Improved MRNA-loaded lipid nanoparticles and methods of making same

Abstract

The present invention provides an improved lipid nanoparticle formulation that encapsulates mRNA, comprising DEPE as a helper lipid.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 202080062330.7 (the application date of which is July 8, 2020, and the name of the invention is “Improved MRNA-loaded lipid nanoparticles and preparation methods thereof”). The original application is a Chinese national phase application with international application number PCT / US2020 / 041122, which claims priority to U.S. provisional application serial number 62 / 871,513 filed on July 8, 2019. The disclosure of the provisional application is hereby incorporated by reference. Technical Field

[0003] The present invention relates to lipid-mediated mRNA delivery; and lipid compounds and compositions comprising such compounds. In particular, the present invention relates to methods and uses of such compounds and compositions, and to methods for preparing such compounds and compositions. Background Art

[0004] Messenger RNA therapy (MRT) is becoming an increasingly important method for treating or preventing a variety of diseases. MRT involves administering messenger RNA (mRNA) to a subject in need of the therapy to provide for the production of a protein encoded by the mRNA in the subject. Lipid nanoparticles are used to encapsulate mRNA to achieve efficient in vivo delivery of mRNA.

[0005] Many efforts have been made to identify novel methods and compositions that can use lipid nanoparticles to enhance intracellular delivery and / or expression of mRNA, which can be suitable for scalable and cost-effective manufacturing methods. 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 compositions associated with lipid-mediated mRNA delivery.

[0006] The multi-component lipid nanoparticles that have been found to comprise one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids that encapsulate mRNA are particularly effective for realizing the delivery and expression of mRNA in vivo. The emphasis of recent research is to find the novel cationic lipids that are used for mRNA delivery. Other components of multi-component lipid nanoparticles have hardly received attention. The lipid nanoparticles that continue to need to improve mRNA are delivered to realize intracellular delivery and / or expression of mRNA. Meanwhile, it is expected that new lipid nanoparticle formulations maintain or improve safety and tolerability. Summary of the invention

[0007] The present inventors have surprisingly found that the delivery and / or expression of mRNA in vivo can be significantly improved by optimizing the auxiliary lipid component of the multi-component liposomes encapsulating mRNA. In particular, the present invention is based on the following discovery: relative to conventional liposomes comprising dioleoylphosphoethanolamine (DOPE) as one of the auxiliary lipids, 1,2-dierucyl-sn-glycero-3-phosphoethanolamine (DEPE) as an auxiliary lipid present in lipid nanoparticle formulations of encapsulated mRNA comprising one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids can increase the delivery and / or expression of mRNA in vivo by more than two times. Lipid nanoparticles containing DEPE are comparable lipid nanoparticles containing DOPE in terms of safety and tolerability (as assessed by hepatotoxicity markers such as ALT and AST).

[0008] Therefore, one aspect of the present invention is to provide a lipid nanoparticle for delivering mRNA to a subject in need, wherein the lipid nanoparticle comprises one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids for encapsulating mRNA, wherein the one or more auxiliary lipids include 1,2-dierucyl-sn-glyceryl-3-phosphoethanolamine (DEPE). When administered to a subject, the DEPE in the lipid nanoparticle provides enhanced expression of mRNA.

[0009] In an embodiment of the present invention, DEPE 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) is structurally represented by the following structure:

[0010]

[0011] Or represented by the following structure:

[0012]

[0013] In embodiments of the present invention, compared with the expression of the same mRNA from the second lipid nanoparticle with the same lipid component and amount, the expression of mRNA is enhanced more, except that it comprises different one or more auxiliary lipids and does not comprise DEPE. In certain embodiments, the enhanced expression increases twofold or more relative to the second lipid nanoparticle. In some embodiments, the different one or more auxiliary lipids in the second lipid nanoparticle include dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphoethanolamine (POPE) and / or combinations thereof.

[0014] In certain embodiments, DEPE in lipid nanoparticles is present in a concentration of at least 0.5 mol % of total lipid in lipid nanoparticles, for example, in a concentration of 0.5 mol % to 50 mol %, particularly in a concentration of 10 mol % to 45 mol %. More typically, DEPE in lipid nanoparticles is present in a concentration of 25 mol % to 35 mol % of total lipid in lipid nanoparticles.

[0015] In some embodiments, the one or more cationic lipids is or includes cKK-E12.

[0016] In certain embodiments, the one or more cationic lipids is or includes ICE (imidazole cholesteryl ester).

[0017] In certain embodiments, the one or more cationic lipids are or include a cationic lipid of the formula:

[0018]

[0019] or a pharmaceutically acceptable salt thereof;

[0020] Each R 1 and R 2 is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 are independently an ester, thioester, disulfide bond or anhydride group; each L 2 Independently C2-C 10 Aliphatic; each X 1 are independently H or OH; and each R 3 Independently C6-C 20 In a specific embodiment, the one or more cationic lipids are or include the following compounds:

[0021]

[0022] Or a pharmaceutically acceptable salt thereof. In another specific embodiment, the one or more cationic lipids are or include the following compounds:

[0023]

[0024]

[0025] Or a pharmaceutically acceptable salt thereof. In another specific embodiment, the one or more cationic lipids are or include the following compounds:

[0026]

[0027] Or a pharmaceutically acceptable salt thereof. In certain embodiments, one or more PEG-modified lipids are or comprise a polyethylene glycol chain of up to 5 kDa in length covalently linked to a C6-C 20 Alkyl chains of lipids.

[0028] In some embodiments, the lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid further comprise a cationic lipid comprising a C6-C 20 In some embodiments, the cationic lipid comprises one to four alkyl chains each having C8-C 16 In some embodiments, the cationic lipid comprises one to four alkyl chains each having C 10 -C 16 In some embodiments, the cationic lipid comprises one to four alkyl chains each having C 10 -C 14 In some embodiments, the cationic lipid comprises one to four alkyl chains each having C 10 In some embodiments, the cationic lipid comprises one to four alkyl chains each having C 12 In some embodiments, the cationic lipid comprises one to four alkyl chains each having C 16 Length of alkyl chain.

[0029] In some embodiments, the lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid further comprise a cationic lipid comprising one to four strands each having C6-C 20 In some embodiments, the cationic lipid comprises one to four aliphatic chains each having C8-C 16 In some embodiments, the cationic lipid comprises one to four aliphatic chains each having C 10 -C 14 In some embodiments, the cationic lipid comprises one to four aliphatic chains each having C 10 In some embodiments, the cationic lipid comprises one to four aliphatic chains each having C 12 In some embodiments, the cationic lipid comprises one to four aliphatic chains each having C 16 Length of aliphatic chain.

[0030] In some embodiments, the lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid further comprise one or more cationic lipids, which are or include lipidoids. In some embodiments, the lipidoids comprise four aliphatic chains. In some embodiments, the length of each of the four lipidoid aliphatic chains is independently C6-C 20 In some embodiments, each of the four lipid aliphatic chains is independently C8-C 16 In some embodiments, the length of each of the four lipid aliphatic chains is independently C 10 -C 14 In some embodiments, the length of each of the four lipid aliphatic chains is independently C 10 or C 12 In some embodiments, the length of all four lipidoid aliphatic chains is C6. In some embodiments, the length of all four lipidoid aliphatic chains is C8. In some embodiments, the length of all four lipidoid aliphatic chains is C 10 In some embodiments, the length of all four lipid aliphatic chains is C 12 In some embodiments, the length of all four lipid aliphatic chains is C 14 In some embodiments, the length of all four lipid aliphatic chains is C 16 In some embodiments, the length of all four lipid aliphatic chains is C 18 In some embodiments, the length of all four lipid aliphatic chains is 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 C 10 In some embodiments, at least two of the four lipid aliphatic chains are C 12 In some embodiments, at least two of the four lipid aliphatic chains are C 14 In some embodiments, at least two of the four lipid aliphatic chains are C 16 In some embodiments, at least two of the four lipid aliphatic chains are C 18 In some embodiments, at least two of the four lipid aliphatic chains are C 20 .

[0031] In some embodiments, the lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid further comprise a lipidoid comprising a C6-C 20In some embodiments, the lipid comprises an alkyl chain having a length of C8-C 16 In some embodiments, the lipid comprises an alkyl chain having a length of C 10 -C 14 In some embodiments, the lipid comprises an alkyl chain having a length of C 10 In some embodiments, the lipid comprises an alkyl chain having a length of C 12 In some embodiments, the lipid comprises an alkyl chain having a length of C 16 Length of alkyl chain.

[0032] In certain embodiments, the lipid nanoparticles further comprise one or more sterols. In some embodiments, the one or more sterols are or include cholesterol-based lipids, such as cholesterol or PEGylated cholesterol.

[0033] In some embodiments, 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 for encapsulating mRNA. For example, in a typical embodiment of the present invention, the lipid component of lipid nanoparticles includes four types of lipids, and the four types of lipids include cationic lipids (for example, cKK-E12, compound 1, compound 2 or compound 3), PEG-modified lipids (for example, DMG-PEG2K), non-cationic lipids (DEPE) and cholesterol-based lipids (for example, cholesterol). In some embodiments, the ratio of cationic lipids to non-cationic lipids and cholesterol-based lipids to PEG-modified lipids in lipid nanoparticles can be between about 30-60:25-35:20-30:1-15.

[0034] In some embodiments, the lipid components of lipid nanoparticles include cationic lipids (e.g., ICE), PEG-modified lipids (e.g., DMG-PEG2K), non-cationic lipids (DEPE). In some embodiments, the ratio of cationic lipids to non-cationic lipids to PEG-modified lipids can be between about 50-60:45-30:5-10.

[0035] The mRNA delivered by the lipid nanoparticles of the present invention is an mRNA encoding a protein that is translated into a therapeutic protein in vivo. In certain 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 a polypeptide that is not present or lacking in the subject. In certain embodiments, the mRNA encoding a protein encodes a peptide. In some embodiments, the peptide is an antigen.

[0036] In another aspect, the present invention provides a method for improving the delivery of mRNA to a subject in need thereof, the method comprising administering to a subject a lipid nanoparticle encapsulating mRNA, the lipid nanoparticle comprising one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids, wherein the one or more auxiliary lipids comprise 1,2-dicerucyl-sn-glyceryl-3-phosphoethanolamine (DEPE). When administered to a subject, the DEPE in the lipid nanoparticle provides enhanced expression of the mRNA.

[0037] In some embodiments, mRNA encodes a protein that is translated into a therapeutic protein or peptide in vivo. In some embodiments, mRNA encoding a protein or peptide is delivered systemically. In some embodiments, the translated protein or peptide can be detected in the liver 6 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in the liver 12 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in the liver 18 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in the liver 24 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in the liver 36 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in the liver 48 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in the liver 72 hours or longer after administration. In some embodiments, the translated protein or peptide can be detected in serum 6 hours or longer after administration. In some embodiments, the translated protein or peptide is detectable in serum 12 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum 18 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum 24 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum 36 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in serum 48 hours or more after administration. In some embodiments, the translated protein or peptide is detectable in liver 72 hours or more after administration.

[0038] On the other hand, the present invention provides a lipid nanoparticle encapsulating mRNA, the lipid nanoparticle comprising one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids, wherein the one or more auxiliary lipids comprise 1,2-dierucyl-sn-glyceryl-3-phosphoethanolamine (DEPE), the lipid nanoparticle is used in a method for treating or preventing a disease or condition in a subject, wherein the mRNA encodes a peptide, polypeptide or protein suitable for treating or preventing a disease or condition in the subject. In a related aspect, the present invention relates to the use of lipid nanoparticles encapsulating mRNA in the manufacture of a medicament for treating or preventing a disease or condition in a subject, wherein the lipid nanoparticle comprises one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids, wherein the one or more auxiliary lipids comprise 1,2-dierucyl-sn-glyceryl-3-phosphoethanolamine (DEPE), and wherein the mRNA encodes a peptide, polypeptide or protein suitable for treating or preventing a disease or condition in a subject. In one embodiment, the mRNA encodes a polypeptide or protein that is not present or lacking in the subject, wherein the disease or condition is a lack of the 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 condition that can be treated by administering an antibody comprising the light chain or the heavy chain to the subject. In another embodiment, the mRNA encodes a peptide, a polypeptide or a protein, wherein the peptide, a polypeptide or a protein can induce an immune response in the subject to treat or prevent a disease or condition.

