. In some embodiments, the mRNA can be modified with any of the following nucleosides: natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanidine ... The base may be or contain: anosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0135] In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. The non-standard nucleotide residues may include, for example, 5-methyl-cytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thio-uridine ("2sU"). For a discussion of such residues and their incorporation into mRNA, see, for example, U.S. Pat. No. 8,278,036 or WO 2011 / 012316. The mRNA may be RNA, defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of RNA are disclosed in U.S. Pat. App. Pub. No. 2012 / 0195936 and WO 2011 / 012316, both of which are incorporated herein by reference in their entireties. The presence of non-standard nucleotide residues can make an mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only standard residues. In further embodiments, the mRNA can contain one or more non-standard nucleotide residues selected from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, and combinations of these and other nucleobase modifications. Some embodiments can further include additional modifications to the furanose ring or nucleobase. Additional modifications include, for example, sugar modifications or substitutions (e.g., one or more of 2'-O-alkyl modifications, locked 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, a 2'-deoxy-2'-fluoro modification, a 2'-O-methyl modification, a 2'-O-methoxyethyl modification, and a 2'-deoxy modification.In some embodiments, any of these modifications can be present in 0-100% of the nucleotides - for example, individually or in combination, 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, greater than 95% or 100% of the constituent nucleotides.

[0136] Post-synthesis processing Typically, a 5' cap and / or a 3' tail can be added post-synthetically. The presence of a cap is important to provide resistance to nucleases found in most eukaryotic cells. The presence of a "tail" helps protect the mRNA from exonuclease degradation.

[0137] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylate transferase, generating a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in published U.S. Patent Application Publication No. 2016 / 0032356, filed Feb. 27, 2017, and U.S. Provisional Patent Application No. 62 / 464,327, incorporated herein by reference.

[0138] Typically, the tail structure comprises a poly(A) and / or poly(C) tail. The polyA or polyC tail on the 3' end of the mRNA typically comprises at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, 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 polyA or polyC tail comprises about 10-800 adenosine or cytosine nucleotides, respectively (e.g., about 10-200 adenosine or cytosine nucleotides, about 10-300 adenosine or cytosine nucleotides, about 10-400 adenosine or cytosine nucleotides, about 10-500 adenosine or cytosine nucleotides, about 10-550 adenosine or cytosine nucleotides, about 10-600 adenosine or cytosine nucleotides, about 50-600 adenosine or cytosine nucleotides, about 100-600 adenosine or cytosine nucleotides, about 150-600 adenosine or cytosine nucleotides, about 200-60 0 adenosine or cytosine nucleotides, about 250-600 adenosine or cytosine nucleotides, about 300-600 adenosine or cytosine nucleotides, about 350-600 adenosine or cytosine nucleotides, about 400-600 adenosine or cytosine nucleotides, about 450-600 adenosine or cytosine nucleotides, about 500-600 adenosine or cytosine nucleotides, about 10-150 adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides). In some embodiments, the tail structure comprises a combination of poly(A) tails and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure comprises 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 comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0139] As described herein, the addition of a 5' cap and / or a 3' tail facilitates the detection of aborted transcripts produced during in vitro synthesis, since in the absence of capping and / or tailing, the size of premature aborted mRNA transcripts is too small to be detected. Thus, in some embodiments, a 5' cap and / or a 3' tail is added to the synthesized mRNA before the mRNA is tested for purity (the level of aborted transcript present in the mRNA). In some embodiments, a 5' cap and / or a 3' tail is added to the synthesized mRNA before the mRNA is purified as described herein. In other embodiments, a 5' cap and / or a 3' tail is added to the synthesized mRNA after the mRNA is purified as described herein.

[0140] The mRNA synthesized according to the present invention can be used without further purification. In particular, the mRNA synthesized according to the present invention can be used without a step of removing shortmers. In some embodiments, the mRNA synthesized according to the present invention can be further purified. Various methods can be used to purify the mRNA synthesized according to the present invention. For example, purification of the mRNA can be performed using centrifugation, filtration and / or chromatography methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, well known to those skilled in the art. In some embodiments, the mRNA is purified using tangential flow filtration. Suitable purification methods include those described in U.S. Patent Application No. 2016 / 0040154, filed February 27, 2018, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA," U.S. Patent Application No. 2015 / 0376220, PCT Application No. PCT / US18 / 19954, and PCT Application No. PCT / US18 / 19978, filed February 27, 2018, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA," all of which are incorporated herein by reference and can be used to practice the present invention.

[0141] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.

[0142] In some embodiments, the mRNA is purified by centrifugation either before or after capping and tailing, or both before and after capping and tailing.

[0143] In some embodiments, the mRNA is purified by filtration either before or after capping and tailing, or both before and after capping and tailing.

[0144] In some embodiments, the mRNA is purified by tangential flow filtration (TFF) either before or after capping and tailing, or both.

[0145] In some embodiments, the mRNA is purified by chromatography either before, after, or both before and after capping and tailing.

[0146] mRNA characterization The full-length or interrupted transcripts of mRNA can be detected and quantified using any method available in the art. In some embodiments, the 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, the synthesized mRNA molecules are detected using UV absorption spectroscopy with separation by capillary electrophoresis. In some embodiments, the mRNA is first denatured by glyoxal dye before gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, the synthesized mRNA is characterized before capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing.

[0147] In some embodiments, the mRNA produced by the methods disclosed herein contains 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%, less than 0.1% of impurities other than full-length mRNA. Impurities include IVT contaminants, such as proteins, enzymes, free nucleotides, and / or shortmers.

[0148] In some embodiments, the mRNA produced according to the present invention is substantially free of shortmers and interrupted transcripts. In particular, the mRNA produced according to the present invention contains undetectable levels of shortmers or interrupted transcripts by capillary electrophoresis or glyoxal gel electrophoresis. As used herein, the term "shortmer" or "interrupted transcript" refers to any transcript that is less than full length. In some embodiments, a "shortmer" or "interrupted transcript" is less than 100 nucleotides, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length. In some embodiments, the shortmers are detected or quantified after adding a 5'-cap and / or a 3'-polyA tail.

