Improved mrna-loaded lipid nanoparticles and processes of making the same

Abstract

The present invention provides an improved lipid nanoparticle formulation for encapsulating mRNA containing DEPE as a helper lipid.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 871,513 filed July 8, 2019, the disclosures of which are incorporated herein by reference.

[0003] Technology field

[0004] 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 processes for preparing such compounds and compositions. Background Technology

[0005] Messenger RNA therapy (MRT) is becoming an increasingly important approach for the treatment or prevention of various diseases. MRT involves administering messenger RNA (mRNA) to subjects who require therapy to produce proteins encoded by mRNA within their bodies. Lipid nanoparticles can be used to encapsulate mRNA for efficient in vivo delivery.

[0006] Much effort has been made to identify novel methods and compositions capable of enhancing the in vivo delivery and / or expression of mRNA using lipid nanoparticles applicable to scalable and cost-effective manufacturing processes. At the same time, it is also important that any such enhancement of intracellular delivery and / or expression of mRNA maintains or improves the safety and tolerability of compositions associated with lipid-mediated mRNA delivery.

[0007] Multi-component lipid nanoparticles containing one or more cationic lipids encapsulating mRNA, one or more PEG-modified lipids, and one or more helper lipids have been found to be particularly effective in achieving in vivo mRNA delivery and expression. A specific focus of recent research has been the discovery of novel cationic lipids for mRNA delivery. Other components of multi-component lipid nanoparticles receive little to no attention. There is a continuous need to improve the delivery of mRNA via lipid nanoparticles to achieve intracellular delivery and / or expression of mRNA. At the same time, it is desirable to maintain or improve the safety and tolerability of new lipid nanoparticle formulations.

[0008] (Patent Document 1) WO 2018 / 089801 A1

[0009] The inventors have surprisingly discovered that the delivery and / or expression of mRNA in vivo can be dramatically improved by optimizing the helper lipid component of a multicomponent liposome encapsulating mRNA. In particular, the present invention is based on the finding that the presence of 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) as a helper lipid in an mRNA-encapsulated lipid nanoparticle formulation comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids can increase the delivery and / or expression of mRNA in vivo by more than twofold compared to conventional liposomes comprising dioleoylphosphatidylethanolamine (DOPE) as one of the helper lipids. DEPE-containing lipid nanoparticles were similar to DOPE-containing lipid nanoparticles in terms of safety and tolerability (evaluated by hepatotoxicity markers such as ALT and AST).

[0010] Accordingly, one aspect of the present invention is to provide lipid nanoparticles for delivering mRNA to a subject requiring it, wherein the lipid nanoparticles comprise one or more cationic lipids encapsulating mRNA, one or more PEG-modified lipids, and one or more helper lipids, wherein one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). The DEPE within the lipid nanoparticles provides enhancement of mRNA expression when administered to a subject.

[0011] In an embodiment of the present invention, DEPE, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) has the following structure:

[0012]

[0013] Or is structurally represented by the following structure

[0014]

[0015] In an embodiment of the present invention, the enhancement of mRNA expression is enhanced compared to the expression of the same mRNA from a second lipid nanoparticle having the same lipid components and amounts, except that it contains one or more different helper lipids and does not contain DEPE. In a given embodiment, the enhancement of expression is increased by more than twofold compared to the second lipid nanoparticle. In some embodiments, one or more different helper lipids in the second lipid nanoparticle comprise dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoylsn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof.

[0016] In a given embodiment, DEPE in the lipid nanoparticles is present at a concentration of at least 0.5 mol% of the total lipid in the lipid nanoparticles, for example, at a concentration of 0.5 mol% to 50 mol%, and particularly at a concentration of 10 mol% to 45 mol%. More generally, DEPE in the lipid nanoparticles is present at a concentration of 25 mol% to 35 mol% of the total lipid in the lipid nanoparticles.

[0017] In a given embodiment, one or more cationic lipids are cKK-E12 or include the same.

[0018] In a given embodiment, one or more cationic lipids are ICE (imidazole cholesterol ester) or include the same.

[0019] In a given embodiment, one or more cationic lipids are cationic lipids of the following chemical formula:

[0020]

[0021] or a pharmaceutically acceptable salt thereof or comprising the same;

[0022] Each R in the equation 1 and R 2 is independently H or C1-C6 aliphatic; each m is an integer having a value from 1 to 4 independently; each A is independently a covalent bond or an arylene; and each L 1 is independently an ester group, a thioester group, a disulfide group, or an anhydride group; and each L 2 is independently C2-C 10 Aliphatic and; each X 1 is independently H or OH; and each R 3 is independently C6-C 20 is. In a given embodiment, one or more cationic lipids are the following compounds:

[0023]

[0024] (Compound 1)

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

[0026]

[0027] (Compound 2)

[0028] or a pharmaceutically acceptable salt thereof or comprises the same. In another specific embodiment, one or more cationic lipids are the following compounds:

[0029]

[0030] (Compound 3)

[0031] or a pharmaceutically acceptable salt thereof or comprises the same. In a given embodiment, one or more PEG-modified lipids are C6-C 20 It is a poly(ethylene) glycol chain up to 5 kDa in length covalently bonded to a lipid having an alkyl chain of length, or includes such a chain.