[0039] The inventors also found that the use of DEPE as a helper lipid makes it possible to prepare a multi-component formulation that does not form stable liposomes when DOPE is used as one of the helper lipids. Therefore, in another aspect, the present invention provides a method for preparing lipid nanoparticles for 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, wherein the one or more helper lipids include 1,2-dierucyl-sn-glycero-3-phosphoethanolamine (DEPE), and (b) forming lipid nanoparticles from the mixture provided in step (a), wherein the method also includes encapsulating mRNA into lipid nanoparticles, wherein encapsulation can be performed before or after the lipid nanoparticles are formed in step (b). The resulting lipid nanoparticles encapsulating 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 auxiliary lipids selected from dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphoethanolamine (POPE) and combinations thereof. In one embodiment, DEPE is present in the mixture at a concentration of 10 mol % to 50 mol %. In one embodiment, the one or more PEG-modified lipids in the mixture include a polyethylene glycol chain of up to 5 kDa in length, the polyethylene glycol chain being covalently linked to a C6-C 20 In some embodiments, the mixture of one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids also includes one or more lipids, such as cholesterol-based lipids. In one embodiment, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol. In one embodiment, mRNA encodes therapeutic peptides, polypeptides or proteins. In some embodiments, mRNA is encapsulated in preformed lipid nanoparticles. In some embodiments, the method for preparing lipid nanoparticles according to the present invention also includes subjecting lipid nanoparticles to tangential flow filtration (TFF) before and / or after encapsulating mRNA. In some embodiments, the method also includes preparing lipid nanoparticles in trehalose solution.

[0040] Without wishing to be bound by any particular theory, the inventors believe that DEPE derivatives, particularly DEPE derivatives with different lipid chain lengths or compositions, provide the same advantages as described herein for DEPE. For convenience, the aforementioned overview and detailed description of the present invention are only with reference to DEPE. However, it should be understood that slight changes in the lipid chains of DEPE do not affect its superior properties, and such DEPE derivatives are clearly included in the present invention.

[0041] Other features, objects and advantages of the present invention are apparent from the following detailed description, drawings and claims. However, it should be understood that the detailed description, drawings and claims, although indicating embodiments of the present invention, are given only in an illustrative manner and not in a limiting manner. 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

[0042] Figure 1 Depicted are exemplary graphical representations of EPO protein expression in mRNA LNPs containing DEPE and other helper lipids.

[0043] Figure 2 Depicted are exemplary graphical representations of post-administration levels of serum ALT and AST for mRNA LNPs comprising DEPE and other helper lipids. DETAILED DESCRIPTION

[0044] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions, and methods which are intended to be exemplary and illustrative, not limiting in scope.

[0045] definition

[0046] The present invention provides an improved method for producing mRNA encapsulated in lipid nanoparticle (LNP) formulations to produce mRNA therapeutic compositions.

[0047] In order to make the present invention more easily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this specification.

[0048] 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; in some embodiments, an amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, 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 substitutes. Amino acids, including carboxyl and / or amino terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the circulating half-life of a peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. The amino acid may comprise one or more post-translational modifications, such as binding to 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 apparent from the context in which the term is used.

[0049] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a person at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0050] Approximately or approximately: As used herein, when applied to one or more target values, the term "approximately" or "approximately" refers to a value similar to the reference value. In certain embodiments, the term "approximately" or "approximately" refers to a series of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the value, unless otherwise stated or otherwise apparent from the context (unless the number exceeds 100% of the possible value).

[0051] Combination: As used herein, the term "combination" is used interchangeably with mixing or blending. Combination refers to combining discrete LNP particles with different properties in the same solution, such as combining mRNA-LNP and empty LNP to obtain an mRNA-LNP composition. In some embodiments, the combination of two LNPs is performed at a specific ratio of the components being combined. In some embodiments, the resulting composition obtained from the combination has properties different from any one or both of its components.

[0052] Delivery: As used herein, the term "delivery" encompasses local delivery and systemic delivery. For example, delivery of mRNA encompasses situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into the patient's circulatory system (e.g., serum), and is distributed throughout the body and absorbed by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0053] Efficacy: As used herein, the term "efficacy" or grammatical equivalents refers to the improvement of a biologically relevant endpoint associated with the delivery of mRNA encoding a protein or peptide of interest. In some embodiments, the biological endpoint is protection against ammonium chloride challenge at certain time points after administration.

[0054] Encapsulation: As used herein, the term "encapsulation" or grammatical equivalents refers to the process of confining individual mRNA molecules within nanoparticles.

[0055] Expression: As used herein, "expression" of mRNA refers to the translation of mRNA into a peptide (e.g., an antigen), a polypeptide, or a protein (e.g., an enzyme), and may also include post-translational modifications of the peptide, polypeptide, or fully assembled protein (e.g., an enzyme) as indicated by the context. In this application, the terms "expression" and "production" and grammatical equivalents are used interchangeably.

[0056] Improvement, increase or decrease: As used herein, the terms "improvement," "increase," or "decrease," or grammatical equivalents, refer to values ​​relative to a baseline measurement, such as a measurement of the same individual before the start of a treatment described herein, or a measurement of a control sample or subject (or multiple control samples or subjects) in the absence of a treatment described herein. A "control sample" is a sample subjected to the same conditions as a test sample except for the test article. A "control subject" is a subject having the same form of disease as a treated subject and whose age is about the same as the treated subject.

[0057] Impurity: As used herein, the term "impurity" refers to a substance in a defined amount of a liquid, gas, or solid that has a different chemical composition than the target material or compound. Impurities are also known as contaminants.

[0058] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0059] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms 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 living cells (as opposed to, for example, in vitro systems).

[0060] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some of the components with which it was originally associated when it was produced (whether naturally and / or in an experimental setting), and / or (2) produced, prepared and / or manufactured by the hand of man. An isolated substance and / or entity can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were originally associated. In some embodiments, the purity of the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of the purity percentage of isolated substances and / or entities should not include excipients (e.g., buffers, solvents, water, etc.).

[0061] Local distribution or delivery: As used herein, the term "local distribution", "local delivery" or grammatical equivalents refers to tissue-specific delivery or distribution. Typically, local distribution or delivery requires that the peptide or protein (e.g., enzyme) encoded by the mRNA be translated and expressed intracellularly or with limited secretion, which avoids entering the patient's circulatory system.

[0062] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide encoding 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, produced and optionally purified using a recombinant expression system, chemically synthesized, etc. Where appropriate, for example in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, the display direction of mRNA sequences is 5' to 3'. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted 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 bonds).

[0063] Nucleic acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain by a phosphodiester bond. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA and / or cDNA. In addition, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, i.e., analogs with a backbone other than a phosphodiester backbone.

[0064] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition can be administered, for example, for experimental purposes, diagnostic purposes, preventive purposes, cosmetic purposes, 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.

[0065] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0066] 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. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed of an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or an organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid), or by using other methods used in the art (such as ion exchange) to form salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C 1-4alkyl)4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed when appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and arylsulfonates. Other pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using appropriate electrophiles such as alkyl halides to form quaternized alkylated amino salts.

[0067] Potency: As used herein, the term "potency" or grammatical equivalents refers to the expression level of a protein or peptide encoded by an mRNA and / or the resulting biological effect.

[0068] Salt: As used herein, the term "salt" refers to an ionic compound that actually results or may result from a neutralization reaction between an acid and a base.

[0069] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or method that affects the entire body or entire organism. Typically, systemic distribution or delivery is accomplished via the body's circulatory system (e.g., bloodstream). This is in contrast to the definition of "local distribution or delivery."

[0070] 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). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person who presents to a medical provider for disease diagnosis or treatment. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may have or be susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0071] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a characteristic or property of interest in full or near full extent or degree. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0072] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some embodiments, target tissues include those tissues that exhibit disease-associated pathology, symptoms, or characteristics.

[0073] Therapeutic Index: As used herein, "therapeutic index" is the ratio of the concentration of a drug in the blood at which it becomes toxic to the concentration at which it is effective. The larger the therapeutic index, the safer the drug.

[0074] Treatment: As used herein, the term "treat / treatment / treating" refers to any method used to partially or completely relieve, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a particular disease, condition, and / or condition. In order to reduce the risk of developing pathology associated with a disease, treatment can be administered to subjects who do not show signs of the disease and / or only show early signs of the disease.

[0075] Yield: As used herein, the term "yield" refers to the percentage of mRNA recovered after encapsulation compared to the total mRNA as the starting material. In some embodiments, the term "recovery rate" is used interchangeably with the term "yield".

[0076] Aliphatic: As used herein, the term aliphatic refers to C 1- C 40 Hydrocarbons include saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons may be straight chain, branched chain, or cyclic. For example, C1-C 20 Aliphatic can include C1-C 20 Alkyl (e.g., straight or branched C1-C 20 Saturated alkyl), C2-C 20 Alkenyl (e.g., straight or branched C4-C 20 Diene, straight or branched C6-C 20 triene, etc.) and C2-C 20 Alkynyl (e.g., straight-chain or branched C2-C 20 C1-C 20 Aliphatic can include C3-C 20 Cyclic aliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl or C8-C 20In some embodiments, the aliphatic group may include one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester or amide. The aliphatic group is unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group 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', -OCO2R', -NH2, -NHR', -N(R')2, -SR' or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently an unsubstituted C1-C3 alkyl. In an embodiment, the aliphatic is unsubstituted. In an embodiment, the aliphatic does not include any heteroatoms.

[0077] Alkyl: As used herein, the term "alkyl" refers to acyclic straight-chain and branched hydrocarbon groups, for example, "C1-C 20 "Alkyl" refers to an alkyl group having 1 to 20 carbons. The alkyl group can be straight 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-pentyl, hexyl, isohexyl, and the like. Other alkyl groups will be apparent to those skilled in the art in view of the benefits 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', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted 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 alkyl group is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituent groups 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, wherein the prefix represents an -OH group and "alkyl" is as described herein.

[0078] Alkenyl: As used herein, "alkenyl" means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds which may be present 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 includes prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In an embodiment, the alkenyl contains 1, 2 or 3 carbon-carbon double bonds. In an embodiment, the alkenyl contains a single carbon-carbon double bond. In an embodiment, multiple double bonds (e.g., 2 or 3) are conjugated. The alkenyl group can be unconjugated. The alkenyl group may be substituted or substituted with one or more substituents described herein. For example, the alkenyl group 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', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted 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, alkenyl is unsubstituted. In some embodiments, alkenyl is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In some embodiments, alkenyl groups are substituted with -OH groups and may also be referred to herein as "hydroxyalkenyl" groups, wherein the prefix represents an -OH group and "alkenyl" is as described herein.

[0079] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain in a straight or branched configuration having one or more carbon-carbon triple bonds at any stable point along the chain, for example, "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 group contains one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl 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', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted C1-C3 alkyl. In an embodiment, alkynyl is unsubstituted. In an embodiment, alkynyl is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).

[0080] Aryl: The term "aryl," used alone or as part of a larger moiety as in "aralkyl," refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein the ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic, and wherein each ring in the system contains 4 to 7 ring members. In embodiments, aryl groups have 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, aryl groups have 10 ring carbon atoms ("C 10 In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl" also includes ring systems in which an aromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups, wherein the radical or point of attachment is on the aromatic ring, and in such a case, the number of carbon atoms continues to refer to the number of carbon atoms in the aromatic ring system. Exemplary aryl groups include phenyl, naphthyl, and anthracene.

[0081] Arylene: As used herein, the term "arylene" refers to a divalent aromatic group (ie, having two points of attachment to the molecule). Exemplary arylene groups include phenylene (eg, unsubstituted phenylene or substituted phenylene).

[0082] Compositions of the present invention

[0083] In some embodiments, the invention provides compositions comprising LNP and mRNA, which, when administered to a subject, induce significantly higher levels of mRNA expression in vivo without changing the subject's tolerance or stress level. Tolerance or stress are determined by the rise of liver enzymes aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT). In some embodiments, specific formulations provide manufacturing advantages, such as the ease of manufacturing process, for example, utilizing common preformed LNP stock solutions, etc.

[0084] The observations of the present invention show that when the mRNA-LNP formed by mixing mRNA with preformed empty LNP in step (a) is further combined with the preformed LNP in step (b) to form an mRNA-LNP composition, the effectiveness of the resulting composition is significantly increased compared to the mRNA-LNP of step (a). This is particularly noteworthy because even when the preformed LNP is empty (i.e., does not contain mRNA) and contains the same lipid component as the mRNA-LNP, an increase in effectiveness is observed. In addition, even when the preformed LNP contains only neutral lipids known to be weak promoters of polynucleotide transfection, an increase in the expression of the protein encoded by the mRNA is observed.

[0085] Therefore, the fact that increased efficacy of mRNA-LNP compositions can be achieved without compromising in vivo tolerability is a distinct advantage of the methods of the present invention in terms of therapeutic design.

[0086] This aspect of the invention allows for at least two significant advantages, (i) providing lower amounts of mRNA per dose of an mRNA therapeutic composition, or reducing the frequency of dosing, to achieve the same biological effect, thereby increasing the therapeutic index of the composition; and (ii) developing a simple, flexible, scalable and / or high-throughput manufacturing method in which one or more preformed LNPs can be prepared in batches and used in multiple mixing and combining steps to achieve the desired formulation as described in the present invention.

[0087] The present invention provides a method, wherein the mRNA-LNP prepared by mixing mRNA with preformed empty LNP is further combined with preformed LNP, wherein the obtained mRNA-LNP composition of the present invention causes the in vivo expression of the protein encoded by mRNA to increase. In some aspects, the method is a manufacturing method, and the method comprises the following steps: (a) preformed empty LNP is mixed with mRNA under conditions allowing the formation of mRNA-LNP; (b) the mRNA-LNP formed in step (a) is combined with preformed LNP, thereby manufacturing a composition of lipid nanoparticles comprising encapsulated mRNA. In some embodiments, the lipid nanoparticle comprises at least cationic lipids, non-cationic lipids and PEG-modified lipids. In some embodiments, the lipid nanoparticle can include neutral lipids, with or without cationic lipids.