[0149] mRNA solution In some embodiments, mRNA can be provided in a solution that is mixed with lipid solution so that mRNA can be encapsulated in lipid nanoparticles.Suitable mRNA solution can be any aqueous solution that contains the mRNA to be encapsulated at various concentrations.For example, suitable mRNA solution can contain mRNA at a concentration of about 0.01mg / ml, 0.05mg / ml, 0.06mg / ml, 0.07mg / ml, 0.08mg / ml, 0.09mg / ml, 0.1mg / ml, 0.15mg / ml, 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.7mg / ml, 0.8mg / ml, 0.9mg / ml, or 1.0mg / ml or greater. In some embodiments, a suitable mRNA solution may be about 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 The mRNA may be contained at a concentration 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 a concentration of up to about 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.

[0150] Typically, a suitable mRNA solution may also contain a buffer and / or salt. Generally, the buffer may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. In some embodiments, a suitable concentration of the buffer may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, a suitable concentration of a buffering agent is about 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.

[0151] Exemplary salts include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, a suitable concentration of salt in an 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. A suitable salt concentration in an mRNA solution is about or greater than 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.

[0152] In some embodiments, a suitable mRNA solution can have a pH in the range of about 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 can have a pH of about 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.

[0153] A variety of methods can be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA can be dissolved directly in a buffer solution as described herein. In some embodiments, mRNA solutions can be made by mixing mRNA stock solutions with a buffer solution before mixing with lipid solution for encapsulation. In some embodiments, mRNA solutions can be made by mixing mRNA stock solutions with a buffer solution immediately before mixing with lipid solution for encapsulation. In some embodiments, suitable mRNA stock solutions can contain mRNA in water at a concentration of about 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 more.

[0154] In some embodiments, the mRNA stock solution is mixed with the buffer using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0155] Typically, the buffer is mixed at a flow rate that exceeds the flow rate of the mRNA stock solution. For example, the buffer is mixed at a flow rate that is at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the flow rate of the mRNA stock solution. In some embodiments, the buffer is mixed at a flow rate in the range of between about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or more.

[0156] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml / min (e.g., about 5-50 ml / min, about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or more.

[0157] Delivery Vehicle The stable lipid nanoparticle formulations described herein are suitable as delivery vehicles for mRNA.

[0158] As used herein, the terms "delivery vehicle," "introduction vehicle," "nanoparticle" or grammatical equivalents are used interchangeably.

[0159] The delivery vehicle is formulated into a pharmacological composition that is combined with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or mixed with suitable excipients.Techniques for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition.A particular delivery vehicle is selected based on its ability to facilitate the transfection of nucleic acid into target cells.

[0160] Liposomal Delivery Vehicles In some embodiments, the suitable delivery vehicle is a liposomal delivery vehicle, such as a lipid nanoparticle. As used herein, a liposomal delivery vehicle, such as a lipid nanoparticle, is typically characterized as a microscopic vesicle with an internal aqueous space isolated from the external medium by one or more bilayer membranes. The bilayer membrane of a liposome is typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin, that contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of a liposome is also formed by amphiphilic polymers and surfactants (e.g., polymersomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport the desired mRNA to a target cell or tissue. 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 less distinct lipid components.In some embodiments, one distinct lipid component is a sterol-based cationic lipid.

[0161] Cationic lipids As used herein, the phrase "cationic lipid" refers to any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0162] Cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2010 / 144740, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include: [ka] The cationic lipid has the compound structure: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and pharma- ceutically acceptable salts thereof.

[0163] Other cationic lipids suitable for use in the compositions and methods of the invention include the ionizable cationic lipids described in WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise an ionizable cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof, 1 and R 2 is hydrogen, optionally substituted, variably saturated or unsaturated C 1 ~C 20 Alkyl and optionally substituted, variably saturated or unsaturated C 6 ~C 20 acyl; L 1 and L 2 is hydrogen, optionally substituted C 1 ~C 30 Alkyl, optionally substituted, variably unsaturated C 1 ~C 30Alkenyl, and optionally substituted C 1 ~C 30 alkynyl; m and o are each independently selected from the group consisting of 0 and any positive integer (e.g., m is 3); n is 0 or any positive integer (e.g., n is 1). In certain embodiments, the compositions and methods of the present invention include: [ka] The cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") has the compound structure: [ka] The cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") has the compound structure: [ka] and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002"), and pharma-ceutically acceptable salts thereof.

[0164] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids described as amino alcohol lipidoids in WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include: [ka] The present invention includes cationic lipids having the compound structure:

[0165] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include: [ka] The present invention includes cationic lipids having the compound structure:

[0166] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include: [ka] The present invention includes cationic lipids having the compound structure:

[0167] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharma- ceutically acceptable salts thereof.

[0168] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise cationic lipids having the following formula: [ka] or a pharma- ceutically acceptable salt thereof (wherein R L Each instance of is independently an optionally substituted C 6 ~C 40 In certain embodiments, the compositions and methods of the present invention include [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] The present invention includes cationic lipids having the compound structure:

[0169] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof (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 Aare 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 are 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. In certain embodiments, the compositions and methods of the invention include [ka] The present invention includes a cationic lipid, "Target 23," having the compound structure:

[0170] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid having the compound structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0171] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following formula: [ka] or a pharma- ceutical acceptable salt thereof (wherein each R 1 and R 2 are independently H or C 1 ~C 6 each m is independently an integer having a value from 1 to 4; each A is independently a covalent bond or arylene; each L 1 is independently an ester, thioester, disulfide, or anhydride group; each L 2 are independent, C 2 ~C 10 is aliphatic; each X 1 is independently H or OH; each R 3 are independent, C 6 ~C 20 In some embodiments, the compositions and methods of the present invention include a cyclic alkyl group having the following formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0172] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In certain embodiments, the cationic lipid of the compositions and methods of the invention is [ka] The present invention includes cationic lipids having the compound structure:

[0173] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0174] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0175] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof (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-;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 a direct bond; G 1 and G 2 each independently represents an unsubstituted C 1 ~C 12 Alkylene or C 1 ~C 12 Alkenylene; G 3 is C 1 ~C 24 Alkylene, C 1 ~C 24 Alkenylene, C 3 ~C 8 Cycloalkylene, C 3 ~C 8 Cycloalkenylene; R a is H or C 1 ~C 12 R is alkyl; 1 and R 2 are each independently, C 6 ~C 24 Alkyl or C 6 ~C 24 alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;R 4 is C 1 ~C 12 R is alkyl; 5 is H or C 1 ~C 6 alkyl; and x is 0, 1 or 2.

