IMPROVED mRNA-LOADED LIPID NANOPARTICLES AND PROCESSES OF MAKING THE SAME

Abstract

To provide an improved lipid nanoparticle formulation encapsulating mRNA comprising DEPE as a helper lipid.SOLUTION: In one aspect, a lipid nanoparticle for delivery of mRNA to a subject in need thereof comprises one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids encapsulating the mRNA, where the one or more helper lipids comprises 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 871,513, filed July 8, 2019, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to lipid-mediated mRNA delivery, and to lipid compounds and compositions comprising such compounds. In particular, the present invention relates to methods and uses of such compounds and compositions, as well as processes for making such compounds and compositions. [Background technology]

[0003] Messenger RNA therapy (MRT) has become an increasingly important approach to the treatment or prevention of various diseases. MRT involves the administration of messenger RNA (mRNA) to a subject in order to provide for the production of the protein encoded by the mRNA in the body of the subject in need of therapy. Lipid nanoparticles can be used to encapsulate the mRNA for efficient in vivo delivery of the mRNA.

[0004] Much effort has been devoted to identifying novel methods and compositions that can enhance intracellular delivery and / or expression of mRNA using lipid nanoparticles, which can be adapted to scalable and cost-effective manufacturing processes.At the same time, it is important that any such enhancement of intracellular delivery and / or expression of mRNA also maintains or improves the safety and tolerability of the composition associated with lipid-mediated mRNA delivery.

[0005] Multicomponent lipid nanoparticles, including one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids that encapsulate mRNA, have been found to be particularly effective in achieving delivery and expression of mRNA in vivo. A specific focus of research in recent years has been the discovery of new cationic lipids for mRNA delivery. Other components of multicomponent lipid nanoparticles have received little or no attention. There is a continuing need to improve lipid nanoparticle mRNA delivery to achieve intracellular delivery and / or expression of mRNA. At the same time, it is desirable for new lipid nanoparticle formulations to maintain or improve safety and tolerability. Summary of the Invention

[0006] The inventors have surprisingly found that by optimizing the helper lipid component of the multi-component liposomes encapsulating mRNA, the delivery and / or expression of mRNA in vivo can be dramatically improved. Specifically, the present invention shows that the presence of 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) as a helper lipid in an mRNA-encapsulating lipid nanoparticle formulation comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids can increase the delivery and / or expression of mRNA in vivo by more than two-fold compared to conventional liposomes containing dioleoylphosphatidylethanolamine (DOPE) as one of the helper lipids. DEPE-containing lipid nanoparticles were comparable to DOPE-containing lipid nanoparticles in terms of safety and tolerability (assessed by hepatotoxicity markers such as ALT and AST).

[0007] Thus, it is an aspect of the present invention to provide lipid nanoparticles for delivering mRNA to a subject in need thereof, the lipid nanoparticles comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids that encapsulate the mRNA, the one or more helper lipids being 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine. DEPE in lipid nanoparticles provides enhanced expression of mRNA when administered to a subject.

[0008] In an embodiment of the invention, DEPE 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) has the following structure: [ka] or structurally represented by the following structure: [ka] .

[0009] In an embodiment of the present invention, the enhanced expression of mRNA is higher than that of the same mRNA from a second lipid nanoparticle that contains one or more different helper lipids and has the same lipid components and amounts except that it does not contain DEPE.In certain embodiments, the enhanced expression is increased by more than 2-fold compared to the second lipid nanoparticle.In some embodiments, the one or more different helper lipids in the second lipid nanoparticle include dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof.

[0010] In certain embodiments, DEPE in the lipid nanoparticle is present at a concentration of at least 0.5 molar percent of the total lipid in the lipid nanoparticle, for example, at a concentration of 0.5 molar percent to 50 molar percent, particularly at a concentration of 10 molar percent to 45 molar percent.More typically, DEPE in the lipid nanoparticle is present at a concentration of 25 molar percent to 35 molar percent of the total lipid in the lipid nanoparticle.

[0011] In certain embodiments, the one or more cationic lipids is or comprises cKK-E12.

[0012] In certain embodiments, the one or more cationic lipids are ICE (imidazole cholesterol). terol esters) or contains the same.

[0013] In certain embodiments, the one or more cationic lipids are cationic lipids of the following formula: [ka] or a pharma- ceutically acceptable salt thereof; In the formula, R 1 and R 2 are each independently H or a C1-C6 aliphatic group, each m is independently an integer having a value of 1 to 4, each A is independently a covalent bond or an arylene group, and L 1 are each independently an ester, thioester, disulfide, or anhydride group; L 2 are independent of each other, C2-C 10 Aliphatic, X 1 are each independently H or OH; R 3 Each independently, C6-C 20 In certain embodiments, the one or more cationic lipids are selected from the group consisting of the following compounds: [ka] or a pharma- ceutically acceptable salt thereof. In another embodiment, the one or more cationic lipids are selected from the group consisting of the following compounds: [ka] or a pharma- ceutically acceptable salt thereof. In another embodiment, the one or more cationic lipids are selected from the group consisting of the following compounds: [ka]

[0014] or a pharma- ceutically acceptable salt thereof. In certain embodiments, the one or more PEG-modified lipids are C6-C 20 The polypeptide is or comprises a poly(ethylene) glycol chain of up to 5 kDa chain length covalently attached to a lipid having an alkyl chain of up to 5 kDa chain length.

[0015] In some embodiments, the lipid nanoparticles encapsulating mRNA and containing DEPE as a helper lipid are 20 In some embodiments, the cationic lipids each comprise an alkyl chain of a chain length of C8-C 16 In some embodiments, the cationic lipids each comprise 1 to 4 alkyl chains having a chain length of C 10 -C 16 In some embodiments, the cationic lipids each comprise 1 to 4 alkyl chains having a chain length of C 10 -C 14 In some embodiments, the cationic lipids each comprise 1 to 4 alkyl chains having a chain length of C 10 In some embodiments, the cationic lipids each comprise 1 to 4 alkyl chains having a chain length of C 12 In some embodiments, the cationic lipids each comprise 1 to 4 alkyl chains having a chain length of C 16 It contains 1 to 4 alkyl chains with a chain length of

[0016] In some embodiments, the lipid nanoparticles encapsulating mRNA and containing DEPE as a helper lipid are each C6-C 20 In some embodiments, the cationic lipids include cationic lipids that contain 1-4 aliphatic chains with a chain length of C8-C 16 In some embodiments, the cationic lipids each comprise 1 to 4 aliphatic chains having a chain length of C 10 -C 14 In some embodiments, the cationic lipids each comprise 1 to 4 aliphatic chains having a chain length of C 10 In some embodiments, the cationic lipids each comprise 1 to 4 aliphatic chains having a chain length of C 12In some embodiments, the cationic lipids each comprise 1 to 4 aliphatic chains having a chain length of C 16 It contains an aliphatic chain of a chain length of

[0017] In some embodiments, the lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid also comprise one or more cationic lipids that are or comprise a lipidoid. In some embodiments, the lipidoid comprises four aliphatic chains. In some embodiments, the four lipidoid aliphatic chains are each independently selected from the group consisting of C6-C 20 In some embodiments, the four lipidoid aliphatic chains are each independently a chain length of C8-C 16 In some embodiments, the four lipidoid aliphatic chains are each independently a chain length of C 10 -C 14 In some embodiments, the four lipidoid aliphatic chains are each independently a chain length of C 10 or C 12 In some embodiments, all four lipidoid aliphatic chains are C6 in chain length. In some embodiments, all four lipidoid aliphatic chains are C8 in chain length. In some embodiments, all four lipidoid aliphatic chains are C 10 In some embodiments, all four lipidoid aliphatic chains are of chain length C 12 In some embodiments, all four lipidoid aliphatic chains are of chain length C 14 In some embodiments, all four lipidoid aliphatic chains are of chain length C 16 In some embodiments, all four lipidoid aliphatic chains are of chain length C 18 In some embodiments, all four lipidoid aliphatic chains are All are C 20 In some embodiments, at least two of the four lipidoid aliphatic chains are C6 in length. In some embodiments, at least two of the four lipidoid aliphatic chains are C8 in length. In some embodiments, at least two of the four lipidoid aliphatic chains are C10 In some embodiments, at least two of the four lipidoid aliphatic chains have a chain length of C 12 In some embodiments, at least two of the four lipidoid aliphatic chains have a chain length of C 14 In some embodiments, at least two of the four lipidoid aliphatic chains have a chain length of C 16 In some embodiments, at least two of the four lipidoid aliphatic chains have a chain length of C 18 In some embodiments, at least two of the four lipidoid aliphatic chains have a chain length of C 20 is the chain length.

[0018] In some embodiments, the lipid nanoparticles encapsulating mRNA and containing DEPE as a helper lipid are 20 In some embodiments, the lipidoids each include an alkyl chain having a chain length of C8-C 16 In some embodiments, the lipidoids each comprise an alkyl chain having a chain length of C 10 -C 14 In some embodiments, the lipidoids each comprise an alkyl chain having a chain length of C 10 In some embodiments, the lipidoids each comprise an alkyl chain having a chain length of C 12 In some embodiments, the lipidoids each comprise an alkyl chain having a chain length of C 16 It contains an alkyl chain of a chain length of

[0019] In certain embodiments, the lipid nanoparticles further comprise one or more sterols. In some embodiments, the one or more sterols are or comprise a cholesterol-based lipid, such as cholesterol or PEGylated cholesterol.

[0020] In some embodiments, the lipid nanoparticles include one or more cationic lipids, one or more PEG-modified lipids, one or more non-cationic lipids, and one or more cholesterol-based lipids that encapsulate mRNA. For example, in a typical embodiment of the present invention, the lipid components of the lipid nanoparticles include four types of lipids, including a cationic lipid (e.g., cKK-E12, Compound 1, Compound 2, or Compound 3), a PEG-modified lipid (e.g., DMG-PEG2K), a non-cationic lipid (DEPE), and a cholesterol-based lipid (e.g., cholesterol). In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol-based lipid:PEG-modified lipid in the lipid nanoparticles can be about 30-60:25-35:20-30:1-15.

[0021] In some embodiments, the lipid component of the lipid nanoparticles comprises a cationic lipid (e.g., ICE), a PEG-modified lipid (e.g., DMG-PEG2K), and a non-cationic lipid (DEPE). In some embodiments, the ratio of cationic lipid:non-cationic lipid:PEG-modified lipid can be about 50-60:45-30:5-10.

[0022] The mRNA delivered by the lipid nanoparticles of the present invention is a protein-encoding mRNA that is translated in vivo into a therapeutic protein. In certain embodiments, the protein-encoding mRNA encodes a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an antibody light chain or an antibody heavy chain. In some embodiments, the therapeutic polypeptide is a polypeptide that is absent or deficient in a subject. In certain embodiments, the protein-encoding mRNA encodes a peptide. In some embodiments, the peptide is an antigen.

[0023] In another aspect, the present invention provides a method for improved delivery of mRNA to a subject in need thereof, the method comprising administering to the subject lipid nanoparticles encapsulating the mRNA, the lipid nanoparticles comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids. and wherein the one or more helper lipids include 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). The DEPE in the lipid nanoparticles provides enhanced expression of mRNA when administered to a subject.

[0024] In some embodiments, the mRNA encodes a protein that is translated in vivo into a therapeutic protein or peptide. In some embodiments, the mRNA encoding the protein or peptide is delivered systemically. In some embodiments, the translated protein or peptide is detectable in the liver at 6 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in the liver at 12 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in the liver at 18 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in the liver at 24 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in the liver at 36 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in the liver at 48 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in the liver at 72 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum at 6 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum at 12 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum at 18 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum at 24 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum at 36 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum at 48 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in liver at 72 hours or more after administration.

[0025] In a further aspect, the present invention provides a lipid nanoparticle encapsulating an mRNA comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein the one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), for use in a method for treating or preventing a disease or disorder in a subject, the mRNA encoding a peptide, polypeptide, or protein suitable for treating or preventing a disease or disorder in a subject. In a related aspect, the present invention relates to the use of a lipid nanoparticle encapsulating an mRNA in the manufacture of a medicament for treating or preventing a disease or disorder in a subject, the lipid nanoparticle comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein the one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), the mRNA encoding a peptide, polypeptide, or protein suitable for treating or preventing a disease or disorder in a subject. In one embodiment, the mRNA encodes a polypeptide or protein that is absent or deficient in the subject, and the disease or disorder is a deficient polypeptide or protein. In another embodiment, the mRNA encodes an antibody light chain or an antibody heavy chain, and the subject suffers from a disease or disorder that can be treated by administering to the subject an antibody that comprises said light chain or said heavy chain. In a further embodiment, the mRNA encodes a peptide, polypeptide, or protein, and said peptide, polypeptide, or protein can induce an immune response in said subject to treat or prevent a disease or disorder.