[0088] In some embodiments, the mRNA encodes a protein or peptide.

[0089] In some embodiments, the preformed LNP in step (b) is an empty LNP. In some embodiments, the preformed LNP in step (b) comprises mRNA. In some embodiments, the preformed LNP in step (b) comprises mRNA encoding a protein or peptide. In some embodiments, the preformed LNP in step (b) comprises the same mRNA encoding the same protein or polypeptide as in the mRNA-LNP formed in step (a). In some embodiments, the preformed LNP in step (b) comprises different mRNAs encoding proteins or polypeptides different from the mRNA-LNP formed in step (a).

[0090] In some embodiments, the empty LNP in step (a) and the preformed LNP in step (b) are different heterogeneous lipid nanoparticles.For example, the empty LNP in step (a) can include cationic lipid HGT-5003, and the preformed LNP in step (b) includes cationic lipid ICE.In another example, the empty LNP in step (a) can include cationic lipid ICE, and the preformed LNP in step (b) includes cationic lipid DOTAP.In another example, the empty LNP in step (a) can include cationic lipid HGT-4001, and the preformed LNP in step (b) includes cationic lipid ckk-E12.The various lipids applicable to LNP and the method for generating the lipid are described in the following corresponding chapters, and any combination of the lipids forming LNP is contained herein.

[0091] In one embodiment, the mRNA-LNP composition generated in step (b) may include the first lipid nanoparticle and the second lipid nanoparticle; wherein the first lipid nanoparticle and the second lipid nanoparticle have the same lipid composition, wherein at least some of the first lipid nanoparticles include mRNA. In one embodiment, the mRNA-LNP composition generated in step (b) may include the first lipid nanoparticle and the second lipid nanoparticle; wherein the first lipid nanoparticle and the second lipid nanoparticle have different lipid compositions. For example, the mRNA-LNP composition may include the first lipid nanoparticle containing cationic lipid ICE and the second lipid nanoparticle containing cationic lipid DOTAP. In some embodiments, the mRNA-LNP composition generated in step (b) may include the first lipid nanoparticle containing cationic lipid C12-200 and the second lipid nanoparticle containing cationic lipid DLinKC2DMA. Therefore, any combination of various lipids suitable for producing LNPs as described in the corresponding sections below is contemplated herein.

[0092] In some embodiments, the empty LNP of step (a) or the preformed LNP of step (b) do not contain cationic lipids. In some embodiments, the empty LNP or the preformed LNP contain neutral lipids and / or PEG-modified lipids.

[0093] Lipid Nanoparticles (LNP)

[0094] The present invention particularly provides a composition for mRNA therapeutic agents, wherein mRNA is encapsulated in a delivery vehicle for effective cellular uptake and processing in vivo. As used herein, the terms "delivery vehicle," "transport vehicle," "nanoparticle" or grammatical equivalents are used interchangeably. The delivery vehicle can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing agents, or formulated in a pharmaceutical composition in which the delivery vehicle is mixed with a suitable excipient. The technology for preparing and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., the latest edition. Specific delivery vehicles are selected according to their ability to promote transfection of nucleic acids to target cells.

[0095] In some embodiments, suitable delivery vehicles are liposome delivery vehicles, such as lipid nanoparticles (LNP) or liposomes. In some embodiments, liposomes include one or more cationic lipids. In some embodiments, liposomes include 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, liposomes include one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids. In some embodiments, liposomes include no more than four different lipid components. In some embodiments, liposomes include no more than three different lipid components. In some embodiments, a different lipid component is a cationic lipid based on sterol. In a typical embodiment, LNP of the present invention is a liposome encapsulating mRNA. The lipid component of suitable liposomes includes 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, the lipid component of suitable liposomes includes cationic lipids (e.g., ICE) based on sterols, non-cationic lipids (DEPE) and PEG-modified lipids (e.g., DMG-PEG2K).

[0096] In some embodiments, DEPE in lipid nanoparticles provides enhanced expression of mRNA encoding proteins or peptides, particularly compared to lipid nanoparticles containing DOPE, the lipid nanoparticles containing DOPE are originally the same in composition (in terms of the molar ratio of lipid components and individual lipid components) as in lipid nanoparticles containing DEPE. In some embodiments, relative to lipid nanoparticles containing DOPE, the expression of mRNA encoding proteins delivered by lipid nanoparticles containing DEPE is enhanced by at least two times. The enhanced expression of mRNA can be determined by administering lipid nanoparticles containing DEPE and lipid nanoparticles with the same formulation of different auxiliary lipids (e.g., DOPE) to test animals (e.g., mice), such as by tail vein injection, and monitoring the expression of mRNA at one or more time points (e.g., 4, 6, 8, 12, 18 or 24 hours after administration).

[0097] In some embodiments, mRNA encodes a protein that is translated into a therapeutic protein in vivo. 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 lacking 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.

[0098] In some embodiments, the lipid nanoparticles containing DEPE of the present invention are safe and tolerable when administered to a subject. For example, the lipid nanoparticles containing DEPE of the present invention do not cause any discernible hepatotoxicity when administered to a subject. Suitable markers for assessing hepatotoxicity are ALT and AST.

[0099] Cationic lipids

[0100] As used herein, the phrase "cationic lipid" refers to any of a variety of lipid species having a net positive charge at a selected pH, such as physiological pH.

[0101] Suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO 2010 / 144740, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids, (6Z, 9Z, 28Z, 31Z)-triacontahedral-6, 9, 28, 31-tetraene-19-yl 4-(dimethylamino) butyrate, and the cationic lipid has the following compound structure:

[0102]

[0103] and pharmaceutically acceptable salts thereof.

[0104] Other suitable cationic lipids for the compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of one of the following formulas:

[0105]

[0106] or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently selected from the group consisting of hydrogen, optionally substituted different saturated or unsaturated C1-C 20 alkyl and optionally substituted different saturated or unsaturated C6-C 20Acyl; wherein L1 and L2 are each independently selected from the group consisting of hydrogen, optionally substituted C1-C 30 Alkyl, optionally substituted different unsaturated C1-C 30 Alkenyl and optionally substituted C1-C 30 Alkynyl; wherein m and o are each independently selected from the group consisting of: zero and any positive integer (e.g., wherein m is three); and wherein n is zero or any positive integer (e.g., wherein n is one). In certain embodiments, the compositions and methods of the present invention include a cationic lipid (15Z, 18Z)-N, N-dimethyl-6-((9Z, 12Z)-octadec-9,12-dien-1-yl) tetracos-15,18-dien-1-amine ("HGT5000") having the following compound structure:

[0107]

[0108] and pharmaceutically 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)-octadec-9,12-dien-1-yl)tetracos-4,15,18-trien-1-amine ("HGT5001") having the following compound structure:

[0109]

[0110] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid and (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadec-9,12-dien-1-yl)tetracos-5,15,18-trien-1-amine ("HGT5002"), wherein the cationic lipid has the following compound structure:

[0111]

[0112] and pharmaceutically acceptable salts thereof.

[0113] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids described as amino alcohol lipids in International Patent Publication WO2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure:

[0114]

[0115] and pharmaceutically acceptable salts thereof.

[0116] In some embodiments, the lipidoids used in the compositions and methods of the present invention include those synthesized by reacting commercially available amines with lipophilic acrylates, acrylamides, or epoxides. In some embodiments, the lipidoids are derived from amine 86 (N,N-bis(2-hydroxyethyl)ethylenediamine) and amine 87 (N-(3-aminopropyl)diethylamine). Lipidoids have several advantages as a potential new class of nucleic acid delivery agents: (i) the chemical methods used to synthesize lipidoids are simple and economical, (ii) structural diversity libraries have been developed, and (iii) the correlation between the structure and function of the delivery system can be constructed by screening large data sets accumulated from lipidoid libraries. The simplicity of these reactions allows the construction of a structurally diverse lipidoid library by varying the type of amine and the length and type (acrylamide / acrylate / epoxide) of the tail (or carbon arm chain).

[0117] In some embodiments, the lipidoid comprises about 2-20 carbon arms. In some embodiments, the lipidoid comprises about 5-18 carbon arms. In some embodiments, the lipidoid comprises about 10-16 carbon arms. In some embodiments, the lipidoid comprises about 10-14 carbon arms. In some embodiments, the lipidoid comprises about 10-12 carbon arms. In some embodiments, the lipidoid comprises about 10 carbon arms. In some embodiments, the lipidoid comprises about 12 carbon arms. In some embodiments, the lipidoid comprises about 14 carbon arms. In some embodiments, the lipidoid comprises about 16 carbon arms.

[0118] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structures:

[0119]

[0120] and pharmaceutically acceptable salts thereof.

[0121] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structures:

[0122]

[0123] and pharmaceutically acceptable salts thereof.

[0124] Other suitable cationic lipids for use in the compositions and methods of the invention include cationic lipids having the general formula 14,25-di-tridecyl-15,18,21,24-tetraaza-octatriacontane and pharmaceutically acceptable salts thereof.

[0125] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publications WO2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the formula:

[0126]

[0127] or a pharmaceutically acceptable salt thereof, wherein R L Each instance of is independently optionally substituted C6-C 40 In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0128]

[0129] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0130]

[0131] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0132]

[0133] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0134]

[0135] and pharmaceutically acceptable salts thereof.

[0136] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0137]

[0138] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R A R is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; and each R B Independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen. In certain embodiments, the compositions and methods of the present invention include cationic lipid "target 23" having the following compound structure:

[0139]

[0140] and pharmaceutically acceptable salts thereof.

[0141] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structures:

[0142]

[0143] Or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0144]

[0145] Or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0146]

[0147] or a pharmaceutically acceptable salt thereof.

[0148] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in U.S. Provisional Patent Application Serial No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the formula:

[0149]

[0150] or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or a C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 are independently an ester, thioester, disulfide bond or anhydride group; each L 2 Independently C2-C 10 Aliphatic; each X 1 are independently H or OH; and each R 3 Independently C6-C 20 In some embodiments, the compositions and methods of the invention include a cationic lipid having the formula:

[0151]

[0152] Or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0153]

[0154] Or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0155]

[0156] or a pharmaceutically acceptable salt thereof.

[0157] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids such as J.McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5: 4277, which are incorporated herein by reference. In certain embodiments, the cationic lipids of the compositions and methods of the present invention include cationic lipids with the following compound structures:

[0158]

[0159] and pharmaceutically acceptable salts thereof.

[0160] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structures:

[0161]

[0162] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0163]

[0164] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0165]

[0166] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0167]

[0168] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0169]

[0170] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0171]

[0172] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0173]

[0174] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0175]

[0176] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0177]

[0178] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0179]

[0180] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0181]

[0182] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0183]

[0184] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0185]

[0186] and pharmaceutically acceptable salts thereof.

[0187] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structures:

[0188]

[0189] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0190]

[0191] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0192]

[0193] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0194]

[0195] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0196]

[0197] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0198]

[0199] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0200]

[0201] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0202]

[0203] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0204]

[0205] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0206]

[0207] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0208]

[0209] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0210]

[0211] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0212]

[0213] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0214]

[0215] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0216]

[0217] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0218]

[0219] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0220]

[0221] and pharmaceutically acceptable salts thereof.

[0222] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the formula:

[0223]

[0224] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a-、-OC(=O)NR a -or-NR a C(=O)O-; and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O- or direct bond; G 1 and G 2 Each is independently unsubstituted C1-C 12 Alkylene or C1-C 12 Alkenylene; G 3 It is C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a Is H or C1-C 12 Alkyl; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 Yes H, OR 5 、CN、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 It is C1-C 12 Alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0225] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure:

[0226]

[0227] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0228]

[0229] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0230]

[0231] and pharmaceutically acceptable salts thereof.

[0232] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the compositions and methods of the present invention include compounds having one of the following formulae:

[0233]

[0234]

[0235] and pharmaceutically acceptable salts thereof. For any of the four general formulae, R4 is independently selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of: -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; and n is 1, 2 or 3. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0236]

[0237] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0238]

[0239] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0240]

[0241] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0242]

[0243] and pharmaceutically acceptable salts thereof.

[0244] Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publications WO2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structures:

[0245]

[0246] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0247]

[0248] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0249]

[0250] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure:

[0251]

[0252] and pharmaceutically acceptable salts thereof.

[0253] Other suitable cationic lipids for use in the compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:

[0254]

[0255] wherein R1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino) and pyridyl; wherein R2 is selected from the group consisting of one of the following two general formulae:

[0256]

[0257] and wherein R3 and R4 are each independently selected from the group consisting of: optionally substituted different saturated or unsaturated C6-C 20 alkyl and optionally substituted different saturated or unsaturated C6-C 20 Acyl; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more). In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4001" having the following compound structure:

[0258]

[0259] In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4002" having the following compound structure:

[0260]

[0261] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4003" having the following compound structure:

[0262]

[0263]

[0264] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4004" having the following compound structure:

[0265]

[0266] In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4005" having the following compound structure:

[0267]

[0268] and pharmaceutically acceptable salts thereof.

