Improved process for preparing MRNA-supported lipid nanoparticles

Abstract

To provide an improved process of preparing MRNA-loaded lipid nanoparticles.SOLUTION: The present invention provides an improved method for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a method of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a solution of pre-formed lipid nanoparticles and mRNA. In particular, encapsulating mRNA by combining pre-formed lipid nanoparticles with mRNA results in formed particles that exhibit unexpectedly efficient in vivo delivery of the mRNA and surprisingly potent expression of proteins and / or peptides that the mRNA encodes.SELECTED DRAWING: None

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 refers to a sequence listing (submitted electronically on November 10, 2017 as a text (.txt) file named "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 Art

[0003] Messenger RNA therapy (MRT) has become an increasingly important approach for the treatment of various diseases. MRT involves administering messenger RNA (mRNA) to a patient who needs treatment, and the mRNA produces a protein encoded by the mRNA in the patient's body. Generally, lipid nanoparticles are used to encapsulate the mRNA in order to efficiently deliver the mRNA in vivo.

[0004] To improve lipid nanoparticle delivery, many attempts have focused on identifying new lipids or specific lipid compositions that can affect intracellular delivery and / or expression of mRNA, for example, 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 Problems

[0005] The present invention provides, in particular, an improved process for preparing mRNA-supported lipid nanoparticles. Specifically, the present invention encapsulates mRNA by combining pre-formed lipid nanoparticles with mRNA, resulting in formed particles that exhibit unexpectedly efficient in vivo delivery of mRNA, as well as remarkably potent expression of the proteins and / or peptides encoded by the mRNA.

[0006] Compared to conventional processes, the processes of the present invention described herein provide mRNA delivered by lipid nanoparticles with higher potency and better efficacy, thereby shifting the therapeutic index in the positive direction and providing additional advantages such as lower cost, better patient compliance, and a more patient-friendly dosing plan. The mRNA-carrying 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, intra-articular, subarachnoid, inhalation (respiration), subcutaneous, intravitreous, and ophthalmic administration.

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

[0008] The process of the present invention also provides excellent encapsulation efficiency, mRNA recovery rate, and homogeneous particle size.

[0009] Accordingly, in one embodiment, the present invention provides a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles and forming mRNA-encapsulated lipid nanoparticle encapsulations, comprising the step of mixing a solution containing pre-formed lipid nanoparticles with a solution containing mRNA. As used herein, pre-formed lipid nanoparticles are substantially mRNA-free. In some embodiments, pre-formed lipid nanoparticles are referred to as empty lipid nanoparticles.

[0010] In some embodiments, the process according to the present invention includes the step of heating (or maintaining) one or more solutions to a temperature higher than the ambient temperature (i.e., applying heat from a heat source to the solutions), wherein one or more solutions are solutions containing pre-formed lipid nanoparticles, solutions containing mRNA, and mixed solutions containing mRNA encapsulated by lipid nanoparticles. In some embodiments, the process includes the step of heating one or both of the mRNA solution and the pre-formed lipid nanoparticle solution before the mixing step. In some embodiments, the process includes heating one or more of the solutions containing pre-formed lipid nanoparticles, solutions containing mRNA, and solutions containing mRNA encapsulated by lipid nanoparticles during the mixing step. In some embodiments, the process includes the step of heating the mRNA encapsulated by lipid nanoparticles after the mixing step. In some embodiments, the temperature at which one or more solutions are heated (or 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 at which one or more solutions are heated is in the range of 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 at which one or more solutions are heated is higher than the ambient temperature is about 65°C.

[0011] In some embodiments, the process according to the present invention includes maintaining one or more solutions containing pre-formed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing 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 includes maintaining one or both of the mRNA solution and the pre-formed lipid nanoparticle solution at ambient temperature before the mixing step. In some embodiments, the process includes maintaining one or more solutions containing pre-formed lipid nanoparticles, a solution containing mRNA, and a solution containing mRNA encapsulated by lipid nanoparticles at ambient temperature during the mixing step. In some embodiments, the process includes 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 solutions are 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 solutions are maintained is in the range of approximately 15–35°C, approximately 15–30°C, approximately 15–25°C, approximately 15–20°C, approximately 20–35°C, approximately 25–35°C, approximately 30–35°C, approximately 20–30°C, approximately 20–30°C, approximately 25–30°C, or 20–25°C. In some embodiments, the ambient temperature at which one or more solutions are maintained is 20–25°C.

[0012] In some embodiments, the process according to the present invention includes a step of mixing a solution containing pre-formed lipid nanoparticles with a solution containing mRNA to form mRNA-encapsulating lipid nanoparticles, carried out at ambient temperature.

[0013] In some embodiments, pre-formed 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. Pre-formed lipid nanoparticles are formed by mixing these lipids. Therefore, in some embodiments, pre-formed lipid nanoparticles include one or more cationic lipids, one or more helper lipids, and one or more PEG lipids. In some embodiments, pre-formed lipid nanoparticles also contain one or more cholesterol lipids.

[0014] In some embodiments, one or more cationic lipids include 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, and DLin-K -Selected from the group consisting of 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 aminolipids. Aminolipids suitable for use in the present invention include those described in International Publication No. 2017180917, which is incorporated herein by reference. Typical aminolipids 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-diene-1-amine (L608), and compound 18. Other aminolipids include compounds 2, 23, 27, 10, and 20. Further aminolipids suitable for use in the present invention include those described in International Publication No. 2017112865, which is incorporated herein by reference. Typical aminolipids in International Publication No. 2017112865 include compounds of one of the following formulas: (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), as well as the compounds of paragraphs

[0185] ,

[0201] , and

[0276] . In some embodiments, cationic lipids suitable for use in the present invention include those described in International Publication No. 2016118725, which is incorporated herein by reference. Typical cationic lipids in International Publication No. 2016118725 include KL22 and KL25, among others. In some embodiments, cationic lipids suitable for use in the present invention include those described in International Publication No. 2016118724, which is incorporated herein by reference. Examples of cationic lipids in International Publication No. 2016118725 include KL10, 1,2-diglinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[0016] In some embodiments, one or more noncationic 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)).

[0017] In some embodiments, one or more PEG-modified lipids are C6-C 20 It contains poly(ethylene) glycol chains, up to 5 kDa in length, covalently bonded to lipids having long alkyl chains.

[0018] In some embodiments, pre-formed 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 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, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have a size in the range of 50 to 150 nm. In some embodiments, substantially all of the purified nanoparticles have a size in the range of 50 to 150 nm. In some embodiments, more than approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 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.

[0019] In some embodiments, the process according to the present invention results in an encapsulation rate of approximately 90%, 95%, 96%, 97%, 98%, or more than 99%. In some embodiments, the process according to the present invention results in a recovery of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of mRNA.

[0020] In some embodiments, pre-formed 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 volute pump. In some embodiments, the process using the pump system is carried out on a large scale. For example, in some embodiments, the process includes mixing a solution of at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of mRNA with a solution of pre-formed lipid nanoparticles using a pump described herein to produce mRNA encapsulated in lipid nanoparticles. In some embodiments, the process of mixing mRNA with pre-formed 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 pre-formed lipid nanoparticles is mixed at flow rates ranging from approximately 25 to 75 ml / min, approximately 75 to 200 ml / min, approximately 200 to 350 ml / min, approximately 350 to 500 ml / min, approximately 500 to 650 ml / min, approximately 650 to 850 ml / min, or approximately 850 to 1000 ml / min. In some embodiments, the solution containing pre-formed lipid nanoparticles is mixed at a flow rate of approximately 50 ml / min, 100 ml / min, 150 ml / min, 200 ml / min, 250 ml / min, 300 ml / min, 350 ml / min, 400 ml / min, 450 ml / min, 500 ml / min, 550 ml / min, 600 ml / min, 650 ml / min, 700 ml / min, 750 ml / min, 800 ml / min, 850 ml / min, 900 ml / min, 950 ml / min, or 1000 ml / min.

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

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

[0024] In some embodiments, the process according to the present invention includes the step of first producing 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 has 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 flow rates in the range of approximately 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 flow rates of approximately 220 ml / min, approximately 600 ml / min, approximately 1200 ml / min, approximately 2400 ml / min, approximately 3600 ml / min, approximately 4800 ml / min, or approximately 6000 ml / min.

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

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

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

[0029] In another embodiment, the present invention provides a composition of lipid nanoparticles encapsulating mRNA produced by a process described herein. In some embodiments, a substantial amount of lipid nanoparticles are pre-formed. 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 pre-formed. In some embodiments, the present invention provides a composition comprising purified lipid nanoparticles, wherein more than 90% of the purified lipid nanoparticles have individual particle sizes less than 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or less than about 50 nm), and more than 70% of the purified lipid nanoparticles each encapsulate mRNA within the individual particles. In some embodiments, approximately 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles have individual particle sizes less than approximately 150 nm (e.g., approximately 145 nm, approximately 140 nm, approximately 135 nm, approximately 130 nm, approximately 125 nm, approximately 120 nm, approximately 115 nm, approximately 110 nm, approximately 105 nm, approximately 100 nm, approximately 95 nm, approximately 90 nm, approximately 85 nm, approximately 80 nm, approximately 75 nm, approximately 70 nm, approximately 65 nm, approximately 60 nm, approximately 55 nm, or less than approximately 50 nm). In some embodiments, substantially all of the purified lipid nanoparticles have individual particle sizes less than 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, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or less than about 50 nm). In some embodiments, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have sizes in the range of 50 to 150 nm.In some embodiments, substantially all of the purified nanoparticles have a size in the range of 50–150 nm. In some embodiments, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 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 each contain mRNA encapsulated within individual particles. In some embodiments, substantially all of the purified lipid nanoparticles each contain mRNA encapsulated within individual particles. In some embodiments, the composition according to the present invention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

[0031] In some embodiments, the pre-formed lipid nanoparticles include one or more cationic lipids, one or more helper lipids, and one or more PEG lipids. In some embodiments, each individual lipid nanoparticle also includes one or more cholesterol-based lipids. In some embodiments, the one or more cationic lipids include 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 -Selected from the group consisting of 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 aminolipids. Aminolipids suitable for use in the present invention include those described in International Publication No. 2017180917, which is incorporated herein by reference. Typical aminolipids 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-diene-1-amine (L608), and compound 18. Other aminolipids include compounds 2, 23, 27, 10, and 20. Further aminolipids suitable for use in the present invention include those described in International Publication No. 2017112865, which is incorporated herein by reference. The exemplary aminolipids in International Publication No. 2017112865 include compounds of one of the following formulas: (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), and the compounds of paragraphs

[0185] ,

[0201] , and

[0276] . In some embodiments, cationic lipids suitable for use in the present invention include those described in International Publication No. 2016118725, which is incorporated herein by reference. Typical cationic lipids in International Publication No. 2016118725 include KL22 and KL25, among others. In some embodiments, cationic lipids suitable for use in the present invention include those described in International Publication No. 2016118724, which is incorporated herein by reference. Typical cationic lipids in International Publication No. 2016118725 include KL10, 1,2-diglinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[0033] In some embodiments, one or more noncationic 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)).