[0026] The inventors also found that by using DEPE as a helper lipid, they were able to prepare multi-component formulations that do not form stable liposomes when DOPE is used as one of the helper lipids. It has also been found that it is possible to formulate the lipid nanoparticles.Therefore, in still another aspect, the present invention provides a method for preparing lipid nanoparticles that encapsulate mRNA, the method comprises: (a) providing a mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, the one or more helper lipids comprising 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); and (b) forming lipid nanoparticles from the mixture provided in step (a), the method further comprises encapsulating mRNA in lipid nanoparticles, and the encapsulation can be carried out before or after the formation of lipid nanoparticles in step (b).The obtained lipid nanoparticles that encapsulate mRNA are stable. In one embodiment, the method for preparing lipid nanoparticles according to the present invention specifically excludes the use of one or more helper lipids selected from dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and combinations thereof. In one embodiment, DEPE is present in the mixture at a concentration of 10 mole percent to 50 mole percent. In one embodiment, one or more PEG-modified lipids in the mixture are C6-C 20 The lipids include poly(ethylene) glycol chains of up to 5 kDa chain length covalently attached to lipids having alkyl chains of up to 5 kDa chain length. In one embodiment, the mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids further includes one or more sterols, such as a cholesterol-based lipid. In one embodiment, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol. In one embodiment, the mRNA encodes a therapeutic peptide, polypeptide, or protein. In some embodiments, the mRNA is encapsulated in a preformed lipid nanoparticle. In some embodiments, the method for preparing lipid nanoparticles according to the present invention includes subjecting the lipid nanoparticles to tangential flow filtration (TFF) before and / or after encapsulation of the mRNA. In some embodiments, the method further includes formulating the lipid nanoparticles in a trehalose solution.

[0027] Without wishing to be bound by any particular theory, the inventors believe that DEPE derivatives, particularly those with varying lipid chain length or composition, provide the same advantages as described herein for DEPE. For convenience, the above summary and detailed description of the invention refer only to DEPE. However, it should be understood that minor changes to the lipid chain of DEPE do not affect its superior properties, and that such DEPE derivatives are expressly included within the present invention.

[0028] Other features, objects, and advantages of the present invention will become apparent in the following detailed description, drawings, and claims. It should be understood, however, that the detailed description, drawings, and claims, while referring to embodiments of the present invention, are given by way of example only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. [Brief description of the drawings]

[0029] [Figure 1] 1 shows an exemplary graphical representation of EPO protein expression in mRNA LNPs containing DEPE and other helper lipids. [Diagram 2] 1 shows an exemplary graphical representation of post-administration levels of serum ALT and AST for mRNA LNPs containing DEPE and other helper lipids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions, and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0031] definition The present invention provides an improved process for producing mRNA encapsulated in lipid nanoparticle (LNP) formulations for producing mRNA therapeutic compositions.

[0032] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, as well as other terms, are set forth throughout the specification.

[0033] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxy-terminal and / or amino-terminal amino acids in a peptide, may be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting their activity. Amino acids may participate in disulfide bonds. An amino acid may include one or post-translational modifications, such as association with one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which it is used.

[0034] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0035] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that includes 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 (except where such number exceeds 100% of possible values), unless otherwise specified or otherwise clear from the context.

[0036] Combining: As used herein, the term "combining" is used interchangeably with mixing or blending. Combining refers to bringing together discrete LNP particles with distinct characteristics in the same solution, e.g., mRNA-LNPs and empty LNPs. It refers to combining NP compositions to obtain mRNA-LNP compositions.In some embodiments, combining two LNPs is performed with a specific ratio of the components to be combined.In some embodiments, the resulting composition obtained from combining has different properties from either one or both of the components.

[0037] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery.For example, the delivery of mRNA encompasses the situation where mRNA is delivered to target tissue, encoded protein or peptide is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and the situation where mRNA is delivered to target tissue, encoded protein or peptide is expressed, secreted into the patient's circulatory system (e.g., serum), distributed throughout the body, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0038] Efficacy: As used herein, the term "efficacy," or grammatical equivalents, refers to the improvement of a biologically relevant endpoint associated with delivery of an mRNA encoding a relevant protein or peptide. In some embodiments, the biological endpoint is protection against ammonium chloride challenge at a certain time point after administration.

[0039] Encapsulation: As used herein, the term "encapsulation," or grammatical equivalents, refers to the process of confining individual mRNA molecules within a nanoparticle.

[0040] Expression: As used herein, "expression" of mRNA refers to the translation of mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme), and may also include post-translational modification of a peptide, polypeptide, or fully assembled protein (e.g., an enzyme), as indicated by the context. In this application, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0041] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual before the start of a treatment described herein, or a measurement in a control sample or subject (or control samples or subjects) in the absence of a treatment described herein. A "control sample" is a sample that is subjected to the same conditions as the test sample, except for the test article. A "control subject" is a subject that suffers from the same form of disease as the subject being treated and is approximately the same age as the subject being treated.

[0042] Impurity: As used herein, the term "impurity" refers to a limited amount of a substance in a liquid, gas, or solid that differs from the chemical composition of the target substance or compound. Impurities are also called contaminants.

[0043] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0044] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0045] Isolated: As used herein, the term "isolated" means (1) a substance that is free of components with which it is originally associated when produced (whether in nature and / or in an experimental setting). It refers to substances and / or entities that are separated from at least some and / or (2) artificially produced, prepared, and / or manufactured. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which they were initially associated. In some embodiments, 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 more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of percent purity of an isolated substance and / or entity should not include excipients (eg, buffers, solvents, water, etc.).

[0046] Local distribution or local delivery: As used herein, the term "local distribution", "local delivery" or grammatical equivalents refer to tissue-specific delivery or distribution.Typically, local distribution or local delivery requires that the peptide or protein (e.g., enzyme) encoded by mRNA is translated and expressed within cells or with limited secretion to avoid entry into the patient's circulatory system.

[0047] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that codes for at least one peptide, polypeptide, or protein. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, may be produced using recombinant expression systems, and may be optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, mRNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. The mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA may be modified with natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodour ... The base may be or contain a base selected from the group consisting of C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0048] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA. Additionally, the terms "nucleic acid," "DNA," "RNA," and / or similar terms refer to nucleic acids. Includes acid analogs, ie, analogs having other than a phosphodiester backbone.

[0049] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include prenatal and postnatal forms.

[0050] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" refers to a material that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, inflammatory irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0051] Pharmaceutically acceptable salts: 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 those derived from suitable inorganic and organic acids and bases.The examples of pharmaceutically acceptable non-toxic acid addition salts are the salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or the salts of amino groups formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or the salts of amino groups formed using other methods used in the art such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lanthanide, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N-terminated salts. + (C 1-4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations, and amine cations, where appropriate, formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, for example, alkyl halides to form quaternized alkylated amino salts.

[0052] Potency: As used herein, the term "potency" or grammatical equivalents refers to the level of expression of the protein or peptide encoded by the mRNA and / or the resulting biological effect.

[0053] Salt: As used herein, the term "salt" refers to an ionic compound that results or can result from the neutralization reaction between an acid and a base.

[0054] Systemic distribution or delivery: As used herein, the terms "systemic distribution", "systemic delivery", or grammatical equivalents, refer to a delivery or distribution mechanism or approach that affects the entire body or organism. Typically, systemic distribution or delivery is accomplished via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery".

[0055] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, and refers to a human who visits a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may suffer from or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0056] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or reach completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0057] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease being treated. In some embodiments, the target tissue includes tissue that exhibits pathology, symptoms, or characteristics associated with the disease.

[0058] Therapeutic index: As used herein, "therapeutic index" is the ratio of the concentration in the blood where a drug is toxic and the concentration where it is effective. The larger the therapeutic index, the safer the drug.

[0059] Treating: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of the disease and / or who exhibit only early signs of the disease, for the purpose of reducing the risk of developing conditions associated with the disease.

[0060] 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".

[0061] Aliphatic: As used herein, the term aliphatic refers to 1- C 40 Aliphatic refers to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic can be linear, branched, or cyclic. For example, C1-C 20 Aliphatic includes C1-C 20 Alkyl (e.g., linear or branched C1-C 20 Saturated alkyl), C2-C 20 Alkenyl (e.g., linear or branched C4-C 20 Dienyl, linear or branched C6-C 20 trienyl, etc.), and C2-C 20 Alkynyl (e.g., linear or branched C-C 20 alkynyl). 20 Aliphatic includes C3-C 20 Cycloaliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl, or C8-C 20 In certain embodiments, the aliphatic can include one or more cyclic aliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and optionally, It may be substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. Aliphatic groups are unsubstituted or substituted with one or more substituents as described herein. For example, an aliphatic may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCOR', -NH2, -NHR', -N(R')2, -SR', or -SOR', where each instance of R' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C3 alkyl, or a C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently unsubstituted C1-C3 alkyl. In some embodiments, the aliphatic is unsubstituted. In some embodiments, the aliphatic does not contain any heteroatoms.

[0062] Alkyl: As used herein, the term "alkyl" refers to acyclic straight-chain and branched hydrocarbon groups, e.g., C1-C 20"Alkyl" refers to an alkyl group having 1 to 20 carbons. The alkyl group can be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentylhexyl, isohexyl, and the like. Other alkyl groups will be readily apparent to those of skill in the art given the benefit of this disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCOR2R', -NH2, -NHR', -N(R')2, -SR', or -S02R', where each instance of R' is independently selected from C1-C2H, - ... 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C3 alkyl, or a C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted C1-C3 alkyl. In some embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In some embodiments, the alkyl group is substituted with an -OH group, and may also be referred to herein as a "hydroxyalkyl" group, where the prefix indicates the -OH group and "alkyl" is as described herein.

[0063] Alkenyl: As used herein, "alkenyl" means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, e.g., "C2-C 20"Alkenyl" refers to an alkenyl group having 2 to 20 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, an alkenyl contains one, two, or three carbon-carbon double bonds. In embodiments, an alkenyl contains a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., two or three) are conjugated. An alkenyl group can be unsubstituted or substituted with one or more substituents described herein. For example, an alkenyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -COH, -COR', -CN, -OH, -OR', -OCOR', -OCOR', -NH, -NHR', -N(R')2, -SR', or -SOR', where each instance of R' is independently selected from C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl, or C In some embodiments, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R' is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein). In embodiments, the alkenyl group is substituted with an -OH group, and may also be referred to herein as a "hydroxyalkenyl" group, where the prefix denotes the -OH group and "alkenyl" is as described herein.

[0064] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain in either a linear or branched arrangement with one or more carbon-carbon triple bonds occurring at any stable point along the chain, e.g., "C2-C 20 "Alkynyl" refers to an alkynyl group having 2 to 20 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, the alkynyl contains one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more substituents described herein. For example, an alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -COH, -COR', -CN, -OH, -OR', -OCOR', -OCOR', -NH, -NHR', -N(R')2, -SR', or -SOR', where each instance of R' is independently selected from C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C3 alkyl, or a C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently unsubstituted C1-C3 alkyl. In some embodiments, alkynyl is unsubstituted. In some embodiments, alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein).

[0065] Aryl: The term "aryl," used alone or as part of a larger moiety, as in "aralkyl," refers to a monocyclic, bicyclic, or tricyclic carbon ring system having a total of 6 to 14 ring members, said ring system having a single point of attachment to the rest of the molecule, at least one ring in the system being aromatic, and each ring in the system containing 4 to 7 ring members. In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl," e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl", e.g., anthracyl. "Aryl" also includes ring systems in which the aryl ring is fused with one or more carbocyclic or heterocyclyl groups as defined above, in which the bonding radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms is followed by the number of carbons in the aryl ring system. Examples of aryl include phenyl, naphthyl, and anthracene.

[0066] Arylene: As used herein, the term "arylene" refers to a divalent aryl group (i.e., having two points of attachment to the molecule). Examples of arylene include phenylene (e.g., unsubstituted or substituted phenylene).

[0067] Compositions of the Invention In some embodiments, the present invention provides a composition comprising LNPs and mRNA, which, when administered to a subject, inhibits the proliferation or proliferation of a subject without altering the subject's resistance or stress level. Induce significantly higher levels of mRNA expression in vivo. Tolerance or stress is determined by the elevation of liver enzymes aspartate transaminase (AST) and / or alanine aminotransferase (ALT). In some embodiments, certain formulations provide manufacturing advantages, such as, among others, ease of manufacturing process, for example, utilization of a common preformed LNP stock solution.

[0068] The observations of the present invention revealed that when the mRNA-LNPs formed by mixing mRNA with preformed empty LNPs in step (a) are further combined with preformed LNPs in step (b) to form an mRNA-LNP composition, the potency of the resulting composition is significantly increased compared to the mRNA-LNPs of step (a). This is particularly noteworthy because the increased potency is observed even when the preformed LNPs are empty (i.e., do not contain mRNA) and contain the same lipid components as the mRNA-LNPs. Furthermore, increased expression of the mRNA-encoded protein is observed even when the preformed LNPs contain only neutral lipids, which are known to be poor promoters of polynucleotide transfection.