[0032] In some embodiments, lipid nanoparticles encapsulating mRNA and containing DEPE as a helper lipid are C6-C 20 It also comprises cationic lipids comprising alkyl chain(s) of length C8-C 16 It comprises 1 to 4 alkyl chain(s) of length. In some embodiments, the cationic lipids each have C 10 -C 16 It comprises 1 to 4 alkyl chain(s) of length. In some embodiments, the cationic lipids each have C 10 -C 14 It comprises 1 to 4 alkyl chain(s) of length. In some embodiments, the cationic lipids each have C 10 It comprises 1 to 4 alkyl chain(s) of length. In some embodiments, the cationic lipids each have C 12 It comprises 1 to 4 alkyl chain(s) of length. In some embodiments, the cationic lipids each have C16 It includes 1 to 4 alkyl chain(s) of length.

[0033] In some embodiments, lipid nanoparticles encapsulating mRNA and containing DEPE as a helper lipid also each contain C6-C 20 The cationic lipid comprises a cationic lipid comprising 1 to 4 alkyl chain(s) of length. In some embodiments, the cationic lipid is each C8-C 16 It comprises 1 to 4 aliphatic chain(s) of length. In some embodiments, the cationic lipids are each C 10 -C 14 It comprises 1 to 4 aliphatic chain(s) of length. In some embodiments, the cationic lipids are each C 10 It comprises 1 to 4 aliphatic chain(s) of length. In some embodiments, the cationic lipids are each C 12 It comprises 1 to 4 aliphatic chain(s) of length. In some embodiments, the cationic lipids are each C 16 It includes 1 to 4 aliphatic chain(s) of length.

[0034] In some embodiments, the lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid also comprise a lipidoid or one or more cationic lipids comprising the same. In some embodiments, the lipidoid comprises four aliphatic chains. In some embodiments, each of the four lipidoid aliphatic chains is independently C6-C 20 It is the length. In some embodiments, each of the four lipidoid aliphatic chains is independently C8-C 16 It is the length. In some embodiments, each of the four lipidoid aliphatic chains is independently C 10 -C 14 It is the length. In some embodiments, each of the four lipidoid aliphatic chains is independently C 10 or C 12It is the length. In some embodiments, all four lipidoid aliphatic chains are of C6 length. In some embodiments, all four lipidoid aliphatic chains are of C8 length. In some embodiments, all four lipidoid aliphatic chains are C 10 It is the length. In some embodiments, all four lipidoid aliphatic chains are C 12 It is the length. In some embodiments, all four lipidoid aliphatic chains are C 14 It is the length. In some embodiments, all four lipidoid aliphatic chains are C 16 It is the length. In some embodiments, all four lipidoid aliphatic chains are C 18 It is the length. In some embodiments, all four lipidoid aliphatic chains are C 20 It is a length. In some embodiments, at least two of the four lipidoid aliphatic chains are of C6 length. In some embodiments, at least two of the four lipidoid aliphatic chains are of C8 length. In some embodiments, at least two of the four lipidoid aliphatic chains are C 10 It is a length. In some embodiments, at least two of the four lipidoid aliphatic chains are C 12 It is a length. In some embodiments, at least two of the four lipidoid aliphatic chains are C 14 It is a length. In some embodiments, at least two of the four lipidoid aliphatic chains are C 16 It is a length. In some embodiments, at least two of the four lipidoid aliphatic chains are C 18 It is a length. In some embodiments, at least two of the four lipidoid aliphatic chains are C 20 It is the length.

[0035] In some embodiments, lipid nanoparticles encapsulating mRNA and comprising DEPE as a helper lipid also comprise a lipidoid comprising C6-C20 alkyl chain(s). In some embodiments, the lipidoid comprises C8-C 16It comprises alkyl chain(s) of length C. In some embodiments, the lipidoid is C 10 -C 14 It comprises alkyl chain(s) of length C. In some embodiments, the lipidoid is C 10 It comprises alkyl chain(s) of length C. In some embodiments, the lipidoid is C 12 It comprises alkyl chain(s) of length C. In some embodiments, the lipidoid is C 16 It includes alkyl chain(s) of length.

[0036] In certain embodiments, the lipid nanoparticles further comprise one or more sterols. In some embodiments, one or more sterols are or comprise cholesterol-based lipids, e.g., cholesterol or PEGylated cholesterol.