[0269] Other suitable cationic lipids for use in the compositions and methods of the present invention include cleavable cationic lipids as described in U.S. Provisional Application No. 62 / 672,194, filed May 16, 2018, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, the cationic lipid being any of the general formulas described in U.S. Provisional Application No. 62 / 672,194 or any of structures (1a)-(21a) and (1b)-(21b) and (22)-(237). In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure according to formula (I'),

[0270]

[0271] in:

[0272] R X Independently -H, -L 1 -R 1 or -L 5A -L 5B -B';

[0273] L 1 , L 2 and L 3 Each of the above is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NR L -;

[0274] Each L 4A and L 5A are independently -C(O)-, -C(O)O- or -C(O)NR L -;

[0275] Each L 4B and L 5B Independently C1-C 20 Alkylene, C2-C 20 Alkenylene or C2-C 20 Alkynylidene;

[0276] Each B and B' is NR 4 R 5 or a 5- to 10-membered nitrogen-containing heteroaryl group;

[0277] Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl;

[0278] Each R4 and R 5 are independently hydrogen, C1-C 10 Alkyl; C2-C 10 Alkenyl; or C2-C 10 alkynyl; and

[0279] Each R L are independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl.

[0280] In certain embodiments, the compositions and methods of the present invention include a cationic lipid, wherein the cationic lipid is compound (139) of 62 / 672,194, which has the following compound structure:

[0281]

[0282] In some embodiments, the compositions and methods of the invention include a cationic lipid, N-[l-(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, which is incorporated herein by reference). Other cationic lipids suitable for use in the compositions and methods of the present invention include, for example, 5-carboxysperminylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2-(spermine-carboxyamide)ethyl]-N,N-dimethyl-l-propylammonium ("DOSPA") (Behr et al., Proc. Nat'l Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); l,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); l,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0283] Additional exemplary cationic lipids suitable for use in the compositions and methods of the present invention include: 1,2-distearoyloxy-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-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearoyl-N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyloxyprop-3- 1-(cis, cis-9,12-octadecadienyloxy)propane ("ClinDMA"); 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethylll-(cis, cis-9',1-2'-octadecadienyloxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane ("DOcarbDAP"); 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethylll-(cis, cis-9',1-2'-octadecadienyloxy)propane ("CpLinDMA ... ,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLincarbDAP"); 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA"); (2 R)-2-((8-[(3β)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-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]propane-1-amine ("octyl-CLinDMA(2S)"); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane ("DLin-K-XTC2-DMA");and 2-(2,2-di((9Z,12Z)-octadec-9,1 2-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethylamine ("DLin-KC2-DMA") (see WO 2010 / 042877, which is 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 WO 2005 / 121348). In some embodiments, the one or more cationic lipids comprise at least one of an imidazole, dialkylamino or guanidinium moiety. ;

[0284] In some embodiments, one or more cationic lipids suitable for use in the compositions and methods of the present invention include 2,2-dilinoleyl 1-4-dimethylaminoethyl 1-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-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-di-undecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0285] In some embodiments, the compositions of the invention comprise one or more cationic lipids, which account for at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition (e.g., lipid nanoparticles) by weight. In some embodiments, the compositions of the invention comprise one or more cationic lipids, which account for at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition (e.g., lipid nanoparticles) by mole%. In some embodiments, the compositions of the invention comprise one or more cationic lipids that account for about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition (e.g., lipid nanoparticle) by weight. In some embodiments, the compositions of the invention comprise one or more cationic lipids that account for about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition (e.g., lipid nanoparticle) by mole %.

[0286] In some embodiments, as a replacement of cationic lipids as described herein or in addition to cationic lipids as described herein, cationic lipids based on sterols can be used. Suitable cationic lipids based on sterols are cationic lipids based on sterols containing dialkylamino, imidazoles and guanidine. For example, some embodiments relate to compositions comprising one or more cationic lipids based on sterols comprising imidazoles, such as imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R) -10,13- dimethyl -17- ((R) -6- methylheptane -2- bases) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopentadien [a] phenanthrene -3- base 3- (1H- imidazoles -4- bases) propionic acid ester, as represented by the following structure (I). In certain embodiments, lipid nanoparticles for delivering RNA (e.g., mRNA) encoding a functional protein may comprise one or more imidazole-based cationic lipids, such as imidazole cholesteryl 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) propionate, as represented by the following structure:

[0287]

[0288] In some embodiments, the percentage of cationic lipids in the liposomes 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 lipids account for about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40% of the liposomes by weight. In some embodiments, the cationic lipids (e.g., ICE lipids) account for about 30%, about 35%, about 40%, about 45%, or about 50% of the liposomes by molar ratio.

[0289] Non-cationic lipids

[0290] As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid, which is also referred to herein as a "helper lipid." As used herein, the phrase "anionic lipid" refers to any of a variety of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0291] The present invention relates to mRNA-LNPs comprising one or more non-cationic helper lipids, wherein the one or more non-cationic helper lipids comprise 1,2-dierucyl-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 comprises DEPE and cholesterol.

[0292] Without wishing to be bound by any particular theory, the lipid chain length or composition of some DEPE derivatives different from DEPE are also included in the present invention. For example, the inventors have found that the alkyl chain or alkyne chain of 10-20 carbon in length is particularly suitable for the formation of mRNA-LNP. In some embodiments, the DEPE derivatives with the alkyl chain or alkyne chain of 16-20 carbon in length are particularly preferred. Or, the DEPE derivatives with the alkyl chain or alkyne chain of 10-14 carbon in length, for example, the DEPE derivatives with 10, 12 or 14 carbon, may be particularly suitable for practice of the present invention.

[0293] Other non-cationic lipids or helper lipids that may be included in the mRNA-LNP include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (3-phosphoglycerol). ethanolamine (POPE), 4-(N-maleimidomethyl)-cyclohexane-l-carboxylic acid dioleoyl-phosphatidylethanolamine (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a mixture thereof.

[0294] In some embodiments, such non-cationic lipids can be used alone, but are preferably used in combination with other lipids (e.g., cationic lipids). In some embodiments, non-cationic lipids can account for 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, non-cationic lipids are neutral lipids, i.e., lipids that do not carry a net charge under the conditions of preparing and / or applying the composition. In some embodiments, the percentage of non-cationic lipids in the liposome can be greater than 5%, greater than 10%, greater than 20%, greater than 30% or greater than 40%.

[0295] Cholesterol-based lipids

[0296] In some embodiments, compositions (such as liposome compositions) include one or more cholesterol-based lipids. For example, cholesterol is suitable for practicing the present invention. Other suitable cholesterol-based lipids include, for example, DC-Chol (N, N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao et al., Biochem.Biophys.Res.Comm.179,280 (1991); Wolf et al., BioTechniques23,139 (1997); U.S. Patent No. 5,744,335) or imidazole cholesterol ester (ICE).

[0297] In some embodiments, lipids based on cholesterol can exist with 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 lipids based on cholesterol in lipid nanoparticles can 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 lipids based on cholesterol in lipid nanoparticles can be no more than about 5 mol %, no more than about 10 mol %, no more than about 20 mol %, no more than about 30 mol % or no more than about 40 mol %.

[0298] In some embodiments, cholesterol-based lipids can exist with a weight ratio (wt%) of about 1% to about 30% or about 5% to about 20% of the total lipids present in the liposome. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles can 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 lipids in lipid nanoparticles can be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt% or no more than about 40 wt%.

[0299] PEGylated lipids

[0300] In some embodiments, suitable lipid solutions include one or more PEGylated lipids, also referred to herein as PEG-modified lipids. Suitable PEG-modified or PEGylated lipids for practicing the present invention are 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000 (DMG-PEG2K). For example, the present invention also contemplates the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids (such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl (methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide)). Contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains having a length of up to 2kDa, up to 3kDa, up to 4kDa, or up to 5kDa, covalently attached to a C6-C 20 In some embodiments, the 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 ) of PEG-ceramide. The addition of such components can prevent aggregation of the complexes and can also provide a means of extending circulation life and increasing delivery of lipid-nucleic acid compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1): 235-237), or they can be selected to be rapidly exchanged from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are those with shorter acyl chains (e.g., C 14 or C 18 PEG-ceramide. The PEG-modified phospholipids and derivatized lipids of the present invention may comprise about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% on a molar basis of the total lipids present in the liposomal transfer vehicle.

[0301] In a suitable lipid solution, the phospholipids and derivatized lipids modified by PEG can account for no more than about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of the total lipids by weight or by mole. In some embodiments, in a suitable lipid solution, the lipids modified by PEG can account for about 5% or less of the total lipids by weight or by mole concentration. In some embodiments, in a suitable lipid solution, the lipids modified by PEG can account for about 4% or less of the total lipids by weight or by mole concentration. In some embodiments, in a suitable lipid solution, the lipids modified by PEG usually account for 3% or less of the total lipids by weight or by mole concentration. In some embodiments, in a suitable lipid solution, the lipids modified by PEG usually account for 2% or less of the total lipids by weight or by mole concentration. In some embodiments, in a suitable lipid solution, the lipids modified by PEG usually account for 1% or less of the total lipids by weight or by mole concentration. In some embodiments, in a suitable lipid solution, the PEG-modified lipid accounts for about 1-5%, about 1-4%, about 1-3%, or about 1-2% of the total lipid by weight or molar concentration. In some embodiments, in a suitable lipid solution, the PEG-modified lipid accounts for about 0.01-3% (e.g., about 0.01-2.5%, 0.01-2%, 0.01-1.5%, 0.01-1%) of the total lipid by weight or molar concentration.

[0302] Molar lipid ratio

[0303] According to various embodiments, the selection of cationic lipids, noncationic lipids and / or PEG-modified lipids comprising lipid nanoparticles, and the relative molar ratios of these lipids to each other are based on the characteristics of the selected lipids, the properties of the expected target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the size, charge, pH, pKa, fusogenicity and tolerance of the saturation of the alkyl chain and the selected lipids. Therefore, the molar ratio can be adjusted accordingly.

[0304] Various lipid combinations, i.e., cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, that can be used to prepare and be included in preformed lipid nanoparticles are described in the literature and herein. For example, a 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, cholesterol, and DMG-PEG2K; HGT5001, DPPC, cholesterol, and DMG-PEG2K; or ICE, DEPE, and DMG-PEG2K. 。Other combinations of lipids are described in the art, for example, PCT / US17 / 61100, filed on November 10, 2017, and published as WO 2018 / 089790; entitled “Novel ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA,” PCT / US18 / 21292, filed on March 7, 2018, and published as WO 2018 / 165257, entitled “PolyAnionic Delivery of Nucleic Acids”; PCT / US18 / 36920, filed on June 11, 2018, and entitled “Poly(Phosphoesters)for Delivery of Nucleic Acids”; U.S. Provisional Application 62 / 676,147, filed on May 24, 2018, and entitled “His Cationic Lipids”; U.S. Provisional Application 62 / 677,821, filed May 30, 2018, entitled “Cationic Lipids Comprising a Steroidal Moiety”; U.S. Provisional Application 62 / 677,809, filed May 30, 2018, entitled “Macrocyclic Lipids”; U.S. Provisional Application 62 / 677,818, filed May 30, 2018, entitled “Vitamin K Cationic Lipids”; U.S. Provisional Application 62 / 677,828, filed May 30, 2018, entitled “Vitamin D Cationic Lipids”; U.S. Provisional Application 62 / 677,851, filed May 30, 2018, entitled “Vitamin A Cationic Lipids”; U.S. Provisional Application 62 / 677,855, filed May 30, 2018, entitled “Vitamin E CationicLipids"; the disclosure of said provisional application is hereby incorporated by reference in its entirety.

[0305] In various embodiments, the cationic lipid (e.g., cKK-E12, Compound 1, Compound 2 or Compound 3, C12-200, ICE and / or HGT4003) accounts for about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45% or about 35-40%) of the liposome by molar ratio. In some embodiments, the percentage of cationic lipid (e.g., cKK-E12, Compound 1, Compound 2 or Compound 3, C12-200, ICE and / or HGT4003) is equal to or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55% or about 60% of the liposome by molar ratio.

[0306] In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids can be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 50:25:20:5. In some embodiments, the ratio of sterol lipids to non-cationic lipids to PEG-modified lipids is 50:45:5. In some embodiments, the ratio of sterol lipids to non-cationic lipids to PEG-modified lipids is 50:40:10. In some embodiments, the ratio of sterol lipids to non-cationic lipids to PEG-modified lipids is 55:40:5. In some embodiments, the ratio of sterol lipids to non-cationic lipids to PEG-modified lipids is 55:35:10. In some embodiments, the ratio of sterol lipids to non-cationic lipids to PEG-modified lipids is 60:35:5. In some embodiments, the ratio of sterol lipids to non-cationic lipids to PEG-modified lipids is 60:30:10.