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

[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 with pseudouridine. In some embodiments, one or more nucleotides are modified with 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 for delivering mRNA for in vivo protein synthesis, comprising administering to a subject a composition of lipid nanoparticles encapsulating mRNA produced by a process described herein, wherein the mRNA encodes one or more proteins or peptides of interest.

[0037] In another embodiment, 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 includes the step of 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 cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids are present as pre-formed lipid nanoparticles. In some embodiments, the method is also carried out without the use of citrate.

[0038] In certain embodiments, the method is carried out without using 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 a residual amount of ethanol and / or non-aqueous solvent present in 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 a residual amount of citrate in 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 otherwise stated. In this application, the terms “comprise,” and variations such as “comprising” and “comprises,” are not intended to exclude other additives, ingredients, integers, or processes. In this application, the terms “about” and “approximately” are used synonymously. Both terms are intended to cover any ordinary variations as understood by those skilled in the art.

[0040] Other features, purposes, and advantages of the present invention will become apparent in the following detailed description, drawings, and claims. However, it should be understood that the following detailed description, drawings, and claims illustrate embodiments of the present invention, but are given for illustrative purposes only and are not limiting. 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) A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles, A method comprising mixing a solution containing pre-formed lipid nanoparticles and mRNA so that lipid nanoparticles encapsulating mRNA are formed. (Item 2) The method according to item 1, wherein the solution containing pre-formed lipid nanoparticles and mRNA contains less than 10 mM citrate. (Item 3) The method according to item 1, wherein the solution containing pre-formed lipid nanoparticles and mRNA contains less than 25% of a non-aqueous solvent. (Item 4) The method according to item 1, further comprising heating the lipid nanoparticles and mRNA to a temperature higher than the ambient temperature after mixing. (Item 5) The method according to item 1, wherein the mRNA and / or the pre-formed lipid nanoparticles are heated to a temperature higher than the ambient temperature before mixing. (Item 6) The method according to item 4 or 5, wherein the temperature is approximately 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, or higher. (Item 7) The method according to any one of items 4 to 6, wherein the temperature is in the range of approximately 25 to 70°C, approximately 30 to 70°C, approximately 35 to 70°C, approximately 40 to 70°C, approximately 45 to 70°C, approximately 50 to 70°C, or approximately 60 to 70°C. (Item 8) The method according to any one of items 4 to 7, wherein the temperature is approximately 65°C. (Item 9) The method according to any one of items 1 to 8, wherein the pre-formed lipid nanoparticles are formed by mixing lipids dissolved in ethanol with an aqueous solution. (Item 10) The method according to item 9, wherein the lipid comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids, and PEG lipids. (Item 11) The aforementioned one or more cationic lipids include 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, and DLin-K-XT. The method according to item 10, 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) The method according to item 10, wherein the one or more cationic lipids described above contain target 24. (Item 13) The method according to item 10, wherein the one or more cationic lipids mentioned above contain ICE. (Item 14) The method according to item 10, wherein the one or more cationic lipids include cKK-E12. (Item 15) The method according to item 10, wherein the one or more noncationic 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 aforementioned one or more PEG-modified lipids, C6-C 20 The method according to item 10, comprising a poly(ethylene) glycol chain of up to 5 kDa in length covalently bonded to a lipid having a long alkyl chain. (Item 17) The method according to any one of items 1 to 16, wherein the pre-formed lipid nanoparticles are purified by a tangential flow filtration (TFF) process. (Item 18) The method according to item 17, wherein more than 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. (Item 19) The method according to either item 17 or 18, wherein substantially all of the purified nanoparticles have a size in the range of 75 to 150 nm. (Item 20) The method according to any one of items 17 to 19, wherein more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have a size in the range of 50 to 80 nm. (Item 21) 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) The method described in any one of items 1-21, which results in an encapsulation rate of approximately 90%, 95%, 96%, 97%, 98%, or 99% or higher. (Item 23) The method described in any one of items 1 to 22, which results in a recovery rate of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of mRNA. (Item 24) The method according to any one of items 1 to 23, wherein the pre-formed lipid nanoparticles and mRNA are mixed using a pump system. (Item 25) The method according to item 22, wherein the pump system comprises a pulseless flow pump. (Item 26) The method according to item 23, wherein the pump is a gear pump. (Item 27) The method according to any one of items 1 to 26, wherein the solution containing pre-formed lipid nanoparticles is mixed at a flow rate in the range of approximately 25 to 75 ml / min, approximately 75 to 200 ml / min, approximately 200 to 350 ml / min, approximately 350 to 500 ml / min, approximately 500 to 650 ml / min, approximately 650 to 850 ml / min, or approximately 850 to 1000 ml / min. (Item 28) The method according to any one of items 1 to 27, wherein the solution containing pre-formed lipid nanoparticles is mixed at a flow rate of approximately 50 ml / min, approximately 100 ml / min, approximately 150 ml / min, approximately 200 ml / min, approximately 250 ml / min, approximately 300 ml / min, approximately 350 ml / min, approximately 400 ml / min, approximately 450 ml / min, approximately 500 ml / min, approximately 550 ml / min, approximately 600 ml / min, approximately 650 ml / min, approximately 700 ml / min, approximately 750 ml / min, approximately 800 ml / min, approximately 850 ml / min, approximately 900 ml / min, approximately 950 ml / min, or approximately 1000 ml / min. (Item 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 approximately 25 to 75 ml / min, approximately 75 to 200 ml / min, approximately 200 to 350 ml / min, approximately 350 to 500 ml / min, approximately 500 to 650 ml / min, approximately 650 to 850 ml / min, or approximately 850 to 1000 ml / min. (Item 30) The method according to any one of items 1 to 29, wherein the mRNA is mixed at a flow rate of approximately 50 ml / min, approximately 100 ml / min, approximately 150 ml / min, approximately 200 ml / min, approximately 250 ml / min, approximately 300 ml / min, approximately 350 ml / min, approximately 400 ml / min, approximately 450 ml / min, approximately 500 ml / min, approximately 550 ml / min, approximately 600 ml / min, approximately 650 ml / min, approximately 700 ml / min, approximately 750 ml / min, approximately 800 ml / min, approximately 850 ml / min, approximately 900 ml / min, approximately 950 ml / min, or approximately 1000 ml / min. (Item 31) The method according to any one of items 1 to 30, comprising the step of first preparing an mRNA solution by mixing a citrate buffer with an mRNA stock solution. (Item 32) The method according to item 29, wherein the citrate buffer contains approximately 10 mM citrate, approximately 150 mM NaCl, and a pH of approximately 4.5. (Item 33) The method according to item 29 or 30, wherein the mRNA stock solution contains the mRNA at concentrations of approximately 1 mg / ml, approximately 10 mg / ml, approximately 50 mg / ml, or approximately 100 mg / ml or higher. (Item 34) 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 approximately 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, or 4800 to 6000 ml / min. (Item 35) The method according to any one of items 29 to 32, wherein the citrate buffer is mixed at a flow rate of approximately 220 ml / min, approximately 600 ml / min, approximately 1200 ml / min, approximately 2400 ml / min, approximately 3600 ml / min, approximately 4800 ml / min, or approximately 6000 ml / min. (Item 36) 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 approximately 10 to 30 ml / min, approximately 30 to 60 ml / min, approximately 60 to 120 ml / min, approximately 120 to 240 ml / min, approximately 240 to 360 ml / min, approximately 360 to 480 ml / min, or approximately 480 to 600 ml / min. (Item 37) The method according to any one of items 29 to 34, wherein the mRNA stock solution is mixed at a flow rate of approximately 20 ml / min, approximately 40 ml / min, approximately 60 ml / min, approximately 80 ml / min, approximately 100 ml / min, approximately 200 ml / min, approximately 300 ml / min, approximately 400 ml / min, approximately 500 ml / min, or approximately 600 ml / min. (Item 38) The method according to any one of items 1 to 37, wherein the lipid nanoparticles encapsulating the mRNA are prepared from the pre-formed lipid nanoparticles in a trehalose solution. (Item 39) The method according to any one of items 1 to 38, wherein the lipid nanoparticles encapsulating the mRNA do not require further downstream processing. (Item 40) A composition of lipid nanoparticles containing mRNA produced by the method described in any one of items 1 to 39. (Item 41) The method or composition according to any one of items 1 to 40, wherein the mRNA comprises one or more modified nucleotides. (Item 42) The method or composition according to any one of items 1 to 41, wherein the mRNA is not modified. (Item 43) The composition according to item 39, wherein the nanoparticles have a size of about 75 nm to 150 nm. (Item 44) The composition according to item 39, wherein the nanoparticles contain PdI in amounts of less than about 0.25 to less than 0.16. (Item 45) The composition according to item 39, wherein the administered preparation contains a concentration of mRNA encapsulated in lipid nanoparticles of approximately 0.016 mg / kg to 1.0 mg / kg. (Item 46) A method for delivering mRNA for in vivo protein production, A method comprising administering to a subject a composition of lipid nanoparticles containing mRNA produced by the method described in 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 to a subject any one of the compositions described in items 40 to 45. [Brief explanation of the drawing]

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

[0042] [Figure 1] Figure 1 shows a schematic diagram of the encapsulation process (Process A) of exemplary lipid nanoparticle mRNA, 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 the exemplary lipid nanoparticle mRNA encapsulation process (Process B), which involves mixing mRNA dissolved in aqueous buffer with pre-formed empty lipid nanoparticles using a pump system.