[0037] In some embodiments, the lipid nanoparticles comprise one or more cationic lipids that encapsulate mRNA, one or more PEG-modified lipids, one or more non-cationic lipids, and one or more cholesterol-based lipids. For example, in a typical embodiment of the present invention, the lipid component of the lipid nanoparticles comprises four types of lipids, including cationic lipids (e.g., cKK-E12, Compound 1, Compound 2, or Compound 3), PEG-modified lipids (e.g., DMG-PEG2K), non-cationic lipids (DEPE), and cholesterol-based lipids (e.g., cholesterol). In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) of the lipid nanoparticles may be between about 30 to 60:25 to 35:20 to 30:1 to 15.

[0038] In some embodiments, the lipid components of the lipid nanoparticles include cationic lipids (e.g., ICE), PEG-modified lipids (e.g., DMG-PEG2K), and non-cationic lipids (DEPE). In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) may be between about 50 to 60:45 and 30:5 to 10.

[0039] The mRNA delivered by the lipid nanoparticles of the present invention is mRNA that encodes a protein that is translated into a therapeutic protein in vivo. In certain embodiments, the mRNA that encodes the 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 absent or deficient in the subject. In certain embodiments, the mRNA that encodes the protein encodes a peptide. In some embodiments, the peptide is an antigen.

[0040] In another aspect, the present invention provides a method for improving the delivery of mRNA to a subject requiring such improvement, the method comprising the step of administering to a subject lipid nanoparticles encapsulating mRNA comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). DEPE within the lipid nanoparticles provides enhancement of mRNA expression when administered to the subject.

[0041] In some embodiments, the mRNA encodes a protein that is translated into a therapeutic protein or peptide in vivo. In some embodiments, the mRNA encoding the protein or peptide is delivered systematically. In some embodiments, the translated protein or peptide is detectable at least 6 hours after administration. In some embodiments, the translated protein or peptide is detectable at least 12 hours after administration. In some embodiments, the translated protein or peptide is detectable at least 18 hours after administration. In some embodiments, the translated protein or peptide is detectable at least 24 hours after administration. In some embodiments, the translated protein or peptide is detectable at least 36 hours after administration. In some embodiments, the translated protein or peptide is detectable at least 48 hours after administration. In some embodiments, the translated protein or peptide is detectable at least 72 hours after administration. In some embodiments, the translated protein or peptide is detectable in serum at least 6 hours after administration. In some embodiments, the translated protein or peptide is detectable in serum after 12 hours or more have elapsed since administration. In some embodiments, the translated protein or peptide is detectable in serum after 18 hours or more have elapsed since administration. In some embodiments, the translated protein or peptide is detectable in serum after 24 hours or more have elapsed since administration. In some embodiments, the translated protein or peptide is detectable in serum after 36 hours or more have elapsed since administration. In some embodiments, the translated protein or peptide is detectable in serum after 48 hours or more have elapsed since administration. In some embodiments, the translated protein or peptide is detectable in serum after 72 hours or more have elapsed since administration.

[0042] In a further embodiment, the present invention provides lipid nanoparticles encapsulating mRNA comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein, for use in a method for treating or preventing a disease or disorder in a subject, one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), and the mRNA encodes a peptide, polypeptide, or protein suitable for treating or preventing a disease or disorder in a subject. In a related aspect, the present invention relates to the use of lipid nanoparticles encapsulating mRNA in the manufacture of a medicine for treating or preventing a disease or disorder in a subject, wherein the lipid nanoparticles comprise one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein the one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), and the mRNA encodes a peptide, polypeptide, or protein suitable for treating or preventing a disease or disorder in a subject. In one embodiment, the mRNA encodes a polypeptide or protein that is absent or deficient in the subject, wherein the disease or disorder is a deficiency of said polypeptide or protein. In another embodiment, the mRNA encodes an antibody light chain or antibody heavy chain, and the subject suffers from a disease or disorder that can be treated by administering an antibody containing said light chain or said heavy chain to the subject. In a further embodiment, the mRNA encodes a peptide, polypeptide, or protein, wherein the peptide, polypeptide, or protein can induce an immune response in the subject to treat or prevent a disease or disorder.