[0069] Thus, the fact that increased potency of mRNA-LNP compositions is achievable without compromising in vivo tolerability is a significant advantage of the methods of the present invention from the perspective of therapeutic design.

[0070] This aspect of the invention allows for at least two important advantages: (i) providing a lower amount of mRNA in an mRNA therapeutic composition per dose or reducing the frequency of administration to achieve the same biological effect, thereby increasing the therapeutic index of the composition, and (ii) developing an easy, flexible, scalable, and / or high-throughput manufacturing process in which one or more preformed LNPs can be prepared in bulk and made available for multiple mixing and combination steps to achieve the desired formulations described in the invention.

[0071] The present invention provides a process in which the mRNA-LNP prepared by mixing the mRNA with the preformed empty LNP is further combined with the preformed LNP, and the resulting mRNA-LNP composition of the present invention results in increased in vivo expression of the mRNA-encoded protein. In some aspects, the process is a manufacturing process comprising: (a) mixing the preformed empty LNP with the mRNA under conditions that allow the formation of the mRNA-LNP; and (b) mixing the mRNA-LNP formed in step (a) with the preformed LNP, thereby producing a composition comprising lipid nanoparticles encapsulating the mRNA. In some embodiments, the lipid nanoparticles comprise at least cationic lipids, non-cationic lipids, and PEG-modified lipids. In some embodiments, the lipid nanoparticles may comprise neutral lipids with or without cationic lipids.

[0072] In some embodiments, the mRNA encodes a protein or peptide.

[0073] In some embodiments, the preformed LNPs in step (b) are empty LNPs. In some embodiments, the preformed LNPs in step (b) comprise mRNA. In some embodiments, the preformed LNPs in step (b) comprise mRNA encoding a protein or peptide. In some embodiments, the preformed LNPs in step (b) comprise the same mRNA encoding the same protein or polypeptide as the mRNA-LNPs formed in step (a). In some embodiments, the preformed LNPs in step (b) comprise a different mRNA encoding a different protein or polypeptide than in the mRNA-LNPs formed in step (a).

[0074] In some embodiments, the empty LNPs of step (a) and the preformed LNPs of step (b) are LNPs are distinct heterogeneous lipid nanoparticles. For example, the empty LNPs in step (a) may comprise cationic lipid HGT-5003, and the preformed LNPs in step (b) comprise cationic lipid ICE. In another example, the empty LNPs in step (a) may comprise cationic lipid ICE, and the preformed LNPs in step (b) comprise cationic lipid DOTAP. In yet another example, the empty LNPs in step (a) may comprise cationic lipid HGT-4001, and the preformed LNPs in step (b) comprise cationic lipid ckk-E12. Various lipids suitable for LNPs and methods for producing them are described in the following respective sections, and any combination of lipids that form LNPs is contemplated herein.

[0075] In one embodiment, the mRNA-LNP composition produced in step (b) may comprise a first lipid nanoparticle and a second lipid nanoparticle, the first lipid nanoparticle and the second lipid nanoparticle having the same lipid composition, and at least some of the first lipid nanoparticles comprise mRNA. In one embodiment, the mRNA-LNP composition produced in step (b) may comprise a first lipid nanoparticle and a second lipid nanoparticle, the first lipid nanoparticle and the second lipid nanoparticle having distinct lipid compositions. For example, the mRNA-LNP composition may comprise a first lipid nanoparticle comprising the cationic lipid ICE, and a second lipid nanoparticle comprising the cationic lipid DOTAP. In some embodiments, the mRNA-LNP composition produced in step (b) may comprise a first lipid nanoparticle comprising the cationic lipid C12-200, and a second lipid nanoparticle comprising the cationic lipid DLinKC2DMA. Thus, any combination of various lipids suitable for producing LNPs as described in the respective sections below is contemplated herein.

[0076] In some embodiments, either the empty LNPs of step (a) or the preformed LNPs of step (b) do not contain cationic lipids, hi some embodiments, either the empty LNPs or the preformed LNPs contain neutral lipids and / or PEG-modified lipids.

[0077] Lipid Nanoparticles (LNPs) The present invention provides, inter alia, compositions for mRNA therapeutics, where the mRNA is encapsulated in a delivery vehicle for efficient cellular uptake and processing in vivo. As used herein, the terms "delivery vehicle", "implantation vehicle", "nanoparticle", or grammatical equivalents are used interchangeably. The delivery vehicle may be formulated in a pharmaceutical composition in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or mixed with suitable excipients. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based on its ability to facilitate transfection of nucleic acids into target cells.

[0078] In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, such as a lipid nanoparticle (LNP) or liposome. In some embodiments, the liposome comprises one or more cationic lipids. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, the liposome comprises four or fewer distinct lipid components. In some embodiments, the liposome comprises three or fewer distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid. In an exemplary embodiment, the LNP of the present invention is a liposome that encapsulates mRNA. Suitable liposomal lipid components include cationic lipids (e.g., cKK-E12, Compound 1, Compound 2, or Compound 3), non-cationic lipids (DEPE), cholesterol-based lipids (e.g., cholesterol), and PEG-modified lipids (DMG-PEG2K). Alternatively, suitable liposomal lipid components include sterol-based cationic lipids (e.g., ICE), non-cationic lipids (DEPE), and PEG-modified lipids (e.g., DMG-PEG2K).

[0079] In some embodiments, DEPE in lipid nanoparticles provides enhanced expression of mRNA encoding a protein or peptide, compared to DOPE-containing lipid nanoparticles that are otherwise identical in composition (with respect to lipid components and molar ratios of individual lipid components) to DEPE-containing lipid nanoparticles. In some embodiments, the expression of the mRNA-encoded protein delivered by DEPE-containing lipid nanoparticles is enhanced at least 2-fold compared to DOPE-containing lipid nanoparticles. Enhancement of mRNA expression can be determined by administering, for example, by tail vein injection, to test animals (e.g., mice) DEPE-containing lipid nanoparticles and identically formulated lipid nanoparticles with different helper lipids (e.g., DOPE) and monitoring mRNA expression at one or more time points (e.g., 4, 6, 8, 12, 18, or 24 hours after administration).

[0080] In some embodiments, the mRNA encodes a protein that is translated in vivo into a therapeutic protein. In some embodiments, the mRNA encoding a protein encodes a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an antibody light chain or an antibody heavy chain. In some embodiments, the therapeutic polypeptide is absent or deficient in the subject to which the mRNA is administered. In some embodiments, the mRNA encoding a protein encodes a peptide. In some embodiments, the peptide is an antigen.

[0081] In some embodiments, the DEPE-containing lipid nanoparticles of the present invention are safe and tolerable when administered to a subject.For example, the DEPE-containing lipid nanoparticles of the present invention do not cause any discernible hepatotoxicity when administered to a subject.Preferred markers for assessing hepatotoxicity are ALT and AST.

[0082] Cationic lipids As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0083] Suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2010 / 144740, which is incorporated herein by reference.In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate: [ka] and pharma- ceutically acceptable salts thereof.

[0084] Other suitable cationic lipids for use in the compositions and methods of the invention include the ionizable cationic lipids described in International Patent Publication No. WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids that are ionizable. and a cationic lipid having one of the following formulas: [ka] or a pharma- ceutically acceptable salt thereof, wherein R and R are each independently hydrogen, an optionally substituted variably saturated or unsaturated C-C 20 Alkyl, and optionally substituted variably saturated or unsaturated C6-C 20acyl; L and L are each independently selected from the group consisting of hydrogen, optionally substituted C-C 30 Alkyl, optionally substituted variably unsaturated C1-C 30 Alkenyl, and optionally substituted C-C 30 alkynyl, m and o are each independently selected from the group consisting of zero and any positive integer (e.g., m is 3), and n is zero or any positive integer (e.g., n is 1). In certain embodiments, the compositions and methods of the present invention include a cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-l-amine ("HGT5001") having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002"): [ka] and pharma- ceutically acceptable salts thereof.

[0085] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids described as aminoalcohol lipidoids in International Patent Publication No. WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0086] In some embodiments, lipidoids used in the compositions and methods of the invention are synthesized by reacting commercially available amines with lipophilic acrylates, acrylamides, or epoxides. In some embodiments, lipidoids are derived from amine 86 (N,N-bis(2-hydroxyethyl)ethylenediamine) and amine 87 (N-(3-aminopropyl)diethanamine). Lipidoids have several advantages as a potential new class of nucleic acid delivery reagents: (i) the chemistry used to synthesize lipidoids is simple and economical, (ii) libraries of structural diversity have already been developed, and (iii) correlations between the structure and function of delivery systems can be constructed from large data sets accumulated from screening libraries of lipidoids. The simplicity of these reactions has made it possible to construct structurally diverse libraries of lipidoids by varying the type of amine, as well as the chain length and type (acrylamide / acrylate / epoxide) of the tail (or carbon arm chain).

[0087] In some embodiments, the lipidoid comprises about 2-20 carbon arm chains. In some embodiments, the lipidoid comprises about 5-18 carbon arm chains. In some embodiments, the lipidoid comprises about 10-16 carbon arm chains. In some embodiments, the lipidoid comprises about 10-14 carbon arm chains. In some embodiments, the lipidoid comprises about 10-12 carbon arm chains. In some embodiments, the lipidoid comprises about 10 carbon arm chains. In some embodiments, the lipidoid comprises about 12 carbon arm chains. In some embodiments, the lipidoid comprises about 14 carbon arm chains. In some embodiments, the lipidoid comprises about 16 carbon arm chains.

[0088] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. WO 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0089] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0090] Other suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharma- ceutically acceptable salts thereof.

[0091] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication Nos. 2013 / 063468 and 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein R L each instance of is independently an optionally substituted C6-C 40 In certain embodiments, the compositions and methods of the present invention provide a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and The method includes providing a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0092] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and R A are each independently hydrogen, optionally substituted C alkyl, optionally substituted C alkenyl, optionally substituted C alkynyl, optionally substituted C carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C aryl, optionally substituted 5-14 membered heteroaryl or halogen; R B each independently represents hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, or optionally substituted 5-14 membered heteroaryl. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, "Target 23," having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0093] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0094] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 and R 2 are each independently H or a C1-C6 aliphatic group, each m is independently an integer having a value of 1 to 4, each A is independently a covalent bond or an arylene group, and L 1 are each independently an ester, thioester, disulfide, or anhydride group; L 2 are independent of each other, C2-C 10 Aliphatic, X 1 are each independently H or OH; R 3 Each independently, C6-C 20 In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0095] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, MCKing, Cell, 1999, 144:1311-1315, which are incorporated herein by reference. 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277. In certain embodiments, the cationic lipid of the compositions and methods of the present invention is a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0096] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0097] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0098] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 or L 2 One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, L 1 or L 2 -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond, G 1 and G 2 each independently is an unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene, G 3 But, C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a But H or C1-C 12 is alkyl, R 1 and R 2 Each independently, C6-C24 Alkyl or C6-C 24 alkenyl, R 3 But, H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and R 4 But, C1-C 12 is alkyl, R 5 is H or C1-C6 alkyl and x is 0, 1, or 2.

