An improved process for preparing mRNA-loaded lipid nanoparticles

Abstract

The present invention provides improved methods for lipid nanoparticle formulations and mRNA encapsulation. In some embodiments, the present invention provides a method for encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising mixing a solution of mRNA with preformed lipid nanoparticles. In particular, the present invention demonstrates that encapsulating mRNA by combining preformed lipid nanoparticles with mRNA results in formed particles that exhibit unexpectedly efficient in vivo delivery of the mRNA and surprisingly strong expression of the protein and / or peptide encoded by the mRNA.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 420,413, filed November 10, 2016, and U.S. Provisional Application No. 62 / 580,155, filed November 1, 2017, the disclosures of which are incorporated herein by reference. Sequence Listing

[0002] This specification references a Sequence Listing (submitted electronically on November 10, 2017 as a text (.txt) file entitled "MRT-1246WO_SL"). The text file was created on November 10, 2017 and is 17,482 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference. [Background technology]

[0003] Messenger RNA therapy (MRT) is becoming an increasingly important approach to the treatment of various diseases. MRT involves administering messenger RNA (mRNA) to patients in need of treatment, resulting in the production of the protein encoded by the mRNA within the patient's body. For efficient in vivo delivery of mRNA, lipid nanoparticles are typically used to encapsulate the mRNA.

[0004] To improve lipid nanoparticle delivery, many attempts have focused on identifying new lipids or specific lipid compositions that can affect the intracellular delivery and / or expression of mRNA in various types of mammalian tissues, organs, and / or cells (e.g., mammalian hepatocytes). However, these existing approaches are costly, time-consuming, and unpredictable. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides, inter alia, an improved process for preparing mRNA-loaded lipid nanoparticles. In particular, the present invention encapsulates mRNA by combining preformed lipid nanoparticles with mRNA, resulting in formed particles that exhibit unexpectedly efficient in vivo delivery of the mRNA and surprisingly strong expression of the proteins and / or peptides encoded by the mRNA.

[0006] Compared with conventional processes, the inventive process described herein provides mRNA delivered by lipid nanoparticles with higher potency and better efficacy, thereby positively shifting the therapeutic index and offering additional advantages such as lower cost, better patient compliance, and more patient-friendly dosing regimens. The mRNA-loaded lipid nanoparticle formulations provided by the present invention can be successfully delivered in vivo for more potent and effective protein expression via different administration routes, such as intravenous, intramuscular, intraarticular, intrathecal, inhalation (respiratory), subcutaneous, intravitreal, and ophthalmic.

[0007] The process of the present invention can be carried out using a pumping system and is therefore scalable, allowing for improved particle formation / formulation in quantities sufficient for conducting, for example, clinical trials and / or commercial sales. A variety of pumping systems may be used to carry out the present invention, including, but not limited to, pulseless flow pumps, gear pumps, peristaltic pumps, and centrifugal pumps.

[0008] The process of the present invention also results in excellent encapsulation efficiency, mRNA recovery, and uniform particle size.

[0009] Thus, in one aspect, the present invention provides a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising mixing a solution containing preformed lipid nanoparticles with a solution containing mRNA to form lipid nanoparticles encapsulating mRNA. As used herein, preformed lipid nanoparticles are substantially free of mRNA. In some embodiments, preformed lipid nanoparticles are referred to as empty lipid nanoparticles.

[0010] In some embodiments, processes according to the present invention include heating (or maintaining) one or more solutions to a temperature above ambient temperature (i.e., applying heat from a heat source to the solutions), where the one or more solutions are a solution containing preformed lipid nanoparticles, a solution containing mRNA, or a mixed solution containing mRNA encapsulated by lipid nanoparticles. In some embodiments, the process includes heating one or both of the mRNA solution and the preformed lipid nanoparticle solution prior to the mixing step. In some embodiments, the process includes heating one or more of the solution containing preformed lipid nanoparticles, the solution containing mRNA, and the solution containing mRNA encapsulated by lipid nanoparticles during the mixing step. In some embodiments, the process includes heating the mRNA encapsulated by lipid nanoparticles after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated (or one or more of the solutions is maintained) is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature to which one or more of the solutions are heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature above ambient temperature to which one or more of the solutions are heated is about 65° C.

[0011] In some embodiments, processes according to the present invention comprise maintaining one or more of a solution comprising preformed lipid nanoparticles, a solution comprising mRNA, and a mixed solution comprising mRNA encapsulated by lipid nanoparticles at ambient temperature (i.e., without applying heat from a heat source to the solutions). In some embodiments, the process comprises maintaining one or both of the mRNA solution and the preformed lipid nanoparticle solution at ambient temperature prior to the mixing step. In some embodiments, the process comprises maintaining one or more of the solution comprising preformed lipid nanoparticles, the solution comprising mRNA, and the solution comprising mRNA encapsulated by lipid nanoparticles at ambient temperature during the mixing step. In some embodiments, the process comprises maintaining the mRNA encapsulated by lipid nanoparticles at ambient temperature after the mixing step. In some embodiments, the ambient temperature at which one or more of the solutions is maintained is about 35°C, 30°C, 25°C, 20°C, or 16°C or less. In some embodiments, the ambient temperature at which one or more of the solutions are maintained ranges from about 15-35° C., about 15-30° C., about 15-25° C., about 15-20° C., about 20-35° C., about 25-35° C., about 30-35° C., about 20-30° C., about 20-30° C., about 25-30° C., or 20-25° C. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is 20-25° C.

[0012] In some embodiments, the process according to the present invention comprises mixing a solution containing preformed lipid nanoparticles with a solution containing mRNA to form lipid nanoparticles encapsulating the mRNA, carried out at ambient temperature.

[0013] In some embodiments, the preformed lipid nanoparticles are formed by mixing lipids dissolved in ethanol with an aqueous solution. In some embodiments, the lipids include one or more cationic lipids, one or more helper lipids, and one or more PEG lipids. In some embodiments, the lipids also include one or more cholesterol lipids. The preformed lipid nanoparticles are formed by mixing these lipids. Thus, in some embodiments, the preformed lipid nanoparticles include one or more cationic lipids, one or more helper lipids, and one or more PEG lipids. In some embodiments, the preformed lipid nanoparticles also contain one or more cholesterol lipids.

[0014] In some embodiments, the one or more cationic lipids are selected from the group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (imidazole based), HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K. -XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24), N1GL, N2GL, V1GL, and combinations thereof.

[0015] In some embodiments, one or more cationic lipids are amino lipids. Amino lipids suitable for use in the present invention include those described in International Publication No. 2017180917, which is incorporated herein by reference. Exemplary amino lipids in International Publication No. 2017180917 include those described in paragraph

[0744] , such as DLin-MC3-DMA (MC3), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), and compound 18. Other amino lipids include compound 2, compound 23, compound 27, compound 10, and compound 20. Additional amino lipids suitable for use in the present invention include those described in International Publication No. 2017112865, which is incorporated herein by reference. Exemplary amino lipids in WO2017112865 include compounds according to one of formulas (I), (Ial)-(Ia6), (1b), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), as well as the compounds in paragraphs

[0185] ,

[0201] , and

[0276] . In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118725, which is incorporated herein by reference. Exemplary cationic lipids in WO2016118725 include those such as KL22 and KL25. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118724, which is incorporated herein by reference. Examples of cationic lipids in WO2016118725 include KL10, 1,2-diglycoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[0016] In some embodiments, the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)).

[0017] In some embodiments, the one or more PEG-modified lipids are C6-C 20 They contain poly(ethylene) glycol chains up to 5 kDa in length covalently attached to lipids with long alkyl chains.

[0018] In some embodiments, the preformed lipid nanoparticles are purified by a tangential flow filtration (TFF) process. In some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size of less than about 150 nm (e.g., less than about 145 nm, less than about 140 nm, less than about 135 nm, less than about 130 nm, less than about 125 nm, less than about 120 nm, less than about 115 nm, less than about 110 nm, less than about 105 nm, less than about 100 nm, less than about 95 nm, less than about 90 nm, less than about 85 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a size of less than 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size in the 50-150 nm range. In some embodiments, substantially all of the purified nanoparticles have a size in the 50-150 nm range. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size in the 80-150 nm range, hi some embodiments, substantially all of the purified nanoparticles have a size in the 80-150 nm range.

[0019] In some embodiments, processes according to the invention result in greater than about 90%, 95%, 96%, 97%, 98%, or 99% encapsulation. In some embodiments, processes according to the invention result in greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

[0020] In some embodiments, the preformed lipid nanoparticles and mRNA are mixed using a pump system. In some embodiments, the pump system includes a pulseless flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the process using the pump system is performed on a large scale. For example, in some embodiments, the process includes using a pump described herein to mix at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of a solution of mRNA with a solution of preformed lipid nanoparticles to produce mRNA encapsulated in lipid nanoparticles. In some embodiments, the process of mixing mRNA with preformed lipid nanoparticles provides a composition according to the present invention containing at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

[0021] In some embodiments, the solution containing the preformed lipid nanoparticles is mixed at a flow rate ranging from about 25-75 ml / min, about 75-200 ml / min, about 200-350 ml / min, about 350-500 ml / min, about 500-650 ml / min, about 650-850 ml / min, or about 850-1000 ml / min. In some embodiments, the solution containing preformed lipid nanoparticles is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min.

[0022] In some embodiments, the mRNA is mixed in the solution at a flow rate ranging from about 25-75 ml / min, about 75-200 ml / min, about 200-350 ml / min, about 350-500 ml / min, about 500-650 ml / min, about 650-850 ml / min, or about 850-1000 ml / min. In some embodiments, the mRNA is mixed in the solution at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min.

[0023] In some embodiments, the process according to the present invention involves first generating a pre-formed lipid nanoparticle solution by mixing lipids dissolved in ethanol with a citrate buffer.

[0024] In some embodiments, the process includes first generating an mRNA solution by mixing a citrate buffer with an mRNA stock solution. In certain embodiments, a suitable citrate buffer contains about 10 mM citrate, about 150 mM NaCl, and a pH of about 4.5. In some embodiments, a suitable mRNA stock solution contains mRNA at a concentration of about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml or more.

[0025] In some embodiments, the citrate buffer is mixed at a flow rate in the range of 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, or 4800-6000 ml / min. In some embodiments, the citrate buffer is mixed at a flow rate of about 220 ml / min, about 600 ml / min, about 1200 ml / min, about 2400 ml / min, about 3600 ml / min, about 4800 ml / min, or about 6000 ml / min.

[0026] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min. In some embodiments, the mRNA stock solution is mixed at a flow rate of about 20 ml / min, about 40 ml / min, about 60 ml / min, about 80 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml / min, about 400 ml / min, about 500 ml / min, or about 600 ml / min.

[0027] In some embodiments, lipid nanoparticles encapsulating mRNA are prepared using preformed lipid nanoparticles by mixing an aqueous solution containing mRNA with an aqueous solution containing preformed lipid nanoparticles. In some embodiments, the aqueous solution containing mRNA and / or the aqueous solution containing preformed lipid nanoparticles is an aqueous solution containing a pharmaceutically acceptable excipient, including, but not limited to, one or more of trehalose, sucrose, lactose, and mannitol.

[0028] In some embodiments, non-aqueous solvents such as ethanol and / or citrate are absent (i.e., below detectable levels) in either or both of the solution containing mRNA and the solution containing preformed lipid nanoparticles during the addition of mRNA to the preformed lipid nanoparticles. In some embodiments, either or both of the solution containing mRNA and the solution containing preformed lipid nanoparticles are buffer exchanged to remove either or both of the non-aqueous solvents such as ethanol and citrate before mixing the mRNA into the preformed lipid nanoparticles. In some embodiments, either or both of the solution containing mRNA and the solution containing preformed lipid nanoparticles contain only citrate, which cannot be removed during mixing of the mRNA into the preformed lipid nanoparticles. In some embodiments, either or both of the solution containing mRNA and the solution containing preformed lipid nanoparticles contain only non-aqueous solvents such as ethanol, which cannot be removed. In some embodiments, one or both of the solutions containing mRNA and the solution containing preformed lipid nanoparticles contain less than about 10 mM (e.g., less than about 9 mM, about 8 mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM, or about 1 mM) citrate present during the addition of mRNA to the preformed lipid nanoparticles. In some embodiments, one or both of the solutions containing mRNA and the solution containing preformed lipid nanoparticles contain less than about 25% (e.g., less than about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%) of a non-aqueous solvent such as ethanol present during the addition of mRNA to the preformed lipid nanoparticles. In some embodiments, the solution containing lipid nanoparticles encapsulating mRNA does not require further downstream processing (e.g., buffer exchange and / or further purification steps) after the preformed lipid nanoparticles and mRNA are mixed to form the solution.

[0029] In another aspect, the present invention provides compositions of lipid nanoparticles encapsulating mRNA produced by the processes described herein. In some embodiments, a substantial amount of the lipid nanoparticles are preformed. In some embodiments, at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the lipid nanoparticles are preformed. In some embodiments, the present invention provides a composition comprising purified lipid nanoparticles, wherein greater than about 90% of the purified lipid nanoparticles have an individual particle size of less than about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm), and greater than about 70% of the purified lipid nanoparticles encapsulate mRNA within each individual particle. In some embodiments, greater than about 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles have an individual particle size of less than about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified lipid nanoparticles have an individual particle size of less than about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size in the 50-150 nm range.In some embodiments, substantially all of the purified nanoparticles have a size in the range of 50-150 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size in the range of 80-150 nm. In some embodiments, substantially all of the purified nanoparticles have a size in the range of 80-150 nm.