[0043] The inventors have also discovered that using DEPE as a helper lipid allows for the formulation of multi-component formulations that do not form stable liposomes when DOPE is used as one of the helper lipids. Accordingly, in another aspect, the present invention provides a method for preparing lipid nanoparticles that encapsulate 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 one or more helper lipids comprises 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); and (b) forming lipid nanoparticles from the mixture provided in step (a), wherein the method further comprises the step of encapsulating mRNA into the lipid nanoparticles, the encapsulation may be performed before or after forming the lipid nanoparticles in step (b). The resulting lipid nanoparticles that encapsulate mRNA are stable. In one embodiment, a method for preparing lipid nanoparticles according to the present invention explicitly excludes the use of one or more helper lipids selected from the following: dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and combinations thereof. In one embodiment, DEPE is present in the mixture at a concentration of 10 mol% to 50 mol%. In one embodiment, one or more PEG-modified lipids in the mixture are C6-C 20It comprises a poly(ethylene) glycol chain up to 5 kDa in length covalently attached to a lipid having an alkyl chain of length. In one embodiment, the mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids further comprises one or more sterols, such as cholesterol-based lipids. In one embodiment, one or more cholesterol-based lipids are cholesterol and / or PEGylated cholesterol. In one embodiment, the mRNA encodes a therapeutic peptide, polypeptide, or protein. In some embodiments, the mRNA is encapsulated in pre-formed lipid nanoparticles. In some embodiments, a method for preparing lipid nanoparticles according to the present invention further comprises the step of purifying the lipid nanoparticles by tangential flow filtration (TFF) before and / or after encapsulation of the mRNA. In some embodiments, the method further comprises the step of formulating the lipid nanoparticles in a trehalose solution.

[0044] Without being bound by any particular theory, DEPE derivatives, specifically DEPE derivatives or compositions having lipid chains of various lengths, are considered to provide the same advantages as those described herein for DEPE. For convenience, the foregoing summary of the invention and specific details for carrying out the invention refer only to DEPE. However, it should be understood that minor variations in the lipid chains of DEPE do not affect the excellent properties of DEPE, and that such DEPE derivatives are explicitly included in the invention.

[0045] Other features, objects, and advantages of the present invention will become apparent from the following description, drawings, and claims regarding the practice of the invention. However, it should be understood that while the description, drawings, and claims regarding the practice of the invention represent embodiments of the invention, they are given merely for illustrative purposes and not to be limiting. It will be apparent to those skilled in the art that various variations and modifications may be made within the scope of the invention. Brief explanation of the drawing

[0046] Fig. 1 This illustrates an exemplary graphic representation of EPO protein expression in mRNA LNPs containing DEPE and other helper lipids. Fig. 2 This shows an exemplary graphic representation of serum ALT and AST levels after administration to mRNA LNP containing DEPE and other helper lipids. Specific details for implementing the invention

[0047] The following embodiments and modes thereof are described and illustrated with systems, compositions, and methods that are exemplary, illustrated, and do not limit the category. .

[0048] definition

[0049] The present invention provides an improved process for producing mRNA encapsulated in a lipid nanoparticle (LNP) formulation for producing an mRNA therapeutic composition.

[0050] To facilitate a better understanding of the present invention, specific terms are defined as follows. Additional definitions of the following terms and other terms are described throughout this specification.

[0051] amino acidsAs used herein, the term “amino acid” in its broadest sense refers to any compound and / or substance that may be included in a polypeptide chain. In some embodiments, the amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid, and in some embodiments, the amino acid is a D-amino acid; and in some embodiments, the amino acid is an L-amino acid. “Standard amino acid” refers to any one of the 20 standard L-amino acids found primarily in naturally occurring peptides. “Non-standard amino acid” refers to any amino acid other than the standard amino acid, regardless of whether it is produced synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses, but is not limited to, chemically modified amino acids including salts, amino acid derivatives (e.g., amides), and / or substituents. Amino acids, including carboxy-terminal amino acids and / or amino-terminal amino acids of a peptide, may be modified by methylation, amidation, acetylation, or by protecting groups, and / or by substitution with other chemical functional groups that can alter the cyclic half-life of the peptide without adversely affecting the activity of the peptide. Amino acids may participate in disulfide bonds. Amino acids, for example, one or more chemical entities ( for example , methyl group, acetate group, acetyl group, phosphate group, formyl moieties, isoprenoid group, sulfate group, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties etc.It may include one or more post-translational modifications such as ). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to free amino acids and / or amino acid residues of peptides. Whether it refers to free amino acids or residues of peptides will become clear from the context in which the term is used.

[0052] animal : As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any stage of development. In some embodiments, “animal” refers to a non-human animal at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primates and / or pigs). In some embodiments, the animal includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a genetically modified animal, a genetically engineered animal, and / or a clone.

[0053] Approximately or about : As used herein, the terms “approximately” or “about” refer to a value similar to a specified reference value when applied to one or more values ​​of interest. In certain embodiments, the terms “approximately” or “about” indicate a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less in any direction (greater than or less than) the stated reference value, unless otherwise stated or otherwise evident from the context (except where such numbers exceed 100% of possible values).

[0054] Combining:As used herein, the term “combination” is used interchangeably with “mixing” or “combination.” Combination refers to combining discrete LNP particles having distinct characteristics in the same solution to obtain an mRNA-LNP composition, for example, combining mRNA-LNP and empty LNP. In some embodiments, combining two LNPs is performed in a specific ratio of the components being combined. In some embodiments, the composition produced by the combination has characteristics distinct from either or both of its components.