[0099] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and The method includes providing a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0100] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the invention is a compound of one of the following formulas: [ka] and pharma- ceutically acceptable salts thereof. In any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), and heterocycle, where n is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention provide a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0101] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication Nos. 2017 / 173054 and 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and The method includes providing a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0102] Other suitable cationic lipids for use in the compositions and methods of the invention include the cleavable cationic lipids described in International Patent Publication No. WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; and wherein R2 is selected from the group consisting of one of the following two formulae: [ka] wherein R and R each independently represent an optionally substituted variably saturated or unsaturated C-C 20 Alkyl and optionally substituted variably saturated or unsaturated C6-C 20 acyl, where n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4001" having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4002" having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4003" having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4004" having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4005" having the following compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0103] Other suitable cationic lipids for use in the compositions and methods of the invention include the cleavable cationic lipids described in U.S. Provisional Application No. 62 / 672,194, filed May 16, 2018, and incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include a cationic lipid having any of the general formulas or any of structures (1a)-(21a) and (1b)-(21b) and (22)-(237) described in U.S. Provisional Application No. 62 / 672,194. In certain embodiments, the compositions and methods of the invention include a cationic lipid having a structure according to formula (I'): [ka] During the ceremony, R X Independently, -H, -L 1 -R 1 , or -L 5A -L 5B -B', L 1 , L 2 , and L 3 each independently is a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L - and L 4A and L 5A each independently is -C(O)-, -C(O)O-, or -C(O)NR L - and L 4B and L 5B are each independently, C1-C 20 Alkylene, C2-C 20 Alkenylene, or C2-C 20is alkynylene, B and B' are each NR 4 R 5 or 5-10 membered nitrogen-containing heteroaryl; R 1 , R 2 , and R 3 Each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, R 4 and R 5 each independently represents hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, or C2-C 10 is alkynyl, R L each independently represents hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, or C2-C 20 Includes cationic lipids that are alkynyl. In certain embodiments, the compositions and methods of the present invention include a cationic lipid that is compound (139) of 62 / 672,194, having the following compound structure: [ka]

[0104] In some embodiments, the compositions and methods of the invention include the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"). (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355), incorporated herein by reference. Other cationic lipids suitable for the compositions and methods of the invention include, for example, 5-carboxyspermylglycine dioctadecylamide (COTMA). arboxyspermylglycinedioctadecylamide ("DOGS"), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium ("DOSPA") (Behr et al. 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-dimethylammonium-propane ("DODAP"), 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0105] Additional exemplary cationic lipids suitable for the compositions and methods of the present invention also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"), N-dioleyl-N,N-dimethylammonium diethyl ammonium chloride ("DODAC"), N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(l,2-dimethylethyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-l-(cis,cis-9,12-octadecadienooxy)propane ("CLinDMA"); 2-[5'-(cholest-5-ene-3-beta-oxybutane-4-yl)-1-(cis,cis-9,12-octadecadienooxy)propane ("CLinDMA"); 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); l,2-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA");(2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan- 1-amine ("Octyl-CLinDMA(2S)"); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"), and 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, incorporated herein by reference; Semple et al., Nature Biotech. 28:172-176 (2010)). (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); International Patent Publication No. 2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole moiety, a dialkylamino moiety, or a guanidinium moiety;

[0106] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention are The substances include 2,2-dilinoleyl-1-4-dimethylaminoethyl-1-[1,3]-dioxolane ("XTC"), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100"), and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0107] In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., as measured by weight of the lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., as measured by mole % of the lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition, e.g., measured by weight of the lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition, e.g., measured by molar % of the lipid nanoparticles.

[0108] In some embodiments, sterol-based cationic lipids may be used instead of or in addition to the cationic lipids described herein.Suitable sterol-based cationic lipids are dialkylamino-containing sterol-based cationic lipids, imidazole-containing sterol-based cationic lipids, and guanidinium-containing sterol-based cationic lipids. For example, certain embodiments are directed to compositions comprising one or more sterol-based cationic lipids comprising imidazole, such as the imidazole cholesterol ester or "ICE" lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as shown by the following structure (I): In certain embodiments, lipid nanoparticles for delivery of RNA (e.g., mRNA) encoding a functional protein may include one or more imidazole-based cationic lipids, such as the imidazole cholesterol ester or "ICE" lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, shown by the following structure: [ka]

[0109] In some embodiments, the percentage of cationic lipid in the liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. In some embodiments, the cationic lipid comprises about 30-50% by weight of the liposome (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%). In some embodiments, the cationic lipid (e.g., ICE lipid) comprises about 30%, about 35%, about 40%, about 45%, or about 50% by molar ratio of the liposome.

[0110] Non-cationic lipids As used herein, the term "non-cationic lipid" refers to any neutral lipid, zwitterionic lipid, or anionic lipid, also referred to herein as "helper lipid".As used herein, the term "cationic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0111] The present invention relates to mRNA-LNPs that include one or more non-cationic helper lipids, including 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). In some embodiments, DEPE is the only non-cationic helper lipid in the mRNA-LNP. In other embodiments, the helper lipid portion of the mRNA-LNP includes DEPE and cholesterol.

[0112] Without wishing to be bound by any particular theory, certain DEPE derivatives that differ from DEPE in their lipid chain length or composition are also included within the present invention. For example, the inventors have found that alkyl or alkene chains with a carbon chain length of 10-20 are particularly suitable for forming mRNA-LNPs. In some embodiments, DEPE derivatives having alkyl or alkene chains with a carbon chain length of 16-20 are particularly preferred. Alternatively, DEPE derivatives having alkyl or alkene chains with a carbon chain length of 10-14, such as those with 10, 12, or 14 carbons, may be particularly suitable for the practice of the present invention.

[0113] Other non-cationic or helper lipids that can be included in mRNA-LNPs include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), 1 -Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides , gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or mixtures thereof.

[0114] In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other lipids, such as cationic lipids. In some embodiments, the non-cationic lipids may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipids present in the liposome. In some embodiments, the non-cationic lipids are neutral lipids, i.e., lipids that do not carry a net charge under the conditions in which the composition is formulated and / or administered. In some embodiments, the percentage of non-cationic lipids in the liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0115] Cholesterol-Based Lipids In some embodiments, the composition (e.g., liposome composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for carrying out the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), or imidazole cholesterol ester (ICE).

[0116] In some embodiments, the cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0117] In some embodiments, the cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be about 5 wt% or less, about 10 wt% or less, about 20 wt% or less, about 30 wt% or less, or about 40 wt% or less.

[0118] PEGylated lipids In some embodiments, the suitable lipid solution comprises one or more PEGylated lipids, also referred to herein as PEG-modified lipids. A suitable PEG-modified or PEGylated lipid for carrying out the present invention is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2K). For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethyleneglycol)-2000] (C8 PEG-2000 ceramide), is also contemplated by the present invention. The contemplated PEG-modified lipids are C6-C 20 In some embodiments, the poly(ethylene glycol) chains include, but are not limited to, polyethylene glycol chains 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 having an alkyl chain of up to 2 kDa in length. In the present case, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, particularly useful exchangeable lipids are those with shorter acyl chains (e.g., C 14 or C 18 ) The addition of such moieties can prevent aggregation of the complex and can also increase the circulatory lifetime and provide a means for increasing the delivery of lipid-nucleic acid compositions to target tissues (Klibanov et al., 2003). al. (1990) FEBS Letters, 268(1):235-237). Alternatively, these components can be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Certain useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 The PEG-modified phospholipids and derivatized lipids of the present invention may comprise a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome import vehicle.

[0119] The PEG-modified phospholipids and derivatized lipids may comprise no more than about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% of the total lipids in a suitable lipid solution, by weight or molar concentration. In some embodiments, the PEG-modified lipids may comprise no more than about 5% of the total lipids in a suitable lipid solution, by weight or molar concentration. In some embodiments, the PEG-modified lipids may comprise no more than about 4% of the total lipids in a suitable lipid solution, by weight or molar concentration. In some embodiments, the PEG-modified lipids typically comprise no more than 3% of the total lipids in a suitable lipid solution, by weight or molar concentration. In some embodiments, the PEG-modified lipids typically comprise no more than 2% of the total lipids in a suitable lipid solution, by weight or molar concentration. In some embodiments, the PEG-modified lipids typically comprise no more than 1% of the total lipids in a suitable lipid solution, by weight or molar concentration. In some embodiments, the PEG-modified lipid comprises about 1-5%, about 1-4%, about 1-3%, or about 1-2% by weight or molar concentration of the total lipid in a suitable lipid solution. In some embodiments, the PEG-modified lipid comprises 0.01-3% (e.g., about 0.01-2.5%, 0.01-2%, 0.01-1.5%, 0.01-1%) of the total lipid in a suitable lipid solution.

[0120] Molar lipid ratio According to various embodiments, the cationic lipid, non-cationic lipid, and / or PEG-modified lipid that comprise lipid nanoparticles are selected, and the relative molar ratio of these lipids to each other is selected based on the characteristics of the selected lipid, the nature of the intended target cell, and the characteristics of the mRNA to be delivered.Further considerations include, for example, the saturation degree of alkyl chain, and the size, charge, pH, pKa, fusogenicity, and tolerability of the selected lipid.Therefore, the molar ratio can be adjusted accordingly.

[0121] The various combinations of lipids, namely cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, that can be used to prepare preformed lipid nanoparticles and that can be included therein are described in the literature and in this specification.For example, suitable lipid solution can contain cKK-E12, DEPE, cholesterol, and DMG-PEG2K, C12-200, DEPE, cholesterol, and DMG-PEG2K, HGT5000, DEPE, cholesterol, and DMG-PEG2K, HGT5001, DEPE, cholesterol, and DMG-PEG2K, cKK-E12, DPPC, cholesterol, and DMG-PEG2K, C12-200, DPPC, cholesterol, and DMG-PEG2K, HGT5000, DPPC, chol, and DMG-PEG2K, HGT5001, DPPC, cholesterol, and DMG-PEG2K, or ICE, DEPE, and DMG-PEG2K. Further combinations of lipids are described in the art, e.g., in a publication entitled "Novel ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA." PCT / US17 / 61100, filed November 10, 2017, published as WO 2018 / 089790, entitled “PolyAnionic Delivery of Nucleic Acids,” PCT / US18 / 21292, filed March 7, 2018, published as WO 2018 / 165257, entitled “Poly (Phosphoesters) for Delivery of Nucleic U.S. Provisional Application No. 62 / 676,147, filed May 24, 2018, entitled "Thioester Cationic Lipids," U.S. Provisional Application No. 62 / 677,821, filed May 30, 2018, entitled "Cationic Lipids Comprising a Steroidal Moiety," U.S. Provisional Application No. 62 / 677,809, filed May 30, 2018, entitled "Macrocyclic Lipids," U.S. Provisional Application No. 62 / 677,818, filed May 30, 2018, entitled "Vitamin K Cationic Lipids," U.S. Provisional Application No. 62 / 677,818, filed May 30, 2018, entitled "Vitamin D Cationic Lipids," No. 62 / 677,828, filed May 30, 2018, entitled "Vitamin A Cationic Lipids," U.S. Provisional Application No. 62 / 677,851, filed May 30, 2018, entitled "Vitamin A Cationic Lipids," and U.S. Provisional Application No. 62 / 677,855, filed May 30, 2018, entitled "Vitamin E Cationic Lipids," the disclosures of which are incorporated herein by reference in their entireties.

[0122] In various embodiments, the cationic lipid (e.g., cKK-E12, compound 1, compound 2, or compound 3, C12-200, ICE, and / or HGT4003) constitutes about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the proportion of the cationic lipid (e.g., cKK-E12, compound 1, compound 2, or compound 3, C12-200, ICE, and / or HGT4003) is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% or more of the liposome by molar ratio.

[0123] In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol-based lipid:PEG-modified lipid may be about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol-based lipid:PEG-modified lipid is about 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol-based lipid:PEG-modified lipid is about 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol-based lipid:PEG-modified lipid is about 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol-based lipid:PEG-modified lipid is about 50:25:20:5. In some embodiments, the ratio of sterol lipid:non-cationic lipid:PEG-modified lipid is 50:45:5. In some embodiments, the ratio of sterol lipids:non-cationic lipids:PEG-modified lipids is 50:40:10. In some embodiments, the ratio of sterol lipids:non-cationic lipids:PEG-modified lipids is 55:40:5. In some embodiments, the ratio of sterol lipids:non-cationic lipids:PEG-modified lipids is 55:35:10. In some embodiments, the ratio of sterol lipids:non-cationic lipids:PEG-modified lipids is 60:35:5. In some embodiments, the ratio of sterol lipids:non-cationic lipids:PEG-modified lipids is 60:30:10.

[0124] In some embodiments, liposomes suitable for the present invention comprise ICE and DEPE in a molar ratio of ICE:DEPE of greater than 1:1. The molar ratio is less than 2.5:1. In some embodiments, the molar ratio of ICE:DEPE is between 1:1 and 2.5:1. In some embodiments, the molar ratio of ICE:DEPE is approximately 1.5:1. In some embodiments, the molar ratio of ICE:DEPE is approximately 1.7:1. In some embodiments, the molar ratio of ICE:DEPE is approximately 2:1. In some embodiments, liposomes suitable for the present invention comprise ICE and DMG-PEG-2K in a molar ratio of ICE:DMG-PEG-2K of greater than 10:1. In some embodiments, the molar ratio of ICE:DMG-PEG-2K is less than 16:1. In some embodiments, the molar ratio of ICE:DMG-PEG-2K is approximately 12:1. In some embodiments, the molar ratio of ICE:DMG-PEG-2K is approximately 14:1. In some embodiments, liposomes suitable for the present invention comprise DEPE and DMG-PEG-2K in a molar ratio of DEPE:DMG-PEG-2K greater than 5:1. In some embodiments, the molar ratio of DEPE:DMG-PEG-2K is less than 11:1. In some embodiments, the molar ratio of DEPE:DMG-PEG-2K is approximately 7:1. In some embodiments, the molar ratio of DEPE:DMG-PEG-2K is approximately 10:1.

[0125] In some embodiments, liposomes suitable for the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of 50:45:5 ICE:DEPE:DMG-PEG-2K. In some embodiments, liposomes suitable for the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of 50:40:10 ICE:DEPE:DMG-PEG-2K. In some embodiments, liposomes suitable for the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of 55:40:5 ICE:DEPE:DMG-PEG-2K. In some embodiments, liposomes suitable for the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of 55:35:10 ICE:DEPE:DMG-PEG-2K. In some embodiments, liposomes suitable for the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 60:35:5. In some embodiments, liposomes suitable for the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 60:30:10.