[0176] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0177] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the invention has the following formula: [ka] and pharma- ceutically acceptable salts thereof. For any one of these four formulas, R 4 Ha-(CH 2 ) n Q and -(CH 2 ) n CHQR; Q is -OR, -OH, -O(CH 2 ) n N(R) 2 , -OC(O)R, -CX 3 , -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O) 2 R, -N(H)S(O) 2R, -N(R)C(O)N(R) 2 , -N(H)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 heterocycle; and n is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] The present invention includes cationic lipids having the compound structure:

[0178] Other cationic lipids suitable for use in the compositions and methods of the invention include those described in WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having a compound structure: [ka] The present invention includes cationic lipids having the compound structure:

[0179] Other cationic lipids suitable for use in the compositions and methods of the invention include the cleavable cationic lipids described in WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cleavable cationic lipid having the following formula: [ka] Cationic lipids (In the formula, R 1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; R 2 is the following two expressions: [ka] and R 3 and R 4 is an optionally substituted, variably saturated or unsaturated C 6 ~C 20Alkyl and optionally substituted, variably saturated or unsaturated C 6 ~C 20 acyl; 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 include [ka] In certain embodiments, the compositions and methods of the present invention include a cationic lipid, "HGT4001," having the compound structure: [ka] The cationic lipid "HGT4002" (also referred to herein as "Guan-SS-Chol") has the compound structure: and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise: [ka] In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid, "HGT4003," having the compound structure: [ka] In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid, "HGT4004," having the compound structure: [ka] The present invention includes a cationic lipid "HGT4005" having the compound structure:

[0180] Other cationic lipids suitable for use in the compositions and methods of the invention include the cleavable cationic lipids described in U.S. Provisional Patent Application No. 62 / 672,194, filed May 16, 2018, and incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include a cationic lipid having any of the general formulas described in U.S. Provisional Patent Application No. 62 / 672,194 or any of structures (1a)-(21a) and (1b)-(21b) and (22)-(237). In certain embodiments, the compositions and methods of the invention include a cationic lipid having a structure according to formula (I'): [ka] (In the formula, R X are independent, -H, -L 1 -R 1 , or -L 5A -L 5B -B'; L 1 , L 2 , and L 3 each independently is a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L - and; Each L 4A and L 5A are independently -C(O)-, -C(O)O-, or -C(O)NR L - and; Each L 4B and L 5B are independent, C 1 ~C 20 Alkylene;C 2 ~C 20 Alkenylene; or C 2 ~C 20 alkynylene; Each B and B' is NR 4 R 5 or a 5-10 membered nitrogen-containing heteroaryl; Each R 1 , R 2 , and R 3 are independent, C 6 ~C 30Alkyl, C 6 ~C 30 Alkenyl, or C 6 ~C 30 is alkynyl; Each R 4 and R 5 are independently hydrogen, C 1 ~C 10 Alkyl;C 2 ~C 10 alkenyl; or C 2 ~C 10 is alkynyl; Each R L are independently hydrogen, C 1 ~C 20 Alkyl, C 2 ~C 20 Alkenyl, or C 2 ~C 20 alkynyl) Includes.

[0181] In certain embodiments, the compositions and methods of the present invention comprise: [ka] The cationic lipid is compound (139), which has the compound structure 62 / 672,194.

[0182] In some embodiments, the compositions and methods of the invention comprise the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA") (Feigner et al., Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, incorporated herein by reference). Other cationic lipids suitable for the compositions and methods of the invention include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium ("DOSPA") (Behr et al., Proc. Nat. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, incorporated herein by reference). Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761; 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0183] 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 N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholest-5-ene-3-beta-o N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA");(2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyh3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane- 1-amine ("Octyl-CLinDMA(2S)"); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (International Publication WO 2010 / 042877; Semple et al., Nature 2010, 133:1111-1111, incorporated herein by reference). 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); WO 2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole, dialkylamino, or guanidinium moiety. In some embodiments, the 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");

[0184] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The compound includes a cationic lipid, TL1-04D-DMA, having the compound structure:

[0185] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The cationic lipid is GL-TES-SA-DME-E18-2 having the compound structure:

[0186] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The compound includes a cationic lipid, SY-3-E14-DMAPr, having the compound structure:

[0187] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The compound includes a cationic lipid, TL1-01D-DMA, having the compound structure:

[0188] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The compound includes a cationic lipid, TL1-10D-DMA, having the compound structure:

[0189] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The cationic lipid is GL-TES-SA-DMP-E18-2 having the compound structure:

[0190] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The cationic lipid is HEP-E4-E10 having the compound structure:

[0191] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include: [ka] The cationic lipid is HEP-E3-E10 having the compound structure:

[0192] In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., lipid nanoparticles, measured by weight. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., lipid nanoparticles, measured as mol%. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition, e.g., lipid nanoparticles, measured by weight. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition, e.g., lipid nanoparticles, measured as mol%.

[0193] Non-cationic / helper lipids In some embodiments, the liposomes provided contain one or more non-cationic ("helper") lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine (DPPG), dioleoyl ... amine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof.