[0044] [Figure 3]Figure 3 shows the typical activity of human ornithine transcarbamylase (hOTC) protein (related to citrulline production) expressed in the liver of OTC spfash mice 24 hours after a single 0.5 mg / kg dose of hOTC mRNA encapsulated in lipid nanoparticles prepared by process A or process B. Before use, lipid nanoparticles prepared by process A and process B were stored (i) T=0 months (fresh, unfrozen) or (ii) T=2.5 months in a frozen state at -80°C.

[0045] [Figure 4] Figure 4 shows the typical activity of expressed hOTC protein (related 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 prepared by Process A or Process B using different pump combinations. Lipid nanoparticle formulations prepared by Process B were prepared using (1) a gear pump, (2) a peristaltic pump, (3) a peristaltic pump at a low flow rate, and (4) 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, using either naked hASS1 mRNA (containing lipofectamine) or hASS1 mRNA encapsulated in lipid nanoparticles (without lipofectamine) generated by process A or process B.

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

[0049] [Figure 8] Figure 8 shows an example of bioluminescence images of wild-type mice 24 hours after intravitreous 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 with polyvinyl alcohol and encapsulated in lipid nanoparticles prepared by process B.

[0051] [Figure 10] Figure 10 shows examples 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 the hOTC protein (related to citrulline production) expressed 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] Figure 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 doses of hEPO mRNA encapsulated in lipid nanoparticles prepared by Process B. The administration routes used were intradermal, subcutaneous, and intramuscular delivery.

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

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

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

[0058] [Figure 17]Figure 17 shows examples of plasma ammonia levels in Spfash mice after ammonia loading with NH4Cl followed by treatment with different dose levels of hOTC mRNA-supported lipid nanoparticles, each prepared via Process B.

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

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

[0061] [Figure 20] Figure 20 shows a comparison of hOTC mRNA copy numbers in tested RNA in OTCspfash mice 24 hours after single intravenous administration (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 prepared by Process A or Process B.

[0062] [Figure 21]Figure 21 shows plasma ammonia results 40 minutes after ammonia loading 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 of hOTC mRNA lipid nanoparticles produced by Process B.

[0063] [Figure 22] Figure 22 shows the 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 of hOTC mRNA lipid nanoparticles prepared by Process B.

[0064] [Figure 23] Figure 23 shows the hOTC protein activity measured by maintained 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 hOTC protein (related to citrulline production) expressed in the liver of OTC sphash mice 24 hours after a single intravenous administration of hOTC mRNA at different dose levels encapsulated in lipid nanoparticle formulations prepared by process A or process B.

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

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

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

[0069] [Figure 28] Figure 28 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA formulations prepared by Process A and Process B, using HGT 5001 as the cationic lipid.

[0070] [Figure 29] Figure 29 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA formulations produced by Process A and Process B, which were formulated using ICE as a cationic lipid.

[0071] [Figure 30]Represents the hEPO protein expression after delivery of lipid nanoparticle mRNA formulations prepared by Process A and Process B, formulated using 30CKK-E12 as the cationic lipid.

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

[0073] [Figure 32] Figure 32 represents the hEPO protein expression after delivery of lipid nanoparticle mRNA formulations prepared by Process A and Process B, formulated using HGT4003 as the cationic lipid. **Mode for Carrying Out the Invention**

[0074] Definitions To more readily understand the present invention, some 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 a radical of a straight-chain or branched saturated hydrocarbon group having 1 to 20 carbon atoms ("C 1-20 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms (C 1-3 alkyl). Examples of C 1-3 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). Examples of C 8-12 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 12Examples include the following. The prefix "n-" (linear) refers to an unbranched alkyl group. For example, n-C8 alkyl refers to -(CH2)7CH3, and nC 10 Alkyl refers to groups such as -(CH2)9CH3.

[0076] Amino Acids: As used herein, the term “amino acid” means, in its broadest sense, any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, amino acids have the general structure H2N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are synthetic amino acids, in some embodiments, amino acids are D-amino acids, and in some embodiments, amino acids are L-amino acids. “Standard amino acids” means any of the 20 standard L-amino acids commonly found in naturally occurring peptides. “Non-standard amino acids” means any amino acid other than standard amino acids, whether synthetically prepared or obtained from natural sources. As used herein, “synthetic amino acids” includes, but is not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids containing carboxy-terminal and / or amino-terminal amino acids in peptides may be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the cyclic half-life of the peptide without adversely affecting its activity. Amino acids may be involved in disulfide bonds. Amino acids may include unimodifications or post-translational modifications, such as association with one or more chemical components (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 this term refers to a free amino acid or a peptide residue will be clear from the context in which the term is used.

[0077] Animals: As used herein, the term “animal” means any member of the animal kingdom. In some embodiments, “animal” means a human at any stage of development. In some embodiments, “animal” means a non-human animal at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or helminths. In some embodiments, animals may be transgenic animals, genetically modified animals, and / or clones.

[0078] Abbreviation or About: As used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​similar to the given reference values. In certain embodiments, unless otherwise specified or evident from the context (except where such numbers exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​in either direction (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.

[0079] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, mRNA delivery encompasses situations where mRNA is delivered to a target tissue, the encoded protein or peptide is expressed, and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations where mRNA is delivered to a target tissue, 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] Effectiveness: As used herein, the term “effectiveness” or its grammatical equivalent refers to an improvement in a biologically relevant endpoint related to the delivery of mRNA encoding the relevant protein or peptide. In some embodiments, the biological endpoint is protection against ammonium chloride loading at a specific point in time after administration.

[0081] Encapsulation: As used herein, the term “encapsulation” or its grammatical equivalent refers to the process of encapsulating 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 modifications of peptides, polypeptides, or fully assembled proteins (e.g., enzymes), as indicated by the context. In this use, the terms “expression” and “production” and their grammatical equivalents are used interchangeably.

[0083] Improvement, Increase, or Reduction: As used herein, the terms “improve,” “increase,” or “reduce,” or their grammatical equivalents, refer to baseline measurements, such as measurements taken in the same individual before the initiation of the treatment described herein, or measurements taken in a control sample or subject (or multiple control samples or subjects) that have not received the treatment described herein. A “control sample” is a sample subjected to the same conditions as the test sample, except for the test article. A “control subject” is a subject with the same disease form as the subject receiving treatment and of approximately the same age as the subject receiving treatment.

[0084] Impurities: As used herein, the term “impurity” refers to a limited amount of substance in a liquid, gas, or solid, which is not part of the chemical composition of the target substance or compound. Impurities are also called contaminants.

[0085] in vitro: As used herein, the term “in vitro” means an event that occurs in an artificial environment, such as in a test tube or reaction vessel, or in a cell culture medium, rather than within a multicellular organism.

[0086] in vivo: As used herein, the term “in vivo” means events occurring within multicellular organisms such as humans and non-human animals. In the context of cell types, the term may be used to mean events occurring within living cells (for example, as an opposite to “in vitro”).

[0087] Isolated: As used herein, the term “isolated” means (1) separated from at least a portion of the components that were associated with them when they were first produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared and / or manufactured by human hands. Isolated substances and / or elements may 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 first associated. In some embodiments, the isolated active substance is ultrapure of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of the percent purity of an isolated substance and / or element should not include excipients (e.g., buffers, solvents, water, etc.).

[0088] Local distribution or local delivery: As used herein, “local distribution,” “local delivery,” or any grammatically equivalent terms refer to tissue-specific delivery or distribution. Typically, local distribution or local delivery requires that a peptide or protein (e.g., an enzyme) encoded in mRNA be translated and expressed within a cell or along with a limited secretion that prevents it from entering the patient’s circulatory system.

[0089] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that codes for at least one peptide, polypeptide, or protein. As used herein, mRNA includes both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems, or optionally purified, chemically synthesized, etc. If necessary, for example, in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs with chemically modified bases or sugars, or skeletal modifications. mRNA sequences are presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, mRNA is derived from natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine) This includes C5-propynyluridine, C5-propynylcytidine, 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., phosphorothioates and 5'-N-phosphoramidite bonds).

[0090] Nucleic Acid: As used herein, the term “nucleic acid” means, in its broadest sense, any compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain via phosphodiester bonds. In some embodiments, “nucleic acid” means individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” means a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” includes RNA, as well as single-stranded DNA 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 a structure other than a phosphodiester backbone.

[0091] Patient: As used herein, the terms “patient” or “subject” mean any living organism to which the provided composition may be administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. Humans include prenatal and postnatal forms.

[0092] Pharmacopoeia: As used herein, “pharmacopoeia” means a substance that, within reasonable medical judgment, is suitable for use in contact with human and animal tissues in proportion to a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0093] pharmaceutically acceptable salts: pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids, as well as inorganic and organic bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts include salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, and rhynchophosphate. Examples include ctobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Suitable salts derived from bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4Examples include alkyl) tetra salts. Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. 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 aryl sulfonates, as appropriate. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines, which form quaternary alkylated amino salts using a suitable electrophile (e.g., alkyl halides).

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

[0095] Salt: As used herein, the term “salt” means an ionic compound that is or may be produced as a result of a neutralization reaction between an acid and a base.

[0096] Whole-body distribution or whole-body delivery: As used herein, the terms “whole-body distribution,” “whole-body delivery,” or their grammatical equivalents mean a mechanism or approach of delivery or distribution that affects the whole body or the entire organism. Typically, whole-body distribution or whole-body delivery is accomplished through the body’s circulatory system, e.g., blood flow. Compare with the definition of “local distribution or local delivery.”