[0126] polymer In some embodiments, suitable delivery vehicles are formulated using polymers as carriers, alone or in combination with other carriers, including various lipids as described herein. Thus, in some embodiments, liposomal delivery vehicles as used herein also encompass nanoparticles containing polymers. Suitable polymers may include, for example, polyacrylates, polyalkoxyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethyleneimine (PEI). When PEI is included, the PEI may be a branched PEI having a molecular weight in the range of 10-40 kDa, for example, a branched PEI of 25 kDa (Sigma#408727).

[0127] Messenger RNA (mRNA) The present invention can be used to encapsulate any mRNA. mRNA is typically thought of as a type of RNA that carries information from DNA to the ribosome. Typically, in eukaryotes, mRNA processing involves adding a "cap" on the 5' end and a "tail" on the 3' end. A typical cap is the 7-methylguanosine cap, which is a guanosine linked via a 5'-5'-triphosphate bond to the first transcribed nucleotide. The presence of a cap is important to provide resistance to nucleases found in most eukaryotic cells. The addition of the tail is typically a polyadenylation event, This adds a polyadenylyl moiety to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from exonuclease degradation. Messenger RNA is translated by ribosomes into a series of amino acids that make up proteins.

[0128] mRNA can be synthesized by any of a variety of known methods. For example, mRNA according to the present invention can be synthesized through in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template that contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0129] In some embodiments, in vitro synthesized mRNA can be purified prior to formulation and encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis.

[0130] The present invention may be used to formulate and encapsulate mRNAs of various lengths. In some embodiments, the present invention may be used to formulate and encapsulate in vitro synthesized mRNAs of lengths 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 some embodiments, the present invention may be used to formulate and encapsulate in vitro synthesized mRNAs of lengths ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0131] The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA that typically contains one or more modifications that enhance stability. In some embodiments, the modifications are selected from modified nucleotides, modified sugar phosphate backbones, and 5' and / or 3' untranslated regions.

[0132] In some embodiments, the modification of the mRNA may include a modification of the nucleotides of the RNA. The modified mRNA according to the present invention may include, for example, a backbone modification, a sugar modification, or a base modification. In some embodiments, the mRNA may contain naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thio O-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleosides, etc. The nucleotides of purines and pyrimidines can be synthesized as modified nucleotide analogs or derivatives, such as 7-deazaguanosine, methylphosphonate, 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. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0133] Typically, mRNA synthesis involves the addition of a "cap" on the 5' end and a "tail" on the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0134] Thus, in some embodiments, the mRNA comprises a 5' cap structure. The 5' cap is typically added as follows: First, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates. Then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, resulting in a 5'5'5 triphosphate bond. The 7-nitrogen of guanine is then methylated by methyltransferase. 2'-O-methylation can also occur at the first and / or second bases after the 7-methylguanosine triphosphate residue. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA, where m refers to a 2'-O methyl residue.

[0135] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect the stability or translation of the mRNA, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0136] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the positional stability of the mRNA in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50-500 nucleotides in length or more.

[0137] While mRNA resulting from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and / or animals.

[0138] The present invention can be used to formulate and encapsulate mRNAs encoding various 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).Exemplary mRNA sequences disclosed herein are listed below:

[0139] Formation of lipid nanoparticles (LNPs) Also provided is a method for preparing lipid nanoparticles encapsulating mRNA, the method comprising: (a) providing a mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, the one or more helper lipids including 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); and (b) forming lipid nanoparticles from the mixture provided in step (a), the method further comprising encapsulating mRNA in the lipid nanoparticles, the encapsulation can be performed before or after the formation of the lipid nanoparticles in step (b). The resulting lipid nanoparticles encapsulating mRNA are stable (e.g., maintain the same encapsulation of mRNA before and after freeze-thawing, or maintain within 10% of the same encapsulation of mRNA before and after freeze-thawing). In one embodiment, the method for preparing lipid nanoparticles according to the present invention specifically excludes the use of one or more helper lipids selected from dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and combinations thereof.

[0140] Various encapsulation processes are described in published US Patent Application No. 2011 / 0244026, published US Patent Application No. 2016 / 0038432, published US Patent Application No. 2018 / 0153822, published US Patent Application No. 2018 / 0125989, and US Provisional Patent Application No. 62 / 877,597, filed July 23, 2019, and may be used to practice the present invention, all of which are incorporated herein by reference. As used herein, Process A refers to the conventional method of encapsulating mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in US 2016 / 0038432. As used herein, Process B or "remixing" refers to a process of encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with the mRNA, as described in US 2018 / 0153822. "Step-down remixing" or "step-up remixing" is an improved process based on the "remixing" process, as described in US Provisional Patent Application No. 63 / 021,319. "Step-down remixing" involves mixing a suspension of preformed empty lipid nanoparticles with batches of a solution of mRNA that are added sequentially. Each addition of a batch of mRNA solution results in an intermediate mixture with a different molar ratio of cationic lipid:mRNA ("N / P") (starting with a high N / P ratio), which is reduced to a lower N / P ratio in the final formulation. In "step-up remixing", starting with an equimolar ratio of cationic lipid:mRNA, a suspension of preformed empty lipid nanoparticles is added in batches to the mRNA solution. For example, four batches of preformed empty lipid nanoparticles are added until a ratio of 4 (cationic lipid):1 (mRNA) is reached.

[0141] In one embodiment, DEPE is present in the mixture at a concentration of 10 mole percent to 50 mole percent. More typically, DEPE is present in the mixture at a concentration of 25 mole percent to 35 mole percent of the total lipid in the mixture. In one embodiment, one or more PEG-modified lipids in the mixture are C6-C 20In one embodiment, the mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids further comprises one or more sterols, such as a cholesterol-based lipid. In one embodiment, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol. In some embodiments, the ratio of cationic lipid:helper lipid:cholesterol-based lipid:PEG-modified lipid can be about 30-60:25-35:20-30:1-15, respectively.

[0142] In some embodiments, the empty preformed lipid nanoparticles are dissolved in ethanol. The lipids are formed by mixing the lipids with an aqueous solution (lipid solution). In some embodiments, the lipids include one or more cationic lipids, one or more non-cationic lipids, and one or more PEG lipids. In some embodiments, the lipids also include one or more cholesterol lipids. In some embodiments, the lipids are present in an ethanol stock solution. Preformed lipid nanoparticles are formed by mixing the lipids. Generally, in some embodiments, the lipid solution containing dissolved lipids and an aqueous or buffer solution are mixed into the solution so that the lipids can form nanoparticles without mRNA (i.e., empty preformed lipid nanoparticles).

[0143] Lipid solution According to the present invention, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulation of mRNA. In some embodiments, the suitable lipid solution is ethanol-based. For example, the suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, the suitable lipid solution is isopropyl alcohol-based. In another embodiment, the suitable lipid solution is dimethyl sulfoxide-based. In another embodiment, the suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[0144] Suitable lipid solutions may contain a mixture of desired lipids at various concentrations.For example, suitable lipid solutions may contain a mixture of desired lipids at a total concentration of about 0.01mg / mL, 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, 0.06mg / mL, 0.07mg / mL, 0.08mg / mL, 0.09mg / mL, about 0.1mg / mL, 0.5mg / mL, 1.0mg / mL, 2.0mg / mL, 3.0mg / mL, 4.0mg / mL, 5.0mg / mL, 6.0mg / mL, 7.0mg / mL, 8.0mg / mL, 9.0mg / mL, 10mg / mL, 15mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL, or 100mg / mL. In some embodiments, a suitable lipid solution may contain a total concentration of the desired lipid in the range of about 0.1-100 mg / mL, 0.5-90 mg / mL, 1.0-80 mg / mL, 1.0-70 mg / mL, 1.0-60 mg / mL, 1.0-50 mg / mL, 1.0-40 mg / mL, 1.0-30 mg / mL, 1.0-20 mg / mL, 1.0-15 mg / mL, 1.0-10 mg / mL, 1.0-9 mg / mL, 1.0-8 mg / mL, 1.0-7 mg / mL, 1.0-6 mg / mL, or 1.0-5 mg / mL. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of up to about 100 mg / mL, 90 mg / mL, 80 mg / mL, 70 mg / mL, 60 mg / mL, 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL, or 10 mg / mL.

[0145] Any desired lipids can be mixed in any ratio suitable for encapsulating mRNA. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids) and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids) and one or more PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including one or more neutral lipids, one or more helper lipids and one or more PEGylated lipids.

[0146] In some embodiments, the empty (i.e., mRNA-free) preformed lipid nanoparticle formulations used in making the nanoparticle formulations of the invention are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% triglyceride. In some embodiments, the addition of mRNA to the empty lipid nanoparticles results in a final formulation that does not require downstream purification or processing and can be stably stored in frozen form.

[0147] Formation of mRNA-LNPs As used herein, the process for the formation of mRNA-loaded lipid nanoparticles (mRNA-LNPs) is used interchangeably with the term "mRNA encapsulation" or grammatical variations thereof. In some embodiments, mRNA-LNPs are formed by mixing an mRNA solution with lipid nanoparticles, and the mRNA solution and / or lipid solution are heated to a predetermined temperature higher than ambient temperature prior to mixing (see U.S. Patent Application Publication No. 14 / 790,562, entitled "Encapsulation of messenger RNA," filed July 2, 2015, and U.S. Provisional Patent Application No. 62 / 020,163, filed July 2, 2014, the disclosures of which are incorporated herein in their entirety).

[0148] Typically, any desired lipids can be mixed in any ratio suitable for forming mRNA-LNP. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids) and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids) and one or more PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more neutral lipids, one or more helper lipids, and one or more PEGylated lipids.

[0149] In some embodiments, the mRNA solution and the preformed lipid nanoparticle solution are mixed in a solution so that the mRNA is encapsulated in the lipid nanoparticle.This solution is also called formulation or encapsulation solution.The process for encapsulating the mRNA by mixing the preformed lipid nanoparticle with the mRNA has already been described in a previous invention published as WO2018 / 089801 and filed as PCT / US17 / 61113 on November 10, 2017, which was filed simultaneously with US patent application Ser. No. 15 / 809,68, both of which are entitled "Improved Process of Preparing mRNA-Loaded Lipid Nanoparticles".The entire contents of that application are incorporated herein by reference.

[0150] Suitable formulations or encapsulating solutions include a solvent such as ethanol. For example, suitable formulations or encapsulating solutions include about 10% ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, or about 40% ethanol. In some embodiments, suitable formulations or encapsulating solutions include a solvent such as isopropyl alcohol. For example, suitable formulations or encapsulating solutions include about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40% isopropyl alcohol.

[0151] In some embodiments, a suitable formulation or encapsulation solution includes a solvent such as dimethyl sulfoxide. For example, a suitable formulation or encapsulation solution includes about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40% dimethyl sulfoxide.

[0152] In some embodiments, suitable formulations or encapsulation solutions may also include buffers or salts. Exemplary buffers may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, the empty preformed lipid nanoparticle formulations used in making this novel nanoparticle formulation may be stably frozen in 10% trehalose solution.

[0153] In some embodiments, ethanol, citrate buffer, and other destabilizing agents are not present during the addition of mRNA, so the formulation does not require any further downstream processing.In some embodiments, the lipid nanoparticle formulation prepared by this novel process comprises pre-formed lipid nanoparticles in a trehalose solution.The lack of destabilizing agents and the stability of the trehalose solution increase the ease of scaling up the formulation and producing lipid nanoparticles encapsulating mRNA.

[0154] mRNA solution mRNA can be provided in a solution to be mixed with lipid solution so that mRNA can be encapsulated in lipid nanoparticles.Suitable mRNA solution can be any aqueous solution that contains the mRNA to be encapsulated at various concentrations less than 1mg / mL.For example, suitable mRNA solution can contain mRNA at or below about 0.01mg / mL, 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, 0.06mg / mL, 0.07mg / mL, 0.08mg / mL, 0.09mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL, or 1.0mg / mL concentration.

[0155] Typically, a suitable mRNA solution may also include a buffer and / or salt. In general, the buffer may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, a suitable concentration of the buffer may be in the range of about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, a suitable concentration of buffering agent is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or more.

[0156] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentrations of salt in the mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. Suitable salt concentrations in the mRNA solution are about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or more.

[0157] 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 in the range 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 It may have a pH of 6.5 or less.

[0158] Various methods may be used to prepare an mRNA solution suitable for the present invention. In some embodiments, the mRNA may be dissolved directly in a buffer solution as described herein. In some embodiments, the mRNA solution may be produced by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, the mRNA solution may be produced by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration of about 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or 1.6 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, or 5.0 mg / mL or more.