[0194] In some embodiments, such non-cationic lipids are used alone, but are preferably used in combination with other lipids, such as cationic lipids. In some embodiments, the non-cationic lipids may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipids present in the liposome. In some embodiments, the non-cationic 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 non-cationic lipids in the liposomes can be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0195] Cholesterol-based lipids In some embodiments, the liposomes provided include one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al., BioTechniques vol. 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid may comprise a molar ratio of about 2% to about 30%, or about 5% to about 20%, of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticles can be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0196] PEG modified lipid The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including, for example, N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), alone or preferably in combination with other lipid formulations, including delivery vehicles (e.g., lipid nanoparticles), is also contemplated by the present invention. Contemplated PEG-modified lipids include, but are not limited to, C6 ~C 20 These include polyethylene glycol chains of up to SkDa length covalently attached to lipids with long alkyl chains. The addition of such moieties can also provide a means to prevent complex aggregation, increase circulatory lifetime, and increase delivery of lipid-nucleic acid compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they are selected to rapidly exchange from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 The PEG-modified phospholipid and derivatized lipid of the present invention can constitute a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome-introduced vehicle.

[0197] According to various embodiments, the selection of cationic lipid, non-cationic lipid, and / or PEG-modified lipid that constitutes lipid nanoparticles, and the relative molar ratio of these lipids to each other are based on the characteristics of the selected lipid, the nature of the intended target cell, and the characteristics of the MCNA to be delivered.Additional considerations include, for example, the saturation of alkyl chain, and the size, charge, pH, pKa, membrane fusogenicity and toxicity of the selected lipid.Therefore, the molar ratio can be adjusted accordingly.

[0198] polymer In some embodiments, suitable delivery vehicles are formulated using polymers as carriers, alone or in combination with other carriers, including various lipids, as described herein. Thus, in some embodiments, liposome delivery vehicles, as used herein, also encompass nanoparticles containing polymers. Suitable polymers include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethyleneimine (PEI). When PEI is present, it can be a branched PEI with a molecular weight in the range of 10-40 kDa, for example, a 25 kDa branched PEI (Sigma No. 408727).

[0199] Liposomes suitable for use according to the present invention The liposome suitable for the present invention can comprise any one or more of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids and / or polymers described herein in various ratios.As a non-limiting example, suitable liposome formulations can comprise a combination selected from 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 ICE, DOPE and DMG-PEG2K.

[0200] In various embodiments, the cationic lipid (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) constitutes 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 liposome by molar ratio. In some embodiments, the percentage of cationic lipid (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) is or exceeds about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome by molar ratio.

[0201] In some embodiments, the ratio of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids is between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids is approximately 50:25:20:5.

[0202] 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 that are 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 via nebulization. Of 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.

[0203] 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 non-cationic lipid components, cholesterol as helper lipid components, and one of DMG-PEG2K as PEG-modified lipid components. In some embodiments, the molar ratios of cationic lipid, non-cationic lipid, cholesterol and PEG-modified lipid can be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the molar ratios of cationic lipid, non-cationic lipid, cholesterol and PEG-modified lipid are approximately 40:30:20:10, respectively. In some embodiments, the molar ratio of cationic lipid, non-cationic lipid, cholesterol, and PEG-modified lipid is approximately 40:30:25:5, respectively. In some embodiments, the molar ratio of cationic lipid, non-cationic lipid, cholesterol, and PEG-modified lipid is approximately 40:32:25:3, respectively. In some embodiments, the molar ratio of cationic lipid, non-cationic lipid, cholesterol, and PEG-modified lipid is approximately 50:25:20:5.

[0204] In some embodiments, the lipid components of the 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 lipid to non-cationic lipid to PEG-modified lipid is approximately 60:35:5.

[0205] Ratio of separate lipid components In embodiments where the lipid nanoparticle comprises three or less than three 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 It is.

[0206] In some embodiments, each of "x," "y," and "z" represents the molar percentage of three separate components of the lipid, and the ratios are molar ratios.

[0207] In some embodiments, "x," "y," and "z" each represent the weight percentage of three separate components of lipid, and the ratios are weight ratios.

[0208] In some embodiments, the lipid component (1), represented by the variable "x", is a sterol-based cationic lipid.

[0209] In some embodiments, lipid component (2), represented by the variable "y", is a helper lipid.

[0210] In some embodiments, the lipid component (3), represented by the variable "z", is a PEG lipid.

[0211] In some embodiments, the variable "x", which represents the molar percentage of lipid component (1) (e.g., sterol-based cationic lipid), 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%.

[0212] In some embodiments, the variable "x", which represents the molar percentage of lipid component (1) (e.g., sterol-based cationic lipid), is about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10% or less. In embodiments, the variable "x" is about 65%, about 60%, about 55%, about 50%, about 40% or less.

[0213] In some embodiments, the variable "x", which represents the molar percentage of lipid component (1) (e.g., sterol-based cationic lipid), 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%.

[0214] In some embodiments, the variable "x", which represents the weight percentage of lipid component (1) (e.g., sterol-based cationic lipid), 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%.

[0215] In some embodiments, the variable "x", which represents the weight percentage of lipid component (1) (e.g., sterol-based cationic lipid), is about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10% or less. In embodiments, the variable "x" is about 65%, about 60%, about 55%, about 50%, about 40% or less.

[0216] In some embodiments, the variable "x", which represents the weight percentage of the lipid component (1) (e.g., sterol-based cationic lipid), 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%.

[0217] In some embodiments, the variable "z" representing the molar percentage of lipid component (3) (e.g., PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% or less. In embodiments, the variable "z" representing the molar percentage of lipid component (3) (e.g., PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, the variable "z", representing the molar percentage of lipid component (3) (e.g., a PEG lipid), is from about 1% to about 10%, from about 2% to about 10%, from about 3% to about 10%, from about 4% to about 10%, from about 1% to about 7.5%, from about 2.5% to about 10%, from about 2.5% to about 7.5%, from about 2.5% to about 5%, from about 5% to about 7.5%, or from about 5% to about 10%.

