RU2832672C2 - Improved method of producing lipid nanoparticles loaded with mrna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and molecular biology. Disclosed is an improved method of forming lipid nanoparticles and encapsulating mRNA, comprising a step of heating lipid nanoparticles with encapsulated mRNA in a solution to formulate a medicinal product.

EFFECT: invention is applicable for providing a higher degree or efficiency of encapsulation when producing mRNA-LNP compared to traditional approaches.

27 cl, 5 dwg, 3 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to U.S. Provisional Patent Application Serial No. 62/847,837, filed May 14, 2019, which is incorporated herein by reference in its entirety for all purposes.

LEVEL OF TECHNOLOGY

[2] Messenger RNA therapy (MRT) is becoming an increasingly important approach for the treatment of a number of diseases. MRT involves the administration of messenger RNA (mRNA) to a patient in need of therapy to ensure that the protein encoded by the mRNA is produced in the patient's body. Lipid nanoparticles are typically used to encapsulate mRNA for efficient delivery of mRNA in vivo .

[3] To improve delivery via lipid nanoparticles, much effort has been focused on identifying new lipids or specific lipid compositions that can influence intracellular delivery and/or mRNA expression, such as in various types of mammalian tissues, organs, and/or cells (e.g., mammalian liver cells). However, such existing approaches are expensive, time-consuming, and unpredictable.

BRIEF DESCRIPTION OF THE INVENTION

[4] The present invention provides, among other things, further improved methods for producing mRNA-loaded lipid nanoparticles (mRNA-LNPs). The present invention is based on the unexpected discovery that, following a process for encapsulating messenger RNA (mRNA) in LNPs, comprising mixing one or more lipids in a lipid solution with one or more mRNAs in an mRNA solution to form mRNA encapsulated in LNPs (mRNA-LNPs) in a LNP formation solution (e.g., Method A, which is further described below), the further steps of exchanging the LNP formation solution for a drug product formulation solution and heating the mRNA-LNPs in the drug product formulation solution provide the unexpected advantage of significantly increasing the encapsulation efficiency of the mRNA-LNPs, i.e., the amount or percentage of mRNA encapsulated in the LNPs (i.e., the degree or efficiency of encapsulation). The present invention is particularly useful for providing a higher degree or efficiency of encapsulation in the production of mRNA-LNPs compared to conventional approaches.

[5] Compared with conventional approaches, the method of the present invention described herein provides higher encapsulation efficiency and accordingly can provide higher activity and better efficacy of mRNA delivered by lipid nanoparticles, thereby shifting the therapeutic index in a positive direction and providing additional advantages such as lower cost, better patient compliance and more convenient dosing regimens for the patient. The mRNA-loaded lipid nanoparticle formulations provided by the present invention can be successfully delivered in vivo to provide more active and efficient protein expression by various routes of administration such as intravenous, intramuscular, intra-articular, intrathecal, inhalation (respiratory), subcutaneous, intravitreal and intraocular.

[6] The method of the present invention can be carried out using a pump system and is therefore scalable, making it possible to improve the formation/formulation of particles in quantities sufficient, for example, to conduct clinical trials and/or commercial sale. Various pump systems can be used to practice the present invention, including, but not limited to, pulseless flow pumps, gear pumps, peristaltic pumps, and centrifugal pumps.

[7] The method of the present invention results in superior encapsulation efficiency and uniform particle size.

[8] Thus, in one aspect, the present invention provides a method of encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), comprising the steps of (a) mixing one or more lipids in a lipid solution with one or more mRNAs in an mRNA solution to form mRNA encapsulated in LNPs (mRNA-LNPs) in an LNP formation solution; (b) exchanging the LNP formation solution with a drug product formulation solution to form mRNA-LNPs in the drug product formulation solution; and (c) heating the mRNA-LNPs in the drug product formulation solution, wherein the encapsulation efficiency for the mRNA-LNPs obtained in step (c) exceeds the encapsulation efficiency for the mRNA-LNPs obtained in step (b).

[9] In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat from a heat source to the solution.

[10] In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat from a heat source to the solution and the solution is maintained at a temperature greater than ambient temperature for 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 40 seconds or more, 50 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, 35 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, 90 minutes or more, 100 minutes or more, or 120 minutes or more. In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat from a heat source to the solution and the solution is maintained at a temperature above ambient temperature for 120 minutes or less, 100 minutes or less, 90 minutes or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less. In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat from a heat source to the solution and the solution is maintained at a temperature above ambient temperature for 10-20 minutes. In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat to the solution from a heat source and the solution is maintained at a temperature above ambient temperature for 20 to 90 minutes. In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat to the solution from a heat source and the solution is maintained at a temperature above ambient temperature for 30 to 60 minutes. In some embodiments, in step (c), the solution for formulating the drug product is heated by applying heat to the solution from a heat source and the solution is maintained at a temperature above ambient temperature for about 15 minutes. In some embodiments, the temperature to which the solution for formulating the drug product is heated (or at which the solution for formulating the drug product is maintained) is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or more. In some embodiments, the temperature to which the solution is heated to formulate the drug product 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 above ambient temperature to which the solution is heated to formulate the drug product is about 65°C.

[11] In some embodiments, in step (a), the formation of lipid nanoparticles is achieved by mixing lipids dissolved in a lipid solution comprising ethanol with mRNA dissolved in an aqueous mRNA solution. In some embodiments, in step (a), the one or more lipids comprise one or more cationic lipids, one or more helper lipids, and one or more PEG-modified lipids (also referred to as PEG lipids). In some embodiments, the lipids also comprise one or more cholesterol-based lipids. The formation of mRNA-LNPs is achieved by mixing the lipid solution and the mRNA solution. Accordingly, in some embodiments, the LNPs comprise one or more cationic lipids, one or more helper lipids, and one or more PEG lipids. In some embodiments, the LNPs also comprise one or more cholesterol-based lipids.

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

[13] In some embodiments, one or more cationic lipids are aminolipids. Aminolipids suitable for use in the present invention include those described in WO2017180917, which is incorporated herein by reference. Illustrative aminolipids in WO2017180917 include those described in paragraph [0744], such as DLin-MC3-DMA (MC3), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), and Compound 18. Other aminolipids include Compound 2, Compound 23, Compound 27, Compound 10, and Compound 20. Additional aminolipids suitable for use in the present invention include those described in WO2017112865, which is incorporated herein by reference. Exemplary aminolipids in WO2017112865 include a compound according to one of formulae (I), (Ia1)-(Ia6), (lb), (II), (Ila), (III), (IIIa), (IV), (17-1), (19-1), (19-11), and (20-1) and compounds of paragraphs [00185], [00201], [0276]. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118725, which is incorporated herein by reference. Exemplary cationic lipids of WO2016118725 include lipids such as KL22 and KL25. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118724, which is incorporated herein by reference. Exemplary cationic lipids from WO2016118725 include lipids such as KL10, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

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

[15] In some embodiments, one or more PEG-modified lipids comprise a polyethyleneglycol chain of up to 5 kDa covalently attached to a lipid with alkyl chain(s) of _C6 - _C20 length.

[16] In some embodiments, after step (a), the mRNA-LNP is purified using a tangential flow filtration (TFF) method. In some embodiments, greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified mRNA-LNPs have a size of less than about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified mRNA-LNPs have a size of less than 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified mRNA-LNPs have a size in the range of 50 to 150 nm. In some embodiments, substantially all of the purified mRNA-LNPs have a size in the range of 50 to 150 nm. In some embodiments, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified mRNA-LNPs have a size in the range of 80 to 150 nm. In some embodiments, substantially all of the purified nanoparticles have a size in the range of 80 to 150 nm.

[17] In some embodiments, the method of the present invention results in an encapsulation efficiency after step (c) that is increased by at least 5% or more compared to the encapsulation efficiency after step (b). In some embodiments, the method of the present invention results in an encapsulation efficiency after step (c) that is increased by at least 10% or more compared to the encapsulation efficiency after step (b). In some embodiments, the method of the present invention results in an encapsulation efficiency after step (c) that is increased by at least 15% or more compared to the encapsulation efficiency after step (b). In some embodiments, the method of the present invention results in an encapsulation efficiency after step (c) that is increased by at least 20% or more compared to the encapsulation efficiency after step (b). In some embodiments, the method of the present invention results in an encapsulation efficiency after step (c) that is increased by at least 25% or more compared to the encapsulation efficiency after step (b).

[18] In some embodiments, the method of the present invention provides an increase in the encapsulation amount of 5% encapsulation or greater when comparing the encapsulation value after step (b) to the encapsulation value after step (c). In some embodiments, the method of the present invention provides an increase in the encapsulation amount of 10% encapsulation or greater when comparing the encapsulation value after step (b) to the encapsulation value after step (c). In some embodiments, the method of the present invention provides an increase in the encapsulation amount of 15% encapsulation or greater when comparing the encapsulation value after step (b) to the encapsulation value after step (c). In some embodiments, the method of the present invention provides an increase in the encapsulation amount of 20% encapsulation or greater when comparing the encapsulation value after step (b) to the encapsulation value after step (c). In some embodiments, the method of the present invention provides an increase in encapsulation index of 25% encapsulation or more when comparing the encapsulation value after step (b) to the encapsulation value after step (c).

[19] In some embodiments, the method of the present invention results in greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA after step (c).

[20] In some embodiments, the method of the present invention results in an encapsulation rate after step (c) of greater than about 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the method of the present invention results in an mRNA recovery after step (c) of greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[21] In some embodiments, the lipid solution and the mRNA solution are mixed using a pump system. In some embodiments, the pump system comprises a pulse-free flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the method using the pump system is performed on a large scale. For example, in some embodiments, the method comprises using the pumps described herein to mix 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 lipid solution comprising one or more cationic lipids, one or more auxiliary lipids, and one or more PEG-modified lipids. In some embodiments, the method of mixing a lipid solution and an mRNA solution provides a composition of the present invention that contains at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA after step (c).

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

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

[24] In some embodiments, the lipid solution comprises a non-aqueous solvent, such as an organic solvent. In some embodiments, the lipid solution comprises an alcohol. In some embodiments, the lipid solution comprises ethanol. In some embodiments, the method of the present invention includes the step of first dissolving one of the lipids in the lipid solution. In some embodiments, the method of the present invention includes the step of first dissolving one of the lipids in a lipid solution containing ethanol.

[25] In some embodiments, the mRNA solution is an aqueous solution. In some embodiments, the mRNA solution comprises citrate. In some embodiments, the mRNA solution is a citrate buffer. In some embodiments, the method of the present invention includes the step of first dissolving the mRNA in an aqueous solution. In some embodiments, the method of the present invention includes the step of first dissolving the mRNA in an aqueous solution containing citrate.

[26] In some embodiments, the method of the present invention comprises the step of mixing a lipid solution comprising lipids in ethanol with an mRNA buffer comprising mRNA dissolved in citrate buffer. In some embodiments, the LNP formation solution comprises ethanol and citrate.