[0097] Subject: As used herein, the term “subject” means human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is human. The subject may be a patient, meaning a human being who visits a medical facility for the diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject may be susceptible to or potentially have a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0098] Substantial: As used herein, the term “substantial” means a qualitative state that indicates all or nearly all range or degree of the characteristics or properties of the subject of interest. Those skilled in the art of the biological field will understand that it is rare, if any, for biological and chemical phenomena to be completed and / or reach the stage of completion, or to achieve or avoid absolute results. Thus, the term “substantial” is used herein to capture the inherent lack of completeness in many biological and chemical phenomena.

[0099] Target tissue: As used herein, the term “target tissue” means any tissue affected by the disease to be treated. In some embodiments, target tissue includes tissue exhibiting disease-related conditions, symptoms, or characteristics.

[0100] Treatment: As used herein, the terms “to treat,” “treatment,” or “treating” mean any method used to partially or completely reduce, remit, alleviate, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of, one or more signs or features of a particular disease, disorder, and / or condition. Treatment may also be performed on subjects who are not presenting with symptoms of the disease, and / or who are presenting only the initial symptoms of the disease, in order to reduce the risk of developing a condition associated with the disease.

[0101] Yield: As used herein, the term “yield” refers to the percentage of mRNA recovered after inclusion compared to the total mRNA as the initiating material. In some embodiments, the term “recovered” is used interchangeably with the term “yield.”

[0102] Detailed explanation The present invention provides lipid nanoparticle formulations and improved processes for mRNA encapsulation. In some embodiments, the present invention provides a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising the steps of forming lipids on pre-formed lipid nanoparticles (i.e., forming in the absence of mRNA), and then combining the pre-formed lipid nanoparticles with mRNA. In some embodiments, the novel formulation process results in mRNA formulations with potentially better tolerability, higher potency (peptide or protein expression), and higher efficacy (improvement of biologically relevant endpoints) compared to identical mRNA formulations prepared without the step of pre-forming lipid nanoparticles (e.g., by directly combining lipids with mRNA), both in vitro and in vivo. The higher the potency and / or efficacy of such formulations, the lower the dose and / or the less frequently the drug product can be administered. In some embodiments, the present invention features improved lipid formulations comprising cationic lipids, helper lipids, and PEG or PEG-modified lipids.

[0103] In some embodiments, the encapsulation efficiency obtained from the lipid nanoparticle formulations and manufacturing methods is approximately 90%. Achieving high encapsulation efficiency is crucial for nucleic acid delivery, as it ensures protection of the drug substance and reduces in vivo activity loss. Furthermore, a remarkable result for lipid nanoparticle formulations prepared by the novel methods of the present invention is the significantly higher transfection efficiency observed in vitro.

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

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

[0106] mRNA can be synthesized according to any of the various known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is often carried out using a linear or circular DNA template containing a promoter, a group of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAseI, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0107] In some embodiments, the mRNA synthesized in vitro may be purified before formulation and inclusion 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 mRNA of various lengths. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA longer than approximately 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 some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA in the range of approximately 1–20 kb, approximately 1–15 kb, approximately 1–10 kb, approximately 5–20 kb, approximately 5–15 kb, approximately 5–12 kb, approximately 5–10 kb, approximately 8–20 kb, or approximately 8–15 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, mRNA modification may include modification of RNA nucleotides. Modified mRNA according to the present invention may include, for example, skeletal modification, sugar modification, or base modification. 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, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine Denine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydro Xymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetate methyl ester, uracil-5-oxy They may be synthesized as modified nucleotide analogs or derivatives of purines and pyrimidines such as cyacetic acid (v), 1-methyl-psoidouracil, queusin, beta-D-mannosyl-queusin, wybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine, and inosine.Preparations of such analogues are known to those skilled in the art from U.S. Patents No. 4,373,071, No. 4,401,796, No. 4,415,732, No. 4,458,066, No. 4,500,707, No. 4,668,777, No. 4,973,679, No. 5,047,524, No. 5,132,418, No. 5,153,319, No. 5,262,530 and No. 5,700,642 (their disclosures 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 mRNA from exonuclease degradation.

[0112] Therefore, in some embodiments, mRNA contains 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 guanylyltransferase to form a 5'5' triphosphate bond; and then, the 7-nitrogen of guanine is methylated by methyltransferase. 2'-O-methylation may occur at the first and / or second bases following 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 includes 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 iron-responsive elements. In some embodiments, the 5' untranslated region may be a nucleotide of about 50 to 500 in length.

[0114] In some embodiments, the 3' untranslated region includes one or more of the following: a polyadenylation signal, a protein binding site that affects the stability of mRNA position in a cell, or one or more miRNA binding sites. In some embodiments, the 3' untranslated region may be about 50 to 500 nucleotides in length or longer.

[0115] While mRNA derived from in vitro transcription reactions is preferred in some embodiments, other sources of mRNA, including mRNA produced from bacteria, fungi, plants, and / or animals, are intended to be within the scope of the present invention.

[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 Code Sequence Codon-Optimized Human ASS1 Code Sequence Codon-Optimized Human CFTR Code Sequence Comparison of Codon-Optimized Human CFTR Effect Code Sequences Codon-Optimized Human PAH Code Sequence (Sequence ID 5)

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

[0118] mRNA may be provided in a solution to be mixed with a lipid solution so that the mRNA can be encapsulated in lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations. For example, a suitable mRNA solution may contain mRNA at concentrations higher than approximately 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml. In some embodiments, a suitable mRNA solution is approximately 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml, 0.0 mRNA may be contained in concentrations within the range of 5-0.8 mg / ml, 0.05-0.7 mg / ml, 0.05-0.6 mg / ml, 0.05-0.5 mg / ml, 0.05-0.4 mg / ml, 0.05-0.3 mg / ml, 0.05-0.2 mg / ml, 0.05-0.1 mg / ml, 0.1-1.0 mg / ml, 0.2-0.9 mg / ml, 0.3-0.8 mg / ml, 0.4-0.7 mg / ml, or 0.5-0.6 mg / ml. In some embodiments, a suitable mRNA solution may contain mRNA at concentrations of up to approximately 5.0 mg / ml, 4.0 mg / ml, 3.0 mg / ml, 2.0 mg / ml, 1.0 mg / ml, 0.09 mg / ml, 0.08 mg / ml, 0.07 mg / ml, 0.06 mg / ml, or 0.05 mg / ml.

[0119] Typically, a suitable mRNA solution may also contain buffers and / or salts. Generally, buffers may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, suitable concentrations of buffers may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, appropriate concentrations of the buffer are approximately 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or higher.

[0120] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable salt concentrations in mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. Suitable salt concentrations in mRNA solution are about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or higher.

[0121] In some embodiments, a suitable mRNA solution may have a pH in the range of approximately 3.5–6.5, 3.5–6.0, 3.5–5.5, 3.5–5.0, 3.5–4.5, 4.0–5.5, 4.0–5.0, 4.0–4.9, 4.0–4.8, 4.0–4.7, 4.0–4.6, or 4.0–4.5. In some embodiments, a suitable mRNA solution may have a pH of approximately 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5 or less.

[0122] Various methods may be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA may be dissolved directly in the buffer solutions described herein. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with a buffer solution before mixing it with a lipid solution for mounting. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with a buffer solution immediately before mixing it with a lipid solution for mounting. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at concentrations of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or higher.

[0123] In some embodiments, the mRNA stock solution is mixed with a buffer using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[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 of at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, or 20× faster than that of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate of about 100 to 6000 ml / min (e.g., 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, 4800 to 6000 ml / min, or 60 to 420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of approximately 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or more.

[0125] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10 to 600 ml / min (e.g., approximately 5 to 50 ml / min, approximately 10 to 30 ml / min, approximately 30 to 60 ml / min, approximately 60 to 120 ml / min, approximately 120 to 240 ml / min, approximately 240 to 360 ml / min, approximately 360 to 480 ml / min, or approximately 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of approximately 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or more. Lipid solution

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

[0127] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at total concentrations of approximately 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100 mg / ml or more. In some embodiments, a suitable lipid solution may contain a desired lipid in a total concentration ranging from approximately 0.1 to 100 mg / ml, 0.5 to 90 mg / ml, 1.0 to 80 mg / ml, 1.0 to 70 mg / ml, 1.0 to 60 mg / ml, 1.0 to 50 mg / ml, 1.0 to 40 mg / ml, 1.0 to 30 mg / ml, 1.0 to 20 mg / ml, 1.0 to 15 mg / ml, 1.0 to 10 mg / ml, 1.0 to 9 mg / ml, 1.0 to 8 mg / ml, 1.0 to 7 mg / ml, 1.0 to 6 mg / ml, or 1.0 to 5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids with a total concentration of up to approximately 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, or 10 mg / ml.

[0128] Any desired lipids can be mixed in any ratio suitable for mRNA encapsulation. In some embodiments, the suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and / or 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. Several cationic lipids are described in the literature, and many are commercially available. Cationic lipids particularly suitable for use in the compositions and methods of the present invention include those described in International Patent Publication 2010 / 053572 (specifically, C12-200 as described in paragraph

[0225] ) and International Patent Publication 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- This includes ionizable cationic lipids described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (incorporated herein by reference), such as liene-l-amine (HGT5001) and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-triene-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)aminobutyl)piperazine-2,5-dione (OF-02).

[0131] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include cationic lipids described in International Publication No. 2015 / 184256 (incorporated herein by reference) titled "Biodegradable Lipids for Nucleic Acid Delivery," such as 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxan-2,5-dione (target 23) and 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxan-2,5-dione (target 24).

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

[0133] In some implementations, each R 2 R is independently hydrogen, methyl, or ethyl. In some embodiments, each R 2 R is independently hydrogen or methyl. In some embodiments, each R 2 It 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 implementations, each R L C is independent 8-12 It is alkyl. In some embodiments, each R L nC 8-12 It is alkyl. In some embodiments, each R L C is independent 9-11 It is alkyl. In some embodiments, each R L nC 9-11 It is alkyl. In some embodiments, each R L C is independent 10 It is alkyl. In some embodiments, each R L nC 10 It is alkyl.

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

[0138] In some implementations, each R 2 is hydrogen, each q is independently 3-5, each R' is hydrogen, and each R L C is independent8-12 It is alkyl.