[0218] In some embodiments, the variable "z" representing the weight percentage of lipid component (3) (e.g., PEG lipid) is about 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 lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, the variable "z", representing the weight percentage of lipid component (3) (e.g., PEG lipid), is from about 1% to about 10%, from about 2% to about 10%, from about 3% to about 10%, from about 4% to about 10%, from about 1% to about 7.5%, from about 2.5% to about 10%, from about 2.5% to about 7.5%, from about 2.5% to about 5%, from about 5% to about 7.5%, or from about 5% to about 10%.

[0219] For compositions having three and only three distinct lipid components, the variables "x," "y," and "z" can be any combination as long as the sum of the three variables totals 100% of the total lipid content.

[0220] Formation of liposomes encapsulating mRNA The liposome-introducing vehicle for use in the compositions of the present invention can be prepared by various techniques currently known in the art. The liposome for use in the compositions provided can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by dissolving selected lipids in a suitable solvent on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film in the vessel or spray drying. Aqueous phase can then be added to the vessel with a vortex motion, which results in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenizing, sonicating or extruding the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0221] In certain embodiments, the provided composition comprises liposomes in which mRNA is associated on both surfaces of the liposome and encapsulated in the liposome.For example, during the preparation of the composition of the present invention, cationic liposomes can be associated with mRNA through electrostatic interaction.For example, during the preparation of the composition of the present invention, cationic liposomes can be associated with mRNA through electrostatic interaction.

[0222] 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, the mRNA is encapsulated in one or more liposomes that differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligand, and / or combinations thereof. In some embodiments, one or more liposomes can have different compositions of sterol-based cationic lipids, neutral lipids, PEG-modified lipids, and / or combinations thereof. In some embodiments, one or more liposomes can have different molar ratios of cholesterol-based cationic lipids, neutral lipids, and PEG-modified lipids used to make the liposomes.

[0223] The method of incorporating a desired mRNA into liposomes is often referred to as "loading". Exemplary methods are described in Lasic et al., FEBS Lett., vol. 312:255-258, 1992, which is incorporated herein by reference. The nucleic acid incorporated into the liposome can be located completely or partially within the interior space of the liposome, within the bilayer membrane of the liposome, or completely or partially associated with the outer surface of the liposome membrane. The incorporation of a nucleic acid into a liposome is also referred to herein as "encapsulation", where the nucleic acid is contained completely within the interior space of the liposome. The purpose of incorporating an mRNA into a delivery vehicle such as a liposome is often to protect the nucleic acid from an environment that may contain enzymes or chemicals that degrade the nucleic acid and / or the system or receptor, causing rapid excretion 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 the mRNA to a target cell or tissue.

[0224] Suitable liposomes according to the present invention can be made in various sizes. In some embodiments, the provided liposomes can be smaller than liposomes that encapsulate known mRNA. In some embodiments, the reduction in size of liposomes is associated with more efficient delivery of mRNA. The selection of suitable liposome size can take into account the site of target cells or tissues, and to some extent, the application for which the liposomes are made.

[0225] In some embodiments, liposomes of appropriate size are selected to promote the systemic distribution of the antibody encoded by mRNA.In some embodiments, it may be desirable to restrict the transfection of mRNA to a certain cell or tissue.For example, to target hepatocytes, liposomes can be sized so that their dimensions are smaller than the fenestrated endothelial layer that lines hepatic sinusoids in the liver.In such a case, liposomes can easily penetrate such endothelial fenestrated areas to reach the target hepatocytes.

[0226] Alternatively or additionally, liposomes can be sized such that the dimensions of the liposomes are of a diameter sufficient to limit or explicitly avoid distribution to certain cells or tissues.

[0227] A variety of alternative methods known in the art are available for sizing liposome populations. One such sizing method is described in U.S. Pat. No. 4,737,323, which is incorporated herein by reference. Sonication of the liposome suspension, either by bath or probe sonication, results in a gradual size reduction down to small ULVs less than about 0.05 microns in diameter. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller liposomes. In a typical homogenization procedure, MLVs are recirculated through a standard emulsion homogenizer until selected liposome sizes are observed, typically between about 0.1 and 0.5 microns. Liposome size can be determined by quasi-electric light scattering (QELS), as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), which is incorporated herein by reference. Sonication of the formed liposomes can reduce the average liposome diameter. To guide efficient liposome synthesis, intermittent sonication cycles can be alternated with QELS assessment.

[0228] Therapeutic Uses of the Compositions In one aspect, the present invention provides, inter alia, LNP formulations containing encapsulated mRNA useful for therapeutic purposes. For example, in some embodiments, the LNP-encapsulated mRNA encodes an antibody for the treatment of a disease in a subject, such as an immune disease.

[0229] In some embodiments, the mRNA is codon optimized. Various codon optimization methods are known in the art.

[0230] spray The effectiveness of nebulizing a pharmaceutical composition for pulmonary delivery depends on the size of the small aerosol droplets. In general, the smaller the droplet size, the greater the chance of lung penetration and retention. Large droplets (greater than 10 μm in diameter) are most likely to be deposited in the oral cavity and throat, medium droplets (5-10 μm in diameter) are most likely to be deposited between the mouth and airways, and small droplets (less than 5 μm in diameter) are most likely to be deposited and remain in the lungs.

[0231] Inhaled aerosol droplets with particle sizes between 1 and 5 μm can penetrate the narrow branches of the lower respiratory tract. Larger diameter aerosol droplets are typically absorbed by the epithelial cells lining the oral cavity and are unlikely to reach the lower respiratory tract epithelium and deep alveolar tissue.

[0232] Particle size in an aerosol is generally described with reference to the mass median aerodynamic diameter (MMAD). The MMAD, together with the geometric standard deviation (GSD), statistically describes the particle size distribution of any aerosol based on the weight and size of the particles. Means for calculating the MMAD of an aerosol are well known in the art.