[27] In some embodiments, the method of the present invention includes the step of first preparing an mRNA solution by mixing a citrate buffer with a stock mRNA solution. In certain embodiments, a suitable citrate buffer comprises about 10 mM citrate, about 150 mM NaCl, pH about 4.5. In some embodiments, a suitable stock mRNA solution comprises mRNA at a concentration of about 1 mg/mL, about 10 mg/mL, about 50 mg/mL, or about 100 mg/mL or greater.

[28] In some embodiments, the citrate buffer is mixed at a flow rate in the range of about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, or 4800-6000 ml/minute. In some embodiments, the citrate buffer is mixed at a flow rate of about 220 ml/minute, about 600 ml/minute, about 1200 ml/minute, about 2400 ml/minute, about 3600 ml/minute, about 4800 ml/minute, or about 6000 ml/minute.

[29] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute. In some embodiments, The mRNA stock solution is mixed at a flow rate of approximately 20 ml/minute, approximately 40 ml/minute, approximately 60 ml/minute, approximately 80 ml/minute, approximately 100 ml/minute, approximately 200 ml/minute, approximately 300 ml/minute, approximately 400 ml/minute, approximately 500 ml/minute, or approximately 600 ml/minute.

[30] In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing pharmaceutically acceptable excipients, including but not limited to a cryoprotectant. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing pharmaceutically acceptable excipients, including but not limited to a sugar. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing pharmaceutically acceptable excipients, including but not limited to one or more of trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, in step (b), the solution for formulating the drug product comprises trehalose. In some embodiments, in step (b), the solution for formulating the drug product comprises sucrose. In some embodiments, in step (b), the solution for formulating the drug product comprises mannose. In some embodiments, in step (b), the solution for formulating the drug product comprises lactose. In some embodiments, in step (b), the solution for formulating the drug product comprises mannitol. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of a sugar such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of trehalose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of sucrose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of mannose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of lactose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing 5% to 20% w/v mannitol. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing about 10% w/v sugar, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing about 10% w/v trehalose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing about 10% w/v sucrose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing about 10% w/v mannose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing about 10% w/v lactose. In some embodiments, in step (b), the solution for formulating the drug product is an aqueous solution containing about 10% w/v mannitol.

[31] In some embodiments, the solution for formulating the drug product lacks (i.e., is below detectable levels) one or both of a non-aqueous solvent, such as ethanol, and citrate. In some embodiments, the solution for formulating the drug product lacks citrate (i.e., is below detectable levels). In some embodiments, the solution for formulating the drug product lacks ethanol (i.e., is below detectable levels). In some embodiments, the solution for formulating the drug product contains ethanol but does not contain citrate (i.e., is below detectable levels). In some embodiments, the solution for formulating the drug product contains citrate but does not contain ethanol (i.e., is below detectable levels). In some embodiments, the solution for formulating the drug product contains only residual citrate. In some embodiments, the solution for formulating the drug product contains only residual non-aqueous solvent, such as ethanol. In some embodiments, the drug product formulation solution comprises less than about 10 mM (e.g., less than about 9 mM, about 8 mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM, or about 1 mM) citrate. In some embodiments, the drug product formulation solution comprises less than about 25% (e.g., less than about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%) of a non-aqueous solvent, such as ethanol. In some embodiments, the drug product formulation solution does not require any additional post-processing (e.g., buffer exchange and/or additional purification steps) prior to lyophilization. In some embodiments, the solution for formulating the drug product does not require any additional post-processing (e.g., buffer exchange and/or additional purification steps) prior to administration to a subject.

[32] In some embodiments, the solution for formulating the drug product has a pH of between pH 4.5 and pH 7.5. In some embodiments, the solution for formulating the drug product has a pH of between pH 5.0 and pH 7.0. In some embodiments, the solution for formulating the drug product has a pH of between pH 5.5 and pH 7.0. In some embodiments, the solution for formulating the drug product has a pH greater than pH 4.5. In some embodiments, the solution for formulating the drug product has a pH greater than pH 5.0. In some embodiments, the solution for formulating the drug product has a pH greater than pH 5.5. In some embodiments, the solution for formulating the drug product has a pH greater than pH 6.0. In some embodiments, the solution for formulating the drug product has a pH greater than pH 6.5.

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

[34] In another aspect, the present invention provides a method of delivering mRNA to provide protein production in vivo , comprising administering to a subject a lipid nanoparticle composition encapsulating mRNA obtained by the method described herein, wherein the mRNA encodes one or more proteins or peptides of interest.

[35] In this application, the use of the term "or" means "and/or" unless otherwise specified. As used herein, the term "comprise" and variations of such term such as "comprising" and "comprises" are not intended to exclude other additives, components, integers or steps. As used herein, the terms "about" and "approximately" are used as equivalents. Both terms are understood to encompass any normal variations known to one skilled in the art.

[36] Other features, objects and advantages of the present invention will become apparent from the detailed description, drawings and claims. However, it should be understood that the detailed description, drawings and claims, while indicating embodiments of the present invention, are provided solely by way of illustration and not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[37] Graphic materials are provided for illustrative purposes only and are not limiting.

[38] FIG. 1 shows a schematic diagram of a conventional method for encapsulating LNP-mRNA (Method A), which comprises mixing mRNA dissolved in an aqueous mRNA solution with lipids dissolved in a lipid solution using a pump system to obtain mRNA-LNP in a solution for forming LNPs, and then exchanging the solution for forming LNPs with a solution for formulating a drug product.

[39] FIG. 2 shows a diagram of an exemplary method for encapsulating LNP-mRNA according to the present invention, which comprises mixing mRNA dissolved in an aqueous mRNA solution with lipids dissolved in a lipid solution using a pump system to obtain mRNA-LNP in an LNP formation solution, then replacing the LNP formation solution with a drug product formulation solution, and then heating the drug product formulation solution to ensure an increase in the level of mRNA encapsulation in the LNP.

[40] FIG. 3 shows the difference in encapsulation level before and after the final heating step of mRNA-LNPs in drug product formulation solution for twelve different mRNA-LNPs tested.

[41] FIG. 4 shows the difference in encapsulation before and after the final heating step of mRNA-LNPs in the drug product formulation solution for thirteen different mRNA-LNPs tested.

[42] FIG. 5 shows an illustrative graph of protein expression following pulmonary administration of mRNA encapsulated in lipid nanoparticles prepared by Method A, after a heating step.

DEFINITIONS

[43] To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are provided throughout the description.

[44] Alkyl . As used herein, the term “alkyl” refers to a radical of a linear or branched saturated hydrocarbon group containing from 1 to 20 carbon atoms (“C _1-20 alkyl”). In some embodiments, the alkyl group contains from 1 to 3 carbon atoms (“C _1-3 alkyl”). Examples of C _1-3 alkyl groups include methyl (C ₁ ), ethyl (C ₂ ), n-propyl (C ₃ ), and isopropyl (C ₃ ). In some embodiments, the alkyl group contains from 8 to 12 carbon atoms (“C _8-12 alkyl”). Examples of C _8-12 alkyl groups include, but are not limited to, n -octyl (C ₈ ), n -nonyl (C ₉ ), n -decyl (C ₁₀ ), n -undecyl (C ₁₁ ), n -dodecyl (C ₁₂ ), etc. The prefix " n- " (normal) refers to unbranched alkyl groups. For example, n -C ₈ alkyl refers to -(CH ₂ ) ₇ CH ₃ , n -C ₁₀ alkyl refers to -(CH ₂ ) ₉ CH _{3 ,} etc.

[45]Amino acid. As used herein, the term "amino acid" in its broadest sense refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, the amino acid has the general structure H₂N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any of the standard L-amino acids that are commonly present in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether produced synthetically or from a natural source. As used herein, the term "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and/or substituents. Amino acids, including the carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can alter the peptide's circulating half-life without adversely affecting their activity. Amino acids can participate in disulfide bond formation. Amino acids can contain one or more post-translational modifications, such as association with one or more chemical building blocks (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethyleneglycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

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

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

[48] Delivery . As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and situations in which mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into the patient's circulation (e.g., serum), distributed systemically, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[49] Efficacy . As used herein, the term "efficacy" or grammatical equivalents thereof refers to an improvement in the value of a biologically significant endpoint associated with the delivery of mRNA that encodes the relevant protein or peptide. In some embodiments, the biological endpoint is protection against ammonium chloride injury at certain time points after administration.

[50] Encapsulation . As used herein, the term “encapsulation” or grammatical equivalents refers to the method of enclosing an individual mRNA molecule within a nanoparticle.

[51] Expression . As used herein, the term "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, as indicated by the context, post-translational modification of a peptide, polypeptide, or fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "production" and their grammatical equivalents are used interchangeably.

[52] Improve, increase, or decrease . As used herein, the terms "improve,""increase," or "decrease," or their grammatical equivalents, refer to values expressed relative to a baseline measurement, such as a measurement in the same individual before the treatment described herein begins, or a measurement in a control sample or subject (or multiple control samples or subjects) in the absence of the treatment described herein. A "control sample" is a sample exposed to the same conditions as a test sample, except that it does not contain the test article. The term "control subject" is a subject suffering from the same form of the disease as the subject being treated, who is approximately the same age as the subject being treated.

[53] Impurities . As used in this document, the term "impurities" refers to substances within a limited quantity of liquid, gas, or solid that differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.

[54] In vitro . As used herein, the term " in vitro " refers to events that occur in an artificial environment, such as a test tube or reaction vessel, in a cell culture, etc. , and not in a multicellular organism.

[55] In vivo . As used herein, the term " in vivo " refers to events that occur in a multicellular organism, such as a human or non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur in a living cell (as opposed to, for example, in vitro systems).

[56]Selected. As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was originally associated when produced (in nature and/or under experimental conditions) and/or (2) produced, obtained, and/or manufactured by man. Isolated substances and/or entities 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 it was originally associated. In some embodiments, the isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of purity in percent of isolated substances and/or entities shall not include excipients (e.g., buffer, solvent, water etc.).

[57] Local distribution or delivery . As used herein, the terms "local distribution,""localdelivery," or a grammatical equivalent refer to tissue-specific delivery or distribution. Typically, local distribution or delivery requires that the peptide or protein (e.g., enzyme) encoded by the mRNA be translated and expressed intracellularly or with limited secretion, thereby avoiding entry into the patient's circulation.

[58]Messenger RNA (mRNA). As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one peptide, polypeptide, or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may include one or more coding and non-coding regions. mRNA may be obtained by purification from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, and etc. If necessary, for example In the case of chemically synthesized molecules, mRNA may contain nucleoside analogues, such as those having chemically modified bases or sugars, backbone modifications, and etc. The mRNA sequence is in 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA is or comprises naturally occurring nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogues (eg, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynylcytidine, C-5-propynyluridine, 2-aminoadenosine, C5-bromuridine, C5-fluorouridine, C5-ioduridine, 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 (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose) and/or modified phosphate groups (e.g. phosphorothioates and 5'-N-phosphoramidite bonds).

[59] Nucleic acid . As used herein, the term "nucleic acid" in its broadest sense refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, a "nucleic acid" refers to individual nucleic acid units (e.g., nucleotides and/or nucleosides). In some embodiments, a "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid units. In some embodiments, a "nucleic acid" encompasses RNA, as well as single- and/or double-stranded DNA and/or cDNA. Moreover, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e. analogs having a backbone other than the phosphodiester backbone.