[0030] In some embodiments, more than about 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles encapsulate mRNA within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles encapsulate mRNA within each individual particle. In some embodiments, compositions according to the present invention comprise at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

[0031] In some embodiments, the preformed lipid nanoparticles comprise one or more cationic lipids, one or more helper lipids, and one or more PEG-lipids. In some embodiments, each individual lipid nanoparticle also comprises one or more cholesterol-based lipids. In some embodiments, the one or more cationic lipids are selected from the group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (imidazole-based), HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K. -XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24), N1GL, N2GL, V1GL, and combinations thereof.

[0032] In some embodiments, one or more cationic lipids are amino lipids. Amino lipids suitable for use in the present invention include those described in International Publication No. 2017180917, which is incorporated herein by reference. Exemplary amino lipids in International Publication No. 2017180917 include those described in paragraph

[0744] , such as DLin-MC3-DMA (MC3), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), and compound 18. Other amino lipids include compound 2, compound 23, compound 27, compound 10, and compound 20. Additional amino lipids suitable for use in the present invention include those described in International Publication No. 2017112865, which is incorporated herein by reference. Exemplary amino lipids in WO2017112865 include compounds according to one of formulas (I), (Ial)-(Ia6), (1b), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), and the compounds in paragraphs

[0185] ,

[0201] , and

[0276] . In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118725, which is incorporated herein by reference. Exemplary cationic lipids in WO2016118725 include those such as KL22 and KL25. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118724, which is incorporated herein by reference. Exemplary cationic lipids in WO2016118725 include KL10, 1,2-diglycolyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[0033] In some embodiments, the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)).

[0034] In some embodiments, the one or more cholesterol-based lipids are cholesterol or PEGylated cholesterol. In some embodiments, the one or more PEG-modified lipids are C6-C 20 It comprises a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid having an alkyl chain of 1 kDa.

[0035] In some embodiments, the present invention is used to encapsulate mRNA containing one or more modified nucleotides. In some embodiments, one or more nucleotides are modified to pseudouridine. In some embodiments, one or more nucleotides are modified to 5-methylcytidine. In some embodiments, the present invention is used to encapsulate unmodified mRNA.

[0036] In yet another aspect, the present invention provides a method of delivering mRNA for in vivo protein production, comprising administering to a subject a composition of lipid nanoparticles encapsulating mRNA produced by the processes described herein, wherein the mRNA encodes one or more proteins or peptides of interest.

[0037] In another aspect, the present invention provides a method for encapsulating messenger RNA (mRNA) in lipid nanoparticles, the method being carried out without the use of ethanol. In some embodiments, the method comprises mixing a solution containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids with a solution containing mRNA. In some embodiments, in the solution containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, at least a portion of the one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids are present as preformed lipid nanoparticles. In some embodiments, the method is also carried out without the use of citrate.

[0038] In certain embodiments, the method is performed without the use of any non-aqueous solvent. In some embodiments, there is no detectable ethanol and / or no detectable non-aqueous solvent. In some embodiments, there is no detectable citrate. In some embodiments, there is only residual amounts of ethanol and / or non-aqueous solvent present at less than about 25% of the solution (e.g., less than about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%). In some embodiments, there is only residual amounts of citrate present at less than 10 mM (e.g., less than about 9 mM, about 8 mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM, or about 1 mM).

[0039] In this application, the use of "or" means "and / or" unless stated otherwise. As used in this application, the term "comprise," and variations of terms such as "comprising" 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 synonymously. Both terms are meant to cover any normal fluctuations understood by one of ordinary skill in the art.

[0040] Other features, objects, and advantages of the present invention will become apparent in the following detailed description, drawings, and claims. It should be understood, however, that the following 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 present invention will become apparent to those skilled in the art. The present invention provides, for example, the following items. (Item 1) 1. A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising: A method comprising mixing a solution comprising preformed lipid nanoparticles and mRNA, such that lipid nanoparticles encapsulating the mRNA are formed. (Item 2) 2. The method of claim 1, wherein the solution containing preformed lipid nanoparticles and mRNA contains less than 10 mM citrate. (Item 3) 2. The method of claim 1, wherein the solution comprising preformed lipid nanoparticles and mRNA comprises less than 25% non-aqueous solvent. (Item 4) Item 10. The method of claim 1, further comprising heating the lipid nanoparticles and mRNA to a temperature higher than ambient temperature after mixing. (Item 5) 2. The method of claim 1, wherein the mRNA and / or the preformed lipid nanoparticles are heated to a temperature higher than ambient temperature prior to the mixing. (Item 6) 6. The method of claim 4 or 5, wherein the temperature is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. (Item 7) 7. The method according to any one of items 4 to 6, wherein the temperature is in the range of about 25 to 70°C, about 30 to 70°C, about 35 to 70°C, about 40 to 70°C, about 45 to 70°C, about 50 to 70°C, or about 60 to 70°C. (Item 8) 8. The method according to any one of items 4 to 7, wherein the temperature is about 65°C. (Item 9) 9. The method according to any one of items 1 to 8, wherein the preformed lipid nanoparticles are formed by mixing lipids dissolved in ethanol with an aqueous solution. (Item 10) 10. The method of claim 9, wherein the lipids comprise one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids, and PEG-lipids. (Item 11) The one or more cationic lipids may be selected from the group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (imidazole based), HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XT 11. The method of claim 10, wherein the compound is selected from the group consisting of C2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24), and combinations thereof. (Item 12) 11. The method of claim 10, wherein the one or more cationic lipids comprise target 24. (Item 13) 11. The method of claim 10, wherein the one or more cationic lipids comprise ICE. (Item 14) 11. The method of claim 10, wherein the one or more cationic lipids comprise cKK-E12. (Item 15) Item 11. The method of item 10, wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)). (Item 16) The one or more PEG-modified lipids are 6 -C 20 11. The method of claim 10, comprising a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid having a long alkyl chain. (Item 17) 17. The method of any one of items 1 to 16, wherein the preformed lipid nanoparticles are purified by a tangential flow filtration (TFF) process. (Item 18) 18. The method of claim 17, wherein greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size in the range of 75 to 150 nm. (Item 19) 19. The method of any one of items 17 or 18, wherein substantially all of the purified nanoparticles have a size in the range of 75 to 150 nm. (Item 20) 20. The method of any one of items 17 to 19, wherein greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size in the range of 50 to 80 nm. (Item 21) 19. The method according to any one of items 15 to 18, wherein substantially all of the purified nanoparticles have a size in the range of 75 to 150 nm. (Item 22) 22. The method of any one of items 1 to 21, resulting in an encapsulation rate of greater than about 90%, 95%, 96%, 97%, 98%, or 99%. (Item 23) 23. The method of any one of items 1 to 22, resulting in a recovery of more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of mRNA. (Item 24) 24. The method of any one of items 1 to 23, wherein the preformed lipid nanoparticles and mRNA are mixed using a pump system. (Item 25) 23. The method of claim 22, wherein the pump system comprises a pulseless flow pump. (Item 26) 24. The method of claim 23, wherein the pump is a gear pump. (Item 27) 27. The method of any one of items 1 to 26, wherein the solution containing preformed lipid nanoparticles is mixed at a flow rate in the range of about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min. (Item 28) 28. The method of any one of items 1 to 27, wherein the solution containing preformed lipid nanoparticles is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min. (Item 29) 29. The method according to any one of items 1 to 28, wherein the mRNA is mixed at a flow rate in the range of about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min. (Item 30) 30. The method of any one of items 1 to 29, wherein the mRNA is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min. (Item 31) 31. The method of any one of items 1 to 30, comprising first generating an mRNA solution by mixing a citrate buffer with an mRNA stock solution. (Item 32) 30. The method of claim 29, wherein the citrate buffer comprises about 10 mM citrate, about 150 mM NaCl, a pH of about 4.5. (Item 33) 31. The method of claim 29 or 30, wherein the mRNA stock solution comprises the mRNA at a concentration of about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, about 100 mg / ml or more. (Item 34) 32. The method according to any one of items 29 to 31, wherein the citrate buffer is mixed at a flow rate in the range of about 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, or 4800 to 6000 ml / min. (Item 35) 33. The method according to any one of items 29 to 32, wherein the citrate buffer is mixed at a flow rate of about 220 ml / min, about 600 ml / min, about 1200 ml / min, about 2400 ml / min, about 3600 ml / min, about 4800 ml / min, or about 6000 ml / min. (Item 36) 34. The method according to any one of items 29 to 33, wherein the mRNA stock solution is mixed at a flow rate in the range of about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min. (Item 37) 35. The method of any one of items 29 to 34, wherein the mRNA stock solution is mixed at a flow rate of about 20 ml / min, about 40 ml / min, about 60 ml / min, about 80 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml / min, about 400 ml / min, about 500 ml / min, or about 600 ml / min. (Item 38) 38. The method of any one of items 1 to 37, wherein the lipid nanoparticles encapsulating the mRNA are prepared by dissolving the preformed lipid nanoparticles in a trehalose solution. (Item 39) 39. The method of any one of items 1 to 38, wherein the lipid nanoparticles encapsulating the mRNA do not require further downstream processing. (Item 40) 40. A composition of lipid nanoparticles encapsulating mRNA produced by the method according to any one of items 1 to 39. (Item 41) 41. The method or composition of any one of items 1 to 40, wherein the mRNA comprises one or more modified nucleotides. (Item 42) 42. The method or composition of any one of items 1 to 41, wherein the mRNA is unmodified. (Item 43) Item 39. The composition according to item 39, wherein the nanoparticles have a size of about 75 nm to 150 nm. (Item 44) 40. The composition of claim 39, wherein the nanoparticles comprise a PdI of less than about 0.25 to less than 0.16. (Item 45) 40. The composition of claim 39, wherein the dosage formulation comprises a concentration of mRNA encapsulated in lipid nanoparticles of about 0.016 mg / kg to 1.0 mg / kg. (Item 46) A method for delivering mRNA for in vivo protein production 40. A method comprising administering to a subject a composition of lipid nanoparticles encapsulating mRNA produced by the method of any one of items 1 to 39, wherein the mRNA encodes a protein of interest. (Item 47) A method for delivering mRNA for in vivo protein production A method comprising administering the composition according to any one of items 40 to 45 to a subject. [Brief explanation of the drawings]

[0041] The drawings are for purposes of illustration only and not limitation.

[0042] [Figure 1]Figure 1 shows a schematic diagram of an exemplary lipid nanoparticle mRNA encapsulation process (Process A), which involves mixing mRNA dissolved in aqueous buffer with lipids dissolved in ethanol using a pump system.

[0043] [Figure 2] Figure 2 shows a schematic diagram of an exemplary lipid nanoparticle mRNA encapsulation process (Process B), which involves mixing pre-formed empty lipid nanoparticles with mRNA dissolved in an aqueous buffer using a pump system.

[0044] [Figure 3] Figure 3 shows the typical activity of expressed human ornithine transcarbamylase (hOTC) protein (related to citrulline production) in the liver of OTC spfash mice 24 hours after a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticles produced by Process A or Process B. Prior to use, lipid nanoparticles produced by Process A and Process B were stored in frozen form at -80°C for either (i) T = 0 months (fresh, not frozen) or (ii) T = 2.5 months.

[0045] [Figure 4] Figure 4 shows typical activity of expressed hOTC protein (relative to citrulline production) in the liver of female OTC spfash mice 24 hours after a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B using different pump combinations. Lipid nanoparticle formulations made by Process B were prepared (1) using a gear pump, (2) using a peristaltic pump, (3) using a peristaltic pump at a low flow rate, and (4) using a peristaltic pump at different flow rates of mRNA and empty unformed lipid nanoparticles.

[0046] [Figure 5]Figure 5 shows typical human argininosuccinate synthetase (ASS1) protein expression in 293T cells 16 hours after transfection with either naked hASS1 mRNA (with lipofectamine) or lipid nanoparticle-encapsulated hASS1 mRNA (without lipofectamine) produced by Process A or Process B.

[0047] [Figure 6] Figure 6 shows typical immunohistochemical detection of human cystic fibrosis transmembrane conductance receptor (hCFTR) protein in rat lungs 24 hours after inhalation of hCFTR mRNA lipid nanoparticles prepared by Process B using different cationic lipids. Protein was detected in both bronchial epithelial cells and alveolar regions. Positive (brown) staining was observed in all mRNA lipid nanoparticle test groups compared to the lungs of saline-treated control rats.

[0048] [Figure 7] Figure 7 shows an example of immunohistochemical detection of hCFTR protein in mouse lungs 24 hours after inhalation of hCFTR mRNA lipid nanoparticles prepared by Process B. Protein was detected in both bronchial epithelial cells and alveolar regions. Positive (brown) staining was observed for the mRNA lipid nanoparticle test article group compared to the lungs of saline-treated control mice.

[0049] [Figure 8] Figure 8 shows an example of bioluminescence images of a wild-type mouse 24 hours after intravitreal administration of firefly luciferase (FFL) mRNA encapsulated in lipid nanoparticles prepared by process B.

[0050] [Figure 9] Figure 9 shows an example of bioluminescence images of wild-type mice 24 hours after topical application of eye drops containing FFL mRNA formulated in polyvinyl alcohol and encapsulated in lipid nanoparticles prepared by Process B.

[0051] [Figure 10] Figure 10 shows an example of serum phenylalanine levels in phenylalanine hydroxylase (PAH) knockout (KO) mice before and after treatment with human PAH (hPAH) mRNA encapsulated in lipid nanoparticles prepared by Process B. Serum samples were measured 24 hours after a single subcutaneous administration.