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

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

[0141] In certain embodiments, suitable cationic lipids are 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] As further examples of cationic lipids, the cationic lipid of formula I and [ka] The pharmaceutically acceptable salts include, During the ceremony, R is [ka] ("OF-00") R is [ka] ("OF-01") R is [ka] ("OF-02") or R is [ka] This is ("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, one or more cationic lipids suitable for the present invention may be N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA". (Feigner et al. (Proc. Nat.'l Acad. Sci. 84,7413 (1987); U.S. Patent No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide, i.e. "DOGS", 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-l-propaneaminium, i.e. "DOSPA" (Behr et al. Proc. Nat.'l Acad. Sci. 86,6982 (1989); U.S. Patent No. 5,171,678, U.S. Patent No. 5,334,761), l,2-dioleoyl-3-dimethylammonium-propane, i.e. "DODAP", and l,2-dioleoyl-3-trimethylammonium-propane, i.e. "DOTAP".

[0144] Further exemplary cationic lipids include l,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", l,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-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", and 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-l-(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 "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,l 2-dien-1-yl)-l,3-dioxolane-4-yl)-N,N-dimethylethaneamine (DLin-KC2-DMA)) (International Publication No. 2010 / 042877; Semple et al., Nature Biotech.Examples include 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 WO2005 / 121348A1). In some embodiments, one or more cationic lipids include at least one of an imidazole moiety, a dialkylamino moiety, or a guanidium moiety.

[0145] In some embodiments, one or more cationic lipids are 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-dimethylammoniumpropane), HGT4003 (International Publication No. 2012 / 170889, the teachings thereof are incorporated herein by reference in their entirety), ICE (International Publication No. 2011 Amino alcohol lipidoids such as those disclosed in International Publication No. 61 / 617,468 (Provisional Patent Application No. 61 / 617,468, the teachings thereof are incorporated herein by reference in their entirety), HGT5000 (Provisional Patent Application No. 61 / 617,468, the teachings thereof are incorporated herein by reference in their entirety), or HGT5001 (cis or trans) (Provisional Patent Application No. 61 / 617,468), International Publication No. 2010 / 053572, DOTAP (1,2-dioleyl-3-trimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic DLin-KC2-DMA (Semple, SCet Materials can be selected from (al. "Rational Design of Cationic Lipids for siRNA Delivery" Nature Biotech. 2010, 28, 172-176) and C12-200 (Love, KT 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 aminolipids. Aminolipids suitable for use in the present invention include those described in International Publication No. 2017180917, which is incorporated herein by reference. Examples of aminolipids 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-diene-1-amine (L608), and compound 18. Other aminolipids include compounds 2, 23, 27, 10, and 20. Further aminolipids suitable for use in the present invention include those described in International Publication No. 2017112865, which is incorporated herein by reference. The exemplary aminolipids in International Publication No. 2017112865 include compounds of one of the following formulas: (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1), and the compounds of paragraphs

[0185] ,

[0201] , and

[0276] . In some embodiments, cationic lipids suitable for use in the present invention include those described in International Publication No. 2016118725, which is incorporated herein by reference. Typical cationic lipids in International Publication No. 2016118725 include KL22 and KL25, among others. In some embodiments, cationic lipids suitable for use in the present invention include those described in International Publication No. 2016118724, which is incorporated herein by reference. Examples of cationic lipids in International Publication No. 2016118725 include KL10, 1,2-diglinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

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

[0148] As used herein, the term “noncationic lipid” means any neutral, zwitterionic, or anionic lipid. As used herein, the term “cationic lipid” means any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Noncationic lipids include, but are not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl Examples include oil-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or mixtures thereof.

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

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

[0151] In some embodiments, a suitable lipid solution comprises one or more PEGylated lipids. For example, derivatized lipids such as polyethylene glycol (PEG)-modified phospholipids and derivatized ceramides (PEG-CER) including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide) are also intended by the present invention. The intended PEGylated lipids have a length of C6-C20 This includes, but is not limited to, polyethylene glycol chains of up to 2 kDa, 3 kDa, 4 kDa, or 5 kDa in length covalently bonded to a lipid having an alkyl chain. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, certain useful interchangeable lipids are those with shorter acyl chains (e.g., C 14 or C 18 It is a PEG ceramide that has ).

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

[0153] Various combinations of lipids contained therein, namely cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, can be used to prepare pre-formed lipid nanoparticles and are described in the literature and herein. For example, a suitable lipid solution may 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 relevant technical field, 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 for "Novel ICE-based Lipid Nanoparticle Formulations for mRNA Delivery" (filed November 10, 2017), the full extent of which these disclosures are incorporated herein by reference. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids, including the lipid mixture, and the relative molar ratios of these lipids, are based on the characteristics of the selected lipids, as well as the properties and characteristics of the mRNA to be encapsulated. Further considerations include, for example, the saturation of the alkyl chains of the selected lipids, as well as their size, charge, pH, pKa, fusionability, and toxicity. Thus, the molar ratios may be adjusted accordingly. Formulation and mixing process of pre-formed nanoparticles

[0154] This invention is based on the surprising and unexpected discovery that mixing empty, pre-formed lipid nanoparticles (i.e., lipid nanoparticles formed without mRNA) with mRNA results in potency and efficacy for the resulting encapsulated mRNA.

[0155] In some previously disclosed processes, refer to U.S. Patent Application No. 14 / 790,562 (filed July 2, 2015) and its Provisional Patent Application No. 62 / 020,163 (filed July 2, 2014) for “Messenger RNA Encapsulation” (these disclosures are incorporated herein by reference in their entirety), and in some embodiments, the prior art provides a step of encapsulating messenger RNA (mRNA) in lipid nanoparticles by mixing an mRNA solution and a lipid solution, wherein the mRNA solution and / or lipid solution are heated to a predetermined temperature above ambient temperature before mixing to form lipid nanoparticles containing 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 pre-formed lipid nanoparticles with mRNA, resulting from the order in which these components are added, and the resulting formed particles exhibit improved potency and efficacy. The mixing of the components is achieved by a pump system that maintains a constant lipid / mRNA (N / P) ratio throughout the process and allows for easy scale-up. In some embodiments, the process is carried out on a large scale. For example, in some embodiments, the composition according to the present invention contains 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, for certain cationic lipid nanoparticle formulations of mRNA, heating of the mRNA in citrate buffer is essential for mRNA protection and delivery. In those processes or methods, heating after formulation (after nanoparticle formation) does not increase the encapsulation efficiency of mRNA in lipid nanoparticles, so heating is required to occur before the formulation process (i.e., heating of separate components). In contrast, in some embodiments of the novel processes of the present invention, the order of mRNA heating does not appear to affect the mRNA encapsulation rate. In some embodiments, heating (i.e., maintaining at ambient temperature) one or more of the solution containing pre-formed lipid nanoparticles, the solution containing mRNA, and the mixed solution containing mRNA encapsulated in lipid nanoparticles does not need to be performed before or after the formulation process. Since controlled temperature changes after mixing are easily achievable, this potentially provides a significant advantage for precise scale-up.

[0158] In this novel process, in some embodiments, encapsulating mRNA by mixing it with empty (i.e., mRNA-free) pre-formed lipid nanoparticles (Process B) yields significantly higher potency compared to encapsulating mRNA by mixing it with lipid components only (i.e., not pre-formed in lipid nanoparticles) (Process A). As shown in the following examples, e.g., Tables 3 and 4, the potency of any mRNA encapsulated in lipid nanoparticles tested ranges from 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 may be ethanol. In some embodiments, the aqueous solution may be a citrate buffer.

[0160] As used herein, the term “ambient temperature” means the room temperature or the temperature surrounding the object of interest (e.g., a pre-formed empty lipid nanoparticle solution, an mRNA solution, or a lipid nanoparticle solution containing mRNA) without heating or cooling. In some embodiments, the ambient temperature at which one or more solutions are maintained is about 35°C, 30°C, 25°C, 20°C, or less than 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] Therefore, a predetermined temperature higher than the ambient temperature is typically higher than about 25°C. In some embodiments, a predetermined temperature suitable for the present invention 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, a predetermined temperature suitable for the present invention is 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 or higher. In a particular embodiment, a predetermined temperature suitable for the present invention is about 65°C.

[0162] In some embodiments, the mRNA or a pre-formed 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 pre-formed empty lipid nanoparticle solution are heated separately to predetermined temperatures before mixing. In some embodiments, the mRNA and the pre-formed empty lipid nanoparticle solution are mixed at ambient temperature, and then heated to a predetermined temperature after mixing. In some embodiments, the pre-formed 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 pre-formed empty lipid nanoparticle solution at ambient temperature.

[0163] In some embodiments, the mRNA solution is heated to a predetermined temperature by adding the mRNA stock solution, which is at ambient temperature, to a heated buffer solution in order to achieve a 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 higher than the ambient temperature before mixing. In some embodiments, the lipid solution containing dissolved lipids and the aqueous solution are heated separately to predetermined temperatures 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 of one or more of the solutions containing pre-formed lipid nanoparticles, the solutions containing mRNA, and the mixed solutions containing mRNA encapsulated with lipid nanoparticles does not occur before or after the formulation process.

[0165] In some embodiments, lipid solutions and aqueous or buffered solutions may be mixed using a pump. In some embodiments, mRNA solutions and pre-formed empty lipid nanoparticle solutions may be mixed using a pump. Since encapsulation can occur on a wide range of scales, different types of pumps may be used to adapt to the desired scale. However, generally, it is preferable to use a pulseless flow pump. As used herein, a pulseless flow pump means any pump capable of establishing a stable flow rate and continuous flow. Suitable pump types may include, but are not limited to, gear pumps and centrifugal pumps. Exemplary gear pumps include, but are not limited to, Cole-Palmer or Siener gear pumps. Exemplary centrifugal pumps include, but are not limited to, those manufactured by Granger or Cole-Palmer.