[60]Patient. As used herein, the terms "patient" or "subject" refer to any organism to which the composition provided may be administered, such as, for experimental, diagnostic, prophylactic, cosmetic and/or therapeutic purposes. Typical patients include animals (e.g. mammals such as mice, rats, rabbits, non-human primates and/or humans). In some embodiments, the patient is a human. Humans include intrauterine and postnatal forms.

[61] Pharmaceutically acceptable The term "pharmaceutically acceptable" as used herein refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic reaction or other problem or complication, consistent with a reasonable benefit/risk ratio.

[62] Pharmaceutically acceptable salt . Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts prepared from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of the amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts prepared by other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerol phosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, 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, valerate salts, etc. Salts derived from the corresponding bases include alkali metal, alkaline earth metal, ammonium and N ⁺ ( _C1-4 alkyl) ₄ salts. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium salts, quaternary ammonium salts, and salts of amine cations formed with counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed by quaternization of an amine using a suitable electrophile, such as an alkyl halide, to form a quaternary alkylated amino salt.

[63] Activity . As used herein, the term "activity" or its grammatical equivalents refers to the expression of the protein(s) or peptide(s) encoded by the mRNA and/or the resulting biological effect.

[64] Salt . As used herein, the term "salt" refers to an ionic compound that is or may be the result of a neutralization reaction between an acid and a base.

[65]System distribution or delivery. As used herein, the terms "systemic distribution", "systemic delivery" or their grammatical equivalents refer to a delivery or distribution mechanism or approach that affects the entire body or organism. Typically, systemic distribution or delivery occurs through the body's circulatory system, such as blood flow. This is contrasted with the term "local distribution or local delivery."

[66]Subject. As used in this document, the term “subject” refers to a human being or any non-human animal (e.g., (a mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Humans include fetal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person who seeks diagnosis or treatment of a disease by a health care provider. The term "subject" is used herein interchangeably with "individual" or "patient." A subject may be afflicted with a disease or disorder, or predisposed to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[67] Substantially . As used herein, the term “substantially” refers to the qualitative state of exhibiting a full or nearly full measure or degree of a characteristic or property of interest. One of ordinary skill in biology will recognize that biological and chemical phenomena rarely, if ever, reach completion and/or approach completion, or achieve or avoid an absolute result. Thus, the term “substantially” is used herein to denote the potential lack of completion inherent in many biological and chemical phenomena.

[68] Target tissues . As used herein, the term "target tissues" refers to any tissue affected by a disease being treated. In some embodiments, target tissues include those tissues that exhibit a disease-associated pathology, symptom, or sign.

[69] Treatment . As used herein, the terms "treat,""treatment," or "treating" refer to any method used to partially or completely alleviate, alleviate, mitigate, suppress, prevent, delay the onset, reduce the severity, and/or decrease the frequency of one or more symptoms or signs of a particular disease, disorder, and/or condition. A treatment may be administered to a subject who does not exhibit signs of the disease and/or exhibits only early signs of the disease, for the purpose of reducing the risk of developing pathology associated with the disease.

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

DETAILED DESCRIPTION

[71] The present invention provides an improved method for formulating lipid nanoparticles and encapsulating mRNA. In some embodiments, the present invention provides a method of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising the steps of (a) mixing one or more lipids in a lipid solution with one or more mRNAs in an mRNA solution to form mRNA encapsulated in an LNP (mRNA-LNP) in an LNP formation solution; (b) exchanging the LNP formation solution with a drug product formulation solution to form mRNA-LNPs in the drug product formulation solution; and (c) heating the mRNA-LNPs in the drug product formulation solution. Surprisingly, it has been found that including step (c) in such a method provides a significantly higher level of mRNA-LNP encapsulation compared to encapsulating the same mRNA-LNPs after step (b).

[72] In some embodiments, the new formulation method results in an mRNA formulation with higher activity (peptide or protein expression) and higher efficacy (improvement in a biologically significant endpoint) both in vitro and in vivo with potentially better tolerability compared to the same mRNA formulation prepared without the additional step of heating the mRNA-LNPs in solution to formulate the drug product (step (c)). The higher activity and/or efficacy of such a formulation may allow for a lower dose and/or less frequent dosing of the drug product. In some embodiments, the present invention relates to an improved lipid formulation comprising a cationic lipid, a secondary lipid, and a PEG-modified lipid.

[73] In some embodiments, the resulting level of encapsulation of mRNA-LNP after step (c) is increased by 10% or more compared to the encapsulation efficiency for the same mRNA-LNP after step (b). In some embodiments, the resulting percentage of encapsulation of mRNA-LNP after step (c) is increased by five percentage points or more compared to the percentage of encapsulation for the same mRNA-LNP after step (b). For nucleic acid delivery, achieving high encapsulation efficiency is critical to achieving drug protection and reducing loss of activity in vivo .

[74] The following sections describe various aspects of the present invention in detail. The application of the sections is not intended to limit the present invention. Each section may be applied to any aspect of the present invention.

Messenger RNA (mRNA)

[75] The present invention can be used to encapsulate any mRNA. mRNA is generally considered to be a type of RNA that carries information from DNA to the ribosome. Typically, in eukaryotic organisms, mRNA processing involves the addition of a "cap" at the 5' end and a "tail" at the 3' end. A typical cap is a 7-methylguanosine cap, which is a guanosine attached via a 5'-5' triphosphate bond to the first nucleotide to be transcribed. The presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells. The addition of the tail is typically a polyadenylation event, in which a polyadenylate moiety is added to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from degradation by exonuclease. Messenger RNA is translated by ribosomes into a series of amino acids that make up protein.

[76] mRNAs can be synthesized according to any of a number of known methods. For example, mRNAs according to the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor. The exact conditions will vary depending on the particular application.

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

[78] The present invention can be used to formulate and encapsulate mRNA of varying lengths. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA of about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb or more in length. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA that ranges in length from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb.

[79] The present invention can be used to formulate and encapsulate mRNA that is unmodified or mRNA that contains one or more modifications that typically enhance stability. In some embodiments, the modifications are selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and/or 3' untranslated region.

[80] In some embodiments, mRNA modifications may include modifications of RNA nucleotides. Modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA can 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)), and, as modified nucleotides, analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyladenine, N6-methyladenine, N6-isopentenyladenine, 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-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-hydroxyacetic acid methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 1-methylpseudouracil, queuosine, beta-D-mannosylqueuosine, vibutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and U.S. Patent No. 5,700,642, the disclosures of which are incorporated herein in their entirety by reference.

[81] Typically, mRNA synthesis involves the addition of a cap at the 5' end and a tail at the 3' end. The presence of the cap is important to ensure resistance to nucleases present in most eukaryotic cells. The presence of the tail serves to protect the mRNA from degradation by exonucleases.

[82] Thus, in some embodiments, the mRNAs comprise a 5'-cap structure. The 5'-cap is typically added as follows: first, a terminal RNA 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 phosphates by a guanylyltransferase, forming a 5'5'5-triphosphate linkage, and then the nitrogen atom at position 7 of the guanine is methylated by a methyltransferase. 2'-O-methylation can also occur at the first base and/or the second base after the 7-methylguanosine triphosphate residues. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where m represents 2'-O-methyl residues).

[83] In some embodiments, the mRNAs comprise a 5' and/or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA, such as an iron response element. In some embodiments, the 5' untranslated region can be from about 50 to 500 nucleotides in length.

[84] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect the stability or position of mRNA in a cell, or one or more binding sites for miRNA. In some embodiments, the 3' untranslated region can be from about 50 to 500 nucleotides in length or more.

[85] Although some embodiments may require mRNA obtained from in vitro transcription reactions, it is envisaged that other sources of mRNA are within the scope of the present invention, including mRNA obtained from bacteria, fungi, plants and/or animals.

[86] The present invention can be used to formulate and encapsulate mRNA encoding a variety of proteins. Non-limiting examples of mRNAs suitable for the present invention include mRNAs 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). Listed below are exemplary mRNA sequences disclosed herein.

Codon-optimized coding sequence of human OTC

AUGCUGUUCAACCUUCGGAUCUUGCUGAACAACGCUGCGUUCCGGAAUGGUCACAACUUCAUGGUCCGGAACUUCAGAUGCGGCCAGCCGCUCCAGAACAAGGUGCAGCUCAAGGGGAGGGACCUCCCUCACCC UGAAAAACUUCACCGGAGAAGAGAUCAAGUACAUGCUGUGGCUGUCAGCCGACCUCAAAUUCCGGAUCAAGCAGAAGGGCGAAUACCUUCCUUUGCUGCAGGGAAAGUCCCUGGGGAUGAUCUUCGAGAAGCG CAGCACUCGCACUAGACUGUCAACUGAAACCGGCUUCGCGCUGCUGGGAGGACACCCCUGCUUCCUGACCACCCAAGAUAUCCAUCUGGGUGUGAACGAAUCCCUCACCGACACAGCGCGGGUGCUGUCGUCC AUGGCAGACGCGGUCCUCGCCCGCGUGUACAAGCAGUCUGAUCUGGACACUCUGGCCAAGGAAGCCUCCAUUCCUAUCAUUAAUGGAUUGUCCGACCUCUACCAUCCCAUCCAGAUUCUGGCCGAUUAUCUGA CUCUGCAAGAACAUUACAGCUCCCUGAAGGGGCUUACCCUUUCGUGGAUCGGCGACGGCAACAACAUUCUGCACAGCAUUAUGAUGAGCGCUGCCAAGUUUGGAAUGCACCUCCAAGCAGCGACCCCGAAGGG AUACGAGCCAGACGCCUCCGUGACGAAGCUGGCUGAGCAGUACGCCAAGGAACGGCACUAAGCUGCUGCUCACCAACGACCCUCUCGAAGCCGCCCACGGUGGCAACGUGCUGAUCACCGAUACCUGGAUC UCCAUGGGACAGGAGGAGGAAAAGAAGAAGCGCCUGCAAGCAUUUCAGGGGUACCAGGUGACUAUGAAAACCGCCAAGGUCGCCGCCUCGGACUGGACCUUCUUGCACUGUCUGCCCAGAAAGCCCGAAGAGG UGGACGACGAGGUGUUCUACAGCCCGCGGUCGCUGGUCUUUCCGGAGGCCGAAAACAGGAAGUGGACUAUCAUGGCCGUGAUGGUGUCCCUGCUGACCGAUUACUCCCCGCAGCUGCAGAAACCAAAGUUCUGA ( SEQ ID NO: 1 )

Codon-optimized coding sequence of human ASS1

AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACCAGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUACCUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCCC UGAAGCUGGGCGCCAAGAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUCGUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGACCGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAGGUG GAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACCGGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCCCCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGCUUCAA GGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCACAUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCCGGCCUGU ACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGACAUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUGAAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAACGAGGUGGCC GGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGCUUCAUCGGCAUGAAGAGCCGCGGCAUCUACGACCCCCGCCGGCACCAUCCUGUACCACGCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUGCGCAAGAUCAA GCAGGGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACCGGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAAGGUGCAGGUGAGCGUGCUGAAGGGCCAGGUGUACAUCCUGG GCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUGGUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUCAUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAGGUGACCGCCAAGUGA ( SEQ ID NO: 2 )