[0052] [Figure 11] Figure 11 shows an example of the activity of expressed hOTC protein (related to citrulline production) in the liver of OTC KO spfash mice 24 hours after a single subcutaneous administration of hOTC mRNA encapsulated in lipid nanoparticles prepared by Process B.

[0053] [Figure 12] FIG. 12 shows an example of human ASS1 protein levels measured in the liver of ASS1 KO mice 24 hours after a single subcutaneous administration of hASS1 mRNA encapsulated in lipid nanoparticles prepared by process B.

[0054] [Figure 13] Figure 13 shows examples of measured human erythropoietin (hEPO) protein levels in the serum of treated mice 6 and 24 hours after single administration of different doses of hEPO mRNA encapsulated in lipid nanoparticles prepared by Process B. The routes of administration used were intradermal, subcutaneous, and intramuscular delivery.

[0055] [Figure 14] FIG. 14 shows a comparison of hEPO protein levels measured in the serum of treated mice 6 and 24 hours after a single intradermal administration of hEPO mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B.

[0056] [Figure 15]FIG. 15 shows a comparison of hEPO protein levels measured in the serum of treated mice 6 and 24 hours after a single intramuscular administration of hEPO mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B.

[0057] [Figure 16] FIG. 16 shows an example of the dosing and testing scheme in Spfash mice that participated in an ammonia challenge.

[0058] [Figure 17] FIG. 17 shows examples of plasma ammonia levels in Spfash mice after ammonia challenge with NH 4 Cl and treatment with different dose levels of hOTC mRNA-loaded lipid nanoparticles, each prepared via Process B.

[0059] [Figure 18] Figure 18 shows hOTC protein expression in Spfash mouse liver 24 hours after a single intravenous administration of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B (i.e., 0.5 mg / kg, 0.16 mg / kg, 0.05 mg / kg, or 0.016 mg / kg).

[0060] [Figure 19] Figure 19 shows a comparison of hOTC mRNA copy numbers in liver tissue of OTC mice 24 hours after a single intravenous administration of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B (i.e., 0.5 mg / kg, 0.16 mg / kg, 0.05 mg / kg, or 0.016 mg / kg).

[0061] [Figure 20]Figure 20 shows a comparison of hOTC mRNA copy numbers in the tested RNAs of OTCspfash mice 24 hours after a single intravenous administration of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B (i.e., 0.5 mg / kg, 0.16 mg / kg, 0.05 mg / kg, and 0.016 mg / kg).

[0062] [Figure 21] Figure 21 shows plasma ammonia results 40 minutes after being subjected to an ammonia challenge in wild-type mice (WT), untreated spfash mice (untreated), and spfash mice 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B.

[0063] [Figure 22] Figure 22 shows hOTC protein activity, as measured by citrulline production, in wild-type mice (WT), untreated spfash mice (untreated), and spfash mice 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B.

[0064] [Figure 23] Figure 23 shows hOTC protein activity as measured by sustained low levels of urinary orotic acid production in spfash mice (untreated), spfash mice 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B, and untreated wild-type mice (untreated C57BL / 6).

[0065] [Figure 24] Figure 24 shows the typical activity of expressed hOTC protein (related to citrulline production) in the liver of OTC sphash mice 24 hours after a single intravenous administration of different dose levels of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B.

[0066] [Figure 25] Figure 25 shows immunohistochemical detection of expressed hOTC protein in mouse liver by Western blot after a single intravenous administration of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or by Process B at various dose levels.

[0067] [Figure 26] Figure 26 shows exemplary activity of expressed hOTC protein (with respect to citrulline production) in the liver of OTCspfash mice 24 hours after a single 0.5 mg / kg intravenous administration of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process B compared to those made by Process A.

[0068] [Figure 27] Figure 27(a)-(d) show immunohistochemical detection of hOTC protein in mouse liver tissue 24 hours after administration of hOTC mRNA lipid nanoparticles prepared by Process A or by Process B via immunohistochemical staining. Figure 27(a)-(b) show results for mRNA lipid nanoparticles produced by Process B. Figure 27(c)-(d) show results for mRNA lipid nanoparticles produced by Process A.

[0069] [Figure 28] FIG. 28 depicts hEPO protein expression following delivery of lipid nanoparticle mRNA formulations made by Process A and Process B, formulated using HGT 5001 as the cationic lipid.

[0070] [Figure 29] FIG. 29 depicts hEPO protein expression following delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, formulated using ICE as the cationic lipid.

[0071] [Figure 30] FIG. 30 depicts hEPO protein expression following delivery of lipid nanoparticle mRNA formulations made by Process A and Process B formulated using CKK-E12 as the cationic lipid.

[0072] [Figure 31] FIG. 31 depicts hEPO protein expression following delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, formulated using C12-200 as the cationic lipid.

[0073] [Figure 32] FIG. 32 depicts hEPO protein expression following delivery of lipid nanoparticle mRNA formulations made by Process A and Process B, formulated using HGT4003 as the cationic lipid. DETAILED DESCRIPTION OF THE INVENTION

[0074] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, and other terms, are set forth throughout the specification.

[0075] Alkyl: As used herein, "alkyl" refers to the radical of a straight or branched saturated hydrocarbon group having from 1 to 20 carbon atoms ("C 1-20 In some embodiments, an alkyl group has 1 to 3 carbon atoms (C 1-3 alkyl). C 1-3Examples of alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), and isopropyl (C3). In some embodiments, the alkyl group has 8 to 12 carbon atoms (C 8-12 alkyl). C 8-12 Examples of alkyl groups include, but are not limited to, n-octyl (C8), n-nonyl (C9), n-decyl (C 10 ), n-undecyl (C 11 ), n-dodecyl (C 12 The prefix "n-" (straight chain) refers to an unbranched alkyl group. For example, n-C8 alkyl refers to -(CH2)7CH3, and nC 10 Alkyl refers to -(CH2)9CH3, and so on.

[0076] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxy- and / or amino-terminal amino acids in a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids may participate in disulfide bonds. Amino acids may include mono- or post-translational modifications, such as association with one or more chemical moieties (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to free amino acids and / or amino acid residues of peptides. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which the term is used.

[0077] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically modified animals, and / or clones.

[0078] Approximately or About: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less), unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values).

[0079] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses a situation in which the mRNA is delivered to a target tissue, where the encoded protein or peptide is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and a situation in which the mRNA is delivered to a target tissue, where the encoded protein or peptide is expressed, 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").

[0080] Efficacy: As used herein, the term "efficacy" or grammatical equivalents refers to the improvement of a biologically relevant endpoint associated with delivery of mRNA encoding a relevant protein or peptide. In some embodiments, the biological endpoint is protection against an ammonium chloride challenge at a specific time point after administration.

[0081] Encapsulation: As used herein, the term "encapsulation" or grammatical equivalents refers to the process of confining individual mRNA molecules within nanoparticles.

[0082] Expression: As used herein, "expression" of mRNA refers to the translation of mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme), and may also include post-translational modification of the peptide, polypeptide, or fully assembled protein (e.g., an enzyme), as indicated by the context. In this application, the terms "expression" and "production" and grammatical equivalents are used interchangeably.

[0083] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value that corresponds to a baseline measurement, such as a measurement in the same individual before the initiation of a treatment described herein, or a measurement in a control sample or subject (or control samples or subjects) that has not received the treatment described herein. A "control sample" is a sample that has been subjected to the same conditions as the test sample, with the exception of the test article. A "control subject" is a subject that suffers from the same form of disease as the subject being treated and is approximately the same age as the subject being treated.

[0084] Impurity: As used herein, the term "impurity" refers to a limited amount of a substance in a liquid, gas, or solid that differs from the chemical composition of the target substance or compound. Impurities are also called contaminants.

[0085] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, such as, for example, in a test tube or reaction vessel, in cell culture, etc.

[0086] 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 cell-type systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0087] Isolated: As used herein, the term "isolated" refers to substances and / or elements 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, prepared, and / or manufactured by the hand of man. Isolated substances and / or elements can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were 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 percent purity of isolated substances and / or elements should not include excipients (e.g., buffers, solvents, water, etc.).

[0088] Local distribution or local delivery: As used herein, "local distribution," "local delivery," or grammatical equivalents thereof, refers to tissue-specific delivery or distribution. Typically, local distribution or local delivery requires that the peptide or protein (e.g., an enzyme) encoded by the mRNA be translated and expressed intracellularly or with limited secretion that precludes entry into the patient's circulatory system.

[0089] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one peptide, polypeptide, or protein. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA can contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, or optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. The mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA is selected from 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-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0090] 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, nucleic acids are compounds and / or substances that are 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 comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.

[0091] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions 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. Humans include prenatal and postnatal forms.

[0092] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0093] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lanthanide, and lanthanide. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and ammonium salts. + (C 1-4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations, and amine cations, formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates, as appropriate. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using a suitable electrophile (e.g., alkyl halide) to form quaternized alkylated amino salts.

[0094] Efficacy: As used herein, the term "efficacy" or grammatical equivalents refers to the expression of a protein or peptide encoded by an mRNA and / or the resulting biological effect.

[0095] Salt: As used herein, the term "salt" means an ionic compound that results or can result from the neutralization reaction between an acid and a base.

[0096] Systemic distribution or delivery: As used herein, the term "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or approach that affects the entire body or the entire organism. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery."

[0097] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a 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 may be a patient, which refers to a person who visits a medical institution for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0098] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or reach a state of 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.

[0099] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated. In some embodiments, the target tissue includes tissues that exhibit disease-associated pathology, symptoms, or characteristics.

[0100] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more signs or characteristics of a particular disease, disorder, and / or condition. Treatment may also be administered to subjects who do not exhibit symptoms of a disease and / or who exhibit only early symptoms of a disease in order to reduce the risk of developing conditions associated with the disease.

[0101] Yield: As used herein, the term "yield" refers to the proportion of mRNA recovered after encapsulation compared to the total mRNA as starting material. In some embodiments, the term "recovery" is used interchangeably with the term "yield."

[0102] Detailed Description The present invention provides improved processes for lipid nanoparticle formulations and mRNA encapsulation. In some embodiments, the present invention provides a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising forming lipids into preformed lipid nanoparticles (i.e., in the absence of mRNA) and then combining the preformed lipid nanoparticles with the mRNA. In some embodiments, the novel formulation process results in mRNA formulations with higher potency (peptide or protein expression) and higher efficacy (improvement in biologically relevant endpoints), both in vitro and in vivo, potentially better tolerated than identical mRNA formulations prepared without preforming the lipid nanoparticles (e.g., by directly combining lipids with the mRNA). The higher potency and / or efficacy of such formulations may allow for lower dosages and / or less frequent administration of the drug product. In some embodiments, the present invention features improved lipid formulations comprising a cationic lipid, a helper lipid, and a PEG or PEG-modified lipid.

[0103] In some embodiments, the encapsulation efficiency obtained with the lipid nanoparticle formulation and manufacturing method is about 90%. For nucleic acid delivery, achieving high encapsulation efficiency is important to achieve drug substance protection and reduce loss of activity in vivo. Furthermore, a surprising result of the lipid nanoparticle formulations prepared by the novel method of the present invention is the significantly higher transfection efficiency observed in vitro.

[0104] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section may be applicable to any aspect of the invention. Messenger RNA (mRNA):

[0105] The present invention may be used to encapsulate any mRNA. mRNA is generally considered a type of RNA that carries information from DNA to ribosomes. Typically, in eukaryotes, mRNA processing involves adding a "cap" to the 5' end and a "tail" to the 3' end. A typical cap is the 7-methylguanosine cap, which is a guanosine linked via a 5'-5'-triphosphate attached to the first transcribed nucleotide. The presence of a cap is important for conferring resistance to nucleases found in most eukaryotic cells. The addition of the tail is typically a polyadenylation event, whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this "tail" helps protect the mRNA from exonuclease degradation. Messenger RNA is translated by ribosomes into a series of amino acids that make up proteins.

[0106] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is often performed using a linear or circular DNA template containing a promoter, a set of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. Stringent conditions will vary depending on the specific application.

[0107] In some embodiments, in vitro synthesized mRNA may be purified prior to formulation and encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis.

[0108] The present invention can be used to formulate and encapsulate mRNAs of various lengths. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNAs greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNAs ranging in length from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0109] The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that generally enhance stability, in some embodiments, the modifications are selected from modified nucleotides, modified sugar phosphate backbones, and 5' and / or 3' untranslated regions.

[0110] In some embodiments, modifications of mRNA may include modifications of nucleotides of the RNA. Modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotides such as 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine. Denine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydro) (oxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxy Modified nucleotide analogs or derivatives of purines and pyrimidines, such as cytosine (v), 1-methyl-pseudouracil, queuosine, beta-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine, and inosine, may also be synthesized.The preparation of such analogs is known to those skilled in the art from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0111] mRNA synthesis typically involves the addition of a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap is important for conferring resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0112] Thus, in some embodiments, the mRNA comprises a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to generate a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. 2'-O-methylation can occur at the first and / or second base after the 7-methylguanosine triphosphate residue. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where m represents a 2'-O-methyl residue).

[0113] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes 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 about 50-500 nucleotides in length.

[0114] 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's location in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be about 50-500 nucleotides in length, or longer.

[0115] While mRNA resulting from an in vitro transcription reaction is desirable in some embodiments, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and / or animals.