[0055] transmission As used herein, the term "transfer" includes both local and systemic transfer. For example, the transfer of mRNA includes: a situation in which mRNA is delivered to a target tissue, the encoded protein or peptide is expressed, and is maintained within the target tissue (also referred to as "local distribution" or "local transfer"); and a situation in which mRNA is delivered to a target tissue, the encoded protein or peptide is expressed, is secreted into the patient's circulatory system (e.g., serum), and is distributed systemically and absorbed by other tissues (also referred to as "systemic distribution" or "systemic transfer").

[0056] efficacy As used herein, the term "efficacy" or its grammatical equivalent refers to the improvement of a biologically relevant endpoint in connection with the delivery of mRNA encoding a relevant protein or peptide. In some embodiments, the biological endpoint is protection against ammonium chloride inoculation at a given time after administration.

[0057] Encapsulation: As used herein, the term "encapsulation" or its grammatical equivalent refers to the process of confining individual mRNA molecules within nanoparticles.

[0058] expressionAs used herein, "expression" of mRNA refers to the translation of mRNA into a peptide (e.g., antigen), polypeptide, or protein (e.g., enzyme), and, as indicated by the context, may include post-translational modification of the peptide, polypeptide, or fully assembled protein (e.g., enzyme). In this application, the terms "expression" and "production" and their grammatically equivalent expressions are used interchangeably.

[0059] Improve, increase, or reduce As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to values ​​relative to baseline measurements, e.g., measurements in the same individual prior to the initiation of the treatment described herein, or measurements in a control sample or subject (or multiple control samples or subjects) in the absence of the treatment described herein. A “control sample” is a sample subjected to the same conditions as the test sample, except for the test item. A “control subject” is a subject with the same form of disease as the subject being treated and of approximately the same age as the subject being treated.

[0060] impurities As used herein, the term "impurity" refers to a substance within a confined amount of a liquid, gas, or solid that differs in chemical composition from that of the target substance or compound. Impurities are also referred to as contaminants.

[0061] in vitro As used herein, the term "in vitro ( in vitro "..." refers to events that occur in artificial environments, such as test tubes or reaction vessels, cell cultures, etc., rather than within multicellular organisms.

[0062] In vivo As used herein, the term "in vivo ( in vivo)" refers to events occurring within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within active cells (as opposed to, for example, in vitro systems).

[0063] isolated As used herein, the term “isolated” means (1) isolated from at least some of the combined components when originally produced (whether in a natural or experimental environment) and / or (2) a material and / or entity produced, manufactured and / or made by human hands. The isolated material and / or entity may be isolated to 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 originally combined. In some embodiments, the isolated formulation has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher than about 99%. As used herein, the material is "pure" where there are substantially no other components. As used herein, excipients (e.g., buffers, solvents, water, etc.) should not be included in the calculation of the purity percentage of the isolated material and / or entity.

[0064] Local distribution or delivery : As used herein, the terms “local distribution,” “local delivery,” or their grammatical equivalents refer to tissue-specific delivery or distribution. Generally, local distribution or delivery requires that a peptide or protein (e.g., an enzyme) encoded by mRNA be translated and expressed within a cell, or be secreted in a limited manner so as not to enter the patient’s circulatory system.

[0065] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide encoding at least one polypeptide. 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 using a recombinant expression system and selectively purified, or chemically synthesized. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs such as chemically modified bases or sugars, analogs having backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in the 5’ to 3’ direction. In some embodiments, mRNA is a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); Nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothimidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocitidine, 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-phosphomidite bonds) or include these.

[0066] Nucleic acid:As used herein, the term “nucleic acid” in the broadest sense refers to any compound and / or substance that is or may be incorporated into a polynucleotide chain. In some embodiments, the nucleic acid is a compound and / or substance that is or may be incorporated into a polynucleotide chain via phosphodiester linkages. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain containing individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Additionally, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having a backbone other than a phosphodiester backbone.

[0067] patient: As used herein, the terms “patient” or “subject” refer to any organism to which a composition provided for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes may be administered. A typical patient is an animal ( for example Includes mice, rats, rabbits, non-human primates and / or mammals such as humans. In some embodiments, the patient is human. Humans include prenatal and postnatal forms.

[0068] pharmaceutically acceptable As used herein, the term "pharmaceuticalally acceptable" refers to a substance that, within the scope of thorough medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and corresponds to a reasonable benefit / risk ratio.

[0069] Pharmaceutically acceptable salts : Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. [in the literature on pharmaceutically acceptable salts J. Pharmaceutical SciencesThis is described in detail in

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

[0070] Potency:As used herein, the term "efficacy" or its grammatical equivalent refers to the expression level of a protein(s) or peptide(s) encoded by mRNA and / or the biological effect resulting therefrom.

[0071] salt As used herein, the term "salt" refers to an ionic compound that is or may be produced by a neutralization reaction between an acid and a base.