[0307] In some embodiments, suitable liposomes for use in the present invention comprise ICE and DEPE in an ICE:DEPE molar ratio>1:1. In some embodiments, the ICE:DEPE molar ratio is <2.5:1. In some embodiments, the ICE:DEPE molar ratio is between 1:1 and 2.5:1. In some embodiments, the ICE:DEPE molar ratio is about 1.5:1. In some embodiments, the ICE:DEPE molar ratio is about 1.7:1. In some embodiments, the ICE:DEPE molar ratio is about 2:1. In some embodiments, suitable liposomes for use in the present invention comprise ICE and DMG-PEG-2K in an ICE:DMG-PEG-2K molar ratio>10:1. In some embodiments, the ICE:DMG-PEG-2K molar ratio is <16:1. In some embodiments, the ICE:DMG-PEG-2K molar ratio is about 12:1. In some embodiments, the ICE:DMG-PEG-2K molar ratio is about 14:1. In some embodiments, suitable liposomes for use in the present invention comprise DEPE and DMG-PEG-2K in a DEPE:DMG-PEG-2K molar ratio >5: 1. In some embodiments, the DEPE:DMG-PEG-2K molar ratio is <11: 1. In some embodiments, the DEPE:DMG-PEG-2K molar ratio is about 7: 1. In some embodiments, the DEPE:DMG-PEG-2K molar ratio is about 10: 1.

[0308] In some embodiments, suitable liposomes for use in the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 50:45:5. In some embodiments, suitable liposomes for use in the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 50:40:10. In some embodiments, suitable liposomes for use in the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 55:40:5. In some embodiments, suitable liposomes for use in the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 55:35:10. In some embodiments, suitable liposomes for use in 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, suitable liposomes for use in the present invention comprise ICE, DEPE, and DMG-PEG-2K in a molar ratio of ICE:DEPE:DMG-PEG-2K of 60:30:10.

[0309] polymer

[0310] In some embodiments, polymer is used as carrier, alone or in combination with other carriers including various lipids as described herein, suitable delivery vehicles are prepared.Therefore, in some embodiments, as used herein, liposome delivery vehicles also encompass nanoparticles comprising polymers.Suitable polymers may include, for example, polyacrylates, polyalkyl cyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethyleneimine (PEI).When there is PEI, it may be a branched PEI with a molecular weight range of 10 to 40kDa, for example a branched PEI (Sigma#408727) of 25kDa.

[0311] Messenger RNA (mRNA)

[0312] The present invention can be used to encapsulate any mRNA. mRNA is generally considered to be a type of RNA that carries information from DNA to the ribosome. Typically, in eukaryotic organisms, mRNA processing includes the addition of a "cap" at the 5' end and a "tail" at the 3' end. The typical cap is a 7-methylguanosine cap, which is a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond. The presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells. The addition of the tail is typically a polyadenylation event, whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from exonuclease degradation. The messenger RNA is translated by the ribosome into a series of amino acids that make up proteins.

[0313] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). In short, IVT is typically performed using a linear or circular DNA template comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase and / or RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0314] 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 in the mRNA synthesis process).

[0315] The present invention can be used for allocating and encapsulating mRNA of various lengths. In some embodiments, the present invention can be used for allocating and encapsulating mRNA of in vitro synthesis equal to or greater than about 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, 15kb or 20kb. In some embodiments, the present invention can be used for allocating and encapsulating mRNA of in vitro synthesis in length of about 1-20kb, about 1-15kb, about 1-10kb, about 5-20kb, about 5-15kb, about 5-12kb, about 5-10kb, about 8-20kb or about 8-15kb.

[0316] The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that generally enhance stability. In some embodiments, the modification is selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and / or 3' untranslated regions.

[0317] In some embodiments, the modification of the mRNA may include modification of the nucleotides of the RNA. The modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 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, uracil, dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyl-uracil, N-uracil-5-oxyacetate, 5-methylaminomethyl-uracil, 5-methyl Oxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, Q nucleoside, β-D-mannosyl-Q nucleoside, whitinosine and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. 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. Nos. 5,262,530 and 5,700,642, the entire disclosures of which are incorporated herein by reference.

[0318] Typically, mRNA synthesis includes the addition of a "cap" on the 5' end and a "tail" on the 3' end. The presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0319] Therefore, in some embodiments, the mRNA includes a 5' cap structure. The 5' cap is usually added as follows: first, an RNA terminal phosphatase removes a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylate transferase to produce a 5'5'5 triphosphate bond; and then the 7-nitrogen of guanine is methylated with a methyltransferase. 2'-O-methylation can also occur at the first base and / or the second base after the 7-methylguanosine triphosphate residue. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (wherein m represents a 2'-O-methyl residue).

[0320] In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA, such as an iron response element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides.

[0321] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization stability of the mRNA in the cell, or one or more miRNA binding sites. In some embodiments, the length of the 3' untranslated region can be between 50 and 500 nucleotides or longer.

[0322] While in some embodiments mRNA provided by an in vitro transcription reaction is desirable, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and / or animals.

[0323] The present invention can be used to formulate and encapsulate mRNA encoding various proteins. Non-limiting examples of mRNA suitable for use in the present invention include mRNA encoding spinal motor neuron 1 (SMN), α-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:

[0324] Formation of lipid nanoparticles (LNPs)

[0325] 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, wherein the one or more helper lipids comprise 1,2-dierucyl-sn-glycero-3-phosphoethanolamine (DEPE), and (b) forming lipid nanoparticles from the mixture provided in step (a), wherein the method further comprises encapsulating mRNA into the lipid nanoparticles, wherein the encapsulation can be performed before or after forming the lipid nanoparticles in step (b). The resulting lipid nanoparticles encapsulating mRNA are stable (e.g., maintaining the same mRNA encapsulation before and after freeze-thaw, or maintaining within 10% of the same mRNA encapsulation before and after freeze-thaw). In one embodiment, the method for preparing lipid nanoparticles according to the present invention specifically excludes the use of one or more auxiliary 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.

[0326] Various encapsulation methods are described in disclosed U.S. Application No. US2011 / 0244026, disclosed U.S. Application No. US2016 / 0038432, disclosed U.S. Application No. US2018 / 0153822, disclosed U.S. Application No. US2018 / 0125989 and U.S. Provisional Application No. 62 / 877,597 filed on July 23, 2019, and can be used to practice the present invention, all of which are incorporated herein by reference. As used herein, method A refers to a conventional method for encapsulating mRNA by mixing mRNA with a lipid mixture without first preforming lipids into lipid nanoparticles, as described in US 2016 / 0038432. As used herein, method B or "remixing" refers to a method for encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with mRNA, as described in US2018 / 0153822. "Step-down remixing" or "step-up remixing" is an improved method based on the "remixing" method, as described in U.S. Provisional Application 63 / 021,319. "Step-down remixing" involves mixing a suspension of preformed empty lipid nanoparticles with batches of mRNA solutions added sequentially. Each addition of an mRNA solution batch produces an intermediate mixture with a different cationic lipid to mRNA molar ratio ("N / P"), starting with a high N / P ratio and decreasing to a lower N / P ratio in the final formulation. In "step-up remixing", starting with an equimolar ratio of cationic lipid to mRNA, a suspension of preformed empty lipid nanoparticles is added to the mRNA solution in batches. For example, four batches of preformed empty lipid nanoparticles are added until a ratio of 4 (cationic lipid) to 1 (mRNA) is reached.

[0327] In one embodiment, DEPE is present in the mixture at a concentration of 10 mol % to 50 mol %. More typically, DEPE in the mixture is present at a concentration of 25 mol % to 35 mol % of the total lipid in the mixture. In one embodiment, the one or more PEG-modified lipids in the mixture include a polyethylene glycol chain of up to 5 kDa in length, which is covalently attached to a C6-C 20 In some embodiments, the mixture of one or more cationic lipids, one or more PEG-modified lipids and one or more auxiliary lipids also includes one or more lipids, such as cholesterol-based lipids. In one embodiment, cholesterol-based lipids are cholesterol and / or PEGylated cholesterol. In some embodiments, the ratio of cationic lipids to auxiliary lipids to cholesterol-based lipids and PEG-modified lipids can be between about 30-60:25-35:20-30:1-15 respectively.

[0328] In some embodiments, empty preformed lipid nanoparticles are formed by mixing lipid dissolved in ethanol with an aqueous solution (lipid solution). In some embodiments, lipid includes one or more cationic lipids, one or more non-cationic lipids and one or more PEG lipids. In some embodiments, lipid also contains one or more cholesterol lipids. In some embodiments, lipid is present in an ethanol stock solution. Preformed lipid nanoparticles are formed by mixing those lipids. Generally, in some embodiments, lipid solutions and aqueous solutions or buffer solutions containing dissolved lipids are mixed into the solution so that lipids can form nanoparticles (i.e. empty preformed lipid nanoparticles) without mRNA.

[0329] Lipid solution

[0330] According to the present invention, lipid solution contains the lipid mixture that is suitable for forming lipid nanoparticles for encapsulating mRNA.In some embodiments, suitable lipid solution is based on ethanol.For example, suitable lipid solution can contain the mixture of the desired lipid dissolved in pure ethanol (that is, 100% ethanol).In another embodiment, suitable lipid solution is based on isopropanol.In another embodiment, suitable lipid solution is based on dimethyl sulfoxide.In another embodiment, suitable lipid solution is the mixture of suitable solvent (including but not limited to ethanol, isopropanol and dimethyl sulfoxide).

[0331] Suitable lipid solutions can contain a mixture of the required lipid of various concentrations. For example, suitable lipid solutions can contain a mixture of the required lipid of about 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml or 100 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from 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.

[0332] Any desired lipid can be mixed with any ratio applicable to encapsulation of mRNA.In some embodiments, suitable lipid solution contains the mixture of desired lipid, and the desired lipid includes cationic lipid, auxiliary lipid (for example, non-cationic lipid and / or cholesterol lipid) and / or PEGylated lipid.In some embodiments, suitable lipid solution contains the mixture of desired lipid, and the desired lipid includes one or more cationic lipid, one or more auxiliary lipid (for example, non-cationic lipid and / or cholesterol lipid) and one or more PEGylated lipid.In some embodiments, suitable lipid solution contains the mixture of desired lipid, and the desired lipid includes one or more neutral lipid, one or more auxiliary lipid and one or more PEGylated lipid.

[0333] In some embodiments, empty (i.e., mRNA-free) preformed lipid nanoparticle formulations used to prepare nanoparticle formulations of the invention can be stably frozen in about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose solution. In some embodiments, adding mRNA to empty lipid nanoparticles can produce a final formulation that does not require any downstream purification or processing and can be stably stored in frozen form.

[0334] Formation of mRNA-LNPs

[0335] As used herein, the method for forming the lipid nanoparticle (mRNA-LNP) of mRNA load is used interchangeably with the term "mRNA encapsulation" or its grammatical variants. In some embodiments, mRNA-LNP is formed by mixing an mRNA solution with a lipid solution, wherein before mixing, the mRNA solution and / or the lipid solution are heated to a predetermined temperature higher than ambient temperature (see U.S. Patent Application Serial No. 14 / 790,562, entitled "Encapsulation of messenger RNA", filed on July 2, 2015, and Provisional U.S. Patent Application Serial No. 62 / 020,163, filed on July 2, 2014, the disclosures of which are hereby incorporated by reference in their entirety).

[0336] Generally, any required lipid can be mixed with any ratio suitable for forming mRNA-LNP.In some embodiments, suitable lipid solution contains the mixture of required lipid, and the required lipid includes cationic lipid, auxiliary lipid (for example, non-cationic lipid and / or cholesterol lipid) and / or PEGylated lipid.In some embodiments, suitable lipid solution contains the mixture of required lipid, and the required lipid includes one or more cationic lipid, one or more auxiliary lipid (for example, non-cationic lipid and / or cholesterol lipid) and one or more PEGylated lipid.In some embodiments, suitable lipid solution contains the mixture of required lipid, and the required lipid includes one or more neutral lipid, one or more auxiliary lipid and one or more PEGylated lipid.

[0337] In some embodiments, the mRNA solution and the preformed lipid nanoparticle solution are mixed into the solution so that the mRNA becomes encapsulated in the lipid nanoparticles. Such a solution is also referred to as a formulation or encapsulation solution. The method for encapsulating mRNA by mixing preformed lipid nanoparticles with mRNA has been previously described in an earlier invention filed as PCT / US17 / 61113 on November 10, 2017, published as WO2018 / 089801; and in U.S. patent application serial number 15 / 809,68 filed simultaneously, both of which are titled "Improved Process of Preparing mRNA-Loaded Lipid Nanoparticles". The entire contents of this application are incorporated herein by reference.

[0338] Suitable formulations or encapsulation solutions include solvents such as ethanol. For example, suitable formulations or encapsulation solutions include about 10% ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, or about 40% ethanol. In some embodiments, suitable formulations or encapsulation solutions include solvents such as isopropanol. For example, suitable formulations or encapsulation solutions include about 10% isopropanol, about 15% isopropanol, about 20% isopropanol, about 25% isopropanol, about 30% isopropanol, about 35% isopropanol, or about 40% isopropanol.