[0116] The present invention can be used to formulate and encapsulate mRNA encoding various proteins. Non-limiting examples of mRNA suitable for the present invention include mRNA encoding spinal motor neuron 1 (SMN), alpha-galactosidase (GLA), argininosuccinate synthetase (ASS1), ornithine transcarbamylase (OTC), factor IX (FIX), phenylalanine hydroxylase (PAH), erythropoietin (EPO), cystic fibrosis transmembrane conductance receptor (CFTR), and firefly luciferase (FFL). Examples of mRNA sequences disclosed herein are listed below: Codon-optimized human OTC coding sequence Codon-optimized human ASS1 coding sequence Codon-optimized human CFTR coding sequence Comparison of codon-optimized human CFTR mRNA coding sequences Codon-optimized human PAH coding sequence

[0117] In some embodiments, mRNA suitable for the present invention has a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, mRNA suitable for the present invention comprises a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. mRNA solution

[0118] mRNA can be provided in a solution that can be 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 higher than 1.0mg / ml. 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 to 0.8 mg / ml, 0.05 to 0.7 mg / ml, 0.05 to 0.6 mg / ml, 0.05 to 0.5 mg / ml, 0.05 to 0.4 mg / ml, 0.05 to 0.3 mg / ml, 0.05 to 0.2 mg / ml, 0.05 to 0.1 mg / ml, 0.1 to 1.0 mg / ml, 0.2 to 0.9 mg / ml, 0.3 to 0.8 mg / ml, 0.4 to 0.7 mg / ml, or 0.5 to 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.

[0119] Typically, a suitable mRNA solution may also contain a buffer and / or salt. Common buffers 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 mM to 12 mM. In some embodiments, a suitable concentration of 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 more.

[0120] Exemplary salts can 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 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or greater.

[0121] In some embodiments, a suitable mRNA solution may have a pH in the range of about 3.5 to 6.5, 3.5 to 6.0, 3.5 to 5.5, 3.5 to 5.0, 3.5 to 4.5, 4.0 to 5.5, 4.0 to 5.0, 4.0 to 4.9, 4.0 to 4.8, 4.0 to 4.7, 4.0 to 4.6, or 4.0 to 4.5. In some embodiments, a suitable mRNA solution may 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.

[0122] Various methods may be used to prepare an mRNA solution suitable for the present invention. In some embodiments, mRNA may be directly dissolved in a buffer solution as described herein. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may 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, 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 greater.

[0123] 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.

[0124] Typically, the buffer solution is mixed at a faster rate than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate that is at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x faster than the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate of 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 solution 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 greater.

[0125] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 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. lipid solution

[0126] According to the present invention, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, a suitable lipid solution is ethanol-based. For example, a suitable lipid solution may include a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol-based. In another embodiment, a suitable lipid solution is dimethyl sulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents, including, but not limited to, ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[0127] Suitable lipid solutions can include a mixture of desired lipids at various concentrations, for example, a total concentration of about 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100 mg / ml or more. In some embodiments, a suitable lipid solution may contain a total concentration of desired lipids in the range of about 0.1-100 mg / ml, 0.5-90 mg / ml, 1.0-80 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, or 10 mg / ml.

[0128] Any desired lipids can be mixed in any ratio suitable for encapsulating mRNA. In some embodiments, a suitable lipid solution contains a desired lipid mixture, including cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a desired lipid mixture, including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and one or more PEGylated lipids. cationic lipids

[0129] As used herein, the term "cationic lipid" refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Some cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2010 / 053572 (specifically, C12-200, as described in paragraph

[0225] ) and International Patent Publication No. 2012 / 170930, both of which are incorporated herein by reference. In certain embodiments, cationic lipids suitable for the compositions and methods of the present invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-tocosa ionizable cationic lipids described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (hereby incorporated by reference), such as (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

[0130] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include cationic lipids such as 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02).

[0131] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include those cationic lipids described in WO 2015 / 184256 entitled "Biodegradable Lipids for Delivery of Nucleic Acids," which is incorporated herein by reference, such as 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24).

[0132] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include those cationic lipids described in International Publication No. 2013 / 063468 entitled "Lipid Formulations for Delivery of Messenger RNA" and U.S. Provisional Application No. 2013 / 063468, both of which are incorporated herein by reference. In some embodiments, the cationic lipid comprises a compound of formula I-c1-a: [ka] or a pharmaceutically acceptable salt thereof, wherein: Each R 2 are independently hydrogen or C 1-3 is alkyl; each q is independently 2 to 6; Each R' is independently hydrogen or C 1-3 is alkyl; Each R L is independently C 8-12 It is alkyl.

[0133] In some embodiments, each R 2 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 2 is independently hydrogen or methyl. In some embodiments, each R 2 is hydrogen.

[0134] In some embodiments, each q is independently 3 to 6. In some embodiments, each q is independently 3 to 5. In some embodiments, each q is 4.

[0135] In some embodiments, each R' is independently hydrogen, methyl, or ethyl. In some embodiments, each R' is independently hydrogen or methyl. In some embodiments, each R' is independently hydrogen.

[0136] In some embodiments, each R L is independently C 8-12 In some embodiments, each R L are independently nC 8-12 In some embodiments, each R L is independently C 9-11 In some embodiments, each R L are independently nC 9-11 In some embodiments, each R L is independently C 10 In some embodiments, each R L are independently nC 10 It is alkyl.

[0137] In some embodiments, each R 2 is independently hydrogen or methyl, each q is independently 3 to 5, each R' is independently hydrogen or methyl, and each R L is independently C 8-12 It is alkyl.

[0138] In some embodiments, each R 2 is hydrogen, each q is independently 3 to 5, each R' is hydrogen, and each R L is independently C8-12 It is alkyl.

[0139] In some embodiments, each R 2 is hydrogen, each q is 4, each R' is hydrogen, and each R L is independently C 8-12 It is alkyl.

[0140] In some embodiments, the cationic lipid is a compound of formula Ig: [ka] or a pharmaceutically acceptable salt thereof, wherein each R L is independently C 8-12 In some embodiments, each R L are independently nC 8-12 In some embodiments, each R L is independently C 9-11 In some embodiments, each R L are independently nC 9-11 In some embodiments, each R L is independently C 10 In some embodiments, each R L nC 10 It is alkyl.

[0141] In certain embodiments, a suitable cationic lipid is cKK-E12 or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione). The structure of cKK-E12 is shown below: [ka]

[0142] Further examples of cationic lipids include cationic lipids of formula I: [ka] and pharmaceutically acceptable salts thereof, During the ceremony, R is [ka] ("OF-00") or R is [ka] ("OF-01") or R is [ka] ("OF-02"), or R is [ka] ("OF-03") (See, for example, Fenton, Owen S., et al. “Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.” Advanced materials (2016)).

[0143] In some embodiments, the one or more cationic lipids suitable for the present invention may be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, or "DOTMA." (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS," 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" (Behr et al. Proc. Nat'l Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761), l,2-dioleoyl-3-dimethylammonium-propane or "DODAP," and l,2-dioleoyl-3-trimethylammonium-propane or "DOTAP."

[0144] Further exemplary cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA," 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA," 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA," 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA," N-dioleyl-N,N-dimethylammonium chloride or "DODAC," N,N-distearyl-N,N-dimethylammonium bromide or "DDAB," N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE," 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12 -octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or or "DOcarbDAP", 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane or "DLincarbDAP", l,2-Dilinoleylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane or "DLin- -DMA”, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane or “DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z,12Z)-octadeca-9,l2-dien-1-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (WO 2010 / 042877; Semple et al., Nature Biotech.28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication No. WO2005 / 121348A1). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole moiety, a dialkylamino moiety, or a guanidinium moiety.

[0145] In some embodiments, the one or more cationic lipids are selected from the group consisting of XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012 / 170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011 / 068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61 / 617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61 / 617,468), aminoalcohol lipidoids such as those disclosed in WO 2010 / 053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. "Cationic DLin-KC2-DMA (Semple, SC et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, KP et al."Lipid-like materials for low-dose in vivo gene silencing" PNAS 2010, 107, 1864-1869).

[0146] In some embodiments, one or more cationic lipids are amino lipids. Amino lipids suitable for use in the present invention include those described in International Publication No. 2017180917, which is incorporated herein by reference. Examples of amino lipids in International Publication No. 2017180917 include those described in paragraph

[0744] , such as DLin-MC3-DMA (MC3), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), and compound 18. Other amino lipids include compound 2, compound 23, compound 27, compound 10, and compound 20. Additional amino lipids suitable for use in the present invention include those described in International Publication No. 2017112865, which is incorporated herein by reference. Exemplary amino lipids in WO2017112865 include compounds according to one of formulas (I), (Ial)-(Ia6), (1b), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), and the compounds in paragraphs

[0185] ,

[0201] , and

[0276] . In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118725, which is incorporated herein by reference. Exemplary cationic lipids in WO2016118725 include those such as KL22 and KL25. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118724, which is incorporated herein by reference. Examples of cationic lipids in WO2016118725 include KL10, 1,2-diglycoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[0147] In some embodiments, the cationic lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight or molar of the total lipids in a suitable lipid solution. In some embodiments, the cationic lipids constitute about 30-70% by weight or molar of the total lipid mixture (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%). Non-cationic / Helper Lipids

[0148] As used herein, the term "non-cationic lipid" refers to any neutral lipid, zwitterionic lipid, or anionic lipid. As used herein, the term "cationic lipid" refers to any of a number of lipid species that carry 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), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... and mixtures thereof.

[0149] In some embodiments, non-cationic lipids may comprise at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight or molar of the total lipids in a suitable lipid solution, hi some embodiments, non-cationic lipids comprise about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution, by weight or molar. Cholesterol-based lipids

[0150] In some embodiments, the suitable lipid solution comprises one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in the suitable lipid solution by weight or molar ratio. In some embodiments, the cholesterol-based lipids constitute about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution, by weight or molar. PEGylated lipids

[0151] In some embodiments, suitable lipid solutions include one or more PEGylated lipids. For example, polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), are also contemplated by the present invention. Contemplated PEG-modified lipids are those having a length of C6-C20 These include, but are not limited to, polyethylene glycol chains up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length covalently attached to lipids having alkyl chains of 1 kDa or less. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 ) is a PEG-ceramide.

[0152] PEG-modified phospholipids and derivatized lipids may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% by weight or molar of the total lipids in a suitable lipid solution. In some embodiments, the PEGylated lipids comprise about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution.

[0153] The various combinations of lipids, namely cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, that can be used to prepare preformed lipid nanoparticles are described in literature and herein.For example, suitable lipid solution can contain CKK-E12, DOPE, cholesterol, and DMG-PEG2K; C12-200, DOPE, cholesterol, and DMG-PEG2K; HGT5000, DOPE, cholesterol, and DMG-PEG2K; HGT5001, DOPE, cholesterol, and DMG-PEG2K; cKK-E12, DPPC, cholesterol, and DMG-PEG2K; C12-200, DPPC, cholesterol, and DMG-PEG2K; HGT5000, DPPC, cholesterol, and DMG-PEG2K; HGT5001, DPPC, cholesterol, and DMG-PEG2K; or ICE, DOPE, and DMG-PEG2K. Further combinations of lipids are described in the art, for example, U.S. Patent No. 62 / 420,421 (filed November 10, 2016), U.S. Patent No. 62 / 421,021 (filed November 11, 2016), U.S. Patent No. 62 / 464,327 (filed February 27, 2017), and the PCT application entitled "Novel ICE-Based Lipid Nanoparticle Formulations for Delivery of mRNA" (filed November 10, 2017), the disclosures of which are incorporated herein by reference in their entirety. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids comprising the lipid mixture, as well as the relative molar ratios of these lipids to each other, are based on the characteristics of the selected lipids and the properties and characteristics of the mRNA to be encapsulated. Additional considerations include, for example, the degree of saturation of the alkyl chain of the selected lipid, as well as its size, charge, pH, pKa, fusogenicity, and toxicity. Thus, the molar ratios can be adjusted accordingly. Preformed Nanoparticle Formulation and Mixing Process

[0154] The present invention is based on the surprising and unexpected discovery that mixing mRNA with empty pre-formed lipid nanoparticles (i.e., lipid nanoparticles formed in the absence of mRNA) confers potency and efficacy to the resulting encapsulated mRNA.

[0155] In some previously disclosed processes, see U.S. Patent Application No. 14 / 790,562, filed July 2, 2015, entitled "Encapsulation of Messenger RNA," and U.S. Provisional Patent Application No. 62 / 020,163, filed July 2, 2014, the disclosures of which are incorporated herein in their entireties, in some embodiments, the prior invention provides a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles by mixing an mRNA solution with a lipid solution, wherein the mRNA solution and / or the lipid solution are heated to a predetermined temperature above ambient temperature prior to mixing to form lipid nanoparticles encapsulating the mRNA.

[0156] The present invention relates to a novel method for formulating mRNA-containing lipid nanoparticles. The present invention identifies a novel process for preparing mRNA-containing lipid nanoparticles, which involves combining preformed lipid nanoparticles with mRNA under conditions where the order of addition of these components results in the resulting formed particles exhibiting improved potency and efficacy. The mixing of the components is achieved with a pump system, which maintains a constant lipid / mRNA (N / P) ratio throughout the process and facilitates easy scale-up. In some embodiments, the process is carried out on a large scale. For example, in some embodiments, compositions according to the present invention contain at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

[0157] To achieve high mRNA encapsulation, certain cationic lipid nanoparticle formulations of mRNA require heating of the mRNA in citrate buffer, which is essential for mRNA protection and delivery. In these processes or methods, heating after formulation (after nanoparticle formation) does not increase the encapsulation efficiency of the mRNA in the lipid nanoparticles, so heating is required to occur before the formulation process (i.e., heating the separate components). In contrast, in some embodiments of the novel process of the present invention, the order in which the mRNA is heated does not appear to affect the encapsulation rate of the mRNA. In some embodiments, heating (i.e., maintaining at ambient temperature) one or more of the solution containing preformed lipid nanoparticles, the solution containing mRNA, and the mixed solution containing lipid nanoparticle-encapsulated mRNA does not need to be performed before or after the formulation process. Because controlled temperature changes after mixing are easy to achieve, this potentially offers significant advantages for precise scale-up.