[0072] Systemic distribution or transmission As used herein, the terms "systemic distribution" or "systemic delivery" or their grammatical equivalents refer to a mechanism or approach of delivery or distribution that affects the whole or the entire organism. Generally, systemic distribution or delivery is achieved through the body's circulatory system, such as blood flow, for example. Compare with the definition of "local distribution or delivery."

[0073] subject As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject refers to a human who goes to a healthcare provider for the diagnosis or treatment of a disease and may be a patient. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject may or may not be susceptible to a disease or disorder.

[0074] substantiallyAs used herein, the term “substantially” refers to a qualitative state indicating the full or nearly full range or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely become complete or progress to become complete, or achieve or avoid absolute results (even if they do). Therefore, the term “substantially” is used herein to express the potential lack of completeness inherent in many biological and chemical phenomena.

[0075] Target tissues As used herein, the term "target tissue" refers to any tissue where the disease to be treated has occurred. In some embodiments, target tissue includes tissues exhibiting disease-related pathology, symptoms, or characteristics.

[0076] Treatment Index As used here, the "therapeutic index" is the ratio of the blood drug concentration at which the drug becomes toxic to the concentration at which the drug is effective. The higher the therapeutic index, the safer the drug.

[0077] therapy As used herein, the term "treatment" (treat, treatment, or treating) refers to any method that partially or completely alleviates, improves, relieves, suppresses, prevents, delays the onset of, reduces the severity of, or decreases the frequency of occurrence of one or more symptoms or features of a specific disease, disorder, and / or condition. Treatment may be administered to subjects who show no signs of the disease or only early signs of the disease, for the purpose of reducing the risk of developing disease-related conditions.

[0078] transference numberAs 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" is used interchangeably with the term "yield."

[0079] Aliphatic: As used herein, the term "aliphatic" is C 1- C 40 It refers to hydrocarbons and includes both saturated and unsaturated carbohydrates. Aliphates can be linear, branched, or cyclic. For example, C1-C 20 Aliphates are C1-C 20 Alkyl (e.g., linear or branched C1-C 20 saturated alkyl), C2-C 20 Alkenyl (e.g., linear or branched C4-C 20 Dienyl, linear, or branched C6-C 20 Trienyl, etc.), and C2-C 20 Alkynyl (e.g., linear or branched C2-C 20 It may include alkynyl. C1-C 20 Aliphates are C3-C 20 Circular aliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl, or C8-C 20It may include cycloalkynyl). In a given embodiment, the aliphatic may include one or more cyclic aliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and optionally may be substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. The aliphatic group may not be substituted or may be substituted with one or more substituents as described herein. For example, the aliphatic may be substituted with one or more of halogens, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), and in the formula R' Each instance of is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted C1-C3 alkyl. In an embodiment, the aliphatic is not substituted. In an embodiment, the aliphatic does not contain any heteroatom.

[0080] alkyl: As used herein, the term "alkyl" refers to acyclic linear and branched hydrocarbon groups, for example, "C1-C 20"Alkyl" refers to an alkyl group having 1 to 20 carbons. The alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentylhexyl, isohexyl, etc. Other alkyl groups will be readily apparent to those skilled in the art in consideration of the advantages of this disclosure. The alkyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group may be one or more of halogens, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected). It can be substituted with a substituent, and among the expressions R' Each instance of is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted C1-C3 alkyl. In an embodiment, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In an embodiment, the alkyl group is substituted with an a-OH group, which may be referred to herein as a "hydroxyalkyl" group, where the prefix indicates an -OH group and "alkyl" is as described herein.

[0081] alkenylAs used herein, "alkenyl" means any linear or branched carbon-hydrogen chain having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, for example, "C2-C 20 "Alkenyl" refers to an alkenyl group having 2 to 20 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In an embodiment, the alkenyl comprises 1, 2, or 3 carbon-carbon double bonds. In an embodiment, the alkenyl comprises a single carbon-carbon double bond. In an embodiment, a plurality of double bonds (e.g., 2 or 3) are conjugated. The alkenyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkenyl group may be a halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, It may be substituted with one or more of -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), and in the formula R' Each instance of is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R'is an independently unsubstituted C1-C3 alkyl. In an embodiment, the alkenyl is not substituted. In an embodiment, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In an embodiment, the alkenyl group is substituted with an a-OH group, which may be referred to herein as a "hydroxyalkenyl" group, where the prefix indicates an --OH group, and "alkenyl" is as described herein.