[0339] In some embodiments, suitable formulations or encapsulation solutions include solvents such as dimethyl sulfoxide. For example, suitable formulations or encapsulation solutions include 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.

[0340] In some embodiments, suitable formulations or encapsulation solutions may also contain buffers or salts. Exemplary buffers may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, empty preformed lipid nanoparticle formulations for preparing this novel nanoparticle formulation may be stably frozen in 10% trehalose solution.

[0341] In some embodiments, ethanol, citrate buffer and other destabilizing agents are not present during the addition of mRNA, and thus the formulation does not require any further downstream processing. In some embodiments, the lipid nanoparticle formulation prepared by this novel method comprises preformed 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.

[0342] mRNA solution

[0343] mRNA can be provided in solution to mix with lipid solution so that mRNA can be encapsulated in lipid nanoparticle. Suitable mRNA solution can be any aqueous solution containing mRNA to be encapsulated with various concentrations lower than 1mg / ml. For example, suitable mRNA solution can contain a concentration of or less than 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 mRNA.

[0344] Typically, suitable mRNA solutions can also contain buffer and / or salt. Generally speaking, buffer can include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. In some embodiments, the suitable concentration of buffer can be in the range of about 0.1mM to 100mM, 0.5mM to 90mM, 1.0mM to 80mM, 2mM to 70mM, 3mM to 60mM, 4mM to 50mM, 5mM to 40mM, 6mM to 30mM, 7mM to 20mM, 8mM to 15mM or 9 to 12mM. In some embodiments, a suitable concentration of the buffer is at or greater than 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.

[0345] Exemplary salt can include sodium chloride, magnesium chloride and potassium chloride. In some embodiments, the suitable concentration of salt in the mRNA solution can be in the range of about 1mM to 500mM, 5mM to 400mM, 10mM to 350mM, 15mM to 300mM, 20mM to 250mM, 30mM to 200mM, 40mM to 190mM, 50mM to 180mM, 50mM to 170mM, 50mM to 160mM, 50mM to 150mM or 50mM to 100mM. The salt concentration in the suitable mRNA solution is or is greater than about 1mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM.

[0346] In some embodiments, suitable mRNA solutions can have a pH in the range of about 3.5-6.5, 3.5-6.0, 3.5-5.5., 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. In some embodiments, suitable mRNA solutions can have a pH equal to or not greater than about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5.

[0347] Various methods can be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA can be directly dissolved in a buffer solution as described herein. In some embodiments, mRNA stock solution can be produced by mixing an mRNA stock solution with a buffer solution before being mixed with a lipid solution for encapsulation. In some embodiments, mRNA stock solution can be produced by mixing an mRNA stock solution with a buffer solution immediately before being mixed with a lipid solution for encapsulation. In some embodiments, suitable mRNA stock solution can contain a concentration of or greater than about 0.2mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.8mg / ml, 1.0mg / ml, 1.2mg / ml, 1.4mg / ml, 1.5mg / ml, or 1.6mg / ml, 2.0mg / ml, 2.5mg / ml, 3.0mg / ml, 3.5mg / ml, 4.0mg / ml, 4.5mg / ml or 5.0mg / ml mRNA in water.

[0348] In some embodiments, a pump is used to mix the mRNA stock solution with the buffer solution. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0349] Typically, the buffer solution is mixed at a flow rate greater than the flow rate of the mRNA stock solution. For example, the buffer solution can be mixed at a flow rate greater than at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times greater than the flow rate of the mRNA stock solution. In some embodiments, the method according to the present invention comprises the following steps: first, an mRNA solution is produced by mixing a citrate buffer with an mRNA stock solution. In certain embodiments, a suitable citrate buffer contains about 10mM citrate, about 150mM NaCl, and a pH of about 4.5. In some embodiments, a suitable mRNA stock solution contains an mRNA concentration of or greater than about 1mg / ml, about 10mg / ml, about 50mg / ml or about 100mg / ml.

[0350] In some embodiments, the citrate buffer is mixed at a flow rate ranging from 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.

[0351] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min. In some embodiments, the mRNA stock solution is mixed at a flow rate of about 20 ml / min, about 40 ml / min, about 60 ml / min, about 80 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml / min, about 400 ml / min, about 500 ml / min, or about 600 ml / min.

[0352] In some embodiments, the buffer solution is mixed at a flow rate ranging between about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min.

[0353] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml / min (e.g., about 5-50 ml / min, about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min.

[0354] In some embodiments, a pump system is used to mix preformed lipid nanoparticles and mRNA. In some embodiments, the pump system comprises a pulseless flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the method using a pump system is carried out on a large scale. For example, in some embodiments, the method includes using a pump as described herein to mix a solution of at least about 1mg, 5mg, 10mg, 50mg, 100mg, 500mg or 1000mg 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, the composition containing at least about 1mg, 5mg, 10mg, 50mg, 100mg, 500mg or 1000mg of the encapsulated mRNA.

[0355] In some embodiments, the solution containing preformed lipid nanoparticles is mixed at a flow rate in the range of 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 pre-formed 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.

[0356] In some embodiments, the mRNA is mixed in the solution at a flow rate in the range of 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.

[0357] In some embodiments, a pump system is used to carry out the step of combining the lipid nanoparticles of the mRNA with preformed lipid particles. Such combination can be carried out using a pump. In some embodiments, the lipid nanoparticles of the mRNA are mixed with preformed lipid nanoparticles with a flow velocity within the range of about 25-75ml / minute, about 75-200ml / minute, about 200-350ml / minute, about 350-500ml / minute, about 500-650ml / minute, about 650-850ml / minute or about 850-1000ml / minute. 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.

[0358] In some embodiments, the mixing of lipid nanoparticles and mRNA is performed in the absence of any pump.

[0359] In some embodiments, the method according to the present invention includes a step of heating one or more solutions (i.e., applying heat from a heat source to the solution) to a temperature above ambient temperature (or maintaining at a temperature above ambient temperature), wherein the one or more solutions are a solution comprising preformed lipid nanoparticles, a solution comprising mRNA, and a mixed solution comprising mRNA encapsulated by lipid nanoparticles. In some embodiments, the method includes a step of heating one or both of the mRNA solution and the preformed lipid nanoparticle solution before the mixing step. In some embodiments, the method includes heating one or more of the following during the mixing step: a solution comprising preformed lipid nanoparticles, a solution comprising mRNA, and a solution comprising mRNA encapsulated by lipid nanoparticles. In some embodiments, the method includes a step of heating the mRNA encapsulated by lipid nanoparticles after the mixing step. In some embodiments, the temperature to which the one or more solutions are heated (or the temperature at which the one or more solutions are maintained) is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature to which the one or more solutions are heated is in the range of about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature to which the one or more solutions are heated above ambient temperature is about 65° C.

[0360] In some embodiments, the method according to the present invention includes maintaining one or more of a solution comprising preformed lipid nanoparticles, a solution comprising mRNA, and a mixed solution comprising mRNA encapsulated by lipid nanoparticles at ambient temperature (i.e., without applying heat from a heat source to the solution). In some embodiments, the method includes a step of maintaining one or both of the mRNA solution and the preformed lipid nanoparticle solution at ambient temperature before the mixing step. In some embodiments, the method includes maintaining one or more of the following at ambient temperature during the mixing step: a solution comprising preformed lipid nanoparticles, a solution comprising mRNA, and a solution comprising mRNA encapsulated by lipid nanoparticles. In some embodiments, the method includes a step of maintaining the mRNA encapsulated by lipid nanoparticles at ambient temperature after the mixing step. In some embodiments, the ambient temperature maintained by one or more solutions is or is less than about 35°C, 30°C, 25°C, 20°C, or 16°C. In some embodiments, the ambient temperature maintained by one or more solutions is in the range of about 15-35° C., about 15-30° C., about 15-25° C., about 15-20° C., about 20-35° C., about 25-35° C., about 30-35° C., about 20-30° C., about 25-30° C., or about 20-25° C. In some embodiments, the ambient temperature maintained by one or more solutions is 20-25° C.

[0361] In some embodiments, the method according to the present invention comprises the following steps, performed at ambient temperature: mixing a solution comprising preformed lipid nanoparticles and a solution comprising mRNA to form lipid nanoparticles encapsulating mRNA.

[0362] In some embodiments, greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size less than about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a size less than 150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size within the range of 50-150 nm. In some embodiments, substantially all of the purified nanoparticles have a size within 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 within the range of 80-150 nm. In some embodiments, substantially all of the purified nanoparticles have a size within the range of 80-150 nm.

[0363] In some embodiments, the methods according to the invention result in an encapsulation efficiency greater than about 90%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the methods according to the invention result in an mRNA recovery greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0364] In some embodiments, the lipid nanoparticles of the mRNA encapsulation are combined with the preformed lipid particles in step (b) of the method at 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 method of combining lipid nanoparticles is used as described above for mixing lipid nanoparticles with mRNA. In some embodiments, the lipid nanoparticles of the mRNA encapsulation are combined with the preformed lipid particles in step (b) of the method at a ratio of 20: 1. In some embodiments, the lipid nanoparticles of the mRNA encapsulation are combined with the preformed lipid particles in step (b) of the method at a ratio of 19: 1. In some embodiments, the lipid nanoparticles of the mRNA encapsulation are combined with the preformed lipid particles in step (b) of the method at a ratio of 15: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 10: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 9: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 8: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 7: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 6: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 5: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 4: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 3: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 2: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1: 1. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1: 2. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1: 3.In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:4. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:5. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:6. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:7. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:8. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:9. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1:10. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1: 12. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1: 15. In some embodiments, the lipid nanoparticles encapsulating mRNA are combined with the preformed lipid particles in step (b) of the method at a ratio of 1: 20.

[0365] purification

[0366] In some embodiments, empty preformed lipid nanoparticles or mRNA-LNPs are purified and / or concentrated. Various purification methods can be used. In some embodiments, lipid nanoparticles are purified by tangential flow filtration (TFF) method. In some embodiments, lipid nanoparticles are purified by gravity-based forward flow filtration (NFF). In some embodiments, lipid nanoparticles are purified by any other suitable filtration method. In some embodiments, lipid nanoparticles are purified by centrifugation. In some embodiments, lipid nanoparticles are purified by chromatography.

[0367] Pharmaceutical formulations and therapeutic uses

[0368] Compositions comprising mRNA-LNPs can be formulated in a desired buffer, such as PBS.

[0369] The method according to the present invention produces mRNA-LNP compositions with higher efficacy and effectiveness, thereby allowing lower dosages, thereby moving the therapeutic index in a positive direction. In some embodiments, the method according to the present invention produces homogeneous mRNA-LNPs with small particle sizes (e.g., less than 150 nm).

[0370] Thus, the invention provides compositions comprising mRNA-LNPs as described herein. In some embodiments, the majority of the purified nanoparticles in the composition (i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the purified nanoparticles) have a size of 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 have a size 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). Lipid nanoparticles with a size less than 100 nm are particularly suitable because they can penetrate the liver to perforate and enter hepatocytes. Similarly, lipid nanoparticles with a size of about 100 nm or less are easily aerosolized and can penetrate deeply into the lungs when administered to a subject using aerosolization.

[0371] In addition, more uniform nanoparticles with narrow particle size range are obtained by method of the present invention. For example, in compositions provided by the invention, nanoparticles greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% have a size in the range of about 75-150nm (for example, about 75-145nm, about 75-140nm, about 75-135nm, about 75-130nm, about 75-125nm, about 75-120nm, about 75-115nm, about 75-110nm, about 75-105nm, about 75-100nm, about 75-95nm, about 75-90nm or 75-85nm). In some embodiments, substantially all of the purified nanoparticles have a size in the range of about 75-150 nm (e.g., about 75-145 nm, about 75-140 nm, about 75-135 nm, about 75-130 nm, about 75-125 nm, about 75-120 nm, about 75-115 nm, about 75-110 nm, about 75-105 nm, about 75-100 nm, about 75-95 nm, about 75-90 nm, or 75-85 nm).

[0372] In some embodiments, the nanoparticles in the compositions provided herein have a 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.

[0373] In some embodiments, the compositions according to the invention contain at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, the methods according to the invention result in an mRNA recovery greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0374] In some embodiments, the mRNA in the compositions of the invention maintains greater than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% integrity. In some embodiments, the mRNA has 100% integrity.

[0375] In some embodiments, the composition according to the present invention is formulated so that the combination of a specific dose is applied to a subject. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of about 5mg / kg mRNA or less than 5mg / kg mRNA (i.e., less than 4mg / kg mRNA, less than 3mg / kg, less than 2mg / kg, 1.0mg / kg, 0.6mg / kg, 0.5mg / kg, 0.3mg / kg, 0.016mg / kg, 0.05mg / kg and 0.016mg / kg). In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration less than 4mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration less than 3mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration less than 2mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 1 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 0.6 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 0.5 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 0.3 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 0.2 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 0.1 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is prepared at a dosage concentration of less than 0.0.08 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of less than 0.06 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of less than 0.0.05 mg / kg mRNA lipid nanoparticles. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of less than 0.01 mg / kg mRNA lipid nanoparticles.