[0158] In this novel process, in some embodiments, encapsulating mRNA by mixing the mRNA with empty (i.e., mRNA-free) pre-formed lipid nanoparticles (Process B) results in significantly higher potency compared to encapsulating mRNA by mixing the mRNA with lipid components only (i.e., not pre-formed into lipid nanoparticles) (Process A). As described in the examples below, e.g., Tables 3 and 4, the potency of any mRNA encapsulated in lipid nanoparticles tested is 100% to 1000% higher when prepared by Process B compared to Process A.

[0159] In some embodiments, empty (i.e., mRNA-free) lipid nanoparticles are formed by mixing a lipid solution containing lipids dissolved in a solvent with an aqueous / buffer solution. In some embodiments, the solvent can be ethanol. In some embodiments, the aqueous solution can be a citrate buffer.

[0160] As used herein, the term "ambient temperature" refers to room temperature or the temperature surrounding an object of interest (e.g., a preformed empty lipid nanoparticle solution, an mRNA solution, or an mRNA-containing lipid nanoparticle solution) without heating or cooling. In some embodiments, the ambient temperature at which one or more solutions are maintained is less than about 35°C, 30°C, 25°C, 20°C, or 16°C. In some embodiments, the ambient temperature at which one or more solutions are maintained is in the range of about 15-35°C, about 15-30°C, about 15-25°C, about 15-20°C, about 20-35°C, about 25-35°C, about 30-35°C, about 20-30°C, about 25-30°C, or about 20-25°C. In some embodiments, the ambient temperature at which one or more solutions are maintained is 20-25°C.

[0161] Thus, a predetermined temperature higher than ambient temperature is typically greater than about 25°C. In some embodiments, a predetermined temperature suitable for the present invention is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, a predetermined temperature suitable for the present invention is greater than about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C. In a specific embodiment, a predetermined temperature suitable for the present invention is about 65°C.

[0162] In some embodiments, the mRNA or the preformed empty (i.e., mRNA-free) lipid nanoparticle solution, or both, may be heated to a predetermined temperature higher than ambient temperature before mixing. In some embodiments, the mRNA and the preformed empty lipid nanoparticle solution are heated separately to a predetermined temperature before mixing. In some embodiments, the mRNA and the preformed empty lipid nanoparticle solution are mixed at ambient temperature, but then heated to a predetermined temperature after mixing. In some embodiments, the preformed empty lipid nanoparticle solution is heated to a predetermined temperature and mixed with the mRNA at ambient temperature. In some embodiments, the mRNA solution is heated to a predetermined temperature and mixed with the preformed empty lipid nanoparticle solution at ambient temperature.

[0163] In some embodiments, the mRNA solution is heated to a predetermined temperature by adding an mRNA stock solution at ambient temperature to a heated buffer solution to achieve the desired predetermined temperature.

[0164] In some embodiments, the lipid solution containing dissolved lipids, or the aqueous / buffer solution, or both, may be heated to a predetermined temperature above ambient temperature before mixing. In some embodiments, the lipid solution containing dissolved lipids and the aqueous solution are heated separately to a predetermined temperature before mixing. In some embodiments, the lipid solution containing dissolved lipids and the aqueous solution are mixed at ambient temperature and then heated to a predetermined temperature after mixing. In some embodiments, the lipid solution containing dissolved lipids is heated to a predetermined temperature and mixed with the aqueous solution at ambient temperature. In some embodiments, the aqueous solution is heated to a predetermined temperature and mixed with the lipid solution containing dissolved lipids at ambient temperature. In some embodiments, heating one or more of the solution containing preformed lipid nanoparticles, the solution containing mRNA, and the mixed solution containing mRNA encapsulated in lipid nanoparticles does not occur before or after the formulation process.

[0165] In some embodiments, the lipid solution and the aqueous or buffer solution can be mixed using a pump. In some embodiments, the mRNA solution and the pre-formed empty lipid nanoparticle solution can be mixed using a pump. Because the encapsulation process can occur over a wide range of scales, different types of pumps can be used to accommodate the desired scale. However, it is generally desirable to use a flow pump that is smaller than a pulse. As used herein, a pulseless flow pump refers to any pump that can establish a stable flow rate and continuous flow. Suitable pump types can include, but are not limited to, gear pumps and centrifugal pumps. Exemplary gear pumps include, but are not limited to, Cole-Parmer or Ziegner gear pumps. Exemplary centrifugal pumps include, but are not limited to, those manufactured by Granger or Cole-Parmer.

[0166] The mRNA solution and the preformed empty lipid nanoparticle solution can be mixed at various flow rates.Typically, the mRNA solution can be mixed at a faster rate than that of the lipid solution.For example, the mRNA solution can be mixed at a rate that is at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x or 20x faster than that of the lipid solution.

[0167] The appropriate flow rate for mixing can be determined based on a scale. In some embodiments, the mRNA solution is mixed at a flow rate of about 40-400 ml / min, 60-500 ml / min, 70-600 ml / min, 80-700 ml / min, 90-800 ml / min, 100-900 ml / min, 110-1000 ml / min, 120-1100 ml / min, 130-1200 ml / min, 140-1300 ml / min, 150-1400 ml / min, 160-1700 ml / min, 170-1800 ml / min, 180-1900 ml / min, 190-2000 ml / min, 200-2100 ml / min, 210-2200 ml / min, 220-2300 ml / min, 230-2400 ml / min, 240-2500 ml / min, 250-2600 ml / min, 260-2700 ml / min, 270-2800 ml / min, 280-2900 ml / min, 290-3000 ml / min, 300-3100 ml / min, 320-3300 ml / min, 340-3500 ml / min, 350-3600 ml / min, 360-3700 ml / min, 370-3800 ml / min, 380-3900 ml / min, 390-4000 ml / min, 400-4100 ml / min, 420-4300 ml / min, 440-4500 ml / min, 450-4600 ml / min, 460-4700 ml / min In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml / min, about 500 ml / min, about 1000 ml / min, about 2000 ml / min, about 3000 ml / min, about 4000 ml / min, or about 5000 ml / min.

[0168] In some embodiments, the lipid solution or preformed lipid nanoparticle solution is mixed at a flow rate ranging from about 25-75 ml / min, 20-50 ml / min, 25-75 ml / min, 30-90 ml / min, 40-100 ml / min, 50-110 ml / min, 75-200 ml / min, 200-350 ml / min, 350-500 ml / min, 500-650 ml / min, 650-850 ml / min, or 850-1000 ml / min. In some embodiments, the lipid solution is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min.

[0169] Generally, in some embodiments, a lipid solution containing dissolved lipids and an aqueous or buffer solution are mixed into a solution so that the lipids can form nanoparticles (or empty pre-formed lipid nanoparticles) without mRNA. In some embodiments, an mRNA solution and a pre-formed lipid nanoparticle solution are mixed into a solution so that the mRNA is encapsulated in the lipid nanoparticles. Such a solution is also called a formulation or encapsulation solution. Suitable formulation or encapsulation solutions include a solvent such as ethanol. For example, suitable formulation or encapsulation solutions include about 10% ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, or about 40% ethanol.

[0170] In some embodiments, a suitable formulation or encapsulating solution includes a solvent such as isopropyl alcohol. For example, a suitable formulation or encapsulating solution includes about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40% isopropyl alcohol.

[0171] In some embodiments, a suitable formulation or encapsulating solution comprises a solvent such as dimethyl sulfoxide. For example, a suitable formulation or encapsulating solution comprises about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40% dimethyl sulfoxide.

[0172] In some embodiments, suitable formulations or encapsulating solutions may also contain buffers or salts. Exemplary buffers may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, the empty preformed lipid nanoparticle formulations used in making this novel nanoparticle formulation can be stably frozen in 10% trehalose solution.

[0173] In some embodiments, the empty (i.e., mRNA-free) preformed lipid nanoparticle formulations used in making the novel nanoparticle formulations can be stably frozen in about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose solution. In some embodiments, the addition of mRNA to the empty lipid nanoparticles results in a final formulation that does not require downstream purification or processing and can be stably stored in frozen form.

[0174] In some embodiments, ethanol, citrate buffer, and other destabilizing agents are not present during the addition of mRNA, so the formulation does not require further downstream processing. In some embodiments, the lipid nanoparticle formulation prepared by this novel process consists of pre-formed lipid nanoparticles in a trehalose solution. The absence of destabilizing agents and the stability of the trehalose solution increase the ease of scaling up the formulation and producing lipid nanoparticles encapsulating mRNA. purification

[0175] In some embodiments, empty preformed lipid nanoparticles or lipid nanoparticles containing mRNA are purified and / or concentrated. Various purification methods may be used. In some embodiments, lipid nanoparticles are purified using tangential flow filtration. Tangential flow filtration (TFF), also known as crossflow filtration, is a type of filtration in which the material to be filtered passes tangentially through the filter rather than through it. In TFF, undesired permeate passes through the filter, while desired retentate passes along the filter and is collected downstream. It is important to note that the desired material is typically contained in the retentate in TFF, the opposite of what is typically encountered in conventional dead-end filtration.

[0176] Depending on the material to be filtered, TFF is typically used for either microfiltration or ultrafiltration. Microfiltration is typically defined as when the filter has a pore size of 0.05 μm to 1.0 μm (inclusive), while ultrafiltration typically involves filters with pore sizes less than 0.05 μm. The pore size also determines the nominal molecular weight cutoff (NMWL), also known as the molecular weight cutoff (MWCO), for a particular filter, with microfiltration membranes typically having an NMWL greater than 1,000 kilodaltons (kDa), and microfiltration membranes and ultrafiltration filters having an NMWL of 1 kDa to 1,000 kDa.

[0177] A major advantage of tangential flow filtration is that non-permeable particles (sometimes called "filter cake") that would otherwise agglomerate and plug the filter during conventional "dead-end" filtration are instead carried along the surface of the filter. This advantage allows tangential flow filtration to be widely used in industrial processes requiring continuous operation, as downtime is significantly reduced since the filter generally does not need to be removed and cleaned.

[0178] Tangential flow filtration can be used for several purposes, including concentration and diafiltration. Concentration is a process in which solvent is removed from a solution while solute molecules are retained. To effectively concentrate a sample, a membrane with a NMWL or MWCO substantially smaller than the molecular weight of the solute molecules to be retained is used. Generally, one skilled in the art can select a filter with a NMWL or MWCO that is 3-6 times smaller than the molecular weight of the target molecule.

[0179] Diafiltration is a fractionation process whereby small, undesired particles pass through a filter while large, desired nanoparticles are retained in the retentate without changing the concentration of those nanoparticles in the solution. Diafiltration is often used to remove salts or reaction buffers from a solution. Diafiltration can be either continuous or discontinuous. In continuous diafiltration, the diafiltrate solution is added to the sample feed at the same rate as the filtrate is produced. In discontinuous diafiltration, the solution is first diluted and then concentrated to a starting concentration. Discontinuous diafiltration may be repeated until the desired nanoparticle concentration is reached.

[0180] The purified and / or concentrated lipid nanoparticles can be formulated in a desired buffer, such as, for example, PBS. Nanoparticles encapsulating the provided mRNA

[0181] The process according to the present invention results in higher potency and efficacy, thereby allowing for lower doses, thereby positively shifting the therapeutic index. In some embodiments, the process according to the present invention results in uniform and small particle sizes (e.g., less than 150 nm), and significantly improved encapsulation efficiency and / or mRNA recovery compared to conventional technology processes.

[0182] Thus, the present invention provides compositions comprising the purified nanoparticles described herein. In some embodiments, a majority of the purified nanoparticles in the composition, i.e., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles, have a size of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all of the purified nanoparticles have a size of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0183] Furthermore, more uniform nanoparticles having a narrow particle size range are achieved by the processes of the present invention, for example, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles in the compositions provided by the present invention have a size in the range of about 75-150 nm (e.g., about 75-145 nm, about 75-140 nm, about 75-135 nm, about 75-130 nm, about 75-125 nm, about 75-120 nm, about 75-115 nm, about 75-110 nm, about 75-105 nm, about 75-100 nm, about 75-95 nm, about 75-90 nm, or 75-85 nm). In some embodiments, substantially all of the purified nanoparticles have a size in the range of about 75 to 150 nm (e.g., about 75 to 145 nm, about 75 to 140 nm, about 75 to 135 nm, about 75 to 130 nm, about 75 to 125 nm, about 75 to 120 nm, about 75 to 115 nm, about 75 to 110 nm, about 75 to 105 nm, about 75 to 100 nm, about 75 to 90 nm, or 75 to 85 nm).

[0184] In some embodiments, the nanoparticles in the compositions provided herein have a molecular size dispersity, or molecular size heterogeneity measure (PDI), of less than about 0.23 (e.g., less than about 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, or 0.08). In certain embodiments, the PDI is less than about 0.16.

[0185] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in the compositions provided herein encapsulate mRNA within each individual particle, hi some embodiments, substantially all of the purified lipid nanoparticles in the composition encapsulate mRNA within each individual particle.