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

[0167] The appropriate flow rate for mixing can be determined based on the scale. In some embodiments, the mRNA solution is mixed at approximately 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, and 150-1400 ml. The mixture is mixed at flow rates in the range of 160-1500 ml / min, 170-1600 ml / min, 180-1700 ml / min, 150-250 ml / min, 250-500 ml / min, 500-1000 ml / min, 1000-2000 ml / min, 2000-3000 ml / min, 3000-4000 ml / min, or 4000-5000 ml / min. In some embodiments, the mRNA solution is mixed at flow rates of approximately 200 ml / min, approximately 500 ml / min, approximately 1000 ml / min, approximately 2000 ml / min, approximately 3000 ml / min, approximately 4000 ml / min, or approximately 5000 ml / min.

[0168] In some embodiments, the lipid solution or pre-formed lipid nanoparticle solution is mixed at flow rates in the range of approximately 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. In some embodiments, the lipid solution is mixed at a flow rate of approximately 50 ml / min, 100 ml / min, 150 ml / min, 200 ml / min, 250 ml / min, 300 ml / min, 350 ml / min, 400 ml / min, 450 ml / min, 500 ml / min, 550 ml / min, 600 ml / min, 650 ml / min, 700 ml / min, 750 ml / min, 800 ml / min, 850 ml / min, 900 ml / min, 950 ml / min, or 1000 ml / min.

[0169] Generally, in some embodiments, a lipid solution containing dissolved lipids and an aqueous or buffer solution are mixed in 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 in a solution so that the mRNA is encapsulated within the lipid nanoparticles. Such solutions are also called formulations or encapsulation solutions. A suitable formulation or encapsulation solution contains a solvent such as ethanol. For example, a suitable formulation or encapsulation solution may contain 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 mounting solution includes a solvent such as isopropyl alcohol. For example, a suitable formulation or mounting solution may contain 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, the suitable formulation or encapsulation solution contains a solvent such as dimethyl sulfoxide. For example, the suitable formulation or encapsulation solution contains 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, the appropriate formulation or encapsulation solution may also include 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, pre-formed lipid nanoparticle formulations used in preparing this novel nanoparticle formulation can be stably frozen in a 10% trehalose solution.

[0173] In some embodiments, the empty (i.e., mRNA-free) pre-formed lipid nanoparticle formulations used to prepare the novel nanoparticle formulations can be stably frozen in approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% trehalose solutions. In some embodiments, adding mRNA to the empty lipid nanoparticles yields 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 destabilizers are absent during mRNA addition, and therefore 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 destabilizers and the stability of the trehalose solution increase the ease of scaling up the formulation and producing mRNA-encapsulated lipid nanoparticles. purification

[0175] In some embodiments, empty, pre-formed 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 called cross-flow filtration, is a type of filtration in which the substance to be filtered passes tangentially to the filter rather than through it. In TFF, undesirable permeates pass through the filter, while desirable retained substances pass along the filter and are collected downstream. It is important to note that the desired substances are usually contained in the retained substances in TFF and are the opposite of what is typically encountered in conventional total filtration.

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

[0177] The main advantage of tangential flow filtration is that it carries impermeable particles (sometimes called a "filtration cake") along the surface of the filter instead of agglomerating and clogging it during conventional "total" filtration. This advantage significantly reduces downtime because the filter generally does not need to be removed and cleaned, making tangential flow filtration widely usable in industrial processes requiring continuous operation.

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

[0179] Dialysis filtration is a fractionation process in which small, undesirable particles pass through a filter, while larger, desired nanoparticles are retained within the retainer without changing the concentration of those nanoparticles in the solution. Dialysis filtration is often used to remove salts or reaction buffers from a solution. Dialysis filtration can be continuous or discontinuous. In continuous dialysis filtration, the dialysis filtration solution is added to the sample feed at the same rate as the filtrate is produced. In discontinuous dialysis filtration, the solution is first diluted and then concentrated to a starting concentration. Discontinuous dialysis filtration may be repeated until the desired nanoparticle concentration is reached.

[0180] Purified and / or concentrated lipid nanoparticles can be formulated in a desired buffer, such as PBS. Nanoparticles containing the provided mRNA

[0181] The process according to the present invention yields higher potency and efficacy, thereby enabling lower doses and thereby shifting the therapeutic index in a positive direction. In some embodiments, the process according to the present invention yields homogeneous and small particle size (e.g., less than 150 nm), as well as significantly improved encapsulation efficiency and / or mRNA recovery rate, compared to processes of the conventional art.

[0182] Accordingly, the present invention provides compositions comprising purified nanoparticles as described herein. In some embodiments, the 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 homogeneous nanoparticles having a narrower particle size range are achieved by the process of the present invention. For example, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles in the composition provided by the present invention 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 95 nm, about 75 to 90 nm, or 75 to 85 nm). In some embodiments, substantially all of the purified nanoparticles 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 75–90 nm, or 75–85 nm).

[0184] In some embodiments, the polydispersity index (PDI), a measure of the molecular size dispersity or molecular size heterogeneity, of the nanoparticles in the composition provided by the present invention is 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, more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in the composition provided by the present invention encapsulate mRNA within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles in the composition encapsulate mRNA within each individual particle.

[0186] In some embodiments, the composition according to the present invention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, the process according to the present invention results in a recovery of more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the mRNA.

[0187] In some embodiments, the composition according to the present invention is formulated for administration to a subject. In some embodiments, the composition of mRNA-lipid nanoparticles described herein is formulated at a dose concentration of less than 1.0 mg / kg mRNA-lipid nanoparticles (e.g., 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 result in higher potency and efficacy. In some embodiments, the dose is reduced by about 70%, 65%, 60%, 55%, 50%, 45%, or 40%.

[0188] In some embodiments, the efficacy of lipid nanoparticles encapsulating mRNA 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%) when prepared by process B compared to process A, and greater than 1000%. [Examples]

[0189] While specific compounds, compositions, and methods of the present invention have been described in detail according to specific embodiments, the following examples are for illustrative purposes only and are not intended to limit the present invention. lipid material

[0190] The formulations described in the following examples, unless otherwise specified, comprise a multi-component lipid mixture in varying proportions, 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 an exemplary lipid nanoparticle formulation process for encapsulating mRNA. As used herein, Process A refers to a method of encapsulating mRNA by mixing mRNA with a lipid mixture without first pre-forming the lipids into lipid nanoparticles. Compared to Process B, which is described below, Process A does not involve the pre-formation of lipid nanoparticles.

[0192] An exemplary formulation process A is shown in Figure 1. In this process, in some embodiments, an ethanol-lipid solution and an mRNA buffer solution were prepared separately. A solution of lipid mixtures (such as cationic lipids, helper lipids, zwitterionic lipids, and PEG lipids) was prepared by dissolving them in ethanol. The mRNA solution was prepared by dissolving mRNA in citrate buffer, yielding mRNA at a concentration of 0.0833 mg / ml in 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 examples, 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 dialysfiltration 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 pre-formed lipid nanoparticles

[0193] This embodiment illustrates exemplary process B for encapsulating mRNA. As used herein, process B refers to the process of encapsulating messenger RNA (mRNA) by mixing it with pre-formed lipid nanoparticles. A wide range of different conditions, such as varying temperatures (i.e., heating or non-heating of the mixture), buffers, and concentrations, may be used in process B. 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 flows formed empty lipid nanoparticles, which was a self-assembly process. The resulting formulation mixture consisted of empty lipid nanoparticles in a citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process for buffer exchange. The resulting suspension of pre-formed empty lipid nanoparticles was then mixed with mRNA using a pump system. For certain cationic lipids, heating the mixed solution resulted in a higher proportion of mRNA-containing lipid nanoparticles and a higher total mRNA yield.

[0195] In addition, the effect of the presence of citrate buffer during mRNA addition in process B was studied. Table 1 shows an example of the encapsulation efficiency of lipid nanoparticle formulations in process B with citrate buffer (Ph4.5). A decrease in mRNA encapsulation efficiency was observed when citrate buffer was present during the mixing of pre-formed empty lipid nanoparticles and mRNA. In the presence of citrate buffer, the encapsulation efficiency of lipid nanoparticle formulations in process B was less than 60%. The encapsulation efficiency of lipid nanoparticle formulations prepared by process B without citrate buffer was 90% or higher. 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 generated by process B was unexpectedly more effective than that generated by process A.

[0197] In this example, OTC spf ashMice were administered a single dose of 0.5 mg / kg 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. The formulations were first tested immediately after mixing without storage, and then again after mixing, and stored 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 prepared by process A or process B. ash Show a mouse.

[0199] Generally, the activity of expressed hOTC proteins can be evaluated using citrulline production. As shown in Figure 3, OTC spf ash The citrulline activity mediated by hOTC protein expressed in mouse liver was measured 24 hours after a single administration of lipid nanoparticle mRNA preparations prepared by Process A and Process B, respectively. Graph (i) in Figure 3 shows the citrulline activity attributable to hOTC expressed immediately after mixing the preparations and without storage time, after delivery of the lipid nanoparticle mRNA preparations by Process A and Process B, respectively. Graph (ii) in Figure 3 shows the citrulline activity attributable to hOTC expressed after storing the preparation mixture at -80°C for 2.5 months, after delivery of the lipid nanoparticle mRNA preparations by Process A and Process B, respectively.

[0200] The results shown in Figure 3 indicate that the formulation prepared by process B with pre-formed empty lipid nanoparticles exhibited 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 formulations produced by process A and process B showed 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 shows that lipid nanoparticles produced by Process B with different parameters lead to citrulline activity comparable to that seen in wild-type mice when delivered to spf ash 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 were prepared using different types of pumps and made by Process B.

[0203] Figure 4 shows the typical activity of hOTC protein (for citrulline production) expressed in the livers of OTC spf ash mice 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. Lipid nanoparticle formulations made by Process B were prepared using (1) a gear pump, (2) a peristaltic pump, (3) a peristaltic pump at a lower flow rate, and (4) different flow rates of mRNA and empty preformed lipid nanoparticles.

[0204] In some embodiments, lipid nanoparticle formulations by 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, the activity of expressed hOTC proteins can be evaluated using citrulline production. As shown in Figure 4, OTC spf ash The citrulline activity of hOTC protein in mouse liver was measured 24 hours after a single dose of lipid nanoparticle mRNA preparations prepared by either process A or process B, which have different parameters.