[0376] In certain embodiments, the amount of mRNA required for achieving 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 for achieving a therapeutic effect is reduced by at least two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, twelve times, fifteen times, twenty times or twenty-five times or more.

[0377] Therefore, in certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a purified mRNA encoding a peptide or polypeptide, the therapeutic composition being used for delivery to a human subject or treating a human subject. In some embodiments, the therapeutic composition comprising purified mRNA is used for delivery to a subject's lungs or lung cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition, the therapeutic composition comprising purified mRNA encoding an endogenous protein that may be defective or non-functional in a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition, the therapeutic composition comprising purified mRNA encoding an endogenous protein that may be defective or non-functional in a subject.

[0378] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or treatment of a subject's lung or lung cells. In certain embodiments, the present invention can be used to prepare a method for mRNA encoding the cystic fibrosis transmembrane conductance regulator CFTR. CFTR mRNA is delivered to the lungs of a subject in need of a therapeutic composition for the treatment of cystic fibrosis.

[0379] In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or treatment of a subject's liver or hepatocytes. Such peptides and polypeptides may include those associated with urea cycle disorders, lysosomal storage diseases, glycogen storage diseases, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder, for which delivery to or treatment of the liver or hepatocytes with enriched full-length mRNA provides a therapeutic benefit.

[0380] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding proteins associated with urea cycle disorders. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding argininosuccinate synthetase 1 protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding carbamoyl phosphate synthetase I protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding argininosuccinate lyase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding argininosuccinate lyase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding arginase protein.

[0381] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding proteins associated with lysosomal storage diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding alpha galactosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding glucocerebrosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding isocyanate-2-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding iduronidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding N-acetyl-α-D-glucosaminidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding heparan N-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding galactosamine-6 ​​sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding β-galactosidase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding lysosomal lipase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising purified mRNA encoding transcription factor EB (TFEB).

[0382] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding proteins associated with glycogen storage diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding acid alpha-glucosidase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding glucose-6-phosphatase (G6PC) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding liver glycogen phosphorylase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding muscle phosphoglycerate mutant enzyme protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding glycogen debranching enzyme.

[0383] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding proteins related to amino acid metabolism. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding phenylalanine hydroxylase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding glutaryl-CoA dehydrogenase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding propionyl-CoA carboxylase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding oxalase alanine-glyoxylate aminotransferase.

[0384] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding proteins associated with lipid metabolism or fibrotic diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding mTOR inhibitors. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding ATPase phospholipid transport 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding one or more NF-κB inhibitors, such as one or more of I-κBα, interferon-related developmental regulator 1 (IFRD1) and Sirtuin 1 (SIRT1). In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding PPAR-γ protein or active variants.

[0385] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding proteins associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding methylmalonyl-CoA mutant enzyme proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding methylmalonyl-CoA epimerase proteins.

[0386] In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA, which can provide a therapeutic beneficial effect when delivered to the liver or treated. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding ATP7B protein (also known as Wilson disease protein). In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding porphobilinogen deaminase. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding one or more coagulase, such as factor VIII, factor IX, factor VII and factor X. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding human hemochromatosis (HFE) protein.

[0387] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or treatment of a cardiovascular disease or cardiovascular cell in a subject. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a vascular endothelial growth factor A protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a relaxin protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a bone morphogenetic protein-9 protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a bone morphogenetic protein-2 receptor protein.

[0388] In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide, the therapeutic composition is used to deliver to or treat a subject's muscle or muscle cell. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a dystrophin. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide, the therapeutic composition is used to deliver to or treat a subject's myocardium or myocardial cell. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein regulating one or both of potassium channels and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein regulating a Kv7.1 channel in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein regulating a Nav1.5 channel in muscle tissue or muscle cells.

[0389] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide, the therapeutic composition being used to deliver to or treat the nervous system or nervous system cells of a subject. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a survival motor neuron 2 protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a human mitochondrial protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a CLN3 protein.

[0390] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide, the therapeutic composition is used to deliver to or treat the blood or bone marrow or blood cells or bone marrow cells of a subject. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding beta globin. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding Bruton's tyrosine kinase protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding one or more coagulase, such as factor VIII, factor IX, factor VII, and factor X.

[0391] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for delivery to or treatment of a subject's kidney or renal cells. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding type IV collagen α5 chain (COL4A5) protein.

[0392] In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a purified mRNA encoding a peptide or polypeptide, the therapeutic composition is used to deliver to or treat the eye or eye cell of a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a purified mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a purified mRNA encoding a retinal chitin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a purified mRNA encoding a retinal pigment epithelium-specific 65kDa (RPE65) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a purified mRNA encoding a centrosomal protein (CEP290) of 290kDa.

[0393] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide, the therapeutic composition being used to deliver a vaccine to a subject or a subject's cell or to treat with a vaccine. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen from an infectious agent such as a virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen from an influenza virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a respiratory syncytial virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a rabies virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a rotavirus. In certain embodiments, the present invention provides methods for preparing 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 methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from human papillomaviruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from herpes simplex viruses, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from human immunodeficiency viruses, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from human metapneumoviruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from human parainfluenza viruses, 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 methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from human parainfluenza viruses, 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 methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from malaria viruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding antigens from Zika viruses. In certain embodiments, the invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding an antigen from Chikungunya virus.

[0394] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen associated with a subject's cancer or an antigen identified from a subject's cancer cell. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen determined from a subject's own cancer cell, i.e., providing a personalized cancer vaccine. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding an antigen expressed from a mutant KRAS gene.

[0395] In certain embodiments, the present invention provides a method for preparing 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 independent mRNA-LNPs in step (b) of the method include mRNA encoding antibody light and heavy chains. In some embodiments, the mRNA-LNP composition of the present invention may include a combination of different LNPs containing different lipid compositions and mRNA encapsulating the light or heavy chains of the antibody. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody against CD3. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody against CD19.

[0396] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding an immunomodulator. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding interleukin 12. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding interleukin 23. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding interleukin 36γ. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding one or more constitutively active variants of stimulators of interferon genes (STING) proteins.

[0397] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding endonucleases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding RNA-guided DNA endonucleases proteins, such as Cas 9 proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding meganuclease proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding transcription activator-like effector nuclease proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising purified mRNA encoding zinc finger nuclease proteins.

[0398] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising purified mRNA encoding a protein for treating an eye disease. In some embodiments, the method is used to prepare a therapeutic composition comprising purified mRNA encoding a retinoschisis protein.

[0399] Example

[0400] Although certain compounds, compositions and methods of the present invention have been specifically described according to certain embodiments, the following examples are intended to illustrate the present invention only and are not intended to limit the present invention. Although certain compounds, compositions and methods of the present invention have been specifically described according to certain embodiments, the following examples are intended to illustrate the present invention only and are not intended to limit the present invention.

[0401] Unless otherwise stated, the mRNA-LNP test preparations described in the following examples contain mRNA encapsulated in a multi-component lipid mixture having various ratios 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 mRNA).

[0402] Example 1. Preparation of mRNA

[0403] In vitro transcription of mRNA

[0404] Unless otherwise described, mRNA was synthesized via in vitro transcription (IVT) using T7 polymerase or SP6 polymerase. Briefly, in SP6 polymerase IVT reactions, for each gram of transcribed mRNA, reactions containing 20 mg of linearized double-stranded DNA plasmid and RNA polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 5 mM NTP, 10 mM DTT, and reaction buffer (10x-250 mM Tris-HCl, pH 7.5, 20 mM spermidine, 50 mM NaCl) were prepared with RNase-free water and then incubated at 37°C for 60 min. The mRNA was then incubated for 60 min at 37°C by adding DNase I and DNase I buffer (10x-100 mM Tris-HCl, 5 mM MgCl2, and 25 mM CaCl 2， The reaction was quenched at 4% paraformaldehyde (pH 7.6) to facilitate digestion of double-stranded DNA template in preparation for purification.

[0405] 5' capping of mRNA

[0406] Unless otherwise described, IVT-transcribed mRNAs are capped on their 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 into the nascent RNA strand as the first "base". The cap analog can be Cap 0, Cap 1, Cap 2, m6 A m or a non-natural cap. Alternatively, a 5' N 7 Uncapped and purified in vitro transcribed (IVT) mRNA is enzymatically modified after IVT to include a cap by adding a methyl group to the 2'O position of the penultimate nucleotide using a 2'O-methyltransferase to generate a Cap 1 structure as described by Fechter, P.; Brownlee, GG "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249.

[0407] 3' tailing of mRNA

[0408] Unless otherwise described, IVT-transcribed mRNAs are tailed on their 3' end by including a tailing template in a linearized plasmid that tails the mRNA as part of the IVT reaction or in a subsequent enzymatic step. For tailing as part of the IVT reaction, a poly-T or similar tailing feature is incorporated into the pDNA template so that a poly-A tail or similar appropriate tail is formed on the mRNA as part of the IVT process. Alternatively, a poly-A tail can be enzymatically added to the 3' end of the IVT-generated mRNA after the IVT reaction, for example using a poly-A polymerase.

[0409] Example 2. Preparation of lipid nanoparticles (LNP) and encapsulation of mRNA

[0410] LNP preparation and encapsulation of mRNA in LNPs comprising compound 3 as a cationic lipid were performed according to method B. Method B is further described in U.S. Published Patent Application No. US2018153822, which is incorporated herein by reference for all purposes. As described above, the LNP formulation is a multi-component lipid mixture comprising one or more cationic lipids, one or more auxiliary 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 the mRNA obtained as described in Example 1.

[0411] As used herein, Method A refers to a general method in which LNPs are formed from a multi-component mixture of lipids, and mRNA is encapsulated in those forming LNPs in a single step.

[0412] Method B refers to the method for encapsulating mRNA by mixing preformed LNP with mRNA.Preformed LNP is first prepared by instantaneously mixing a multi-component lipid mixture dissolved in a solvent such as ethanol with a citrate buffer in the absence of mRNA.The mixing of two streams leads to the formation of empty lipid nanoparticles, which is a self-assembly process.The resulting formulation provides empty lipid nanoparticles in a citrate buffer containing alcohol, which is subjected to buffer exchange (e.g., by tangential flow filtration (TFF)) to provide empty lipid nanoparticles in a 10% weight / volume trehalose solution buffer.Then, empty lipid nanoparticles and mRNA in the mixed aqueous solution are formed to form the mRNA encapsulated in the lipid nanoparticles.

[0413] Specifically, to prepare empty lipid nanoparticles, DEPE or DOPE was used as a helper lipid in the ratios described in Table 2-1, along with Compound 3 as a cationic lipid, DMG-PEG2K as a PEG-modified lipid, and cholesterol.

[0414] Table 2-1. Lipid nanoparticle formation using DEPE and DOPE

[0415]

[0416] Surprisingly, it was found that these multicomponent lipid mixtures with compound 3 as the cationic lipid could not be formulated using DOPE as the non-cationic helper lipid, but stable liposomes were formed when DEPE was used instead of DOPE as the non-cationic helper lipid.

[0417] Example 3. Enhancement of in vivo production of mRNA in LNPs using DEPE

[0418] This example demonstrates that the titer of LNP-encapsulated mRNA is unexpectedly increased using LNPs comprising DEPE as a helper lipid.

[0419] The mRNA encoding EPO was synthesized as described in Example 1. Using method B as described in Example 2, mRNA was encapsulated into LNPs containing different auxiliary lipids but otherwise identical (see Table 3-1 below). Specifically, each LNP of encapsulated mRNA includes different auxiliary lipids, but includes the same cationic lipid (compound 1), the same PEG-modified lipid (DMG-PEG2K), the same sterol compound (cholesterol), those lipids of the same molar ratio, the same mRNA (EPOmRNA), the same N / P ratio=4 (i.e., the molar ratio of cationic lipid to mRNA), the same mRNA concentration (0.2mg / mL), and prepared according to the same method (method B). The characteristics of the obtained mRNA-LNP are provided in Table 3-1.

[0420] Table 3-1. Characteristics of EPO mRNA-LNP

[0421]

[0422] Each of the four test preparations containing mRNA encapsulated in LNPs with different auxiliary lipids (1-4 in Table 3-1) was administered intravenously to mice by tail vein injection (n=5, CD-1 mice 6-8 weeks old) at a dose of 1 mg / kg mRNA in a dose volume of 5 mL / kg. Six hours after administration, mid-term whole blood was collected by tail clipping. 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 kits (R&D Systems Catalog 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 provided in Table 3-2, respectively and in Figure 1 and Figure 2 Depicted graphically in .