[0186] In some embodiments, compositions according to the invention include at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, processes according to the invention result in greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

[0187] In some embodiments, the composition according to the present invention is formulated for administration to a subject at a dose.In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of less than 1.0 mg / kg mRNA lipid nanoparticle (for example, 0.6 mg / kg, 0.5 mg / kg, 0.3 mg / kg, 0.016 mg / kg). 0.05 mg / kg, and 0.016 mg / kg.In some embodiments, the dose is reduced due to the unexpected finding that lower doses produce higher efficacy and effectiveness.In some embodiments, the dose is reduced by about 70%, 65%, 60%, 55%, 50%, 45% or 40%.

[0188] In some embodiments, the efficacy of mRNA-encapsulated lipid nanoparticles produced by Process B is greater than 100% (i.e., greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 600%, greater than 700%, greater than 800%, or greater than 900%) to greater than 1000% when prepared by Process B compared to Process A. [Example]

[0189] While certain compounds, compositions, and methods of the present invention have been specifically described in accordance with certain embodiments, the following examples serve merely to illustrate the invention and are not intended to be limiting thereof. lipid material

[0190] The formulations described in the examples below, unless otherwise specified, contain multi-component lipid mixtures in varying ratios using one or more cationic lipids, helper lipids (e.g., non-cationic lipids, and / or cholesterol lipids), and PEGylated lipids designed to encapsulate various nucleic acid materials, as previously discussed. Example 1. Lipid Nanoparticle Formulation Process A

[0191] This example illustrates the exemplary lipid nanoparticle preparation process for encapsulating mRNA.As used herein, process A refers to the method of encapsulating mRNA by mixing mRNA with a mixture of lipids without first preforming lipid into lipid nanoparticles.Compared to process B described below, process A does not involve preforming lipid nanoparticles.

[0192] An exemplary formulation process A is shown in Figure 1. In this process, in some embodiments, an ethanolic lipid solution and a buffer solution of mRNA were prepared separately. A solution of a mixture of lipids (such as cationic lipids, helper lipids, zwitterionic lipids, and PEG lipids) was prepared by dissolving in ethanol. An mRNA solution was prepared by dissolving mRNA in a citrate buffer to obtain an mRNA concentration of 0.0833 mg / ml in a citrate buffer at pH 4.5. As shown in Figure 1, both mixtures were then heated to 65°C before mixing. These two solutions were then mixed using a pump system. In some instances, the two solutions were mixed using a gear pump system. In certain embodiments, the two solutions were mixed using a "T" junction (or "Y" junction). The mixture was then purified by diafiltration in a TFF process. The resulting formulation was concentrated and stored at 2-8°C until further use. Example 2. Lipid Nanoparticle Formulation Process B Using Preformed Lipid Nanoparticles

[0193] This example illustrates an exemplary Process B for encapsulating mRNA. As used herein, Process B refers to a process for encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with the mRNA. A wide range of different conditions may be used for Process B, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations. The exemplary conditions described in this and other examples are for illustrative purposes only.

[0194] An exemplary formulation process B is shown in Figure 2. In this process, in some embodiments, lipids dissolved in ethanol and citrate buffer were mixed using a pump system. The instantaneous mixing of the two streams resulted in the formation of empty lipid nanoparticles, a self-assembly process. The resulting formulation mixture was empty lipid nanoparticles in a citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process to perform buffer exchange. The resulting suspension of preformed empty lipid nanoparticles was then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution after mixing resulted in a higher proportion of lipid nanoparticles containing mRNA and a higher total yield of mRNA.

[0195] Additionally, the effect of the presence of citrate buffer during the addition of mRNA in Process B was investigated. Table 1 shows an example of the encapsulation efficiency of lipid nanoparticle formulation Process B with citrate buffer (pH 4.5). When citrate buffer was present during the mixing of preformed empty lipid nanoparticles and mRNA, a decrease in the encapsulation efficiency of mRNA was observed. In the presence of citrate buffer, the encapsulation efficiency of lipid nanoparticle formulation Process B was less than 60%. The encapsulation efficiency of lipid nanoparticle formulations prepared by Process B without citrate buffer was greater than 90%. Table 1. Encapsulation efficiency of lipid nanoparticle formulations using Process B with and without citrate buffer. [Table 1] Example 3.spf ash In vivo activity of hOTC expressed in mice

[0196] This example demonstrates that mRNA delivered via lipid nanoparticles produced by Process B was unexpectedly more effective than that produced by Process A.

[0197] In this example, OTC spf ashMice were administered a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticles prepared by Process A or Process B. Liver tissue from these mice was analyzed for citrulline production 24 hours after administration. Formulations were first tested immediately after mixing without storage, as well as after mixing the formulations and storing them at -80°C for 2.5 months.

[0198] Figure 3 shows the OTC spf 24 hours after a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. ash A mouse is shown.

[0199] Generally, citrulline production can be used to assess the activity of the expressed hOTC protein. As shown in Figure 3, OTC spf ash Citrulline activity due to expressed hOTC protein in mouse liver was measured 24 hours after a single administration of lipid nanoparticle mRNA formulations made by Process A and Process B, respectively. Graph (i) in Figure 3 shows the citrulline activity due to expressed hOTC after delivery of lipid nanoparticle mRNA formulations by Process A and Process B, respectively, immediately after mixing the formulations and without storage. Graph (ii) in Figure 3 shows the citrulline activity due to expressed hOTC after delivery of lipid nanoparticle mRNA formulations by Process A and Process B, respectively, after storing the formulation mixtures at -80°C for 2.5 months.

[0200] The results shown in Figure 3 indicate that the formulation prepared by Process B with pre-formed empty lipid nanoparticles resulted in approximately three times the citrulline activity of the hOTC protein compared to the formulation prepared by Process A. As evidenced by the similarity of the results shown in graphs (i) and (ii), both the formulations produced by Process A and Process B demonstrated stability and functionality after extended storage at -80°C. Example 4. spf under different Process B parameters ash In vivo activity of hOTC expressed in mice

[0201] This example demonstrates that lipid nanoparticles produced by Process B with different parameters exhibit spf ash When delivered to mice, it is shown to lead to citrulline activity comparable to that seen in wild-type mice.

[0202] In this example, OTC spf ash Mice were administered a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticles prepared by Process A or Process B. Liver tissue from these mice was analyzed for citrulline production 24 hours after administration. Four different lipid nanoparticle formulations, each prepared using a different type of pump, were made by Process B.

[0203] Figure 4 shows the OTC spf 24 hours after a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. ash Figure 1 shows the typical activity of hOTC protein (for citrulline production) expressed in mouse liver. Lipid nanoparticle formulations made with Process B were prepared (1) using a gear pump, (2) using a peristaltic pump, (3) using a peristaltic pump at a lower flow rate, and (4) using mRNA and empty preformed lipid nanoparticles at different flow rates.

[0204] In some embodiments, lipid nanoparticle formulations according to Process B can be prepared under different process parameters as shown in Table 2. Table 2 [Table 2]

[0205] In some embodiments, different formulations prepared by Process A and Process B formulations numbered 1-4 were tested in vivo.

[0206] Generally, citrulline production can be used to assess the activity of the expressed hOTC protein. As shown in Figure 4, OTC spf ash Citrullin activity of hOTC protein in mouse liver was measured 24 hours after a single administration of lipid nanoparticle mRNA formulations made by either Process A or Process B with different parameters.

[0207] As shown in Figure 4, exemplary data show that different lipid nanoparticle formulations (1-4) made by Process B were ash When administered to mice, the treatment led to striking levels of citrullin activity of hOTC protein comparable to that of wild-type mice. At the same dosage level (0.5 mg / kg) of OTC mRNA, the lipid nanoparticle formulation prepared by Process B showed 2-4 times higher in vivo activity than the formulation prepared by Process A. Example 5. In vitro ASS1 expression in 293T cells

[0208] This example demonstrates that lipid nanoparticles prepared by Process B resulted in unexpectedly high protein expression in transfected cells.

[0209] Figure 5 shows an example of human ASS1 protein expression in 293T cells 16 hours after transfection with either hASS1 mRNA (with lipofectamine) or lipid nanoparticles encapsulating hASS1 mRNA (without lipofectamine) produced by Process A or Process B.

[0210] In this example, 293T cells were transduced with ASS1 mRNA lipid nanoparticle formulations prepared by Process A or Process B. 1 μg of ASS1 was transduced using lipofectamine, or 10 μg of ASS1 mRNA encapsulated in a lipid nanoparticle formulation was transduced using 10 μg of ASS1 mRNA encapsulated in a lipid nanoparticle formulation. 6 Cells were transduced for 24 hours. ASS1 protein expression was determined by ELISA.

[0211] As shown in Figure 5, the lipid nanoparticle formulation prepared by Process B results in a much higher level of ASS1 protein expression than the formulation prepared by Process A. The level of ASS1 protein expression resulting from transfection with lipid nanoparticles prepared by Process B was comparable to the level resulting from transfection with ASS1 mRNA-Lipofectamine complexes. 10% of the mRNA-Lipofectamine complexes, lipid nanoparticle formulations - Process A, and lipid nanoparticle formulations - Process B were expressed. 6 The ASS1 protein levels per cell were 12.43, 0.43, and 12.11 μg, respectively. The ASS1 protein levels resulting from transfection with the lipid nanoparticle formulation prepared by Process B were 28-fold higher than those from transfection with the lipid nanoparticle formulation prepared by Process A. Example 6. In vivo expression of hCFTR in rat lung

[0212] Figure 6 shows examples of immunohistochemical detection of hCFTR protein in rat lungs 24 hours after inhalation of hCFTR mRNA lipid nanoparticles prepared by Process B using different cationic lipids.

[0213] Male Sprague-Dawley rats were administered, via inhalation, lipid nanoparticle formulations containing hCFTR mRNA prepared by Process B. cKK-E12, ICE, or Target 24 lipids were used as cationic lipids to create the lipid nanoparticle formulations. Fixed lung tissue from these rats was analyzed for the presence of hCFTR protein by immunohistochemical staining.

[0214] Protein was detected in both bronchial epithelial cells and alveolar regions. Positive (brown) staining was observed in all mRNA lipid nanoparticle test article groups, compared to a lack of brown staining in the lungs of saline-treated control rats. Rats were administered via inhalation (i) saline, (ii) a lipid nanoparticle formulation of cKK-E12 lipid prepared by Process B, (iii) a lipid nanoparticle formulation of ICE lipid prepared by Process B, or (iv) a lipid nanoparticle formulation of Target 24 lipid prepared by Process B. Example 7. In vivo expression of hCFTR in mouse lungs

[0215] FIG. 7 shows an example of immunohistochemical detection of hCFTR protein in mouse lungs 24 hours after inhalation of hCFTR mRNA lipid nanoparticles prepared by Process B.

[0216] In this example, C57BL mice were administered via inhalation lipid nanoparticles containing cKK-E12 and containing hCFTR mRNA, prepared by Process B. Fixed lung tissue from these mice was analyzed for the presence of hCFTR protein by immunohistochemical staining.

[0217] Protein was detected in both bronchial epithelial cells and alveolar regions. Positive (brown) staining was observed for the mRNA lipid nanoparticle test article group compared to the absence of brown staining in the lungs of saline-treated control mice. Example 8. In vivo expression of firefly luciferase protein in mice after intravitreal administration

[0218] This example describes an example of a method for administering firefly luciferase (FFL) mRNA-loaded lipid nanoparticles produced by Process B and an example of a method for analyzing firefly luciferase in target tissues in vivo.

[0219] FIG. 8 depicts bioluminescence images of wild-type mice 24 hours after intravitreal administration of FFL mRNA encapsulated in lipid nanoparticles.

[0220] In this example, wild-type mice were treated with lipid nanoparticles encapsulating mRNA encoding FFL produced by Process B via intravitreal administration. A solution containing 5 μg of FFL mRNA lipid nanoparticles was injected into the left eye of the mice. Luminescence was monitored 24 hours after injection.

[0221] The results, shown in Figure 8, demonstrate that significant luminescence was observed in the eyes of these mice, indicative of sufficient production of active FFL protein. Furthermore, sustained FFL activity was maintained for at least 24 hours. Example 9. In vivo expression of firefly luciferase protein in mice after topical ocular application

[0222] This example describes an example of a method for administering firefly luciferase (FFL) mRNA-loaded lipid nanoparticles produced by Process B and an example of a method for analyzing firefly luciferase in target tissues in vivo.

[0223] FIG. 9 depicts bioluminescence images of wild-type mice 24 hours after topical application of eye drops containing FFL mRNA encapsulated in lipid nanoparticles formulated with polyvinyl alcohol.

[0224] In this example, wild-type mice were treated with lipid nanoparticles encapsulating mRNA encoding FFL produced by Process B via topical application (eye drops). A solution containing 5 μg of FFL mRNA lipid nanoparticles formulated with polyvinyl alcohol was applied to the right eye of the mice. Luminescence was monitored 24 hours after application.

[0225] The results, shown in Figure 9, demonstrate that significant luminescence was observed in the eyes of these mice, indicative of sufficient production of active FFL protein. Furthermore, sustained FFL activity was maintained for at least 24 hours. Example 10. In vivo activity of PAH expressed in mice

[0226] In this example, phenylalanine hydroxylase (PAH) knockout (KO) mice were administered a single subcutaneous injection of 20.0 mg / kg hPAH lipid nanoparticles produced by Process B. Phenylalanine levels in mouse serum were measured 24 hours after administration.