[0082] alkynyl As used herein, "alkynyl" means a carbon-hydrogen chain of any linear or branched configuration having one or more carbon-carbon triple bonds occurring 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-inyl, but-2-inyl, but-3-inyl, pent-2-inyl, 3-methylpent-4-inyl, hex-2-inyl, hex-5-inyl, etc. In an embodiment, the alkynyl comprises one carbon-carbon triple bond. The alkynyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group may be substituted with one or more of halogens, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), and During the meal R' Each instance of is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15Alkyl, C1-C 10 It is an alkyl, or C1-C3 alkyl). In an embodiment, R' is an independently unsubstituted C1-C3 alkyl. In an embodiment, the alkynyl is not substituted. In an embodiment, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0083] 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 6 to 14 ring members, wherein the ring system has a single attachment point to the remainder of the molecule, at least one ring of the system is aromatic, and each ring of the system contains 4 to 7 ring members. In an embodiment, the aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C 10 "Aryl"; e.g., naphthyls such as 1-naphthyl and 2-naphthyl. In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 "Aryl"; e.g., anthracyl. "Aryl" also includes ring systems, where, as defined above, the aryl ring is condensed with one or more carboclicly or heterocycllyl groups, and the radical or attachment site is on the aryl ring, in which case the number of carbon atoms continues to specify the number of carbon atoms within the aryl ring system. Exemplary aryls include phenyl, naphthyl, and anthracene.

[0084] arylene: The term "arylene" as used herein refers to a divalent aryl group (i.e., having two attachment sites on the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted phenylene or substituted phenylene).

[0085] Composition of the present invention

[0086] In some embodiments, the present invention provides a composition comprising LNP and mRNA, which, when administered to a subject, induces significantly higher levels of mRNA expression in vivo without altering the subject's tolerance or stress levels. Tolerance or stress is determined by the elevation of the liver enzymes aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT). In some embodiments, a specific formulation provides advantages in manufacturing, for example, ease of manufacturing methods such as using a common pre-formed LNP mother liquor.

[0087] Observations of the present invention have shown that when an mRNA-LNP formed by mixing mRNA with a pre-formed empty LNP in step (a) is further combined with a pre-formed LNP in step (b) to form an mRNA-LNP composition, the efficacy of the resulting composition is significantly increased compared to the mRNA-LNP of step (a). This is particularly noteworthy because increased efficacy is observed even when the pre-formed LNP is empty (i.e., does not contain mRNA) and contains the same lipid components as the mRNA-LNP. Furthermore, an increase in the expression of the protein encoded by the mRNA is observed even when the pre-formed LNP contains only neutral lipids, which are known to be poor promoters of polynucleotide transfection.

[0088] Therefore, the fact that increased efficacy of the mRNA-LNP composition can be achieved without compromising tolerability in vivo is a significant advantage of the method of the present invention in terms of therapeutic design.

[0089] This aspect of the present invention allows for at least two substantial advantages: (i) increasing the therapeutic index of a composition by making it possible to lower the amount of mRNA in the mRNA therapeutic composition per dose or lower the frequency of administration to achieve the same biological effect; and (ii) enabling the development of an easy, flexible, scalable, and / or high-throughput manufacturing method that allows one or more pre-formed LNPs to be prepared in bulk and utilized in a number of mixing and combining steps to achieve a desired formulation such as that described in the present invention.

[0090] The present invention provides a method for further combining mRNA-LNPs, prepared by mixing mRNA with pre-formed empty LNPs, with pre-formed LNPs, wherein the resulting mRNA-LNP composition of the present invention increases the in vivo expression of a protein encoded by mRNA. In some embodiments, this method is a manufacturing method comprising the following steps: (a) mixing pre-formed empty LNPs with mRNA under conditions that allow for the formation of mRNA-LNPs; (b) combining the mRNA-LNPs formed in step (a) with pre-formed LNPs to prepare a composition comprising lipid nanoparticles that encapsulate mRNA. In some embodiments, the lipid nanoparticles comprise at least cationic lipids, non-cationic lipids, and PEG-modified lipids. In some embodiments, the lipid nanoparticles may comprise neutral lipids with or without cationic lipids.

[0091] In some embodiments, mRNA encodes a protein or a peptide.

[0092] In some embodiments, the LNP pre-formed in step (b) is an empty LNP. In some embodiments, the LNP pre-formed in step (b) contains mRNA. In some embodiments, the LNP pre-formed in step (b) contains mRNA encoding a protein or peptide. In some embodiments, the LNP pre-formed in step (b) contains the same mRNA encoding the same protein or polypeptide as that in the mRNA-LNP formed in step (a). In some embodiments, the LNP pre-formed in step (b) contains a different mRNA encoding a different protein or polypeptide than that in the mRNA-LNP formed in step (a).

[0093] In some embodiments, the empty LNP in step (a) and the pre-formed LNP in step (b) are distinct heterogeneous lipid nanoparticles. For example, the empty LNP in step (a) may comprise the cationic lipid HGT-5003, and the pre-formed LNP in step (b) may comprise the cationic lipid ICE. In another example, the empty LNP in step (a) may comprise the cationic lipid ICE, and the pre-formed LNP in step (b) may comprise the cationic lipid DOTAP. In yet another example, the empty LNP in step (a) may comprise the cationic lipid HGT-4001, and the pre-formed LNP in step (b) may comprise the cationic lipid ckk-E12. Various lipids suitable for LNPs and methods for producing them are described in each of the sections below, and any combination of lipids for forming LNPs is considered herein.