[0233] A specific method for calculating MMAD using a cascade impactor was first reported by Mitchell et al. in 1959. The cascade impactor for measuring particle size is composed of a series of jets, each followed by an impact slide, and is based on the principle that particles in a moving air stream will impact on a slide placed in their path if their momentum is sufficient to overcome the drag force exerted by the air stream as it moves around the slide. As the aerosol passes through the impactor, the velocity of the air stream, and therefore the velocity of the dispersed particles, increases because each jet is smaller than the previous one. As a result, smaller particles eventually gain enough momentum to impact the slide, and complete particle size classification of the aerosol is achieved. The improved next-generation impactor used herein to measure the MMAD of the pharmaceutical composition of the present invention was first reported by Marple et al. in 2003 and is widely used in pharmacopoeias.

[0234] Another parameter for describing particle size in aerosol is volume median diameter (VMD). VMD also describes the particle size distribution of aerosol based on the volume of particles. The means for calculating the VMD of aerosol are well known in the art. The specific method used to determine VMD is laser diffraction, which is used herein to measure the VMD of pharmaceutical compositions of the present invention (see, for example, Clark, 1995, Int J Pharm. 115:69-78).

[0235] In some embodiments, the average particle size of the nebulized pharmaceutical composition is about 4 μm to 6 μm, for example, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, or about 6 μm.

[0236] Fine particle fraction (FPF) is defined as the proportion of particles in an aerosol that have a MMAD or VMD less than a specified value. In some embodiments, the FPF of a nebulized pharmaceutical composition having a particle size of less than 5 μm is at least about 30%, more typically at least about 40%, such as at least about 50%, more typically at least about 60%.

[0237] In some embodiments, nebulization is performed such that the average inhalable emitted dose (i.e., the percentage of FPFs with particle sizes less than 5 μm; e.g., as determined by a next-generation impactor with 15 L / min extraction) is at least about 30% of the emitted dose, e.g., at least about 31%, at least about 32%, at least about 33%, at least about 34%, or at least about 35%. In some embodiments, nebulization is performed such that the average inhalable delivered dose (i.e., the percentage of FPFs with particle sizes less than 5 μm; e.g., as determined by a next-generation impactor with 15 L / min extraction) is at least about 15% of the emitted dose, e.g., at least 16% or 16.5% of the emitted dose.

[0238] sprayer Nebulization can be achieved by any nebulizer known in the art. Nebulizers convert liquid into mist, making it easier for the lungs to inhale. Nebulizers are effective for infants, children and adults. Nebulizers can nebulize large doses of inhaled medication. Typically, nebulizers for use with the present invention include a removable mouthpiece. This is important because only RNase-free, clear mouthpieces should be used when administering the pharmaceutical composition of the present invention.

[0239] In some embodiments, the reservoir volume of the sprayer ranges from about 5.0 mL to about 8.0 mL. In some embodiments, the reservoir volume of the sprayer is about 5.0 mL. In some embodiments, the reservoir volume of the sprayer is about 6.0 mL. In some embodiments, the reservoir volume of the sprayer is about 7.0 mL. In some embodiments, the reservoir volume of the sprayer is about 8.0 mL.

[0240] One type of nebulizer is a jet nebulizer, which includes a tube connected to a compressor that flows compressed air or oxygen at high velocity through a liquid medicine, turning it into an aerosol, which is then inhaled by the patient.

[0241] Another type of atomizer is the ultrasonic atomizer, which includes an electronic oscillator that generates high-frequency ultrasonic waves that cause mechanical vibration of a piezoelectric element in contact with a liquid reservoir. The high-frequency vibration of the liquid is sufficient to generate a vapor mist. Exemplary ultrasonic atomizers are the Omron NE-U17 and the Beurer atomizer IH30.

[0242] A third type of nebulizer is a mesh nebulizer, such as a vibrating mesh nebulizer that includes vibrating mesh technology (VMT). VMT nebulizers typically include a mesh / membrane with 1000-7000 holes that vibrates on top of a liquid reservoir, thereby forcing a mist of very fine aerosol droplets through the holes in the mesh / membrane. VMT nebulizers suitable for delivery of the pharmaceutical composition of the present invention include any of the following: eFlow (PARI Medical Ltd.), i-Neb (Respironics Respiratory Drug Delivery Ltd), Nebulizer IH50 (Beurer Ltd.), AeroNeb Go (Aerogen Ltd.), InnoSpire Go (Respironics Respiratory Drug Delivery Ltd), Mesh Nebulizer (Shenzhen Homed Medical Device Co,Ltd.), Portable Nebulizer (Microbase Technology Corporation) and Airworks (Convexity Scientific LLC). In some embodiments, the mesh or membrane of the VMT nebulizer is made to vibrate by a piezoelectric element. In some embodiments, the mesh or membrane of the VMT nebulizer is made to vibrate ultrasonically.

[0243] It has been found that VMT nebulizer is particularly suitable for carrying out the present invention, since it does not affect the integrity of the oligonucleotide in the pharmaceutical composition of the present invention.Typically, at least about 50% of the oligonucleotide in the pharmaceutical composition of the present invention, for example at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, maintains its integrity after nebulization.

[0244] In some embodiments, nebulization is continuous during inhalation and exhalation. More typically, nebulization is breath actuated. Nebulizers suitable for use with the present invention have a nebulization rate of greater than 0.2 mL / min. In some embodiments, the nebulization rate is greater than 0.25 mL / min. In other embodiments, the nebulization rate is greater than 0.3 mL / min. In some embodiments, the nebulization rate is greater than 0.45 mL / min. In typical aspects, the nebulization rate is in the range of 0.2 mL / min to 0.5 mL / min.

[0245] If the spray volume exceeds 10mL, adverse effects may occur in human subjects during treatment. In particular, such adverse effects are more common when administering a volume of more than 20mL. In some embodiments, the spray volume does not exceed 20mL.