[0423] Table 3-2. In vivo experimental plan of Example 3

[0424]

[0425] As shown in Table 3-2 and Figure 1 As shown in, compared with the protein expression from mRNA LNPs that contain other auxiliary lipids but are otherwise identical, the mRNA LNPs that contain DEPE as auxiliary lipids provide significantly higher in vivo protein expression. Specifically, six hours after administration, the mRNA LNPs that contain DEPE provide in vivo protein expression, which is enhanced to more than 100% at the same time point, i.e. more than twice the protein expression from the mRNA LNPs that contain auxiliary lipids other than DEPE (e.g., DOPE, DLOPE, or POPE). Similarly, 24 hours after administration, the mRNA LNPs that contain DEPE provide in vivo protein expression, which is enhanced to more than 100% at the same time point, i.e. more than twice the protein expression from the mRNA LNPs that contain auxiliary lipids other than DEPE (e.g., DOPE, DLOPE, or POPE).

[0426] In addition, as shown in Table 3-2 and Figure 2 As shown in , ALT and AST levels 24 hours after administration were essentially similar for all mRNA LNPs regardless of the helper lipid, indicating that mRNA LNPs comprising DEPE as a helper lipid have similar safety and tolerability to mRNA LNPs comprising helper lipids other than DEPE (e.g., DOPE, DLOPE, or POPE), while being significantly more effective.

[0427] Example 4. Preparation of lipid nanoparticles (LNP) using DEPE or DOPE as auxiliary lipids

[0428] This example illustrates that the use of DEPE as a helper lipid in mRNA encapsulated lipid nanoparticle (mRNA-LNP) formulations can provide up to two times more protein expression of mRNA in vivo relative to conventional liposomes containing (DOPE) as a helper lipid. It was also observed that mRNA-LNPs with DEPE as a helper lipid provided increased encapsulation efficiency compared to the same mRNA-LNPs with DOPE as a helper lipid. Notably, this enhanced expression and this enhanced encapsulation efficiency were observed in a variety of mRNA-LNPs containing different cationic lipids.

[0429] In these studies, mRNA encoding OTC was encapsulated in LNPs containing DEPE or DOPE as a helper lipid and various cationic lipids listed in Table 4. Each cationic lipid described in Table 4 contained a 10 , C 12 , C 14 or C 16 The length of four alkyl chains, as shown in the last two digits in the description of each lipid. The molar ratio of cationic lipid: DMG-PEG2K: cholesterol: helper lipid is about 40:3:25:32. For the in vivo part of the study, 1 mg / kg of each prepared mRNA-LNP was delivered to mice via tail vein injection. At 24 hours, mice were killed, and the in vivo expression of mRNA encoding OTC was evaluated from the liver homogenate of each mouse. Average protein expression is provided in the following table.

[0430] Table 4. Protein expression and encapsulation efficiency of LNPs using DEPE and DOPE as helper lipids

[0431]

[0432] Table 4 confirms that, compared with DOPE, when using DEPE as a helper lipid, encapsulation efficiency is higher. In addition, surprisingly observed that the multi-component lipid mixture with some lipidoids as cationic lipids can not even be prepared using DOPE, but forms stable liposomes when using DEPE as a helper lipid. Table 4 also confirms that, compared with the protein expression of mRNA LNPs that comprise DOPE helper lipids but otherwise identical, the mRNA LNPs that comprise DEPE as helper lipids demonstrate significantly higher in vivo protein expression. When various cationic lipids were used in LNPs, this was also the case.

[0433] Example 5. Enhanced in vivo protein expression by using DEPE as a helper lipid compared to other helper lipids

[0434] This example demonstrates that the use of DEPE as a helper lipid in mRNA encapsulated lipid nanoparticles (mRNA-LNPs) can increase mRNA expression in vivo, across multiple treatments of encapsulation used to prepare mRNA-LNPs, relative to lipid nanoparticles using other types of helper lipids.

[0435] In these studies, mRNA encoding OTC was encapsulated in LNPs (N / P=4) at a molar lipid ratio, comprising DMG-PEG-2000 as a PEG-modified lipid, cDD-TE-4-E12 as a cationic lipid, cholesterol, and one of a variety of different auxiliary lipids, including DEPE, as shown in Tables 5-1, 5-2, 5-3, and 5-4. Each mRNA-LP formulation was prepared using one of four different encapsulation processes: a conventional process for the formulations described in Table 5-1, a remixing process for the formulations described in Table 5-2, a step-up remixing process for the formulations described in Table 5-3, or a step-down remixing process for the formulations described in Table 5-4. The LNP size, polydispersity, and encapsulation percentage of mRNA-LPP were evaluated, and each result is given in the following table. 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, mice were sacrificed and the in vivo expression of mRNA encoding OTC was evaluated from liver homogenates of each mouse. The average protein expression is provided in the table below.

[0436] Table 5-1. mRNA-LNPs with different auxiliary lipids prepared via conventional encapsulation auxiliary agents

[0437]

[0438] Table 5-2. mRNA-LNPs with different helper lipids prepared via remixing encapsulation

[0439]

[0440] Table 5-3. mRNA-LNPs with different helper lipids prepared via step-up remixing encapsulation

[0441]

[0442] Table 5-4. mRNA-LNPs with different helper lipids prepared via step-down and then mixing encapsulation

[0443]

[0444] Table 5-1, table 5-2, table 5-3 and table 5-4 show the protein expression level of mRNA encapsulated in LNP and delivered to mouse group after 24 hours respectively.As shown in the figure, LNP prepared only with auxiliary lipid DOPE or DEPE provides the effectiveness of protein expression in different encapsulation processes.It should be noted that when using DEPE as auxiliary lipid to prepare LNP, protein expression in vivo is the highest, and has nothing to do with the encapsulation method for preparing mRNA-LNP.

[0445] As will be appreciated by those skilled in the art, this significantly enhanced potency yet comparable safety and efficacy of mRNA LNPs comprising DEPE as a helper lipid provides significant advantages for delivering mRNA as a therapeutic agent.

[0446] Equivalent form

[0447] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims

1. A lipid nanoparticle for delivering mRNA to a subject in need thereof, the lipid nanoparticle comprising one or more cationic lipids, one or more PEG-modified lipids and one or more helper lipids encapsulating the mRNA, wherein the one or more helper lipids include 1,2-dicerucyl-sn-glyceryl-3-phosphoethanolamine (DEPE).

2. The lipid nanoparticle of claim 1, wherein administration of the lipid nanoparticle to the subject results in enhanced expression of the mRNA compared to expression of the same mRNA in a second lipid nanoparticle having the same lipid composition and amount, except that the second lipid nanoparticle comprises different one or more helper lipids and does not comprise DEPE.

3. The lipid nanoparticle of claim 2, wherein the different one or more auxiliary 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.

4. A lipid nanoparticle as described in claim 2 or 3, wherein expression is enhanced at least two-fold relative to the second lipid nanoparticle.

5. The lipid nanoparticle of any preceding claim, wherein the DEPE in the lipid nanoparticle is present at a concentration of 10 mol% to 50 mol%.

6. The lipid nanoparticle of any preceding claim, wherein the one or more cationic lipids are or comprise one to four cationic lipids each having C 10 -C 16 Length of alkyl chain.

7. The lipid nanoparticle of any one of claims 1 to 5, wherein the one or more cationic lipids are lipidoids comprising four aliphatic chains.

8. The lipid nanoparticle of claim 7, wherein the one or more cationic lipids are or include a cationic lipid of the formula: or a pharmaceutically acceptable salt thereof; Each R 1 and R 2 is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 are independently an ester, thioester, disulfide bond or anhydride group; each L 2 Independently C2-C 10 Aliphatic; each X 1 are independently H or OH; and each R 3 Independently C6-C 20 Aliphatic.

9. The lipid nanoparticle of claim 8, wherein each R 3 Independently C8-C 16 Aliphatic.

10. The lipid nanoparticle of any preceding claim, wherein the one or more PEG-modified lipids comprise a polyethylene glycol chain of up to 5 kDa in length covalently attached to a C6-C 20 Alkyl chains of lipids.

11. The lipid nanoparticle of any preceding claim, further comprising one or more sterols.

12. The lipid nanoparticle of claim 11, wherein the one or more sterols comprise a cholesterol-based lipid.

13. The lipid nanoparticle of claim 12, wherein the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol.

14. The lipid nanoparticle of any preceding claim, wherein the mRNA is an mRNA encoding a protein that is translated into a therapeutic protein or peptide in vivo.

15. The lipid nanoparticle of claim 14, wherein the mRNA encoding the protein or peptide is delivered systemically and the translated protein or peptide is detectable in the liver or serum 24 hours or more after administration.

16. The lipid nanoparticle of claim 15, wherein the polypeptide is a therapeutic polypeptide.

17. The lipid nanoparticle of claim 16, wherein the therapeutic polypeptide is (a) an antibody light chain or an antibody heavy chain, or (b) a polypeptide that is not present or lacking in the subject.

18. The lipid nanoparticle of claim 14, wherein the mRNA encoding a protein encodes a peptide.

19. The lipid nanoparticle of claim 18, wherein the peptide is an antigen.

20. A method for delivering mRNA to a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more auxiliary lipids that encapsulate the mRNA, wherein the one or more auxiliary lipids comprise 1,2-dicerucyl-sn-glycero-3-phosphoethanolamine (DEPE).

21. The method of claim 20, wherein administration of the lipid nanoparticle to the subject results in enhanced expression of the mRNA compared to expression of the same mRNA in a second lipid nanoparticle having the same lipid composition and amount, except that the second lipid nanoparticle comprises a different helper lipid or lipids and does not comprise DEPE.

22. The method of claim 21, wherein the different 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.

23. The lipid nanoparticle of claim 21 or 22, wherein expression is enhanced at least two-fold relative to the second lipid nanoparticle.

24. The lipid nanoparticle of any one of claims 20 to 23, wherein the DEPE in the lipid nanoparticle is present at a concentration of 10 mol % to 50 mol %.

25. The method of any one of claims 20 to 24, wherein the one or more cationic lipids is or comprises cKK-E12.

26. The method of any one of claims 20 to 24, wherein the one or more cationic lipids is or comprises ICE (imidazole cholesteryl ester).

27. The method of any one of claims 20 to 24, wherein the one or more cationic lipids are or include a cationic lipid of the formula: or a pharmaceutically acceptable salt thereof; Each R 1 and R 2 is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 are independently an ester, thioester, disulfide bond or anhydride group; each L 2 Independently C2-C 10 Aliphatic; each X 1 are independently H or OH; and each R 3 Independently C6-C 20 Aliphatic.

28. The method of claim 27, wherein the one or more cationic lipids is or comprises Compound 1.

29. The method of any one of claims 20 to 28, wherein the one or more PEG-modified lipids are or comprise a polyethylene glycol chain of up to 5 kDa in length, covalently attached to a C6-C 20 Alkyl chains of lipids.

30. The method of any one of claims 20 to 29, further comprising one or more sterols.

31. The method of claim 30, wherein the one or more sterols comprise a cholesterol-based lipid.

32. The method of claim 31, wherein the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol.

33. The method of any one of claims 21 to 32, wherein the mRNA is an mRNA encoding a protein that is translated into a therapeutic protein in vivo.

34. The method of claim 33, wherein the mRNA encoding a protein encodes a polypeptide.

35. The method of claim 34, wherein the polypeptide is a therapeutic polypeptide.

36. The method of claim 35, wherein the therapeutic polypeptide is (a) an antibody light chain or an antibody heavy chain, or (b) a polypeptide that is absent or deficient in the subject.

37. The method of claim 33, wherein the mRNA encoding a protein encodes a peptide.

38. The method of claim 37, wherein the peptide is an antigen.

39. 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, wherein the one or more helper lipids include 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 the mRNA into the lipid nanoparticles, wherein the encapsulation is performed before or after forming the lipid nanoparticles in step (b).

40. The method of claim 39, wherein the lipid nanoparticles encapsulating the mRNA are stable.

41. The method of claim 39 or 40, wherein the one or more helper lipids do not include 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.

42. The method of any one of claims 39 to 41, wherein the DEPE is present in the mixture at a concentration of 10 mol% to 50 mol%.

43. The method of any one of claims 39 to 42, wherein the one or more PEG-modified lipids in the mixture comprise a polyethylene glycol chain of up to 5 kDa in length covalently attached to a C6-C 20 Alkyl chains of lipids.

44. The method of any one of claims 39 to 43, wherein the mixture further comprises one or more sterols.

45. The method of claim 44, wherein the one or more sterols comprise a cholesterol-based lipid.

46. ​​The method of claim 45, wherein the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol.

47. The method of any one of claims 39 to 46, wherein the mRNA encodes a therapeutic peptide, polypeptide or protein.

48. The method of any one of claims 39 to 47, wherein the mRNA is encapsulated in preformed lipid nanoparticles.

49. The method of any one of claims 39 to 48, wherein the method further comprises subjecting the lipid nanoparticles to tangential flow filtration (TFF) before and / or after encapsulating the mRNA.

50. The method of any one of claims 39 to 49, wherein the method further comprises formulating the lipid nanoparticles in a trehalose solution.

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