[0159] In some embodiments, the mRNA stock solution is mixed with the buffer using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0160] Typically, the buffer solution is mixed at a faster rate than that of the mRNA stock solution. For example, the buffer solution may be mixed at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x faster than that of the mRNA stock solution. In some embodiments, the process according to the invention includes a step of first generating an mRNA solution by mixing a citrate buffer with the mRNA stock solution. In certain embodiments, a suitable citrate buffer comprises about 10 Mm citrate, about 150 Mm NaCl, and a pH of about 4.5. In some embodiments, a suitable mRNA stock solution contains mRNA at a concentration of about 1 mg / mL, about 10 mg / mL, about 50 mg / mL, or about 100 mg / mL or more.

[0161] In some embodiments, the citrate buffer is mixed at a flow rate in the range of about 100-300 mL / min, 300-600 mL / min, 600-1200 mL / min, 1200-2400 mL / min, 2400-3600 mL / min, 3600-4800 mL / min, or 4800-6000 mL / min. In some embodiments, the citrate buffer is mixed at a flow rate of about 220 mL / min, about 600 mL / min, about 1200 mL / min, about 2400 mL / min, about 3600 mL / min, about 4800 mL / min, or about 6000 mL / min.

[0162] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10-30 mL / min, about 30-60 mL / min, about 60-120 mL / min, about 120-240 mL / min, about 240-360 mL / min, about 360-480 mL / min, or about 480-600 mL / min. In some embodiments, the mRNA stock solution is mixed at a flow rate of about 20 mL / min, about 40 mL / min, about 60 mL / min, about 80 mL / min, about 100 mL / min, about 200 mL / min, about 300 mL / min, about 400 mL / min, about 500 mL / min, or about 600 mL / min.

[0163] In some embodiments, the buffer solution is mixed at a flow rate of about 100-6000 mL / min (e.g., about 100-300 mL / min, 300-600 mL / min, 600-1200 mL / min, 1200-2400 mL / min, 2400-3600 mL / min, 3600-4800 mL / min, 4800-6000 mL / min, or 60-420 mL / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 mL / min, 100 mL / min, 140 mL / min, 180 mL / min, 220 mL / min, 260 mL / min, 300 mL / min, 340 mL / min, 380 mL / min, 420 mL / min, 480 mL / min, 540 mL / min, 600 mL / min, 120 The mixture is mixed at flow rates of 0 mL / min, 2400 mL / min, 3600 mL / min, 4800 mL / min, or 6000 mL / min or greater.

[0164] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10-600 mL / min (e.g., about 5-50 mL / min, about 10-30 mL / min, about 30-60 mL / min, about 60-120 mL / min, about 120-240 mL / min, about 240-360 mL / min, about 360-480 mL / min, or about 480-600 mL / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 60 mL / min, 80 mL / min, 100 mL / min, 200 mL / min, 300 mL / min, 400 mL / min, 500 mL / min, or 600 mL / min or greater.

[0165] In some embodiments, the preformed lipid nanoparticles and the mRNA are mixed using a pump system. In some embodiments, the pump system includes a pulseless flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, the suitable pump is a peristaltic pump. In some embodiments, the suitable pump is a centrifugal pump. In some embodiments, the process using the pump system is carried out on a large scale. For example, in some embodiments, the process includes using a pump described herein to mix at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of a solution of mRNA with a solution of preformed lipid nanoparticles to produce mRNA encapsulated in lipid nanoparticles. In some embodiments, the process of mixing mRNA with preformed lipid nanoparticles provides a composition according to the present invention containing at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

[0166] In some embodiments, the solution containing preformed lipid nanoparticles is mixed at a flow rate ranging from about 25-75 mL / min, about 75-200 mL / min, about 200-350 mL / min, about 350-500 mL / min, about 500-650 mL / min, about 650-850 mL / min, or about 850-1000 mL / min. In some embodiments, the solution containing preformed lipid nanoparticles is mixed at a flow rate of about 50 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min, about 800 mL / min, about 850 mL / min, about 900 mL / min, about 950 mL / min, or about 1000 mL / min.

[0167] In some embodiments, the mRNA is mixed in the solution at a flow rate ranging from about 25-75 mL / min, about 75-200 mL / min, about 200-350 mL / min, about 350-500 mL / min, about 500-650 mL / min, about 650-850 mL / min, or about 850-1000 mL / min. In some embodiments, the mRNA is mixed in the solution at a flow rate of about 50 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min, about 800 mL / min, about 850 mL / min, about 900 mL / min, about 950 mL / min, or about 1000 mL / min.

[0168] In some embodiments, the step of combining the lipid nanoparticles encapsulating the mRNA with the preformed lipid particles is performed using a pump system. Such combination may be performed using a pump. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles using a pump system. The nanoparticles are mixed with preformed lipid nanoparticles at flow rates ranging from about 25-75 mL / min mRNA, about 75-200 mL / min, about 200-350 mL / min, about 350-500 mL / min, about 500-650 mL / min, about 650-850 mL / min, or about 850-1000 mL / min. In some embodiments, the mRNA is mixed in the solution at a flow rate of about 50 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min, about 800 mL / min, about 850 mL / min, about 900 mL / min, about 950 mL / min, or about 1000 mL / min.

[0169] In some embodiments, mixing of the lipid nanoparticles and the mRNA is performed without a pump.

[0170] In some embodiments, the process according to the invention comprises heating (or maintaining) one or more solutions to a temperature higher than ambient temperature (i.e., applying heat from a heat source to the solutions), the one or more solutions being a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing mRNA encapsulated by lipid nanoparticles. In some embodiments, the process comprises heating one or both of the mRNA solution and the preformed lipid nanoparticle solution prior to the mixing step. In some embodiments, the process comprises heating one or more of the solution containing preformed lipid nanoparticles, the solution containing mRNA, and the solution containing mRNA encapsulated by lipid nanoparticles during the mixing step. In some embodiments, the process comprises heating the mRNA encapsulated by lipid nanoparticles after the mixing step. In some embodiments, the temperature to which one or more of the solutions are heated (or one or more of the solutions are maintained) is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature to which one or more of the solutions are heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature above ambient to which one or more of the solutions are heated is about 65° C.

[0171] In some embodiments, the process according to the invention comprises maintaining one or more of the solution comprising preformed lipid nanoparticles, the solution comprising mRNA, and the mixed solution comprising mRNA encapsulated by lipid nanoparticles at ambient temperature (i.e., no heat from a heat source is applied to the solution). In some embodiments, the process comprises maintaining one or both of the mRNA solution and the preformed lipid nanoparticle solution at ambient temperature prior to the mixing step. In some embodiments, the process comprises maintaining one or more of the solution comprising preformed lipid nanoparticles, the solution comprising mRNA, and the solution comprising mRNA encapsulated by lipid nanoparticles at ambient temperature during the mixing step. In some embodiments, the process comprises maintaining the mRNA encapsulated by lipid nanoparticles at ambient temperature after the mixing step. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is about 35°C, 30°C, 25°C, 20°C, or 16°C or less. In some embodiments, the ambient temperature at which one or more of the solutions are maintained ranges from about 15-35° C., about 15-30° C., about 15-25° C., about 15-20° C., about 20-35° C., about 25-35° C., about 30-35° C., about 20-30° C., about 25-30° C., or about 20-25° C. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is 20-25° C.

[0172] In some embodiments, the process according to the invention comprises mixing a solution containing preformed lipid nanoparticles and a solution containing mRNA to form lipid nanoparticles encapsulating the mRNA, carried out at ambient temperature.

[0173] In some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size of less than about 150 nm (e.g., less than about 145 nm, less than about 140 nm, less than about 135 nm, less than about 130 nm, less than about 125 nm, less than about 120 nm, less than about 115 nm, less than about 110 nm, less than about 105 nm, less than about 100 nm, less than about 95 nm, less than about 90 nm, less than about 85 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a size of less than 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size in the range of 50-150 nm. In some embodiments, substantially all of the purified nanoparticles have a size in the range of 50-150 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size in the range of 80-150 nm, in some embodiments, substantially all of the purified nanoparticles have a size in the range of 80-150 nm.

[0174] In some embodiments, the process according to the invention results in greater than about 90%, 95%, 96%, 97%, 98%, or 99% encapsulation. In some embodiments, the process according to the invention results in greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

[0175] In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or 1:20. The process of combining lipid nanoparticles is as described above for mixing lipid nanoparticles with mRNA. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 20:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 19:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 15:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 10:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 9:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 8:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 7:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 6:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 5:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 4:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 3:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 3:1. The lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in a ratio of 2:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:1. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:2. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:3. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:4. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:5. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:6. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:7. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:8. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:9. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:10. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:12. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:15. In some embodiments, the lipid nanoparticles encapsulating the mRNA are combined with the preformed lipid particles in step (b) of the process in a ratio of 1:20.

[0176] purification In some embodiments, the empty preformed lipid nanoparticles or mRNA-LNPs are purified and / or concentrated. Various purification methods may be used. In some embodiments, the lipid nanoparticles are purified by a tangential flow filtration (TFF) process. In some embodiments, the lipid nanoparticles are purified by gravity-based dead-end filtration (NFF). In some embodiments, the lipid nanoparticles are purified by any other suitable filtration process. In some embodiments, the lipid nanoparticles are purified by centrifugation. In some embodiments, the lipid nanoparticles are purified by chromatographic methods.

[0177] Pharmaceutical Formulations and Therapeutic Uses A composition comprising the mRNA-LNP may be formulated in a desired buffer, such as, for example, PBS.

[0178] The process according to the invention results in mRNA-LNP compositions of higher potency and efficacy, thereby allowing for lower doses, thereby shifting the therapeutic index in a positive direction. In some embodiments, the process according to the invention results in homogenous mRNA-LNPs with small particle size (e.g., less than 150 nm).

[0179] Thus, the present invention provides compositions comprising the mRNA-LNPs described herein. In some embodiments, the majority of the purified nanoparticles in the composition, i.e., more 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 less than about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all of the mRNA-LNPs are less than about 150 nm (e.g., about 145 nm, about The lipid nanoparticles have a size of about 140nm, about 135nm, about 130nm, about 125nm, about 120nm, about 115nm, about 110nm, about 105nm, about 100nm, about 95nm, about 90nm, about 85nm, or about 80nm. The lipid nanoparticles having a size of less than 100nm are particularly suitable because they can penetrate through the pores of the liver and gain access to liver cells.Similarly, the lipid nanoparticles having a size of about 100nm or less can be easily nebulized and penetrate deep into the lungs when administered to a subject using nebulization.

[0180] Furthermore, more homogeneous nanoparticles with a narrow particle size range are achieved by the process of the present invention, for example, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the nanoparticles in the compositions provided by the present invention have a size in the range of about 75-150 nm (e.g., about 75-145 nm, about 75-140 nm, about 75-135 nm, about 75-130 nm, about 75-125 nm, about 75-120 nm, about 75-115 nm, about 75-110 nm, about 75-105 nm, about 75-100 nm, about 75-95 nm, about 75-90 nm, or 75-85 nm). In some embodiments, substantially all of the purified nanoparticles have a size in the range of about 75 to 150 nm (e.g., about 75 to 145 nm, about 75 to 140 nm, about 75 to 135 nm, about 75 to 130 nm, about 75 to 125 nm, about 75 to 120 nm, about 75 to 115 nm, about 75 to 110 nm, about 75 to 105 nm, about 75 to 100 nm, about 75 to 95 nm, about 75 to 90 nm, or 75 to 85 nm).

[0181] In some embodiments, the nanoparticles in the compositions provided herein have a molecular size dispersity, or molecular size heterogeneity measure (PDI), of less than about 0.23 (e.g., less than about 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, or 0.08). In certain embodiments, the PDI is less than about 0.16.

[0182] In some embodiments, compositions according to the invention include at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, processes according to the invention result in recovery of greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the mRNA.

[0183] In some embodiments, the mRNA in a composition of the invention retains greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% integrity. In some embodiments, the mRNA has 100% integrity.

[0184] In some embodiments, the composition according to the present invention is formulated to administer a specific dose of the composition to a subject. In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of about 5 mg / kg mRNA or less than 5 mg / kg mRNA (i.e., less than 4 mg / kg mRNA, less than 3 mg / kg, less than 2 mg / kg, 1.0 mg / kg, 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 mRNA). In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of less than 4 mg / kg mRNA lipid nanoparticle. In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of less than 3 mg / kg mRNA lipid nanoparticle. In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of less than 2 mg / kg mRNA lipid nanoparticle. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 1 mg / kg of mRNA lipid nanoparticles. The compositions are formulated at a dose concentration of less than 0.6 mg / kg of mRNA lipid nanoparticles. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 0.5 mg / kg of mRNA lipid nanoparticles. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 0.3 mg / kg of mRNA lipid nanoparticles. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 0.2 mg / kg of mRNA lipid nanoparticles. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 0.1 mg / kg of mRNA lipid nanoparticles. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 0.0.08 mg / kg of mRNA lipid nanoparticles. In some embodiments, the compositions of mRNA lipid nanoparticles described herein are formulated at a dose concentration of less than 0.06 mg / kg of mRNA lipid nanoparticles. In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of less than 0.0.05mg / kg of mRNA lipid nanoparticle.In some embodiments, the mRNA lipid nanoparticle composition described herein is formulated at a dose concentration of less than 0.01mg / kg of mRNA lipid nanoparticle.