[0246] In some embodiments, a single dose of the pharmaceutical composition of the present invention can be administered with only one or two refills per nebulization procedure. For example, if the total volume of the pharmaceutical composition administered to a patient is 13 mL, when using a nebulizer with an 8 mL reservoir, only one refill is required to administer the entire volume, whereas when using a nebulizer with a 5 mL reservoir, two refills are required to administer the same volume. In another embodiment, at least three refills are required to administer, for example, a volume of 26 mL per nebulization procedure, and at least three refills are required when using a nebulizer with an 8 mL reservoir. In yet another embodiment, at least four refills are required. For example, at least eight refills are required to deliver 42 mL with a nebulizer with a 5 mL reservoir. Typically, no more than one to three refills are required to administer the pharmaceutical composition of the present invention.

[0247] The pharmaceutical compositions of the invention are typically nebulized at rates ranging from 0.2 mL / min to 0.5 mL / min. Concentrations of 0.5 mg / ml to 0.8 mg / ml (e.g., about 0.6 mg / ml) of oligonucleotide have been found to be particularly suitable, especially when administered with a vibrating mesh nebulizer.

[0248] In some embodiments, the number of nebulizers used during a single nebulization session ranges from 2 to 8. In some embodiments, the number of nebulizers used during a single nebulization session ranges from 1 to 8. In some embodiments, one nebulizer is used during a single nebulization session. In some embodiments, two nebulizers are used during a single nebulization session. In some embodiments, three nebulizers are used during a single nebulization session. In some embodiments, four nebulizers are used during a single nebulization session. In some embodiments, five nebulizers are used during a single nebulization session. In some embodiments, six nebulizers are used during a single nebulization session. In some embodiments, seven nebulizers are used during a single nebulization session. In some embodiments, eight nebulizers are used during a single nebulization session. EXAMPLES

[0249] While certain compounds, compositions and methods of the invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the compounds of the invention. EXAMPLES

[0250] Encapsulation of antibody-encoding mRNA into lipid nanoparticles (LNPs) This example illustrates the encapsulation of an mRNA-encoded antibody in lipid nanoparticles (LNPs) and the preparation of an LNP formulation containing an mRNA encoding the antibody.

[0251] Exemplary LNP formulations were prepared by a conventional process in which oligonucleotides are encapsulated by mixing them with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2016 / 0038432 (Process A).

[0252] An exemplary LNP formulation of anti-IL6R-encoding mRNA was prepared with 10% trehalose (w / v) and an exemplary LNP composition of 5:60:0:35 DMG-PEG_2000 (PEG): Guan-SS-chol (cationic lipid): cholesterol: DOPE (helper lipid). A high encapsulation efficiency of 99.36% was achieved. A particle size of approximately 58 nm was obtained with a polydispersity index of 0.16.

[0253] Another exemplary LNP formulation of anti-IL4Rα-encoding mRNA was prepared using an exemplary LNP composition of 5:60:0:35 DMG-PEG_2000 (PEG): Guan-SS-chol (cationic lipid): cholesterol: DOPE (helper lipid), which also achieved a high encapsulation efficiency of 98%. A particle size of about 60 nm was obtained with a polydispersity index of 0.18. The LNP formulation is described in Table 3 below.

[0254] [Table 3]

[0255] This example demonstrated that lipid nanoparticles with high encapsulation efficiency contained mRNA encoding antibodies such as anti-IL6R and anti-IL4Rα. EXAMPLES

[0256] Pharmacodynamic studies of mRNA-encoded antibodies in mice. This example illustrates a pharmacodynamic study in mice administered with mRNA-encoded antibody.Then, the antibody levels are evaluated in mice administered with mRNA-encoded antibody compared to control mice administered with saline.The study design is outlined in Table 4 below.

[0257] [Table 4]

[0258] Mice were administered messenger RNAs encoding antibodies encapsulated in LNPs directed against various immune targets. Exemplary targets include IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R) or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), other drivers of type 2 inflammation, as well as additional targets. In this example, mRNA-LNPs encapsulating anti-IL6R and anti-IL4Rα were tested.

[0259] CD1 mice approximately 8–10 weeks of age were administered anti-IL6R or anti-IL4Rα encapsulated mRNA-LNPs at a dose of approximately 15 micrograms / animal via intratracheal instillation. Control mice received saline. On day 4, blood was collected for serum preparation. Mice were sacrificed and tissue samples, including bronchoalveolar lavage fluid (BALF), lung and liver, were collected. BALF samples were collected with five successive fluid plunges. The first collected BALF fraction was the cytokine-enriched BALF and BAL cell pellet. Two 8 mm biopsy punches from the left and right whole lungs and liver were frozen in liquid nitrogen.

[0260] Levels of human IgG were measured 72 hours after administration of mRNA therapy in BALF from mice treated with anti-IL6R or anti-IL4Rα antibodies compared to mice that received saline controls. Antibody levels were quantified and the data are shown in Figure 1 and Table 5.

[0261] [Table 5]

[0262] This example shows that mRNA encoding an antibody is delivered and human antibody is expressed in target lung tissue. All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0263] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above specification, but rather is as set forth in the following claims.

Claims

1. 1. A composition comprising one or more mRNAs encoding heavy and light chains of an antibody that binds to a protein target for use in a method of treating an immune disease in a subject in need thereof, the method comprising administering the composition to the subject, wherein the protein target is selected from IL-4 or IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 or IL-5 receptor (IL-5R), IL-6 or IL-6 receptor (IL-6R), IL-9 or IL-9 receptor (IL-9R), IL-13 or IL-13 receptor (IL-13R), IL-25 or IL-25 receptor (IL-25R), or IL-33 or IL-33 receptor (IL-33R, e.g., ST2, IL1RL1), and wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

2. 2. The composition for use of claim 1, wherein the antibody inhibits a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25 or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R) or IL-33 receptor (IL-33R, e.g., ST2, also known as IL1RL1), and optionally the antibody is an anti-IL6R antibody or an anti-IL4Rα antibody.