[0207] As shown in Figure 4, exemplary data were obtained by comparing different lipid nanoparticle formulations (1-4) prepared by process B. ash When administered to mice, the treatment resulted in remarkable levels of citrulline activity in 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 gene-transformed cells.

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

[0210] In this example, 293T cells were transduced using an ASS1 mRNA lipid nanoparticle formulation prepared by process A or process B. Either 1 μg of ASS1 was transduced using lipofectamine, or 10 μg of ASS1 mRNA encapsulated in a lipid nanoparticle formulation was transduced. 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 resulted 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 equivalent to the level resulting from transfection with the ASS1 mRNA-lipofectamine complex. 10 6 The ASS1 protein levels per cell were 12.43, 0.43, and 12.11 μg, respectively. The ASS1 protein levels resulting from transfection using the lipid nanoparticle formulation prepared by Process B were 28 times higher than those from transfection using the lipid nanoparticle formulation prepared by Process A. Example 6. In vivo expression of hCFTR in rat lung

[0212] Figure 6 shows an example 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 Dolly rats were administered via inhalation a lipid nanoparticle formulation containing hCFTR mRNA prepared by Process B. Lipid nanoparticle formulations were prepared using cKK-E12, ICE, or Target 24 lipid as cationic lipids. Fixed lung tissue from these rats was analyzed for the presence of hCFTR protein by immunohistochemical staining.

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

[0215] 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.

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

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

[0218] This example describes an example of a method for administering firefly luciferase (FFL) mRNA-carrying lipid nanoparticles generated by process B, and an example of a method for analyzing firefly luciferase in target tissue in vivo.

[0219] Figure 8 shows bioluminescence images of wild-type mice 24 hours after intravitreous administration of FFL mRNA encapsulated in lipid nanoparticles.

[0220] In this example, wild-type mice were treated with lipid nanoparticles containing mRNA encoding FFL produced by process B via intravitreous 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 indicate significant luminescence in the eyes of these mice, representing 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-carrying lipid nanoparticles generated by process B, and an example of a method for analyzing firefly luciferase in target tissue in vivo.

[0223] Figure 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 containing 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 mouse. Luminescence was monitored 24 hours after application.

[0225] The results shown in Figure 9 indicate significant luminescence in the eyes of these mice, representing 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 the mouse serum were measured 24 hours after administration.

[0227] Figure 10 shows examples 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] The mRNA-derived hPAH protein was shown to be enzymatically active, as demonstrated by measuring the level of serum phenylalanine reduction using a custom ex vivo activity assay. Generally, serum phenylalanine reduction can be used to evaluate the potency (i.e., the 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 preparation prepared by process B delivered subcutaneously. For comparison, serum phenylalanine levels were also measured in PAH KO mice treated with physiological saline.

[0229] The results shown in Figure 10 indicate that subcutaneous injection of the lipid nanoparticle hPAH mRNA formulation resulted in a significant decrease in phenylalanine levels. No significant difference in phenylalanine levels was observed in PAH KO mice treated with saline before and after administration. Example 11. In vivo activity of OTC expressed in mice

[0230] This example involves OTC KOspf treated with physiological saline. ash OTC KO spf treated with subcutaneous administration of hOTC mRNA-supported lipid nanoparticles prepared by mouse and process B. ash This shows a comparison of OTC protein levels in mouse livers.

[0231] As shown in Figure 11, OTC KOspf ash Examples of citrulline production resulting from hOTC protein expression in mouse liver were measured 24 hours after a single subcutaneous administration of lipid nanoparticles containing hOTC mRNA preparations prepared by Process B. In addition, OTC KOspf ash Citrulline production in the liver of mice was measured after injection of physiological saline.

[0232] The results shown in Figure 11 are for subcutaneously injected lipid nanoparticles hOTC prepared by process B. This study demonstrates that the mRNA formulation resulted in significantly greater activity of the hOTC protein expressed 24 hours after administration compared to diseased mice treated with physiological saline. Example 12. In vivo expression of ASS1 in mice

[0233] Figure 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 a lipid nanoparticle formulation containing hASS1 mRNA prepared by Process B.

[0234] Generally, the efficiency of the delivery method can be evaluated using the expressed hASS1 protein level. As shown in Figure 12, exemplary hASS1 protein levels in SAS1 KO mice were measured 24 hours after a single subcutaneous administration of the hASS1 mRNA preparation prepared by Process B. In addition, for comparison, hASS1 protein levels were also measured in ASS1 KO mice treated with physiological saline.

[0235] The results shown in Figure 12 indicate that the subcutaneously injected hASS1 mRNA lipid nanoparticle formulation prepared by Process B resulted in significantly higher hASS1 protein levels in hASS1 KO mice 24 hours after administration, compared to hASS1 KO mice treated with physiological saline. Example 13. In vivo expression of hEPO in mice via various administration routes.

[0236] This example demonstrates a comparison of human EPO (HePO) expression in wild-type mice after administration of hEPO mRNA encapsulated in lipid nanoparticles produced by Process B. This example further illustrates a comparison of the potency 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 doses and time points, regardless of whether it was delivered by the intradermal or intramuscular administration route evaluated.

[0237] In this example, wild-type mice were administered single doses of lipid nanoparticles containing hEPO mRNA produced by Process B at various concentrations (i.e., 1 μg, 10 μg, or 50 μg) via intradermal, subcutaneous, or intramuscular pathways. Serum levels of hEPO protein were measured 6 and 24 hours after administration. Furthermore, wild-type mice were administered hEPO mRNA produced by Process A or Process B via intradermal or intramuscular pathways. Single doses of mRNA-encapsulated lipid nanoparticles at various concentrations (i.e., 1 μg, 10 μg, or 50 μg) were administered. 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 with a single dose of hEPO mRNA preparations prepared by Process B, 6 and 24 hours after administration. The routes compared were intradermal, subcutaneous, or intramuscular injection.

[0239] The efficacy of mRNA via different delivery methods can be evaluated using the levels of hEPO protein in the serum of treated mice. As shown in Figure 13, exemplary hEPO protein levels in mouse serum were evaluated by ELISA 6 and 24 hours after single doses of hEPO mRNA lipid nanoparticle formulations prepared by Process B at 1 μg, 10 μg, and 50 μg. Furthermore, hEPO protein levels from intradermal, subcutaneous, and intramuscular administration routes were compared.

[0240] The results shown in Figure 13 indicate that intramuscular injection of hEPO mRNA lipid nanoparticle formulations generally yielded the highest levels of hEPO protein compared to intradermal and subcutaneous routes. Six hours after 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 prepared by Process A or Process B. Figure 14 shows that, at all doses, formulations prepared by Process B resulted in approximately 2 to 4 times higher hEPO protein expression levels 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 prepared by Process A or Process B. Figure 15 shows that, at all doses, formulations prepared by Process B resulted in approximately 2–4 times higher hEPO protein expression levels compared to formulations prepared by Process A.

[0243] As shown in Figures 14 and 15, hEPO protein levels in mouse serum were evaluated by ELISA 6 and 24 hours after single doses of 1 μg, 10 μg, and 50 μg of hEPO mRNA lipid nanoparticle formulations prepared by Process A or Process B, respectively, administered intradermally and intramuscularly. The results show substantially higher potency of the 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 endothelial system (i.e., myocytes, fibroblasts, macrophages, adipocytes, etc.). Example 14. Protein expression from mRNA lipid nanoparticles in an animal model

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

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

[0246] The test substance was administered as a single dose via tail vein injection. Twenty-four hours after administration, mice were subjected to ammonia loading, and a bolus of ammonium chloride (5 mmol / kg NH4Cl) was administered intraperitoneally. Blood was collected 40 minutes after NH4Cl loading by collecting whole blood aliquots into lithium heparin plasma tubes, which were then treated as plasma, and plasma ammonia was analyzed using an IDEXX Catalyst Dx analyzer. The animals were then sacrificed, their livers were collected, and hOTC expression was evaluated using sandwich ELISA.

[0247] Figure 16 shows a schematic diagram of the ammonia loading portion of the study, which was implemented to replicate the onset of hyperammonemia that patients with OTC deficiency may experience.

[0248] Figure 17 shows ammonia-loaded mice, particularly wild-type mice with normal OTC (WT) and spf mice that did not receive hOTC mRNA lipid nanoparticles (KO). ash Mice and SPF mice that 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 graph shows the plasma ammonia levels of each mouse. As the graph shows, spf mice that did not receive hOTC mRNA lipid nanoparticles (KO) ash In mice, compared to a significant increase in plasma ammonia under identical conditions, 0.5 mg / kg and 0.16 mg / kg of hOTC mRNA lipid nanoparticles produced by Process B achieved statistically significant protection against the development of model hyperammonemia. This data explains 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 ammonium chloride loading for at least 24 hours post-administration.

[0249] Figure 18 and Table 3 show the levels of hOTC protein from the livers of animals sacrificed 24 hours after administration of hOTC mRNA lipid nanoparticles, as measured by sandwich ELISA. As these results show, the hOTC protein expressed from the livers of mice treated with mRNA lipid nanoparticles prepared by process B was approximately 1000% (i.e., 10 times) higher than that from the livers of mice treated with the same mRNA lipid nanoparticles prepared by process A. Table 3 provides specific amounts (as a percentage of total protein) of hOTC 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 shown) 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 mice treated with mRNA lipid nanoparticles prepared by Process A by approximately 700% (approximately 8 times) and up to approximately 1000% (approximately 11 times) at 24 hours post-administration, 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 A compared to those prepared by Process B was 884% (9.65 times) at 24 hours post-administration, across all doses. This data indicates that mRNA lipid nanoparticles produced by Process B are significantly more potent than identical mRNA lipid nanoparticles produced by Process A at 24 hours post-administration, across all doses.

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

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

[0253] In this study, male spf ash Mice were administered 1.0 mg / kg of hOTC mRNA lipid nanoparticles prepared by Process B via a single intravenous tail vein injection. The mouse cohort was removed 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.

[0254] For each removed cohort, the animals were subjected to an ammonia load, and then blood was collected for plasma ammonia (μmol / L) measurement. Subsequently, the animals were euthanized, and citrulline (citrulline μmol / hr / total protein mg) and urinary orotic acid (μmol / creatinine mmol) were measured.