Codon-optimized coding sequence of human CFTR

AUG UAA (SEQ ID NO: 3)

Comparative codon-optimized coding sequence of human CFTR mRNA

AUG UAA (SEQ ID NO: 4)

Codon-optimized coding sequence of human PAH

AUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUCGGCCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUCAGCCUGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGCUGCGCCUGUUCGAGG AGAACGACGUGAACCUGACCCACAUCGAGAGCCGCCCCAGCCGCCUGAAGAAGGACGAGUACGAGUUCUUCACCCACCUGGACAAGCGCAGCCUGCCCGCCCUGACCAACAUCAUCAAGAUCCUGCGCCACGACAUCGGCGCCACCGUGCACGAGCUGAGCCGCGACAAG AAGAAGGACACCGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUGGACCGCUUCGCCAACCAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACCCCGGCUUCAAGGACCCCGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGCCUACAACUACCGCCA CGGCCAGCCCAUCCCCCGCGUGGAGUACAUGGAGGAGGAGAAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGACCUGUACAAGACCCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAAGUACUGCGGCUUCCACGAGGACAACAUCCCCCAGC UGGAGGACGUGAGCCAGUUCCUGCAGACUGCACCGGCUUCCGCCUGCGCCCCGUGGCCGGCCUGCUGAGCAGCCGCGACUUCCUGGGCGGCCUGGCCUUCCGCGUGUUCCACUGCACCCAGUACAUCCGCCACGGCAGCAAGCCCAUGUACACCCCCGAGCCCGACAUC UGCCACGAGCUGCUGGGCCACGUGCCCCUGUUCAGCGACCGCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGGGCGCCCCCGACGAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUGGAGUUCGGCCUGUGCAAGCAGGGCGACAGCAU CAAGGCCUACGGCGCCGGCCUGCUGAGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCCCAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCCAUCCAGAACUACACCGUGACCGAGUUCCAGCCCCUGUACUACGUGGCCGAGAGCUUCAACGACGCCAAGG AGAAGGUGCGCAACUUCGCCGCCACCAUCCCCCGCCCCUUCAGCGUGCGCUACGACCCCUACACCCAGCGCAUCGAGGUGCUGGACAACACCCAGCAGCUGAAGAUCCUGGCCGACAGCAUCAACAGCGAGAUCGGCAUCCUGUGCAGCGCCCUGCAGAAGAUCAAGUAA ( SEQ ID NO: 5 )

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

mRNA solution

[88] The mRNA may be provided in a solution to allow mixing with the lipid solution so that the mRNA can be encapsulated in the lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing the mRNA to be encapsulated in varying concentrations. For example, a suitable mRNA solution may contain mRNA at a concentration of about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml or more. In some embodiments, a suitable mRNA solution may comprise mRNA in a concentration in the range of about 0.01-1.0 mg/mL, 0.01-0.9 mg/mL, 0.01-0.8 mg/mL, 0.01-0.7 mg/mL, 0.01-0.6 mg/mL, 0.01-0.5 mg/mL, 0.01-0.4 mg/mL, 0.01-0.3 mg/mL, 0.01-0.2 mg/mL, 0.01-0.1 mg/mL, 0.05-1.0 mg/mL, 0.05-0.9 mg/mL, 0.05-0.8 mg/mL, 0.05-0.7 mg/mL, 0.05-0.6 mg/mL, 0.05-0.5 mg/mL, 0.05-0.4 mg/mL, 0.05-0.3 mg/mL, 0.05-0.2 mg/mL, 0.05-0.1 mg/mL, 0.1-1.0 mg/mL, 0.2-0.9 mg/mL, 0.3-0.8 mg/mL, 0.4-0.7 mg/mL, or 0.5-0.6 mg/mL. In some embodiments, a suitable mRNA solution may contain mRNA at a concentration of up to about 5.0 mg/mL, 4.0 mg/mL, 3.0 mg/mL, 2.0 mg/mL, 1.0 mg/mL, 0.09 mg/mL, 0.08 mg/mL, 0.07 mg/mL, 0.06 mg/mL, or 0.05 mg/mL.

[89] Typically, a suitable mRNA solution may also contain a buffering agent and/or a salt. In general, buffering agents may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, a suitable concentration of the buffering agent may range from about 0.1 to 100 mM, 0.5 to 90 mM, 1.0 to 80 mM, 2 to 70 mM, 3 to 60 mM, 4 to 50 mM, 5 to 40 mM, 6 to 30 mM, 7 to 20 mM, 8 to 15 mM, or 9 to 12 mM. In some embodiments, a suitable concentration of the buffering agent is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or greater.

[90] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, a suitable concentration of the salts in the mRNA solution may range from about 1 mM to 500 mM, from 5 mM to 400 mM, from 10 mM to 350 mM, from 15 mM to 300 mM, from 20 mM to 250 mM, from 30 mM to 200 mM, from 40 mM to 190 mM, from 50 mM to 180 mM, from 50 mM to 170 mM, from 50 mM to 160 mM, from 50 mM to 150 mM, or from 50 mM to 100 mM. The salt concentration in a suitable mRNA solution is approximately 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 more.

[91] In some embodiments, a suitable mRNA solution may have a pH in the range of about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. In some embodiments, a suitable mRNA solution may have a pH of about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5, or no greater.

[92] Various methods can be used to prepare an mRNA solution suitable for the present invention. In some embodiments, the mRNA can be directly dissolved in the buffer solution described herein. In some embodiments, the mRNA solution can be prepared by mixing a stock solution of mRNA with a buffer solution prior to mixing with the encapsulation lipid solution. In some embodiments, the mRNA solution can be prepared by mixing a stock solution of mRNA with a buffer solution immediately prior to mixing with the encapsulation lipid solution. In some embodiments, a suitable mRNA stock solution may comprise mRNA in water at a concentration of about 0.2 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1.0 mg/mL, 1.2 mg/mL, 1.4 mg/mL, 1.5 mg/mL, or 1.6 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0 mg/mL, 4.5 mg/mL, or 5.0 mg/mL or greater.

[93] In some embodiments, the mRNA solution is prepared by mixing the mRNA stock solution with a buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than the rate of the mRNA stock solution. For example, the buffer solution can be mixed at a rate that is at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute or more.

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

Lipid solution

[95] According to the present invention, the lipid solution comprises a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, the suitable lipid solution is ethanol-based. For example, the suitable lipid solution may comprise a mixture of the 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 another embodiment, the suitable lipid solution is a mixture of suitable solvents, including but not limited to ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[96] A suitable lipid solution may comprise a mixture of the desired lipids at different concentrations. For example, a suitable lipid solution may comprise a mixture of the desired lipids at a total concentration of about 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, or 100 mg/mL or more. In some embodiments, a suitable lipid solution may comprise a mixture of the desired lipids at a total concentration in the range of about 0.1-100 mg/mL, 0.5-90 mg/mL, 1.0-80 mg/mL, 1.0-70 mg/mL, 1.0-60 mg/mL, 1.0-50 mg/mL, 1.0-40 mg/mL, 1.0-30 mg/mL, 1.0-20 mg/mL, 1.0-15 mg/mL, 1.0-10 mg/mL, 1.0-9 mg/mL, 1.0-8 mg/mL, 1.0-7 mg/mL, 1.0-6 mg/mL, or 1.0-5 mg/mL. In some embodiments, a suitable lipid solution may comprise a mixture of the desired lipids at a total concentration of no more than about 100 mg/mL, 90 mg/mL, 80 mg/mL, 70 mg/mL, 60 mg/mL, 50 mg/mL, 40 mg/mL, 30 mg/mL, 20 mg/mL, or 10 mg/mL.

[97] Any desired lipids can be mixed in any ratios suitable for encapsulating mRNA. In some embodiments, a suitable lipid solution comprises a mixture of desired lipids, including cationic lipids, auxiliary lipids (e.g., lipids other than cationic lipids and/or cholesterol-based lipids) and/or PEGylated lipids. In some embodiments, a suitable lipid solution comprises a mixture of desired lipids, including one or more cationic lipids, one or more accessory lipids (e.g., lipids other than cationic and/or cholesterol-based lipids) and one or more PEGylated lipids.

[98] An exemplary lipid mixture for use in the context of the present invention consists of four lipid components: a cationic lipid, a non-cationic lipid (e.g., DSPC, DPPC, DOPE, or DEPE), a cholesterol-based lipid (e.g., cholesterol), and a PEG-modified lipid (e.g., DMG-PEG2K). In some embodiments, the molar ratio of the cationic lipid(s) to the non-cationic lipid(s) to the cholesterol-based lipid(s) to the PEG-modified lipid(s) can be approximately 20-50:25-35:20-50:1-5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is about 20:30:48.5:1.5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is about 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is about 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is about 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5.

[99] In some embodiments, a lipid mixture for use in the context of the present invention may comprise no more than three individual lipid components. In some embodiments, one individual lipid component in such a mixture is a cholesterol-based or imidazole-based cationic lipid. An exemplary lipid mixture may consist of three lipid components: a cationic lipid (e.g., a cholesterol-based or imidazole-based cationic lipid such as ICE, HGT4001, or HGT4002), a non-cationic lipid (e.g., DSPC, DPPC, DOPE, or DEPE), and a PEG-modified lipid (e.g., DMG-PEG2K). The molar ratio of the cationic lipid and the non-cationic lipid and the PEG-modified lipid may be approximately 55-65:30-40:1-15, respectively. In some embodiments, a molar ratio of cationic lipid (e.g., cholesterol-based or imidazole-based lipid such as ICE, HGT4001 or HGT4002) to non-cationic lipid (e.g., DSPC, DPPC, DOPE or DEPE) to PEG-modified lipid (e.g., DMG-PEG2K) of 60:35:5 is particularly suitable for use in the context of the present invention.

Cationic lipids

[100] As used herein, the phrase "cationic lipids" refers to any of a number of lipid compounds that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. In particular, suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Application Publication Nos. WO 2010/053572 (and in particular C12-200, described in paragraph [00225]) and WO 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 an ionizable cationic lipid as described in U.S. Provisional Patent Application Ser. No. 61/617,468, filed March 29, 2012 (incorporated herein by reference), such as, 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-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

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

[102] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include a cationic lipid described in WO 2015/184256 A2 entitled “Biodegradable lipids for delivery of nucleic acids,” which is incorporated herein by reference, such as 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (“Target 23”), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24).

[103] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include the cationic lipid described in WO 2013/063468 and the U.S. Provisional Application entitled “Lipid Formulations for Delivery of Messenger RNA,” both of which are incorporated herein by reference. In some embodiments, the cationic lipid comprises a compound of formula I-c1-a ,

I-c1-a ,

or a pharmaceutically acceptable salt thereof, where

each R ² is independently hydrogen or C _1-3 alkyl;

each q independently equals 2-6;

each R' is independently hydrogen or C _1-3 alkyl;

and each R ^L is independently C _8-12 alkyl.