3. Immune diseases, (i) asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic obstructive pulmonary disease (COPD), systemic sclerosis-interstitial lung disease (SSc-ILD), idiopathic pulmonary fibrosis (IPF), sarcoidosis, or allergy; or (ii) atopic dermatitis, autoimmune dermatitis, asthma with an eosinophilic phenotype or oral corticosteroid-dependent asthma, chronic rhinosinusitis with nasal polyposis, pediatric atopic dermatitis, pediatric asthma, eosinophilic esophagitis, COPD, pruritus nodularis, chronic spontaneous urticaria and bullous pemphigoid, grass pollen allergy, and peanut allergy; or (iii) rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, systemic juvenile arthritis, or severe Covid-19 disease The composition for use according to claim 1 or 2, selected from:

4. Administration is by nebulization, intratracheal delivery, or inhalation, optionally resulting in administration of the mRNA to lung tissue, and optionally (a) administration results in antibody expression for at least about 48 hours, 72 hours, 96 hours, or 120 hours; or (b) low or no systemic exposure of mRNA occurs; A composition for use according to any one of claims 1 to 3.

5. The composition for use according to any one of claims 1 to 3, wherein the administration is performed intravenously or intraperitoneally, optionally resulting in systemic expression of the antibody.

6. (i) the LNPs comprise one or more of a cationic lipid, a non-cationic lipid, and a PEG-modified lipid, and optionally a) the LNP further comprises cholesterol; or b) the LNPs have a molar ratio of cationic lipids to non-cationic lipids to PEG-modified lipids of about 30-60:25-35:1-15, respectively; and / or (ii) the non-cationic lipid is selected from the group consisting of 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-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); and / or (iii) the LNPs comprise DMG-PEG-2000, Guan-SS-Chol, and DOPE, optionally in a ratio of about 5:60:35; A composition for use according to any one of claims 1 to 5.

7. The composition for use according to any one of claims 1 to 6, wherein the heavy and light chains are encoded by a single mRNA, or by separate mRNAs.

8. 8. The composition for use according to any one of claims 1 to 7, wherein the LNPs have a size of 150 nm or less, 100 nm or less, or 75 nm or less, optionally the LNPs have a size of about 60 nm.

9. The composition for use according to any one of claims 1 to 8, wherein one or more mRNAs are unmodified or modified to increase stability, and optionally one or more mRNAs are modified to include modified nucleotides, a cap structure, a polyA tail, a 5' and / or a 3' untranslated region.

10. IL-4 or IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 or IL-5 receptor (IL-5R), IL-6 or IL-6 receptor (IL-6R), IL-9 or IL-9 receptor (IL-9R), IL-13 or IL-13 receptor (IL-13R), IL-25 or IL-25 receptor (IL-25R), or IL-33 or IL-33 receptor (IL-33R, e.g., ST2, IL1RL1). A composition comprising one or more mRNAs encoding heavy and light chains of an antibody that binds to a selected protein target, wherein the one or more mRNAs are encapsulated in a lipid nanoparticle (LNP).

11. 11. The composition of claim 10, wherein the antibody inhibits a protein target selected from IL-4, IL-5, IL-6, IL-9, IL-13, IL-25, or IL-33, IL-4 receptor (IL-4R, e.g., IL-4Rα), IL-5 receptor (IL-5R), IL-6 receptor (IL-6R), IL-9 receptor (IL-9R), IL-13 receptor (IL-13R), IL-25 receptor (IL-25R), or IL-33 receptor (IL-33R, e.g., ST2, IL1RL1).

12. The composition of claim 10 or 11, wherein the antibody is an anti-IL6R antibody or an anti-IL4Rα antibody.

13. One or more mRNAs A. a) an anti-IL6R antibody heavy chain comprising a sequence at least 80% identical to SEQ ID NO: 18 and optionally further comprising a secretory sequence at least 80% identical to SEQ ID NO: 26, optionally the mRNA encoding the anti-IL6R antibody heavy chain is codon-optimized and comprises a sequence at least 80% identical to one of the following sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and / or b) an anti-IL6R antibody light chain comprising a sequence at least 80% identical to SEQ ID NO: 19 and optionally further comprising a secreted sequence at least 80% identical to SEQ ID NO: 26, optionally the mRNA encoding the anti-IL6R antibody light chain is codon-optimized and comprises a sequence at least 80% identical to one of the following sequences: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9; or B. a) an anti-IL4Rα antibody heavy chain comprising a sequence at least 80% identical to SEQ ID NO:20, and optionally further comprising a secretory sequence at least 80% identical to SEQ ID NO:26, optionally the mRNA encoding the anti-IL4Rα antibody heavy chain is codon-optimized and comprises a sequence at least 80% identical to one of the following sequences: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; and / or b) an anti-IL4Rα antibody light chain comprising a sequence at least 80% identical to SEQ ID NO:21 and optionally further comprising a secretory sequence at least 80% identical to SEQ ID NO:26, optionally wherein the mRNA encoding the anti-IL4Rα antibody light chain is codon-optimized and comprises a sequence at least 80% identical to one of the following sequences: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17; To code The composition of claim 12.

14. (i) the LNPs comprise one or more of a cationic lipid, a non-cationic lipid, and a PEG-modified lipid, and optionally a) the LNP further comprises cholesterol; or b) the LNPs have a molar ratio of cationic lipids to non-cationic lipids to PEG-modified lipids of about 30-60:25-35:1-15, respectively; and / or (ii) The non-cationic lipid is selected from the group consisting of 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine ( DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-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); and / or (iii) the LNPs comprise DMG-PEG-2000, Guan-SS-Chol, and DOPE, optionally in a ratio of about 5:60:35; The composition according to any one of claims 10 to 13.

15. The mRNA comprises 5'UTR and 3'UTR sequences, and optionally (i) the 5′UTR comprises a sequence having at least 80% identity to SEQ ID NO:27; and / or (ii) the 3'UTR sequence comprises a sequence having at least 80% identity to SEQ ID NO:28; The composition according to any one of claims 10 to 14.

Images