[0227] Figure 10 shows an example of serum phenylalanine levels in PAH KO mice before and after treatment with human PAH (hPAH) mRNA encapsulated in lipid nanoparticles produced by Process B. Serum samples were measured 24 hours after a single subcutaneous administration.

[0228] We demonstrated that the mRNA-derived hPAH protein was enzymatically active, as demonstrated by measuring serum phenylalanine reduction levels using a custom ex vivo activity assay. Generally, serum phenylalanine reduction can be used to assess the potency (i.e., activity of the expressed PAH protein) and the effectiveness of the delivery method. As shown in Figure 10, typical serum phenylalanine levels in PAH KO mice were measured before and 24 hours after a single dose of the hPAH mRNA formulation prepared by Process B delivered subcutaneously. For comparison, serum phenylalanine levels were also measured in saline-treated PAH KO mice.

[0229] The results, shown in Figure 10, demonstrate that subcutaneously injected lipid nanoparticle hPAH mRNA formulations resulted in a significant reduction in phenylalanine levels. There was no significant difference in phenylalanine levels in saline-treated PAH KO mice before and after administration. Example 11. In vivo activity of expressed OTC in mice

[0230] This example demonstrates saline-treated OTC KOspf ash Mice and OTC KO spf treated with subcutaneous administration of hOTC mRNA-loaded lipid nanoparticles prepared by Process B ash 1 shows a comparison of OTC protein levels in mouse liver.

[0231] As shown in Figure 11, OTC KOspf ash An example of citrulline production as a result of expressed hOTC protein in mouse liver was measured 24 hours after a single subcutaneous administration of lipid nanoparticles encapsulating hOTC mRNA formulations made by Process B. In addition, OTC KOspf ash Citrulline production in the liver of mice was measured after infusion of saline.

[0232] The results shown in Figure 11 demonstrate that subcutaneously injected lipid nanoparticle hOTC mRNA formulations made by Process B resulted in significant activity of expressed hOTC protein 24 hours after administration compared to saline-treated diseased mice. Example 12. In vivo expression of ASS1 in mice

[0233] FIG. 12 shows an example of measured human ASS1 protein levels in the liver of ASS1 KO mice 24 hours after a single subcutaneous administration of a lipid nanoparticle formulation encapsulating hASS1 mRNA made by Process B.

[0234] Generally, the expressed hASS1 protein level can be used to evaluate the efficiency of delivery methods.As shown in Figure 12, exemplary hASS1 protein levels in SAS1KO mice were measured 24 hours after a single subcutaneous administration of the hASS1 mRNA formulation produced by Process B.In addition, for comparison, the hASS1 protein levels were also measured in saline-treated ASS1 KO mice.

[0235] The results shown in Figure 12 demonstrate that subcutaneously injected hASS1 mRNA lipid nanoparticle formulations made by Process B resulted in significant hASS1 protein levels in hASS1 KO mice 24 hours after administration when compared to saline-treated hASS1 KO mice. Example 13. In vivo expression of hEPO in mice via various routes of administration

[0236] This example shows a comparison of expressed human EPO (HePO) in wild-type mice following administration of hEPO mRNA encapsulated in lipid nanoparticles made by Process B. This example further illustrates a comparison of the efficacy of mRNA delivered via lipid nanoparticles produced by Process A and Process B for intradermal and intramuscular administration at various dose levels. mRNA delivered via lipid nanoparticles produced by Process B is shown to be substantially more potent than that produced by Process A at all dosages and time points evaluated, regardless of whether delivered by the intradermal or intramuscular administration route.

[0237] In this example, wild-type mice were administered a single dose of various concentrations (i.e., 1 μg, 10 μg, or 50 μg) of lipid nanoparticles encapsulating hEPO mRNA produced by Process B via the intradermal, subcutaneous, or intramuscular route. Serum levels of hEPO protein were measured 6 and 24 hours after administration. Additionally, wild-type mice were administered a single dose of various concentrations (i.e., 1 μg, 10 μg, or 50 μg) of lipid nanoparticles encapsulating hEPO mRNA produced by Process A or Process B via the intradermal or intramuscular route. Serum levels of hEPO protein were measured 6 and 24 hours after administration.

[0238] Figure 13 shows examples of hEPO protein levels measured in the serum of mice treated 6 and 24 hours after a single dose of an hEPO mRNA formulation made by Process B. The routes compared were administration by intradermal, subcutaneous, or intramuscular injection.

[0239] The hEPO protein levels in the serum of mice after treatment can be used to evaluate the efficacy of mRNA via different delivery methods. As shown in Figure 13, exemplary hEPO protein levels in mouse serum were evaluated by ELISA 6 and 24 hours after a single administration of 1 μg, 10 μg, and 50 μg of hEPO mRNA lipid nanoparticle formulations made by Process B. In addition, hEPO protein levels from intradermal, subcutaneous, and intramuscular administration routes were compared.

[0240] The results, shown in Figure 13, indicate that intramuscularly injected hEPO mRNA lipid nanoparticle formulations generally resulted in the highest levels of hEPO protein when compared with the intradermal and subcutaneous routes. At 6 hours post-administration, hEPO protein levels were slightly higher from subcutaneous administration than from intradermal administration. Comparison of mRNA lipid nanoparticles produced by Process A and Process B for intradermal and intramuscular administration at various dose levels.

[0241] Figure 14 shows a comparison of hEPO protein levels measured in the serum of treated mice 6 and 24 hours after a single intradermal administration of hEPO mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. Figure 14 shows that at all doses, formulations prepared by Process B resulted in approximately 2-4 fold higher hEPO protein level expression compared to formulations prepared by Process A.

[0242] Figure 15 shows a comparison of hEPO protein levels measured in the serum of treated mice 6 and 24 hours after a single intramuscular administration of hEPO mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. Figure 15 shows that at all doses, formulations prepared by Process B resulted in approximately 2-4 fold higher hEPO protein level expression compared to formulations prepared by Process A.

[0243] As shown in Figures 14 and 15, hEPO protein levels in mouse serum were assessed by ELISA 6 and 24 hours after single administration of 1 μg, 10 μg, and 50 μg of hEPO mRNA lipid nanoparticle formulations made by Process A or Process B, via intradermal and intramuscular administration, respectively. The results demonstrate substantially higher potency of mRNA-encapsulated lipid nanoparticles produced by Process B. The higher potency of the Process B formulation was observed to be associated with various cells of the integumentary system (i.e., myocytes, fibroblasts, macrophages, adipocytes, etc.). Example 14. Protein expression from mRNA lipid nanoparticles in animal models

[0244] This example demonstrates significantly improved in vivo protein expression with mRNA delivered via lipid nanoparticles produced by Process B compared to Process A across a wide range of dose levels.

[0245] In this study, male spf ash Mice were treated at four different dose levels (0.50 mg / kg, 0.16 mg / kg, 0.05 mg / kg, and 0.016 mg / kg) with hOTC mRNA lipid nanoparticles made by either Process A or Process B. The test articles used throughout the study were the same except for the indicated differences in lipid nanoparticle manufacturing process (Process A vs. Process B) and dose.

[0246] Test article is administered as a single dose via tail vein injection.24 hours after administration, mice are subjected to ammonia loading, and a bolus injection of ammonium chloride (5mmol / kg NH4Cl) is administered intraperitoneally.40 minutes after NH4Cl loading, blood is collected by collecting whole blood aliquots into lithium heparin plasma tubes, and this is processed into plasma, and plasma ammonia is analyzed using IDEXX Catalyst Dx analyzer.Then, animals are sacrificed, and their livers are taken and evaluated for hOTC expression using sandwich ELISA.

[0247] FIG. 16 shows a schematic diagram of the ammonia challenge portion of the study, which was performed to mimic the hyperammonemic episodes that patients with OTC deficiency may experience.

[0248] FIG. 17 shows the ammonia-loaded mice, specifically wild-type mice with normal mouse OTC (WT), spf mice that did not receive hOTC mRNA lipid nanoparticles (KO). ash Mice and spf received a single dose of 0.5, 0.16, 0.05, or 0.016 mg / kg mRNA lipid nanoparticles produced by Process B ash The plasma ammonia levels of each mouse are shown. As shown in the figure, the spf mice that did not receive hOTC mRNA lipid nanoparticles (KO) ash Compared to the marked elevation of plasma ammonia in mice under the same conditions, 0.5 mg / kg and 0.16 mg / kg of hOTC mRNA lipid nanoparticles made by Process B achieved statistically significant protection from the model hyperammonemia episode. This data demonstrates that mRNA lipid nanoparticles produced by Process B and administered at doses of 0.5 mg / kg and 0.16 mg / kg are effective in protecting against an ammonium chloride challenge for at least 24 hours after administration.

[0249] Figure 18 and Table 3 show hOTC protein levels measured by sandwich ELISA from the livers of animals sacrificed 24 hours after administration of hOTC mRNA lipid nanoparticles. These results show that the hOTC protein expressed from the livers of mice treated with mRNA lipid nanoparticles prepared by Process B was approximately 1000% (i.e., 10-fold) higher than that from the livers of mice treated with the same mRNA lipid nanoparticles prepared by Process A. Table 3 provides the specific amounts of hOTC protein (as a percentage of total protein) expressed from the livers of mice treated with mRNA lipid nanoparticles prepared by Process A and Process B for all doses 24 hours after administration. Table 3. In vivo hOTC protein expression measured 24 hours after administration of different doses (as indicated) of hOTC mRNA lipid nanoparticles formulated by Process A or Process B. [Table 3]

[0250] As shown in Table 3, the amount of hOTC protein expressed from the livers of mice treated with mRNA lipid nanoparticles prepared by Process B exceeded that of livers treated with mRNA lipid nanoparticles prepared by Process A by approximately 700% (approximately 8-fold), and up to approximately 1000% (approximately 11-fold), at 24 hours post-dose across all doses. The overall mean increase in the amount of hOTC protein expressed from the livers of mice treated with mRNA lipid nanoparticles prepared by Process B versus mRNA lipid nanoparticles prepared by Process A was 884% (9.65-fold) at 24 hours post-dose across all dosages. This data demonstrates that mRNA lipid nanoparticles produced by Process B are significantly more potent than identical mRNA lipid nanoparticles produced by Process A at all doses at 24 hours post-dose.

[0251] Figure 19 shows the OTC spf 24 hours after a single intravenous injection of various doses (i.e., 0.5 mg / kg, 0.16 mg / kg, 0.05 mg / kg, and 0.016 mg / kg) of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. ash 20 shows a comparison of hOTC protein levels in mouse liver tissue. As can be seen, the formulation dose produced by Process B resulted in more copies of hOTC mRNA per mg of tissue than the formulation produced by Process A. Figure 20 shows the OTC spf 24 hours after a single intravenous injection of various doses (i.e., 0.5 mg / kg, 0.16 mg / kg, 0.05 mg / kg, and 0.016 mg / kg) of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. ashFigure 1 shows a comparison of hOTC protein amounts in the tested RNAs from mice. As can be seen, the formulation dose produced by Process B resulted in more copies of hOTC mRNA per μg of tested RNA than the formulation produced by Process A. Example 15. Duration of activity of proteins expressed from mRNA lipid nanoparticles in animal models

[0252] In this example, the activity of an exemplary protein expressed in vivo from mRNA lipid nanoparticles was sustained for an extended duration of at least 15 days.

[0253] In this study, male spf ash Mice were administered a single intravenous tail vein injection of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B. Cohorts of mice were removed at each time point: 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration.

[0254] For each withdrawal cohort, animals were subjected to an ammonia challenge, after which blood was collected for plasma ammonia (μmol / L) measurement, followed by sacrifice for citrulline (μmol citrulline / hr / mg total protein) and urinary orotic acid (μmol / mmol creatinine).

[0255] See Figure 16 for a general schematic of ammonia load and plasma ammonia measurements and Example 14 for a description of this test.

[0256] For citrulline measurements, mouse liver homogenates were prepared, diluted with 1x DPBS, and then added to ultrapure water. A predetermined amount of citrulline standard was added to serve as an internal reference. A reaction mixture containing carbamoyl phosphate, ornithine, and triethanolamine was added, and the reaction proceeded for 30 minutes at 37°C. The reaction was stopped using a mixture of phosphoric and sulfuric acids, and diacetyl monoxime was added. Samples were incubated at 85°C for 30 minutes, briefly cooled, and read at 490 nm to quantify citrulline relative to the citrulline standard.

[0257] For urinary orotic acid measurement, orotic acid quantification from animal urine samples was performed via ultra-performance liquid chromatography (UPLC) using an ion-exchange column. Briefly, urine samples were diluted two-fold using RNase-free water, and a portion was loaded onto a ThermoScientific 100x column. Orotic acid was separated using a mobile phase containing acetonitrile and 25 mM ammonium acetate, and orotic acid was quantified using absorbance detection at 280 nm.

[0258] FIG. 21 shows the results of the immunization of wild-type mice (WT), untreated spf mice (n = 1) at 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B. ash Mice (naive), and spf ash For each mouse, plasma ammonia levels in the animals 40 minutes after ammonia challenge are shown. The dashed line represents the mean plasma ammonia level in the wild-type control group (WT). As can be seen from the results shown in the figure, a single administration of hOTC mRNA lipid nanoparticles provides significant protection against hyperammonemia for at least 15 days. Specifically, plasma ammonia levels after challenge are comparable to wild-type levels (WT) or less than untreated levels (untreated) at all time points assessed up to 15 days.