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

[105] In some embodiments, each q is independently 3-6. In some embodiments, each q is independently 3-5. In some embodiments, each q is 4.

[106] 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.

[107] In some embodiments, each R ^L is independently C _8-12 alkyl. In some embodiments, each R ^L is independently n -C _8-12 alkyl. In some embodiments, each R ^L is independently C _9-11 alkyl. In some embodiments, each R ^L is independently n -C _9-11 alkyl. In some embodiments, each R ^L is independently C ₁₀ alkyl. In some embodiments, each R ^L is independently n -C ₁₀ alkyl.

[108] In some embodiments, each R ² is independently hydrogen or methyl; each q is independently 3-5; each R' is independently hydrogen or methyl; and each R ^L is independently C _8-12 alkyl.

[109] In some embodiments, each R ² is hydrogen; each q is independently 3-5; each R' is hydrogen; and each R ^L is independently C _8-12 alkyl.

[110] In some embodiments, each R ² is hydrogen; each q is 4; each R' is hydrogen; and each R ^L is independently C _8-12 alkyl.

[111] In some embodiments, the cationic lipid comprises a compound of formula Ig ,

or a pharmaceutically acceptable salt thereof, wherein each R ^L is independently C _8-12 alkyl. In some embodiments, each R ^L is independently n -C _8-12 alkyl. In some embodiments, each R ^L is independently C _9-11 alkyl. In some embodiments, each R ^L is independently n -C _9-11 alkyl. In some embodiments, each R ^L is independently C ₁₀ alkyl. In some embodiments, each R ^L is n -C ₁₀ alkyl.

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

.

[113] Other suitable cationic lipids include degradable cationic lipids, which are described in International Patent Application Publication No. WO 2012/170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention provide a cationic lipid of the following formula:

where R₁selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., such alkylamino as dimethylamino) and pyridyl; where R₂selected from the group consisting of one of the following two formulas:

and where each of R₃and R₄independently selected from the group consisting of optionally substituted, variably saturated or unsaturated C₆-C₂₀alkyl and optionally substituted, saturated or unsaturated to a varying degree C₆-C₂₀acyl; and where n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty and more). In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4001" having the compound structure

(HGT4001),

and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4002" having the compound structure

(HGT4002),

and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4003" having the compound structure

(HGT4003),

and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4004" having the compound structure

(HGT4004),

and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4005" having the compound structure

(HGT4005),

[114] and its pharmaceutically acceptable salts.

[115] Additional illustrative cationic lipids include lipids of formula I,

and their pharmaceutically acceptable salts,

Where

R represents ("OF-00"),

R represents ("OF-01"),

R represents ("OF-02"), or

R represents ("OF-03")

(See, e.g., Fenton, Owen S., et al. "Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery." Advanced materials (2016)).

[116] In some embodiments, one or more cationic lipids suitable for the present invention can be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA.” (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Patent No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS", 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium or "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), 1,2-dioleoyl-3-dimethylammoniumpropane or "DODAP", 1,2-dioleoyl-3-trimethylammoniumpropane or "DOTAP".

[117] Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-Dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadieneoxy)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", 1,2-N, N'-dilinoleoylcarbamyl-3-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLinDMA", 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA" and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (see WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)) or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO 2005/121348A1). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole, a dialkylamino, or a guanidinium moiety.

[118] In some embodiments, the one or more cationic lipids may be selected from 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-dioleoyl-3-dimethylammoniumpropane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety), or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), amino alcohol based lipidoids such as those disclosed in WO 2010/053572, DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. "Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids" J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S.C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K.T. et al. “Lipid-like materials-dose in vivo gene silencing" PNAS 2010, 107, 1864-1869), N1GL, N2GL, V1GL and their combinations.

[119] In some embodiments, the one or more cationic lipids are aminolipids. Aminolipids suitable for use in the present invention include those described in WO2017180917, which is incorporated herein by reference. Illustrative aminolipids in WO2017180917 include those described in paragraph [0744], such as DLin-MC3-DMA (MC3), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), and Compound 18. Other aminolipids include Compound 2, Compound 23, Compound 27, Compound 10, and Compound 20. Additional aminolipids suitable for use in the present invention include those described in WO2017112865, which is incorporated herein by reference. Exemplary aminolipids in WO2017112865 include a compound according to one of formulae (I), (Ia1)-(Ia6), (lb), (II), (Ila), (III), (IIIa), (IV), (17-1), (19-1), (19-11), and (20-1), and compounds of paragraphs [00185], [00201], [0276]. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118725, which is incorporated herein by reference. Exemplary cationic lipids of WO2016118725 include lipids such as KL22 and KL25. In some embodiments, cationic lipids suitable for use in the present invention include those described in WO2016118724, which is incorporated herein by reference. Exemplary cationic lipids from WO2016118725 include lipids such as KL10, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[120] In some embodiments, the cationic lipids comprise at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipids in a suitable lipid solution by weight or molarity. In some embodiments, the cationic lipid(s) comprise(s) about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid mixture by weight or molarity.

Non-cationic/auxiliary lipids

[121] As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid compounds that carry a net negative charge at a selected pH, such as physiological pH. Lipids other than cationic include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a mixture thereof. In some embodiments, a lipid mixture for use in the context of the present invention may include DSPC as a lipid component other than the cationic component. In some embodiments, a lipid mixture for use in the context of the present invention may include DPPC as a lipid component other than the cationic component. In some embodiments, a lipid mixture for use in the context of the present invention may include DOPE as a lipid component other than the cationic component. In other embodiments, a lipid mixture for use in the context of the present invention may include DEPE as a lipid component other than the cationic component.

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

Cholesterol-based lipids

[123] In some embodiments, a suitable lipid solution comprises one or more cholesterol-based lipids. For example, suitable cationic cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleyl amino propyl) piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997), U.S. Patent No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid(s) constitute(s) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or molarity. In some embodiments, the cholesterol-based lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or molarity.

PEGylated lipids

[124] In some embodiments, a suitable lipid solution comprises one or more PEGylated lipids. For example, the present invention also contemplates the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide). Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa covalently attached to a lipid with _C6 - _C20 alkyl chain(s). In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. For example, a suitable lipid solution may comprise a PEG-modified lipid, such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2K). In some embodiments, particularly useful replacement lipids are PEG-ceramides having shorter acyl chains (e.g., C ₁₄ or C ₁₈ ).

[125] The PEG-modified phospholipid and derivatized lipids can comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipid in a suitable lipid solution by weight or molarity. In some embodiments, the PEG-modified phospholipid and derivatized lipids comprise from about 0% to about 20%, from about 0.5% to about 20%, from about 1% to about 15%, from about 1.5% to about 5% of the total lipid present in the liposomal transport medium carrier. In some embodiments, one or more PEG-modified lipids comprise about 1.5%, about 2%, about 3%, about 4%, or about 5% of the total lipids on a molar basis. In some embodiments, the PEGylated cholesterol-based lipid(s) comprise(s) 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 on a weight or molar basis.

[126] Various combinations of lipids, i.e., cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, that can be used to prepare and are included in the preformed lipid nanoparticles are described in the literature and herein. For example, a suitable lipid solution may comprise 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. Additional lipid combinations are described in the art, such as U.S. Patent Application Ser. No. 62/420,421 (filed November 10, 2016), U.S. Patent Application Ser. No. 62/421,021 (filed November 11, 2016), U.S. Patent Application Ser. No. 62/464,327 (filed February 27, 2017), and a PCT application entitled "Novel ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA" filed November 10, 2017, the disclosures of which are incorporated herein by reference in their entireties. The selection of the cationic lipids, non-cationic lipids and/or PEG-modified lipids that comprise the lipid mixture and the relative molar ratio of such lipids to each other is based on the characteristics of the selected lipid(s) and the nature and characteristics of the mRNA to be encapsulated. Additional factors to be considered include, for example, the saturation of the alkyl chain as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). The molar ratios can thus be adjusted accordingly.

Formation of mRNA-LNP

[127] A method for forming mRNA-encapsulating LNPs (mRNA-LNPs) by mixing the mRNA solution described above with the lipid solution described above to form an LNP forming solution suitable for forming mRNA-LNPs has been previously described. For example, U.S. Patent No. 9,668,980, entitled "Encapsulation of messenger RNA," the entire disclosure of which is incorporated herein in its entirety, provides a method for encapsulating messenger RNA (mRNA) in lipid nanoparticles by mixing an mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are heated to a predetermined temperature above ambient temperature prior to mixing to form lipid nanoparticles that provide encapsulation of mRNA. Alternatively, the mRNA solution and the lipid solution can be mixed into the LNP formation solution, which allows the formation of mRNA-LNPs without heating any one or more of the mRNA solution, the lipid solution, and the LNP formation solution.

[128] In certain cationic lipid and mRNA nanoparticle formulations, the mRNA solution comprises a citrate buffer to achieve increased mRNA encapsulation. In some embodiments, the mRNA solution with the citrate buffer is heated to, for example, 65 degrees Celsius. In these methods or techniques, it is necessary that the heating occur prior to the step of mixing the mRNA solution with the lipid solution (i.e., heating the individual components), since heating after mixing the mRNA solution with the lipid solution (after forming the nanoparticles) has been found to not increase the efficiency of mRNA encapsulation in lipid nanoparticles, and heating the solution to form LNPs has been shown to not increase the efficiency of mRNA encapsulation in lipid nanoparticles. In some embodiments, one or both of the mRNA solution and the lipid solution are maintained and mixed at ambient temperature.

[129] As used herein, the term "ambient temperature" refers to the temperature of a room or the temperature that surrounds an object of interest without heating or cooling. In some embodiments, the ambient temperature at which one or more solutions are maintained is approximately 35°C, 30°C, 25°C, 20°C, or 16°C or less. In some embodiments, the ambient temperature at which the 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 the one or more solutions are maintained is 20-25°C.

[130] Accordingly, the set temperature above the ambient temperature is typically greater than about 25°C. In some embodiments, the set 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 more. In some embodiments, the set temperature suitable for the present invention 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 particular embodiments, the set temperature suitable for the present invention is about 65°C.

[131] In some embodiments, the mRNA solution or the lipid solution, or both, may be heated to a predetermined temperature above ambient temperature prior to mixing. In some embodiments, the mRNA solution and the lipid solution are heated to a predetermined temperature separately prior to mixing. In some embodiments, the mRNA solution and the lipid solution are mixed at ambient temperature and then heated to a predetermined temperature after mixing. In some embodiments, the lipid solution is heated to a predetermined temperature and mixed with the mRNA solution at ambient temperature. In some embodiments, the mRNA solution is heated to a predetermined temperature and mixed with the lipid solution at ambient temperature.