[0185] In certain embodiments, the amount of mRNA required to produce a therapeutic effect is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or 99%. In certain embodiments, the amount of polynucleotide required to produce a therapeutic effect is reduced by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 20-fold, or 25-fold or more.

[0186] Thus, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in the treatment of a human subject. In some embodiments, the therapeutic composition comprising purified mRNA is used for delivery in the lung or lung cells of a subject. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an endogenous protein that may be deficient or non-functional in a subject. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an endogenous protein that may be deficient or non-functional in a subject.

[0187] In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject.In certain embodiments, the present invention is useful in a method for producing mRNA encoding the cystic fibrosis transmembrane conductance regulator, CFTR.CFTR mRNA is delivered to the lung of a subject in need thereof as a therapeutic composition for treating cystic fibrosis.

[0188] In certain embodiments, the present invention provides a method for producing therapeutic compositions, comprising purified mRNA encoding peptides or polypeptides for delivery to or use in the treatment of liver or liver cells of subjects.Such peptides and polypeptides can include those related to urea cycle disorders, those related to lysosomal storage disorders, those related to glycogen storage disorders, those related to amino acid metabolism disorders, those related to lipid metabolism or fibrotic disorders, those related to methylmalonic acidemia, or those related to any other metabolic disorders for which delivery to or treatment with enriched full-length mRNA to liver or liver cells provides therapeutic benefits.

[0189] In certain embodiments, the present invention provides a method for the detection of urea cycle disorders. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an argininosuccinate synthetase 1 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a carbamoyl phosphate synthetase I protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an argininosuccinate lyase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an arginase protein.

[0190] In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a protein associated with lysosomal storage disorders. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding an alpha-galactosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a glucocerebrosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding an iduronate-2-sulfatase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding an iduronidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding an N-acetyl-alpha-D-glucosaminidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a heparan N-sulfatase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a galactosamine-6 ​​sulfatase protein. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding beta-galactosidase protein. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding lysosomal lipase protein. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding transcription factor EB (TFEB).

[0191] In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with a glycogen storage disorder. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an acid alpha-glucosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glucose-6-phosphatase (G6PC) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a liver glycogen phosphorylase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a muscle phosphoglycerate mutase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glycogen debranching enzyme.

[0192] In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein related to amino acid metabolism. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a phenylalanine hydroxylase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glutaryl-CoA dehydrogenase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a propionyl-CoA carboxylase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an oxalase alanine-glyoxylate aminotransferase enzyme.

[0193] In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein related to lipid metabolism or fibrotic disorders. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an mTOR inhibitor. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an ATPase phospholipid transport 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding one or more NF-kappa B inhibitors, such as one or more of I-kappa B alpha, interferon-related development regulator 1 (IFRD1), and sirtuin 1 (SIRT1). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a PPAR-gamma protein or active variant.

[0194] In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a methylmalonyl-CoA mutase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising a purified mRNA encoding a methylmalonyl-CoA epimerase protein.

[0195] In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA, the delivery of which to the liver or treatment of which can provide therapeutic benefit. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding ATP7B protein, also known as Wilson's disease protein. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding porphobilinogen deaminase enzyme. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding one or a clotting enzyme, such as Factor VIII, Factor IX, Factor VII, and Factor X. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding human hemochromatosis (HFE) protein.

[0196] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in treating a cardiovascular condition or cardiovascular cell in a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising purified mRNA encoding a vascular endothelial growth factor A protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising purified mRNA encoding a relaxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising purified mRNA encoding bone morphogenetic protein-9 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising purified mRNA encoding bone morphogenetic protein-2 receptor protein.

[0197] In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in the treatment of muscle or muscle cells of a subject. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a dystrophin protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a frataxin protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in the treatment of muscle or muscle cells of a subject. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a protein that regulates one or both of potassium and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a protein that regulates Kv7.1 channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a protein that regulates Nav1.5 channels in muscle tissue or muscle cells.

[0198] In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in treating the nervous system or nervous system cells of a subject. For example, in certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding survival motor neuron 2 protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding frataxin protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding CLN3 protein.

[0199] In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNAs encoding peptides or polypeptides for delivery to or use in the treatment of blood or bone marrow or blood or bone marrow cells of a subject. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNAs encoding beta-globin proteins. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNAs encoding Bruton's tyrosine kinase proteins. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNAs encoding one or more clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.

[0200] In certain embodiments, the present invention provides methods for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in the treatment of a subject's kidney or kidney cells. Methods are provided for producing a therapeutic composition comprising purified mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.

[0201] In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or use in treating a subject's eye or ocular cells. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a retinoschisin protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a retinal pigment epithelium specific 65 kDa (RPE65) protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding a 290 kDa centrosomal protein (CEP290).

[0202] In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding a peptide or polypeptide for use in delivering or treating a vaccine for a subject or cells of a subject. For example, in certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an antigen from an infectious agent, such as a virus. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an antigen from an influenza virus. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an antigen from a respiratory syncytial virus. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an antigen from a rabies virus. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an antigen from a rotavirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a human papilloma virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a herpes simplex virus, such as herpes simplex virus type 1 or herpes simplex virus type 2. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a human metapneumovirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a malaria virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from a Zika virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen from Chikungunya.

[0203] In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antigen associated with a cancer of a subject or an antigen identified from a cancer cell of a subject.In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antigen determined from a subject's own cancer cell, i.e., for providing a personalized cancer vaccine.In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antigen expressed from a mutant KRAS gene.

[0204] In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In some embodiments, the two separate mRNA-LNPs in step (b) of the process comprise mRNA encoding the light and heavy chains of an antibody. In some embodiments, the mRNA-LNP composition of the present invention may comprise a combination of non-identical LNPs that comprise different lipid compositions and encapsulate mRNA encoding the light or heavy chains of an antibody. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising purified mRNA encoding an antibody against CD3. In certain embodiments, the invention provides methods for producing a therapeutic composition comprising purified mRNA encoding an antibody against CD19.

[0205] In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding an immunomodulatory agent. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding interleukin 12. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding interleukin 23. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding interleukin 36 gamma. In certain embodiments, the present invention provides methods for generating therapeutic compositions comprising purified mRNA encoding one or more constitutively active variants of the stimulator of interferon genes (STING) protein.

[0206] In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising a purified mRNA encoding an endonuclease. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising a purified mRNA encoding an RNA-guided DNA endonuclease protein, such as a Cas 9 protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising a purified mRNA encoding a meganuclease protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising a purified mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the present invention provides a method for generating a therapeutic composition comprising a purified mRNA encoding a zinc finger nuclease protein.

[0207] In certain embodiments, the present invention provides methods for producing a therapeutic composition comprising purified mRNA encoding a peptide or protein for treating an ocular disease. In some embodiments, the method comprises producing a purified mRNA encoding a retinoschisin. A. EXAMPLES

[0208] Although certain compounds, compositions, and methods of the present invention have been specifically described according to certain embodiments, the following examples are intended to serve merely to illustrate the present invention and are not intended to limit it. Although certain compounds, compositions, and methods of the present invention have been specifically described according to certain embodiments, the following examples are intended to serve merely to illustrate the present invention and are not intended to limit it.

[0209] The mRNA-LNP test articles described in the Examples below contain mRNA encapsulated in different ratios of multi-component lipid mixtures using one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids such as DEPE or DOPE), one or more PEGylated lipids, and optionally one or more sterols, such as cholesterol, designed to encapsulate the mRNA, unless otherwise specified.

[0210] Example 1. Preparation of mRNA In vitro transcription of mRNA Unless otherwise stated, mRNA was synthesized via in vitro transcription (IVT) using either T7 or SP6 polymerase. Briefly, for each gram of transcribed mRNA, a reaction containing an RNA polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 5 mM NTPs, 10 mM DTT, and reaction buffer (10x-250 mM Tris-HCl, pH 7.5, 20 mM spiromidin, 50 mM NaCl) and 20 mg of linearized double-stranded DNA plasmid was prepared in RNase-free water and then incubated at 37C for 60 minutes. The reaction was then quenched by the addition of DNase I and DNase I buffer (10x-100 mM Tris-HCl, 5 mM MgCl2 and 25 mM CaCl2, pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification.

[0211] 5' capping of mRNA Unless otherwise stated, IVT transcribed mRNA was either capped at its 5' end by including a cap structure as part of the IVT reaction or in a subsequent enzymatic step. For capping as part of the IVT reaction, a cap analog can be incorporated as the first "base" of the nascent RNA strand. Cap analogs are Cap0, Cap1, Cap2, m6 A m Alternatively, uncapped and purified in vitro transcribed (IVT) mRNA may be enzymatically modified after IVT, e.g., to modify the 5'N cap using guanylate transferase. 7 -methylguanylate cap structure, and Fechter, P.; Brownlee, GG “Recognition of mRNA cap structures by viral and A cap can be included by addition of a methyl group at the 2'O position of the penultimate nucleotide using a 2'O-methyltransferase resulting in a Cap1 structure, as described in "Enzyme-linked cellular proteins" J. Gen. Virology 2005, 86, 1239-1249.

[0212] 3' tailing of mRNA Unless otherwise stated, IVT transcribed mRNA is tailed at its 3' end by including a tail template in a linearized plasmid, which tails the mRNA either as part of the IVT reaction or in a subsequent enzymatic step. For tailing as part of the IVT reaction, a polyA tail or similar suitable tail is added as part of the IVT process. Poly-T or similar tailing functions can be incorporated into the pDNA template so that the tails are formed on the mRNA as part of the pDNA template. Alternatively, poly-A tails can be enzymatically added to the 3' end of the IVT-produced mRNA after the IVT reaction, for example using poly-A polymerase.

[0213] Example 2. Preparation of lipid nanoparticles (LNPs) and encapsulation of mRNA LNP preparation and encapsulation of mRNA in LNPs containing Compound 3 as a cationic lipid were carried out according to Process B. Process B is further described in US Patent Application Publication No. US2018 / 153822, which is incorporated herein by reference for all purposes. As described above, the LNP preparation was a multi-component lipid mixture containing one or more cationic lipids obtained as described in Example 1, one or more helper lipids (e.g., non-cationic lipids such as DEPE or DOPE), one or more PEGylated lipids, and one or more sterols, such as cholesterol, designed to encapsulate mRNA.

[0214] As used herein, Process A refers to the conventional process in which LNPs are formed from a multi-component mixture of lipids and mRNA is encapsulated therein to form the LNP in a single step.

[0215] Process B refers to a process of encapsulating mRNA by mixing preformed LNP with mRNA. Preformed LNP was first prepared by instantaneously mixing a multi-component lipid mixture dissolved in a solvent such as ethanol with citrate buffer in the absence of mRNA. The mixing of these two flows resulted in the formation of empty lipid nanoparticles, which was a self-assembly process. The resulting formulation resulted in empty lipid nanoparticles in a citrate buffer containing alcohol, which was buffer exchanged (e.g., by tangential flow filtration (TFF)) to result in empty lipid nanoparticles in a 10% weight / volume trehalose solution buffer. The empty lipid nanoparticles and mRNA in the aqueous solution were then mixed to form the mRNA encapsulated within the lipid nanoparticles.

[0216] Specifically, to prepare empty lipid nanoparticles, either DEPE or DOPE was used as a helper lipid along with compound 3 as a cationic lipid, DMG-PEG2K as a PEG-modified lipid, and cholesterol in the ratios listed in Table 2-1. [Table 2-1]

[0217] Surprisingly, it was found that these multi-component lipid mixtures containing compound 3 as the cationic lipid could not be formulated using DOPE as the non-cationic helper lipid, but did form stable liposomes when DEPE was used as the non-cationic helper lipid in place of DOPE.

[0218] Example 3. Enhancement of in vivo production of mRNA in LNPs containing DEPE This example demonstrates an unexpected increase in efficacy of LNP-encapsulated mRNA using LNPs containing DEPE as a helper lipid.