[0259] FIG. 22 shows the results of the immunization of wild-type mice (WT), untreated spf mice (n = 1) at 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B. ash Mice (naive), and spf ash Figure 1 shows hOTC protein activity as measured by citrulline production in each of the mice. As can be seen by the results shown in the figure, a single dose of hOTC mRNA lipid nanoparticles results in citrulline levels that exceed or are comparable to wild-type controls (WT), and that far exceed untreated controls (untreated) at all time points evaluated up to 15 days.

[0260] Figure 23 shows untreated spf ash Mice (naive), spf at 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 8 days (day 8), 11 days (day 11), and 15 days (day 15) after administration of 1.0 mg / kg hOTC mRNA lipid nanoparticles produced by Process B ash 1 shows hOTC protein activity as measured by sustained low levels of urinary orotic acid production in untreated spf mice and untreated wild-type mice (untreated C57BL / 6). As can be seen by the results shown in the figures, a single administration of hOTC mRNA lipid nanoparticles resulted in low levels of urinary orotic acid that were lower than or comparable to wild-type controls (untreated C57BL / 6), and untreated spf mice at all time points assessed up to 15 days. ash This resulted in low levels of urinary orotic acid, much less than in untreated mice.

[0261] Taken together, the data in this example demonstrate that a single intravenous administration of exemplary mRNA lipid nanoparticles produced by Process B results in active protein that is active over several measurements for at least 15 days. Example 16. SPF at various doses ash In vivo activity of hOTC expressed in mice

[0262] This example demonstrates that hOTC mRNA delivered via lipid nanoparticles produced by Process B at three different dose levels (1.0 mg / kg, 0.6 mg / kg, and 0.3 mg / kg) was unexpectedly more potent than that produced by Process A at each dose evaluated.

[0263] In this example, OTC spf ash Mice were administered a single intravenous dose (at various concentrations, i.e., 1 mg / kg, 0.6 mg / kg, or 0.3 mg / kg) of hOTC mRNA encapsulated in lipid nanoparticles produced by Process A or Process B. Liver tissue from these mice was analyzed 24 hours after administration of citrulline production.

[0264] Figure 24 shows the OTC spf (spf) 24 hours after a single intravenous administration of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. ash Figure 1 shows the typical activity of expressed hOTC protein (with respect to citrulline production) in the liver of mice. hOTC mRNA was administered at different dosage levels: 1.0 mg / kg, 0.6 mg / kg, and 0.3 mg / kg.

[0265] Generally, citrulline production can be used to assess the activity of the expressed hOTC protein. As shown in Figure 24, OTC spf ash Citrulline activity due to expressed hOTC protein in mouse liver was measured 24 hours after single administration of various dose levels of lipid nanoparticle mRNA formulations made by Process A and Process B. The graph in Figure 24 shows the citrulline activity due to expressed hOTC after delivery of lipid nanoparticle mRNA formulations made by Process A and Process B, formulated according to the above processes.

[0266] The results shown in Figure 24 indicate that the formulation prepared by Process B using pre-formed empty lipid nanoparticles resulted in higher citrulline activity of the hOTC protein when compared to the formulation prepared by Process A.

[0267] Figure 25 shows immunohistochemical detection of hOTC protein in mouse liver by Western blot images after a single intravenous administration of hOTC mRNA produced by Process A or Process B at various dose levels (i.e., 1.0 mg / kg, 0.6 mg / kg, and 0.3 mg / kg). As shown in the figure, at all three doses, the expressed hOTC protein was higher in the group administered with the lipid nanoparticle formulation produced by Process B compared to Process A, as evidenced by the band intensity. Example 17.spf ash In vivo activity of hOTC expressed in mice

[0268] This example demonstrates that hOTC mRNA delivered via lipid nanoparticles produced by Process B was unexpectedly more effective than that produced by Process A.

[0269] Figure 26 shows the OTC spf 24 hours after a single intravenous 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticle formulations made by Process A or Process B. ash 1 shows an exemplary activity of expressed hOTC protein (with respect to citrulline production) in mouse liver.

[0270] Generally, citrulline production can be used to assess the activity of the expressed hOTC protein. As shown in Figure 26, OTC spf ash The citrullin activity of the expressed hOTC protein in mouse liver was measured 24 hours after the dose was administered, and the results show that at equal doses, the formulation prepared by Process B resulted in higher citrullin activity of the hOTC protein compared to the formulation prepared by Process A.

[0271] Figures 27(a)-(d) show the immunohistochemical detection of hOTC protein in mouse liver 24 hours after administration of hOTC mRNA lipid nanoparticles prepared by Process A or Process B via immunohistochemical staining. As can be seen, hOTC protein staining was more intense for the group of mice administered the LMP formulation prepared by Process B (Figures 27(a)-(b)) compared to Process A (Figures 27(c)-(d)). The results shown in Figures 27(a)-(d) are consistent with the higher citrulline production of the formulation prepared by Process B compared to Process A, as shown in Figure 26. Example 18. In vivo expression of mRNA lipid nanoparticle formulations prepared by Process B and Process A using different cationic lipids

[0272] This example demonstrates that EPO mRNA delivered via lipid nanoparticles (composed of a variety of different cationic lipids) produced by Process B was unexpectedly more effective than those produced by Process A.

[0273] In this study, male CD1 mice were administered a single intravenous tail vein injection on day 1 at a dose of 1.0 mg / kg hEPO mRNA lipid nanoparticles prepared using one of five different cationic lipids and produced by Process A or Process B (as described above), respectively.

[0274] Table 4 provides the specific hEPO protein expression levels, as measured by ELISA, measured in the serum of animals sacrificed 6 hours after administration of hEPO mRNA lipid nanoparticles produced by Process A or Process B, each prepared using one of five different cationic lipids. As these results show, hEP protein expressed from mRNA lipid nanoparticles prepared by Process B, as measured in mouse serum, was substantially higher than the same mRNA lipid nanoparticles prepared by Process A across all different cationic lipids evaluated. The percentage increases ranged from 133% to 603%, with consistent increases in potency of greater than 100% observed across the five different lipids tested in the study.

[0275] Figure 28 shows hEPO protein expression after delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, which were manufactured using HGT 5001 as the cationic lipid. Figure 29 shows hEPO protein expression after delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, which were formulated using ICE as the cationic lipid. Figure 30 shows hEPO protein expression after delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, which were formulated using cKK-E12 as the cationic lipid. Figure 31 shows hEPO protein expression after delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, which were formulated using C12-200 as the cationic lipid. Figure 32 shows hEPO protein expression after delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, which were formulated using HGT 4003 as the cationic lipid. As the results in each of these graphs in Figures 28-32 show, the expressed hEPO protein, as measured in mouse serum, from mRNA lipid nanoparticles prepared by Process B was substantially higher than the same mRNA lipid nanoparticles prepared by Process A across all five different cationic lipids evaluated. Table 4. In vivo human EPO protein expression measured in mouse serum 6 hours after administration of hEPO mRNA lipid nanoparticles (across a range of cationic lipids) formulated by Process A or Process B. [Table 4]

[0276] Table 5 shows the structural details of hEPO lipid nanoparticles prepared by Process A or Process B using various cationic lipids. In particular, Table 5 shows the nanoparticle size (nm) and PdI of hEPO lipid nanoparticles prepared by Process A or Process B when different cationic lipids are used. As shown in the table, the nanoparticle size of hEPO mRNA lipid nanoparticles prepared by Process B ranged from approximately 90 nm to 150 nm across all nanoparticles prepared using the five cationic lipids evaluated, while that of those prepared by Process A ranged from approximately 75 nm to 95 nm across all nanoparticles prepared using the five cationic lipids evaluated. Table 5 [Table 5]

[0277] In summary, the data in this example show that across lipid nanoparticles containing a variety of different lipid components, there is a substantial increase in potency for mRNA lipid nanoparticles produced by Process B compared to those produced by Process A. Equal

[0278] 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 description, but rather is as set forth in the following claims.

Claims

1. 1. A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles, said method comprising: mixing a solution containing preformed lipid nanoparticles and mRNA so as to form lipid nanoparticles encapsulating the mRNA; wherein both the mRNA-containing solution and the pre-formed lipid nanoparticle-containing solution contain less than 1 mM citrate, which is present during addition of the mRNA to the pre-formed lipid nanoparticles, and wherein the mixing is performed in the presence of ethanol at a concentration of less than 25% by weight; the preformed lipid nanoparticles comprise one or more cationic lipids, one or more non-cationic lipids, and one or more polyethylene glycol (PEG)-modified lipids; The one or more non-cationic lipids may be DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine). , DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)); DPPG; POPC; POPE; DOPE-mal; DSPE; 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, and 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), The method, wherein the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain up to 5 kDa in length covalently attached to a lipid having an alkyl chain C 6 -C 20 in length.

2. 10. The method of claim 1, wherein the solution comprising preformed lipid nanoparticles and mRNA comprises less than 25% non-aqueous solvent.

3. 10. The method of claim 1, further comprising heating the lipid nanoparticles and mRNA to a temperature higher than ambient temperature after mixing.

4. 10. The method of claim 1, wherein the mRNA and / or the preformed lipid nanoparticles are heated to a temperature above ambient temperature prior to the mixing.

5. 5. The method of claim 3 or 4, wherein the temperature is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher.

6. 6. The method of any one of claims 3 to 5, wherein the temperature is in the range of about 25 to 70°C, about 30 to 70°C, about 35 to 70°C, about 40 to 70°C, about 45 to 70°C, about 50 to 70°C, or about 60 to 70°C.

7. The method of any one of claims 3 to 6, wherein the temperature is about 65°C.

8. The one or more cationic lipids may be selected from the group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinC2DMA, ICE (imidazole based), HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XT 10. The method of claim 1, wherein the hydroxyl group is selected from the group consisting of C2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24), and combinations thereof.

9. The method of claim 1 , wherein the one or more cationic lipids comprise a target 24.

10. 2. The method of claim 1, wherein the one or more cationic lipids comprise ICE.

11. The method of claim 1, wherein the one or more cationic lipids comprise cKK-E12.

12. 2. The method of claim 1, wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)).

13. the preformed lipid nanoparticles are a non-cationic lipid selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)); C ６ -C ２０ PEG-modified lipids comprising poly(ethylene) glycol chains up to 5 kDa in length covalently attached to lipids having long alkyl chains; and Cholesterol lipid, which is cholesterol The method according to any one of claims 1 to 8, comprising:

14. 14. The method of any one of claims 1 to 13, wherein the preformed lipid nanoparticles are purified by a tangential flow filtration (TFF) process.

15. 15. The method of claim 14, wherein greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size in the range of 75-150 nm.

16. 16. The method of claim 14, wherein substantially all of the purified nanoparticles have a size in the range of 75 to 150 nm.

17. 17. The method of any one of claims 14 to 16, wherein greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size in the range of 50-80 nm.

18. 17. The method of any one of claims 13 to 16, wherein substantially all of the purified nanoparticles have a size in the range of 75 to 150 nm.

19. 19. The method of any one of claims 1 to 18, resulting in an encapsulation rate of greater than about 90%, 95%, 96%, 97%, 98%, or 99%.

20. 20. The method of any one of claims 1 to 19, resulting in a recovery of greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of mRNA.

21. 21. The method of any one of claims 1 to 20, wherein the preformed lipid nanoparticles and mRNA are mixed using a pump system.

22. 22. The method of claim 21, wherein the pump system comprises a pulseless flow pump.

23. 23. The method of claim 22, wherein the pump is a gear pump.

24. 21. The method of any one of claims 1 to 20, wherein the solution containing preformed lipid nanoparticles is mixed at a flow rate ranging from about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min.

25. 22. The method of any one of claims 1 to 21, wherein the solution containing preformed lipid nanoparticles is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min.

26. 26. The method of any one of claims 1 to 25, wherein the mRNA is mixed at a flow rate ranging from about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min.

27. 27. The method of any one of claims 1 to 26, wherein the mRNA is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min.

28. 28. The method of any one of claims 1 to 27, comprising first generating an mRNA solution by mixing a citrate buffer with an mRNA stock solution.

29. 27. The method of claim 26, wherein the citrate buffer comprises about 10 mM citrate, about 150 mM NaCl, a pH of about 4.

5.

30. 28. The method of claim 26 or 27, wherein the mRNA stock solution comprises the mRNA at a concentration of about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, about 100 mg / ml or more.

31. 29. The method of any one of claims 26 to 28, wherein the citrate buffer is mixed at a flow rate in the range of about 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, or 4800 to 6000 ml / min.

32. 30. The method of any one of claims 26-29, wherein the citrate buffer is mixed at a flow rate of about 220 ml / min, about 600 ml / min, about 1200 ml / min, about 2400 ml / min, about 3600 ml / min, about 4800 ml / min, or about 6000 ml / min.

33. 31. The method of any one of claims 26-30, wherein the mRNA stock solution is mixed at a flow rate ranging from 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.

34. 32. The method of any one of claims 26-31, wherein the mRNA stock solution is mixed at a flow rate of about 20 ml / min, about 40 ml / min, about 60 ml / min, about 80 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml / min, about 400 ml / min, about 500 ml / min, or about 600 ml / min.

35. 35. The method of any one of claims 1 to 34, wherein the lipid nanoparticles encapsulating the mRNA are prepared with the preformed lipid nanoparticles in a trehalose solution.

36. 36. The method of any one of claims 1 to 35, wherein the lipid nanoparticles encapsulating the mRNA do not require further downstream processing.

37. 33. The method of any one of claims 1 to 32, wherein the mRNA comprises one or more modified nucleotides.

38. 38. The method of any one of claims 1 to 37, wherein the mRNA is unmodified.

39. 10. The method of claim 1, wherein no citric acid is present during the mixing step.

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