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

[133] In some embodiments, the lipid solution containing the dissolved lipids can be heated to a predetermined temperature above ambient temperature prior to mixing. In some embodiments, the lipid solution containing the dissolved lipids is separately heated to a predetermined temperature prior to mixing with the mRNA solution. In some embodiments, the lipid solution containing the dissolved lipids is mixed at ambient temperature with the mRNA solution and then heated to a predetermined temperature after mixing. In some embodiments, the lipid solution containing the dissolved lipids is heated to a predetermined temperature and mixed with the mRNA solution at ambient temperature. In some embodiments, heating of the mRNA solution, the lipid solution, or the LNP formation solution does not occur before or after the step of mixing one or more lipids in the lipid solution with one or more mRNAs in the mRNA solution to form mRNA encapsulated in LNPs (mRNA-LNPs) in the LNP formation solution.

[134] In some embodiments, the mRNA solution and the lipid solution are mixed using a pump. Since the encapsulation procedure with such mixing can occur on a wide range of scales, various types of pumps can be used to achieve the desired scale. However, a pulse-free flow pump is typically required. As used herein, the term "pulse-free flow pump" refers to any pump that can create a continuous flow at a stable flow rate. 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-Parmer or Diener gear pumps. Exemplary centrifugal pumps include, but are not limited to, pumps manufactured by Grainger or Cole-Parmer.

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

[136] Suitable flow rates for mixing can be determined based on scale. In some embodiments, the mRNA solution is mixed at a flow rate of about 40-400 ml/minute, 60-500 ml/minute, 70-600 ml/minute, 80-700 ml/minute, 90-800 ml/minute, 100-900 ml/minute, 110-1000 ml/minute, 120-1100 ml/minute, 130-1200 ml/minute, 140-1300 ml/minute, 150-1400 ml/minute, 160-1500 ml/minute, 170-1600 ml/minute, 180-1700 ml/minute, 150-250 ml/minute, 250-500 ml/minute, 500-1000 ml/minute, 1000-2000 ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000 ml/minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml/minute, about 500 ml/minute, about 1000 ml/minute, about 2000 ml/minute, about 3000 ml/minute, about 4000 ml/minute, or about 5000 ml/minute.

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

Solution for the preparation of a medicinal product

[138] The present invention is based in part on the unexpected discovery that, after mixing a solution of mRNA and a lipid solution to form an LNP formation solution in which LNPs with encapsulated mRNA are formed, and then replacing the LNP formation solution with a solution that is a drug product formulation solution (e.g., 10% trehalose), the level of mRNA encapsulation in the LNPs can be further increased by heating the drug product formulation solution that contains the mRNA-LNPs as well as some free mRNA that was not encapsulated in the LNP formation solution.

[139] The exchange of the solution containing mRNA-LNP from the LNP formation solution to the drug product formulation solution can be accomplished using any of a number of buffer exchange techniques known in the art. For example, in some embodiments, the solution exchange is accomplished by diafiltration. In some embodiments, the step of exchanging the LNP formation solution with the drug product formulation solution to obtain mRNA-LNP in the drug product formulation solution is followed by purification and/or concentration of the mRNA-LNP. Various methods can be used to accomplish the solution exchange along with purification of the mRNA-LNP or concentration of the mRNA-LNP in the solution. In some embodiments, the solution is exchanged and the mRNA-LNP is purified using tangential flow filtration. Tangential flow filtration (TFF), also referred to as cross-flow filtration, is a type of filtration wherein the material to be filtered is passed tangentially across a filter rather than through it. In TFF, the permeate that is not wanted passes through the filter, while the retentate (mRNA-LNP and free mRNA) that is wanted passes along the filter and is collected afterwards. It is important to note that in the case of TFF, the material that is wanted is usually contained in the retentate, which is the opposite of what is typically encountered in traditional dead-end filtration.

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

[141] The primary advantage of tangential flow filtration is that particles to which the filter is impermeable, which would aggregate in the filter and block it (sometimes referred to as "filter residue") in traditional "dead-end" filtration, are instead carried along the surface of the filter. This advantage allows tangential flow filtration to be widely used in industrial processes that require continuous operation, since downtime is greatly reduced because filters typically do not need to be removed and cleaned.

[142] Tangential flow filtration can be used for several purposes, including solvent exchange, concentration, and purification, among others. Concentration is a process in which the solvent is removed from a solution, leaving behind the solute molecules. To effectively concentrate a sample, a membrane with an NMWL or MWCO that is significantly lower than the molecular weight of the solute molecules to be retained is used. Typically, one may select a filter with an NMWL or MWCO that is three to six times lower than the molecular weight of the target molecule(s).

[143] Diafiltration is a fractionation method in which small particles that are not desired are passed through a filter while larger nanoparticles that are desired are retained in the retentate without changing the concentration of these nanoparticles in solution. Diafiltration is often used to remove salts or reaction buffers from a solution. Diafiltration can be either continuous or batch. In continuous diafiltration, the diafiltration solution is added to the feed sample at the same rate as the filtrate is formed. In batch diafiltration, the solution is first diluted and then reconcentrated to the original concentration. Batch diafiltration can be repeated until the desired concentration of nanoparticles is achieved.

[144] The composition of the solution for formulating the drug product may include various components found in drug product formulations. For example, in some embodiments, the solution for formulating the drug product may include a buffer, such as, for example, PBS.

[145] In some embodiments, the solution for formulating the drug product may comprise a buffering agent or a salt. An exemplary buffering agent may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. An exemplary salt may include sodium chloride, magnesium chloride, and potassium chloride.

[146] In some embodiments, the solution for formulating the drug product is an aqueous solution containing pharmaceutically acceptable excipients, including but not limited to a cryoprotectant. In some embodiments, the solution for formulating the drug product is an aqueous solution containing pharmaceutically acceptable excipients, including but not limited to one or more of trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, the solution for formulating the drug product comprises trehalose. In some embodiments, the solution for formulating the drug product comprises sucrose. In some embodiments, the solution for formulating the drug product comprises mannose. In some embodiments, the solution for formulating the drug product comprises lactose. In some embodiments, the solution for formulating the drug product comprises mannitol.

[147] In some embodiments, the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of a sugar such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of trehalose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of sucrose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of mannose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of lactose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising 5% to 20% w/v of mannitol.

[148] In some embodiments, the solution for formulating the drug product is an aqueous solution comprising about 10% w/v of a sugar such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising about 10% w/v trehalose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising about 10% w/v sucrose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising about 10% w/v mannose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising about 10% w/v lactose. In some embodiments, the solution for formulating the drug product is an aqueous solution comprising about 10% w/v mannitol.

[149] In some embodiments, the solution for formulating the drug product does not contain one or both of a non-aqueous solvent, such as ethanol, and citrate. In some embodiments, the solution for formulating the drug product contains only residual citrate. In some embodiments, the solution for formulating the drug product contains only residual non-aqueous solvent, such as ethanol. In some embodiments, the solution for formulating the drug product contains less than about 10 mM (e.g., less than about 9 mM, about 8 mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM, or about 1 mM) citrate. In some embodiments, the solution for formulating a drug product comprises less than about 25% (such as less than about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%) of a non-aqueous solvent, such as ethanol. In some embodiments, the solution for formulating a drug product does not require any additional post-processing (such as buffer exchange and/or additional purification steps and/or additional excipients) prior to lyophilization. In some embodiments, the solution for formulating a drug product does not require any additional post-processing (such as buffer exchange and/or additional purification steps and/or additional excipients) prior to introduction into a sterile container of a vial, syringe, or other vessel. In some embodiments, the solution for formulating a drug product does not require any additional post-processing (such as buffer exchange and/or additional purification steps and/or additional excipients) prior to administration to a subject.

[150] In some embodiments, the solution for formulating the drug product has a pH of between pH 4.5 and pH 7.5. In some embodiments, the solution for formulating the drug product has a pH of between pH 5.0 and pH 7.0. In some embodiments, the solution for formulating the drug product has a pH of between pH 5.5 and pH 7.0. In some embodiments, the solution for formulating the drug product has a pH greater than pH 4.5. In some embodiments, the solution for formulating the drug product has a pH greater than pH 5.0. In some embodiments, the solution for formulating the drug product has a pH greater than pH 5.5. In some embodiments, the solution for formulating the drug product has a pH greater than pH 6.0. In some embodiments, the solution for formulating the drug product has a pH greater than pH 6.5.

[151] In some embodiments, the improved or increased encapsulation rate of mRNA-LNP in the drug product formulation solution after heating is maintained after subsequent freeze-thaw cycles of the drug product formulation solution. In some embodiments, the drug product formulation solution is 10% trehalose and can be frozen stable.

[152] In some embodiments, the mRNA-LNPs in the drug product formulation solution after heating can be frozen in a stable state (e.g., maintaining an increased level of encapsulation) in about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose solution. In some embodiments, the drug product formulation solution does not require any subsequent purification or processing and can be stored in frozen form in a stable state.

Designed mRNA-encapsulating LNPs (mRNA-LNPs)

[153] The method of the present invention results in higher activity and efficacy, which allows for lower doses to be used, thereby shifting the therapeutic index in a positive direction. In some embodiments, the method of the present invention results in uniform particles with small sizes. In some embodiments, the method of the present invention results in uniform particles with small sizes of 200 nm or less. In some embodiments, the method of the present invention results in uniform particles with small sizes of 150 nm or less. In some embodiments, the method of the present invention results in uniform particles with small sizes, as well as a significant increase in encapsulation efficiency and/or mRNA recovery compared to the method of the prior art.

[154] Thus, the present invention provides a composition comprising purified nanoparticles with encapsulated mRNA as described herein. In some embodiments, a major portion of the nanoparticles with encapsulated mRNA in the composition, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles, have a size 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). The exemplary method described herein typically results in lipid nanoparticle compositions in which the lipid nanoparticles have an average size of about 150 nm or less, such as from 75 nm to 150 nm, such as from 100 nm to 150 nm.

[155] Furthermore, the method of the present invention produces uniform nanoparticles with a narrow range of particle sizes. For example, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles in the composition provided by the present invention have a size in the range of about 75-200 nm (e.g., about 75-150 nm, about 75-140 nm, about 75-135 nm, about 75-130 nm, about 75-125 nm, about 75-120 nm, about 75-115 nm, about 75-110 nm, about 75-105 nm, about 75-100 nm, about 75-95 nm, about 75-90 nm, or 75-85 nm). In some embodiments, substantially all of the purified nanoparticles have a size in the range of about 75-200 nm (e.g., about 75-150 nm, about 75-140 nm, about 75-135 nm, about 75-130 nm, about 75-125 nm, about 75-120 nm, about 75-115 nm, about 75-110 nm, about 75-105 nm, about 75-100 nm, about 75-95 nm, about 75-90 nm, or 75-85 nm).

[156] In some embodiments, the dispersion or molecular size inhomogeneity (PDI) measure of the nanoparticles in a composition provided by the present invention is less than about 0.23 (e.g., less than about 0.3, 0.2, 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). The exemplary method described herein typically provides lipid nanoparticle compositions with a PDI of about 0.15 or less, such as from about 0.01 to 0.15.

[157] In some embodiments, more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nanoparticles in a composition provided by the present invention encapsulate mRNA within each individual particle. In some embodiments, substantially all of the nanoparticles in the composition encapsulate mRNA within each individual particle.