[0255] For ammonia loading and plasma ammonia measurement, see Figure 16 for a general schematic diagram, and for a description of this test, see Example 14.

[0256] For citrulline measurement, mouse liver homogenate was prepared, diluted with 1× 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 was allowed to proceed at 37°C for 30 minutes. The reaction was stopped using a mixture of phosphoric acid and sulfuric acid, and diacetyl monooxime was added. The sample was incubated at 85°C for 30 minutes, briefly cooled, and citrulline was quantified relative to the citrulline standard by reading at 490 nm.

[0257] For urinary orotic acid measurement, orotic acid was quantified from animal urine samples via ultra-high-performance liquid chromatography (UPLC) using an ion-exchange column. Briefly, urine samples were diluted 2-fold with RNase-free water, and a portion was loaded onto a ThermoScientific 100x column. Orotic acid was separated by a mobile phase containing acetonitrile and 25 mM ammonium acetate, and quantified by detection based on absorbance at 280 nm.

[0258] Figure 21 shows the results for wild-type mice (WT) and untreated spf mice 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 (untreated), and spf ash For each mouse, the plasma ammonia levels in the animal 40 minutes after ammonia loading are shown. The dashed line represents the mean plasma ammonia levels of the wild-type control group (WT). As seen in the results shown in the figure, a single dose of hOTC mRNA lipid nanoparticles provides significant protection against hyperammonemia for at least 15 days. Specifically, post-load plasma ammonia levels are comparable to wild-type levels (WT) or below untreated levels (untreated) at all time points evaluated up to 15 days.

[0259] Figure 22 shows the results for wild-type mice (WT) and untreated spf mice 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 (untreated), and spf ash The hOTC protein activity, measured by citrulline production in each mouse, is shown. As can be seen from the results shown in the figure, a single dose of hOTC mRNA lipid nanoparticles resulted in citrulline levels exceeding or comparable to wild-type controls (WT), and significantly exceeding those of untreated controls (untreated) at all time points evaluated up to 15 days.

[0260] Figure 23 shows untreated SPF ash In mice (untreated), SPF was measured 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 hOTC protein activity is measured by maintained low levels of urinary orotic acid production in mice and untreated wild-type mice (untreated C57BL / 6), respectively. As seen in the results shown in the figure, a single dose of hOTC mRNA lipid nanoparticles resulted in low levels of urinary orotic acid produced that were lower than or comparable to those of wild-type controls (untreated C57BL / 6), and at all time points evaluated up to 15 days in untreated mice. ash This results in significantly lower levels of urinary orotic acid compared to untreated mice.

[0261] In summary, the data from this embodiment demonstrate that a single intravenous administration of exemplary mRNA lipid nanoparticles produced by process B generates active proteins 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 dosage levels (1.0 mg / kg, 0.6 mg / kg, and 0.3 mg / kg) was unexpectedly more potent than those produced by Process A at each dosage evaluated.

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

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

[0265] Generally, citrulline production can be used to evaluate the activity of the expressed hOTC protein. As shown in Figure 24, OTC spf ash The citrulline activity by the expressed hOTC protein in the livers of OTC spf mice was measured 24 hours after a single administration of lipid nanoparticle mRNA formulations produced by Process A and Process B, respectively, at various dosage levels. The graph in Figure 24 shows the citrulline activity resulting from the expressed hOTC after delivery of lipid nanoparticle mRNA formulations by Process A and Process B formulated for each of the above processes.

[0266] The results shown in Figure 24 indicate that the formulation prepared by process B using pre-formed empty lipid nanoparticles yielded higher citrulline activity of the hOTC protein 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 single intravenous administration of hOTC mRNA prepared 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 prepared by Process B compared to the group administered with Process A, as evidenced by the intensity of the bands. Example 17.spf ash In vivo activity of hOTC expressed in mice

[0268] This example shows hOTC delivered via lipid nanoparticles produced by process B. This indicates that the mRNA was unexpectedly more effective than that generated by process A.

[0269] Figure 26 shows the OTC spf 24 hours after a single intravenous administration of 0.5 mg / kg of hOTC mRNA encapsulated in lipid nanoparticle formulations prepared by Process A or Process B. ash This shows the exemplary activity of hOTC protein (related to citrulline production) expressed in the liver of mice.

[0270] Generally, the activity of expressed hOTC proteins can be evaluated using citrulline production. As shown in Figure 26, OTC spf ash The citrulline activity of hOTC protein expressed in mouse liver was measured 24 hours after dose administration. The results showed that, at the same dose, the formulation prepared by process B resulted in higher citrulline 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 seen in the figures, staining of hOTC protein was stronger in the mouse group administered with the LMP formulation prepared by process B (Figures 27(a)–(b)) compared with process A (Figures 27(c)–(d)). The results shown in Figures 27(a)–(d) are consistent with the higher citrulline production at doses of the formulation prepared by process B compared with 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 (consisting of various different cationic lipids) produced by process B was unexpectedly more effective than that 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 (as described above), prepared using one of five different cationic lipids and manufactured by process A or process B.

[0274] Table 4 shows the specific hEPO protein expression levels measured by ELISA in the serum of animals sacrificed 6 hours after administration of hEPO mRNA lipid nanoparticles, each prepared using one of five different cationic lipids and manufactured by process A or process B. As these results indicate, the hEP protein expressed from mRNA lipid nanoparticles prepared by process B, as measured in mouse serum, was substantially higher than that expressed from the same mRNA lipid nanoparticles prepared by process A across all different cationic lipids evaluated. The increase rates ranged from 133% to 603%, accompanied by a consistent increase of higher potency than 100% observed across the five different lipids tested in the study.

[0275] Figure 28 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA preparations produced by processes A and B using HGT 5001 as the cationic lipid. Figure 29 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA preparations produced by processes A and B using ICE as the cationic lipid. Figure 30 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA preparations produced by processes A and B using cKK-E12 as the cationic lipid. Figure 31 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA preparations produced by processes A and B using C12-200 as the cationic lipid. Figure 32 shows the hEPO protein expression after delivery of lipid nanoparticle mRNA preparations produced by processes A and B using HGT 4003 as the cationic lipid. As the results in each of these graphs (Figures 28-32) show, the hEPO protein expressed from mRNA lipid nanoparticles prepared by Process B, as measured in mouse serum, was substantially higher than that expressed from 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 various 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 was approximately 90 nm to 150 nm across all nanoparticles prepared using the five cationic lipids evaluated, while the nanoparticle size of those prepared by process A was 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 from this embodiment show a substantial increase in the efficacy of mRNA lipid nanoparticles produced by process B compared to process A, across lipid nanoparticles containing various different lipid components. Evenly

[0278] Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein using methods that do not exceed routine experimental techniques. The scope of the invention is not intended to be limited to the foregoing, but rather as described in the following claims.

Claims

1. A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles, The process involves (i) mixing an aqueous solution containing pre-formed lipid nanoparticles having a size in the range of 75 to 150 nm and comprising one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids, and one or more PEG lipids, so that lipid nanoparticles encapsulating mRNA are formed, A method wherein the aqueous solution containing pre-formed lipid nanoparticles and the aqueous solution containing mRNA do not contain ethanol, or contain only trace amounts (less than 1%) of ethanol, and do not contain citric acid, or contain only trace amounts (less than 1 mM) of citric acid.

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

3. The method according to claim 1, wherein the mRNA and / or the pre-formed lipid nanoparticles are heated to a temperature higher than the ambient temperature before mixing.

4. The method according to claim 2 or 3, wherein the temperature is approximately 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, or higher.

5. The method according to any one of claims 2 to 4, 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.

6. The method according to any one of claims 2 to 5, wherein the temperature is approximately 65°C.

7. The one or more cationic lipids mentioned above include 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-DM The method according to claim 1, selected from the group consisting of A, DLin-K-XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxan-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-dioxan-2,5-dione (target 24), and combinations thereof.

8. The method according to claim 1, wherein the one or more cationic lipids include the target 24.

9. The method according to claim 1, wherein the one or more cationic lipids include ICE.

10. The method according to claim 1, wherein the one or more cationic lipids include cKK-E12.

11. The method according to claim 1, wherein the one or more noncationic 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)).

12. The aforementioned one or more PEG-modified lipids, 6 -C 20 The method according to claim 1, comprising a poly(ethylene) glycol chain with a maximum length of 5 kDa covalently bonded to a lipid having a long alkyl chain.

13. The method according to any one of claims 1 to 12, wherein the pre-formed lipid nanoparticles are purified by a tangential flow filtration (TFF) process.

14. The method according to any one of claims 1 to 13, which results in an encapsulation rate of approximately 90%, 95%, 96%, 97%, 98%, or 99% or more.

15. The method according to any one of claims 1 to 14, which yields a recovery rate of more than approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of mRNA.

16. The method according to any one of claims 1 to 15, wherein the lipid nanoparticles encapsulating the mRNA are prepared from the pre-formed lipid nanoparticles in a trehalose solution.

17. The method according to any one of claims 1 to 16, wherein the solution containing mRNA and / or the solution containing pre-formed lipid nanoparticles is an aqueous solution containing one or more of trehalose, sucrose, lactose, and mannitol.

18. The method according to any one of claims 1 to 17, wherein the lipid nanoparticles encapsulating the mRNA do not require further downstream processing.

19. A composition of lipid nanoparticles encapsulating mRNA produced by the method of any one of claims 1 to 18, wherein the lipid nanoparticles are produced by a process comprising mixing an aqueous buffer solution containing mRNA with a solution containing the lipid component of pre-formed lipid nanoparticles dissolved in a non-aqueous solvent to form lipid nanoparticles that encapsulate mRNA. A composition that has higher efficacy than particles.

20. The composition according to claim 19, wherein the efficacy of the lipid nanoparticles is more than 100% higher than that of lipid nanoparticles produced by a process comprising mixing an aqueous buffer solution containing mRNA with a solution containing the lipid component of pre-formed lipid nanoparticles dissolved in a non-aqueous solvent to form lipid nanoparticles that encapsulate mRNA.

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