[0219] The mRNA encoding EPO was synthesized as described in Example 1. Using Process B described in Example 2, the mRNA was encapsulated into LNPs that contained different helper lipids but were otherwise identical (see Table 3-1 below). Specifically, the LNPs encapsulated by each mRNA contained different helper lipids but the same cationic lipid (compound 1), the same PEG-modified lipid (DMG-PEG2K), the same sterol compound (cholesterol), the same molar ratio of those lipids, the same mRNA (EPO mRNA), the same N / P ratio=4 (i.e., the molar ratio of cationic lipid:mRNA), the same mRNA concentration (0.2 mg / mL), and were prepared according to the same process (Process B). The characteristics of the resulting mRNA-LNPs are shown in Table 3-1. [Table 3-1]

[0220] Each of the four test articles (1-4 in Table 3-1) containing mRNA encapsulated in LNPs with different helper lipids was administered intravenously to mice (n=5, 6-8 weeks old CD-1 mice) by tail vein injection at a dose of 1 mg / kg mRNA in a dose volume of 5 mL / kg. At 6 hours after administration, mid-day whole blood was collected by tail graft. At 24 hours after administration, all animals were euthanized, followed by thoracotomy and terminal blood collection. Human erythropoietin (hEPO) levels in serum samples were determined by ELISA kit (R&D system catalogue no. DEP00) according to the manufacturer's instructions. In addition, serum ALT and AST levels were measured by ELISA according to standard techniques. EPO protein expression and ALT / AST results are shown in Table 3-2 and graphically displayed in Figures 1 and 2, respectively. [Table 3-2]

[0221] As shown in Table 3-2 and FIG. 1, mRNA LNPs containing DEPE as a helper lipid resulted in significantly higher in vivo protein expression than those from mRNA LNPs containing other helper lipids but otherwise identical. Specifically, at 6 hours after administration, mRNA LNPs containing DEPE resulted in more than 100% or more than 2-fold enhanced in vivo protein expression than those from mRNA LNPs containing helper lipids other than DEPE, such as DOPE, DLOPE, or POPE, at the same time point. Similarly, at 24 hours after administration, mRNA LNPs containing DEPE resulted in more than 100% or more than 2-fold enhanced in vivo protein expression than those from mRNA LNPs containing helper lipids other than DEPE, such as DOPE, DLOPE, or POPE, at the same time point.

[0222] Furthermore, as shown in Table 3-2 and Figure 2, ALT and AST levels at 24 hours post-administration were substantially similar for all mRNA LNPs regardless of the helper lipid, indicating that mRNA LNPs containing DEPE as a helper lipid have similar safety and tolerability as mRNA LNPs containing helper lipids other than DEPE, such as DOPE, DLOPE, or POPE, and at the same time are significantly more potent.

[0223] Example 4. Preparation of lipid nanoparticles (LNPs) using DEPE or DOPE as helper lipids This example shows that using DEPE as a helper lipid in mRNA-encapsulated lipid nanoparticle (mRNA-LNP) formulations can result in up to a more than two-fold increase in protein expression from mRNA in vivo compared to conventional liposomes containing (DOPE) as a helper lipid. It was also observed that mRNA-LNPs containing DEPE as a helper lipid resulted in increased encapsulation efficiency compared to the same mRNA-LNPs containing DOPE as a helper lipid. Notably, this enhanced expression and this enhanced encapsulation efficiency was observed across a wide variety of mRNA-LNPs containing different cationic lipids.

[0224] In these studies, mRNA encoding OTC was encapsulated in LNPs containing DEPE or DOPE as helper lipids and various cationic lipids listed in Table 4. Each cationic lipid listed in Table 4 has a C cation activity as indicated by the last two digits in each lipid description. 10 , C 12 , C 14 , or C 16 The molar ratio of cationic lipid:DMG-PEG2K:cholesterol:helper lipid was approximately 40:3:25:32. For the in vivo portion of the study, each formulated mR 1 mg / kg of NA-LNP was delivered to mice via tail vein injection. At 24 hours, mice were sacrificed and the in vivo expression of mRNA encoding OTC was evaluated from liver homogenates from each mouse. The average protein expression is provided in the table below. [Table 4]

[0225] Table 4 shows that encapsulation efficiency was higher when DEPE was used as the helper lipid compared to DOPE. Furthermore, we surprisingly observed that multi-component lipid mixtures containing certain lipidoids as cationic lipids could not be formulated using DOPE, but formed stable liposomes when DEPE was used as the helper lipid. Table 4 also demonstrates that mRNA LNPs containing DEPE as the helper lipid showed significantly higher in vivo protein expression compared to protein expression from mRNA LNPs containing DOPE helper lipid but that were otherwise identical. This was true when various cationic lipids were used in the LNPs.

[0226] Example 5. Enhancement of in vivo protein expression by using DEPE as a helper lipid compared to other helper lipids This example shows that using DEPE as a helper lipid in mRNA-encapsulated lipid nanoparticle (mRNA-LNP) formulations can increase mRNA expression in vivo compared to lipid nanoparticles using other types of helper lipids across a range of encapsulation processes used to prepare mRNA-LNPs.

[0227] In these studies, mRNA encoding OTC was encapsulated in LNPs (N / P=4) containing one of several different helper lipids including DMG-PEG-2000 as the PEG-modified lipid, cDD-TE-4-E12 as the cationic lipid, cholesterol, and DEPE in the molar lipid ratios shown in Table 5-1, Table 5-2, Table 5-3, and Table 5-4. Each mRNA-LNP formulation was prepared by using one of four different encapsulation processes: a conventional process for the formulations listed in Table 5-1, a remix process for the formulations listed in Table 5-2, a step-up remix process for the formulations listed in Table 5-3, or a step-down remix process for the formulations listed in Table 5-4. The mRNA-LNPs were evaluated for LNP size, polydispersity, and percent encapsulation, with the results for each shown in the table below. For the in vivo portion of the study, 1 mg / kg of each formulated mRNA-LNP was delivered to mice (n=5) via tail vein injection. At 24 hours, the mice were sacrificed and the in vivo expression of mRNA encoding OTC was assessed from liver homogenates from each mouse. The average protein expression is shown in the table below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0228] Table 5-1, Table 5-2, Table 5-3, and Table 5-4 each show the level of protein expression after 24 hours by mRNA encapsulated in LNPs and delivered to groups of mice. As shown, only LNPs prepared with helper lipids DOPE or DEPE provided efficacy in terms of protein expression across different encapsulation processes. Notably, regardless of the encapsulation process used to prepare mRNA-LNPs, in vivo protein expression was highest when LNPs were prepared with helper lipids DEPE.

[0229] As can be appreciated by one of skill in the art, this significantly increased potency, but similar safety and efficacy, of mRNA LNPs containing DEPE as a helper lipid provides significant advantages for the delivery of mRNA as a therapeutic agent.

[0230] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A composition for delivering mRNA to a subject in need thereof, comprising lipid nanoparticles, said lipid nanoparticles comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, encapsulating said mRNA, said one or more helper lipids comprising 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE).

2. 2. The composition of claim 1, wherein DEPE is present at a concentration of 10 to 50 mole percent of the total lipid in the lipid nanoparticle.

3. The composition of claim 1 or 2, wherein more than 70% of the lipid nanoparticles in the composition have a size in the range of 75 nm to 150 nm.

4. The composition of any one of claims 1 to 3, wherein the composition results in enhanced in vivo expression of the mRNA compared to a reference composition comprising the same lipid nanoparticles except that the helper lipid does not contain DEPE.

5. 5. The composition of any one of claims 1 to 4, wherein the one or more helper lipids comprise dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof.

6. 6. The composition of claim 1, wherein the DEPE is present at a concentration of 25 to 35 mole percent of the total lipid in the lipid nanoparticle.

7. The one or more cationic lipids each have 10 -C 16 7. The composition of claim 1, wherein the alkyl group is or comprises 1 to 4 alkyl chains of a chain length of:

8. 10. The method of claim 1, wherein the one or more cationic lipids are lipidoids containing four aliphatic chains.

8. The composition according to any one of claims 1 to 7.

9. The one or more cationic lipids are cationic lipids of the formula: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof; In the formula, R 1 and R 2 are each independently H or C 1 -C 6 each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene; L 1 are each independently an ester, thioester, disulfide, or anhydride group; L 2 Each independently represents C 2 -C 10 aliphatic, X 1 are each independently H or OH; R 3 Each independently represents C 6 -C 20 The composition of any one of claims 1 to 8, which is aliphatic.

10. R 3 Each independently represents C 8 -C 16 The composition of claim 9 which is aliphatic.

11. The one or more PEG-modified lipids are 6 -C 20 11. The composition of claim 1, comprising a poly(ethylene) glycol chain of up to 5 kDa chain length covalently attached to a lipid having an alkyl chain of up to 5 kDa chain length.

12. The composition of any one of claims 1 to 11, wherein the lipid nanoparticles further comprise one or more sterols, optionally a cholesterol-based lipid, and further optionally, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol.

13. The mRNA is (i) a polypeptide, optionally (a) an antibody light chain or an antibody heavy chain, or (b) a polypeptide that is absent or deficient in said subject; (ii) a peptide; or (iii) Antigen The composition according to any one of claims 1 to 12, which encodes:

14. 1. A composition for use in a subject in need of treatment for a disease or disorder, comprising lipid nanoparticles, said lipid nanoparticles comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, encapsulating mRNA, said one or more helper lipids comprising 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE).

15. The composition of claim 14, wherein DEPE is present at a concentration of 10 to 50 mole percent of the total lipid in the lipid nanoparticle.

16. The composition of claim 14 or 15, wherein more than 70% of the lipid nanoparticles in the composition have a size in the range of 75 nm to 150 nm.

17. The composition contains the same lipid nanoparticles except that the helper lipid does not contain DEPE. The composition of any one of claims 14 to 16, which results in enhanced expression of said mRNA compared to a reference composition comprising said mRNA.

18. 18. The composition of any one of claims 14 to 17, wherein the one or more helper lipids comprise dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof.

19. 19. The composition of any one of claims 14 to 18, wherein DEPE is present at a concentration of 25 to 35 mole percent of the total lipid in the lipid nanoparticle.

20. 20. The composition of any one of claims 14 to 19, wherein the one or more cationic lipids are or comprise cKK-E12 or ICE (imidazole cholesterol ester).

21. The one or more cationic lipids are cationic lipids of the formula: 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof; In the formula, R 1 and R 2 are each independently H or C 1 -C 6 each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene; L 1 are each independently an ester, thioester, disulfide, or anhydride group; L 2 Each independently represents C 2 -C 10 aliphatic, X 1 are each independently H or OH; R 3 Each independently represents C 6 -C 20 The composition of any one of claims 14 to 20, which is aliphatic.

22. The one or more cationic lipids are selected from the group consisting of Compound 1 【Chemistry 3】 22. The composition of claim 21, which is or comprises:

23. The one or more PEG-modified lipids are 6 -C 20 or comprising a poly(ethylene) glycol chain of up to 5 kDa chain length covalently attached to a lipid having an alkyl chain of up to 5 kDa chain length. The composition according to any one of claims 14 to 22.

24. 24. The composition of any one of claims 14 to 23, wherein the lipid nanoparticles further comprise one or more sterols, optionally a cholesterol-based lipid, and further optionally, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol.

25. The mRNA is (i) a peptide or polypeptide, optionally said mRNA encoding a therapeutic polypeptide, optionally said therapeutic polypeptide being (a) an antibody light chain or an antibody heavy chain, or (b) a polypeptide that is absent or deficient in said subject; (ii) a peptide; or (iii) Antigen The composition according to any one of claims 14 to 24, which is an mRNA encoding the

26. 1. A method for preparing lipid nanoparticles that encapsulate mRNA, comprising: (a) providing a mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein the one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); (b) forming lipid nanoparticles from the mixture provided in step (a), The method further comprises encapsulating the mRNA in the lipid nanoparticles, the encapsulation being carried out before or after the formation of the lipid nanoparticles in step (b).

27. 27. The method of claim 26, wherein DEPE is present in the mixture provided in step (a) at a concentration of 10 mole percent to 50 mole percent of the total lipid in the lipid nanoparticles.

28. 28. The method of claim 26 or 27, wherein more than 70% of the lipid nanoparticles in the composition have a size in the range of 75 nm to 150 nm.

29. 29. The method of any one of claims 26 to 28, wherein the one or more helper lipids do not comprise any of dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof.

30. 30. The method of any one of claims 26 to 29, wherein DEPE is present at a concentration of 25 to 35 molar percent of the total lipid in the lipid nanoparticle.

31. The one or more PEG-modified lipids are 6 -C 20 31. The method of any one of claims 26 to 30, comprising a poly(ethylene) glycol chain of up to 5 kDa chain length covalently attached to a lipid having an alkyl chain of up to 5 kDa chain length.

32. 32. The method of any one of claims 26 to 31, wherein the mixture further comprises one or more sterols, optionally wherein the one or more sterols comprise a cholesterol-based lipid, and further optionally wherein the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol.

33. The method of any one of claims 26 to 32, wherein the mRNA encodes a therapeutic peptide or polypeptide.

34. 34. The method of any one of claims 26 to 33, wherein the mRNA is encapsulated in preformed lipid nanoparticles.

35. The method of any one of claims 26 to 34, wherein the method further comprises subjecting the lipid nanoparticles to tangential flow filtration (TFF) before and / or after encapsulation of the mRNA.

36. 36. The method of any one of claims 26 to 35, wherein the method further comprises formulating the lipid nanoparticles in a trehalose solution.

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