[158] In some embodiments, the LNP of the present invention comprises at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, the method of the present invention results in greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

[159] In some embodiments, a composition of the present invention is formulated to allow dosing to a subject. In some embodiments, the mRNA-encapsulated LNP composition described herein is formulated to have a dose concentration of less than about 1.0 mg/kg mRNA-encapsulated 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 discovery that lower doses provide high activity and efficacy. In some embodiments, the dose is reduced by about 70%, 65%, 60%, 55%, 50%, 45%, or 40%.

[160] In some embodiments, the activity of the mRNA-encapsulated LNPs produced according to the present invention is from greater than 100% (i.e., greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 600%, greater than 700%, greater than 800%, or greater than 900%) to greater than 1000% higher when produced by including step (c).

EXAMPLES

[161] Although certain compounds, compositions, and methods of the present invention have been described specifically in accordance with certain embodiments, the following examples serve solely to illustrate the present invention and are not intended to limit it.

Lipid materials

[162] The compositions described in the following example, unless otherwise indicated, comprise a multi-component lipid mixture of varying ratios that utilizes one or more cationic lipids, auxiliary lipids (e.g., non-cationic lipids and/or cholesterol-based lipids), and PEGylated lipids designed to encapsulate various nucleic acid-based materials, as discussed previously.

Example 1. Increased mRNA encapsulation in lipid nanoparticles achieved by an additional heating step of the formulation solution

[163] This example illustrates an exemplary method of the present invention for providing an increased level of encapsulation of mRNA in a lipid nanoparticle by using Method A and then replacing the LNP formulation solution containing mRNA-LNP and free mRNA with a drug product formulation solution and heating such drug product solution. As used herein, the term "Method A" refers to a conventional method of encapsulating mRNA by mixing mRNA with a lipid mixture, such as without first forming lipid nanoparticles from lipids, as described in U.S. Patent Application Serial No. US2018/0008680, the entire contents of which are incorporated by reference.

[164] An exemplary formulation method A is shown in FIG. 1 . In this method, in some embodiments, a lipid solution in which lipids that are components of LNPs are dissolved (e.g., a solution containing ethanol) and an aqueous mRNA solution (containing citrate at a pH of 4.5) are separately prepared. Specifically, the lipid solution (cationic lipid, auxiliary lipids, zwitterionic lipids, PEG lipids, etc.) was prepared by dissolving lipids in ethanol. The mRNA solution was prepared by dissolving mRNA in citrate buffer, thereby obtaining mRNA in citrate buffer at a pH of 4.5. Then, both mixtures were heated to 65°C before mixing. Then, the two solutions were mixed using a pump system to obtain LNPs with encapsulated mRNA in an LNP formation solution containing the lipid mixture solution and the mRNA solution. In some cases, the two solutions were mixed using a gear pump system. In certain embodiments, the two solutions were mixed using a "T" shaped connection section ("Y" shaped connection section).

[165] Then, the LNP formation solution containing mRNA-LNP and free mRNA was diafiltered using the TFF method. As part of this method, the LNP formation solution was removed and replaced with a drug product formulation solution containing 10% trehalose. As can be seen in FIG. 2 , the obtained mRNA-LNP and free mRNA in the drug product formulation solution were then heated to 65°C for 15 minutes. After heating, the mRNA-LNP and free mRNA in the drug product formulation solution were cooled and stored at 2-8°C for subsequent analysis.

[166] The above encapsulation method shown in FIG. 2 was performed for 12 different mRNA-LNPs, as described in more detail in Table 1 below. For each test article, the amount of mRNA encapsulated in the formed LNPs was measured before and after heating in a drug product formulation solution with 10% trehalose using a RiboGreen free RNA assay kit according to published methods, followed by calculation to determine the encapsulated mRNA. In addition, the same assay was used to measure the amount of mRNA encapsulated in the formed LNPs after sequential freeze-thaw cycles to determine whether the increased encapsulation observed as a result of heating the mRNA-LNPs in the drug product remained generally constant upon sequential freeze-thaw cycles of the mRNA-LNPs.

Table 1. mRNA-LNPs obtained according to the present invention

Tested product Cationic lipid Lipid ratio in LNP (cationic lipid: PEG-modified lipid: cholesterol: DOPE) mRNA Encapsulation level before heating, % Encapsulation level after heating, % Encapsulation level after freeze-thaw, % Size (nm)/ PDI before heating Size (nm)/ PDI after heating 1 Cationic Lipid #1 40:1.5:28.5:30 FFL 31.6 78.8 Not tested 220.3/
0.149 236/
0.129 2 Cationic lipid #2 40:3:25:32 OTC 69.9 90.6 Not tested 114.9/
0,1 114.7/0.08 3 Cationic lipid #3 20:1.5:48.5:30 EPO 75 80 Not tested 134/
0.378 125.1/0.213 4 Cationic lipid #3 20:1.5:48.5:30 FFL 54 69 Not tested 145.7/
0.373 133.6/0.207 5 Cationic lipid #4 20:1.5:48.5:30 EPO 35 69 Not tested 125.3/
0.088 130.7/0.106 6 Cationic lipid #4 20:1.5:48.5:30 FFL 25 58 Not tested 134.6/
0.132 137.9/0.117 7 Cationic Lipid No. 5 40:3:25:32 OTC 35 91 67.7 120/
0.20 118.5/0.218 8 Cationic Lipid No. 5 40:5:25:30 OTC 14.2 77.9 64.9 172.2/
0.215 120.3/0.1 9 Cationic Lipid No. 6 40:5:25:30 EPO 58.5 73.1 75.3 116.3/
0.173 117.3/0.15 10 Cationic Lipid No. 6 40:5:25:30 FFL 46.3 52.7 52.2 153.8/
0.168 150.9/0.169 11 Cationic Lipid No. 7 20:1.5:48.5:30 EPO 29.3 77 62.8 161.9/
0.035 141.2/0.024 12 Cationic Lipid No. 7 20:1.5:48.5:30 FFL 13.9 66 55 180.5/
0.028 147.4/0.041

[167] As can be seen from Table 1 and FIG. 3 , the % encapsulation of mRNA encapsulated in the formed LNPs was significantly different after heating in the formulation solution compared to immediately before heating in the same formulation solution in all test articles that were evaluated. Moreover, this increased level of encapsulation was maintained even after sequential freeze-thawing of the mRNA-LNPs in the same formulation solution.

[168] Overall, it is evident from the data in this example that for the mRNA-encapsulated lipid nanoparticles prepared using Method A followed by heating in solution to formulate the drug product, there was a significant increase in the level of encapsulation.

Example 2. In vivo expression of hEPO delivered by mRNA-LNP after heating of the drug product formulation solution

[169] This example confirms that for the lipid nanoparticles with encapsulated mRNA prepared by method A followed by heating in solution to formulate a drug product, there was a significant increase in the encapsulation level. Moreover, the data from this example demonstrate the presence of in vivo expression of human EPO (hEPO) in mice following administration of hEPO mRNA encapsulated in the lipid nanoparticles prepared according to the present invention.

[170] In this example, hEPO mRNA was encapsulated in the lipid nanoparticles shown in Table 2 as described in Example 1. For each test article, the amount of mRNA encapsulated in the formed LNPs was measured before and after heating in a drug product formulation solution of 10 mM citrate in 10% sucrose using the method described in Example 1.

[171] As can be seen from Table 2 , the encapsulation level in % for mRNA encapsulated in the formed LNPs was significantly different after heating in the formulation solution compared to immediately before heating in the same formulation solution in all test articles (each containing different cationic lipids) that were evaluated.

[172] Mice were then given a single dose of 1 μg/30 μL of the hEPO mRNA-encapsulated lipid nanoparticles prepared by method A via intramuscular injection after heating the formulation. Serum hEPO protein levels were measured at 6 hours and 24 hours post-administration.

[173] Serum hEPO protein levels in mice after treatment can be used to assess mRNA activity across different delivery routes. As shown in Table 2 , the lipid nanoparticle formulation with hEPO mRNA administered via intramuscular injection resulted in increased hEPO protein levels.

Table 2. Characteristics and expression of mRNA-LNPs obtained according to the present invention in vivo

Composition Size (nm) PDI Encapsulation efficiency before heating Encapsulation efficiency after heating EPO content after 6 hours (ng/ml) EPO content after 24 hours (ng/ml) MATE-GLA4-E16: DMG-PEG: cholesterol: DOPE 40:1.5:28.5:30 117 0.18 46% 67% 2.89±0.89 1.54±0.33 MATE-Suc2-E18:2: C8PEG2-ceramide: cholesterol: DOPE 40:1.5:28.5:30 122 0.48 50% 73% 5.20±0.39 1.17±0.21 MATE-Suc2-E14: C8PEG2-ceramide: cholesterol: DOPE 40:1.5:13.5:45 119 0.12 63% 75% 10.33±0.74 4.10±0.27

Example 3. In vivo expression of mRNA delivered by pulmonary administration

[174] This example confirms that for the lipid nanoparticles with encapsulated mRNA prepared by method A followed by heating in a solution to formulate a drug product that is applicable to a wide range of cationic lipids, there was a significant increase in the level of encapsulation. Moreover, the data from this example demonstrate the presence of in vivo mRNA expression in mice following pulmonary administration of mRNA encapsulated in the lipid nanoparticles prepared according to the present invention.

[175] In this example, mRNA was encapsulated in the lipid nanoparticles shown in Table 3 as described in Example 1. For each test article, the amount of mRNA encapsulated in the formed LNPs was measured before and after heating in a drug product formulation solution using the method described in Example 1.

Table 3. Characteristics of mRNA-LNPs obtained according to the present invention

Sample Cationic lipid Composition (DMG-PEG2000: cationic lipid: cholesterol: DOPE) Size (nm) PDI Encapsulation efficiency before heating, % Encapsulation efficiency after heating % A VD-3-DMA 5:40:25:30 66.88 0.19 53 80.9 B Cationic Lipid No. 8 5:60:0:35 68 0.127 57 92 C Cationic Lipid No. 9 5:60:0:35 55 0.178 56 77 D Cationic Lipid No. 10 5:40:25:30 72.09 0.13 29 93 E Cationic Lipid No. 11 5:60:0:35 63 0.201 49 86 F TL1-10D-PIP 3:40:25:32 143.2 0.244 63.8 76 G Cationic Lipid No. 12 5:60:0:35 71.9 0.193 58 64 H Cationic Lipid No. 13 5:60:0:35 64.8 0.152 55.0 89.4 I Cationic Lipid No. 14 5:60:0:35 61.1 0.14 53.0 88.2 J Cationic Lipid No. 15 5:60:0:35 55 0.224 58 68 K Cationic Lipid No. 16 5:60:0:35 50 0.171 44 89 L Cationic Lipid No. 17 5:40:25:30 53 0.204 59 89 M Cationic Lipid No. 18 5:40:25:30 50 0.258 55 96

[176] As can be seen from Table 3 and FIG. 4, the encapsulation level in % for mRNA encapsulated in the formed LNPs was significantly different after heating in the formulation solution compared to immediately before heating in the same formulation solution in all test articles (each containing different cationic lipids) that were evaluated.

[177] Mice were then administered 10 μg of mRNA-LNPs prepared by Method A via pulmonary delivery after heating the drug formulation. The fluorescence level of the expressed protein was measured 24 hours after dosing. The protein expression level as a result of the delivered mRNA was measured in units of f/s/ ^cm2 /sr, as shown in FIG. 5. From the data, it is evident that the mRNA-lipid nanoparticle formulation administered via pulmonary delivery resulted in high levels of protein expression.

[178] Overall, it is evident from the data in this example that the mRNA-LNPs obtained according to the present invention result in high encapsulation efficiency, which is reflected in high expression and activity.

EQUIVALENTS

Those skilled in the art will know, or will be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. The scope of the present invention is not intended to be limited by the foregoing description, but is defined by the following claims.

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SEQUENCE LIST

<110> Translate Bio, Inc.

<120> IMPROVED METHOD FOR PRODUCING LIPID NANOPARTICLES,

LOADED mRNA

<130> MRT-2085WO

<140> PCT/US20/32943

<141> 2020-05-14

<150> 62/847,837

<151> 2019-05-14

<160> 5

<170> PatentIn version 3.5

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gaccuccuca cccugaaaaa cuucaccgga gaagagauca aguacaugcu guggcuguca 180

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uucgcgcugc ugggaggaca ccccugcuuc cugaccaccc aagauaucca ucugggugug 360

aacgaauccc ucaccgacac agcgcgggug cugucgucca uggcagacgc gguccucgcc 420

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cugcacagca uuaugaugag cgcugccaag uuuggaaugc accuccaagc agcgaccccg 660

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uuucaggggu accaggugac uaugaaaacc gccaaggucg ccgccucgga cuggaccuuc 900

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gcgguauaca aagaugcaga uuuguauucug cuugauucac cguuuggaua ccucgacgua 1740

uugacagaaa aagaaaucuu cgagucgugc guguguaaac uuauggcuaa uaagacgaga 1800

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gaaggaucgu ccuacuuuua cggcacuuuc ucagaguugc aaaacuugca gccggacuuc 1920

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cccuauaaga augggcaauu gaguaaggua augaucaucg agaacaguca cgugaagaag 3600

gaugacaucu ggccuagcgg gggucagaug accgugaagg accugacggc aaaauacacc 3660

gagggaggga acgcaauccu ugaaaacauc ucguucagca uuagccccgg ucagcgugug 3720

ggguugcucg ggaggaccgg gucaggaaaa ucgacguugc ugucggccuu cuugagacuu 3780

cugaauacag agggugagau ccagaucgac ggcguuucgu gggauagcau caccuugcag 3840

caguggcgga aagcguuugg aguaaucccc caaaaggucu uuaucuuuag cggaaccuuc 3900

cgaaagaauc ucgauccuua ugaacagugg ucagaucaag agauuuggaa agucgcggac 3960

gagguuggcc uucggagugu aauucgagcag uuuccgggaa aacucgacuu uguccuugua 4020

gaugggggau gcguccuguc gcaugggcac aagcagcuca ugugccuggc gcgauccguc 4080

cucucuaaag cgaaaauucu ucucuuggau gaaccuucgg cccaucugga cccgguaacg 4140

uaucagauca ucagaaggac acuuaagcag gcguuugccg acugcacggu gauucucugu 4200

gagcaucgua ucgaggccau gcucgaaugc cagcaauuuc uugucaucga agagaauaag 4260

guccgccagu acgacuccau ccagaagcug cuuaaugaga gaucauuguu ccggcaggcg 4320

auuucaccau ccgauagggu gaaacuuuuu ccacacagaa auucgucgaa gugcaagucc 4380

aaaccgcaga ucgcggccuu gaaagaagag acugaagaag aaguucaaga cacgcgucuu 4440

uaa 4443

<210> 5

<211> 1359

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 5

augagcaccg ccgugcugga gaaccccggc cugggccgca agcugagcga cuucggccag 60

gagaccagcu acaucgagga caacugcaac cagaacggcg ccaucagccu gaucuucagc 120

cugaaggagg aggugggcgc ccuggccaag gugcugcgcc uguucgagga gaacgacgug 180

aaccugaccc acaucgagag ccgccccagc cgccugaaga aggacgagua cgaguucuuc 240

acccaccugg acaagcgcag ccugcccgcc cugaccaaca ucaucaagau ccugcgccac 300

gacaucggcg ccaccgugca cgagcugagc cgcgacaaga agaaggacac cgugcccugg 360

uucccccgca ccauccagga gcuggaccgc uucgccaacc agauccugag cuacggcgcc 420

gagcuggacg ccgaccaccc cggcuucaag gaccccgugu accgcgcccg ccgcaagcag 480

uucgccgaca ucgccuacaa cuaccgccac ggccagccca ucccccgcgu ggaguacaug 540

gaggaggaga agaagaccug gggcaccgug uucaagaccc ugaagagccu guacaagacc 600

cacgccugcu acgaguacaa ccacaucuuc ccccugcugg agaaguacug cggcuuccac 660

gaggacaaca ucccccagcu ggaggacgug agccaguucc ugcagaccug caccggcuuc 720

cgccugcgcc ccguggccgg ccugcugagc agccgcgacu uccugggcgg ccuggccuuc 780

cgcguguucc acugcaccca guacauccgc cacggcagca agcccaugua cacccccgag 840

cccgacaucu gccacgagcu gcugggccac gugccccugu ucagcgaccg cagcuucgcc 900

caguucagcc aggagaucgg ccuggccagc cugggcgccc ccgacgagua caucgagaag 960

cuggccacca ucuacugguu caccguggag uucggccugu gcaagcaggg cgacagcauc 1020

aaggccuacg gcgccggccu gcugagcagc uucggcgagc ugcaguacug ccugagcgag 1080

aagcccaagc ugcugccccu ggagcuggag aagaccgcca ucgaacua caccgugacc 1140

gaguuccagc cccuguacua cguggccgag agcuucaacg acgccaagga gaaggugcgc 1200

aacuucgccg ccaccauccc ccgccccuuc agcgugcgcu acgaccccua cacccagcgc 1260

aucgaggugc uggacaacac ccagcagcug aagauccugg ccgacagcau caacagcgag 1320

aucggcaucc ugugcagcgc ccugcagaag aucaaguaa 1359

<---

Claims (38)

1. A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), comprising the steps of: (a) mixing one or more lipids in a lipid solution with one or more mRNAs in an mRNA solution to form mRNA encapsulated in LNPs (mRNA-LNPs) in a solution to form lipid nanoparticles (LNPs), wherein the lipid solution and/or the mRNA solution are heated to a first temperature above ambient temperature prior to mixing, or wherein the mRNA solution and the lipid solution are mixed at ambient temperature and heated to a first temperature above ambient temperature after mixing, wherein the first temperature is between about 60°C and 70°C; (b) replacing the LNP formation solution with a drug product formulation solution to obtain mRNA-LNP in the drug product formulation solution, wherein the drug product formulation solution is an aqueous solution containing pharmaceutically acceptable excipients; and (c) heating the mRNA-LNP in the solution to formulate the drug product to a second temperature above ambient temperature, wherein the second temperature is between about 60°C and 70°C; wherein the encapsulation efficiency for the mRNA-LNPs obtained in step (c) exceeds the encapsulation efficiency for the mRNA-LNPs obtained in step (b). 2. The method of claim 1, wherein in step (a) the one or more lipids comprise one or more cationic lipids, one or more auxiliary lipids and one or more PEG-modified lipids. 3. The method of claim 2, wherein the lipids further comprise one or more cholesterol-based lipids, such as cholesterol. 4. The method according to any one of the preceding claims, wherein in step (a) the one or more cationic lipids are selected from cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (imidazole-based), HGT5000, HGT5001, HGT4001, HGT4002, HGT4003, HGT4004, HGT4005, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione, 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, N1GL, N2GL, V1GL and combinations thereof. 5. The method according to any one of claims 2 to 4, wherein in step (a) the one or more auxiliary lipids are selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 16-O-monomethyl-PE, 16-O-dimethyl-PE, 18-1-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and combinations thereof. 6. The method of claim 1, wherein in step (a) one or more PEG-modified lipids comprise a polyethyleneglycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length, covalently attached to a lipid with an alkyl chain(s) of _C6 - _C20 in length. 7. The method according to any one of the preceding claims, wherein the lipid component of the lipid solution consists of (a) a cationic lipid, (b) auxiliary lipid, (c) a cholesterol-based lipid and (d) PEG-modified lipid. 8. The method of claim 7, wherein the molar ratio of the cationic lipid, the auxiliary lipid, the cholesterol-based lipid, and the PEG-modified lipid is approximately 20-50:25-35:20-50:1-5. 9. The method according to any one of claims 1-6, wherein the lipid component of the lipid solution consists of (a) a cationic lipid, (b) auxiliary lipid, (c) PEG-modified lipid. 10. The method of claim 9, wherein the cationic lipid is a cholesterol-based lipid or an imidazole-based cationic lipid. 11. The method of claim 9 or 10, wherein the molar ratio of the cationic lipid and the auxiliary lipid and the PEG-modified lipid is approximately 55-65:30-40:1-15. 12. The method according to any of the preceding claims, wherein the mRNA encodes a protein or peptide. 13. The method according to any one of the preceding claims, wherein in step (c) the solution for formulating the medicinal product is heated by applying heat from a heat source to the solution, and the solution is maintained at a second temperature above ambient temperature for 10-20 minutes. 14. The method of claim 13, wherein the second temperature above ambient temperature is approximately 65°C. 15. The method according to any of the preceding claims, wherein the encapsulation efficiency after step (c) exceeds the encapsulation efficiency after step (b) by at least 5% or more. 16. The method according to any one of the preceding claims, wherein the encapsulation efficiency after step (c) is increased by at least 10% or more compared to the encapsulation efficiency after step (b). 17. The method according to any one of the preceding claims, wherein in step (a) the lipid solution comprises lipids dissolved in ethanol. 18. The method according to any of the preceding claims, wherein in step (a) the mRNA solution comprises mRNA dissolved in a citrate buffer. 19. The method according to any of the preceding paragraphs, wherein the solution for composing the medicinal product comprises a cryoprotectant. 20. The method according to any of the preceding paragraphs, wherein the solution for composing the medicinal product additionally contains sugar. 21. The method of claim 20, wherein the sugar is selected from the group consisting of one or more of trehalose, sucrose, mannose, lactose and mannitol. 22. The method according to claim 21, wherein the sugar comprises trehalose. 23. The method according to any one of the preceding claims, wherein in step (b) the solution for formulating the medicinal product is an aqueous solution containing approximately 10% w/v trehalose. 24. The method according to any of the preceding paragraphs, wherein the solution for composing the medicinal product does not contain either ethanol or citrate. 25. The method according to any of the preceding claims, wherein the lipid solution comprises ethanol, the mRNA solution comprises citrate, and the solution for formulating the medicinal product is free of both ethanol and citrate. 26. The method according to any of the preceding claims, wherein the mRNA solution has a pH less than pH 5.0. 27. The method according to any of the preceding claims, wherein the solution for composing the medicinal product has a pH from pH 5.0 to pH 7.0.

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