[0094] In one embodiment, the mRNA-LNP composition produced in step (b) may comprise a first lipid nanoparticle and a second lipid nanoparticle; wherein the first lipid nanoparticle and the second lipid nanoparticle have the same lipid composition, and at least some of the first lipid nanoparticles comprise mRNA. In one embodiment, the mRNA-LNP composition produced in step (b) may comprise a first lipid nanoparticle and a second lipid nanoparticle; wherein the first lipid nanoparticle and the second lipid nanoparticle have distinct lipid compositions. For example, the mRNA-LNP composition may comprise a first lipid nanoparticle comprising the cationic lipid ICE and a second lipid nanoparticle comprising the cationic lipid DOTAP. In some embodiments, the mRNA-LNP composition produced in step (b) may comprise a first lipid nanoparticle comprising the cationic lipid C12-200 and a second lipid nanoparticle comprising the cationic lipid DLinKC2DMA. Accordingly, any combination of various lipids suitable for generating LNPs is considered herein as described in each of the following sections.

[0095] In some embodiments, the empty LNP of step (a) or the pre-formed LNP of step (b) does not contain cationic lipids. In some embodiments, the empty LNP or the pre-formed LNP contains neutral lipids and / or PEG-modified lipids.

[0096] Lipid nanoparticles (LNP)

[0097] The present invention provides, among other things, a composition for an mRNA therapeutic in which mRNA is encapsulated in a delivery vehicle for efficient cellular absorption and in vivo processing. As used herein, the terms "delivery vehicle," "transport vehicle," "nanoparticle," or grammatically equivalent terms are used interchangeably. The delivery vehicle may be formulated in combination with one or more additional nucleic acids, carriers, target ligands, or stabilizing reagents, or formulated as a pharmaceutical composition mixed with suitable excipients. Techniques for the formulation and administration of drugs can be found in the latest edition of the literature ["Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pa.]. A specific delivery vehicle is selected based on its ability to facilitate the transfection of nucleic acids into target cells.

[0098] In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, e.g., lipid nanoparticles (LNPs) or liposomes. In some embodiments, the liposome comprises one or more cationic lipids. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, the liposome comprises four or fewer distinct lipid components. In some embodiments, the liposome comprises three or fewer distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid. In a typical embodiment, the LNP of the present invention is a liposome that encapsulates mRNA. Suitable lipid components of the liposome include cationic lipids (e.g., cKK-E12, compound 1, compound 2, or compound 3), non-cationic lipids (DEPE), cholesterol-based lipids (e.g., cholesterol), and PEG-modified lipids (DMG-PEG2K). Alternatively, suitable lipid components of the liposome include sterol-based cationic lipids (e.g., ICE), non-cationic lipids (DEPE), and PEG-modified lipids (e.g., DMG-PEG2K).

[0099] In some embodiments, DEPE in lipid nanoparticles can enhance the expression of mRNA encoding a protein or peptide compared to lipid nanoparticles that have the same composition (in terms of the molar ratio of lipid components and individual lipid components) as DEPE-containing lipid nanoparticles, particularly except that they contain DOPE. In some embodiments, the expression of mRNA encoding a protein delivered by DEPE-containing lipid nanoparticles is enhanced by at least twofold compared to DOPE-containing lipid nanoparticles. The enhancement of mRNA expression can be determined by administering identically formulated lipid nanoparticles containing DEPE-containing lipid nanoparticles and different helper lipids (e.g., DOPE) to test animals (e.g., mice) (e.g., by tail vein injection) and monitoring mRNA expression at one or more time points (e.g., 4, 6, 8, 12, 18, or 24 hours after administration).

[0100] In some embodiments, the mRNA encodes a protein that is translated into a therapeutic protein in vivo. In some embodiments, the mRNA encoding the protein encodes a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an antibody light chain or an antibody heavy chain. In some embodiments, the therapeutic polypeptide is absent or deficient in the subject to whom the mRNA is administered. In some embodiments, the mRNA encoding the protein encodes a peptide. In some embodiments, the peptide is an antigen.

[0101] In some embodiments, the DEPE-containing lipid nanoparticles of the present invention are safe and tolerable when administered to subjects. For example, the DEPE-containing lipid nanoparticles of the present invention do not cause any distinct hepatotoxicity when administered to subjects. Suitable markers for evaluating hepatotoxicity are ALT and AST.

[0102] cationic lipids

[0103] As used herein, the phrase "cationic lipid" refers to any one of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0104] Cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids 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 comprise cationic lipids, namely (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate having the following compound structure and pharmaceutically acceptable salts thereof:

[0105] .

[0106] Other cationic lipids suitable for use in the compositions and methods of the present invention include ionizable cationic lipids described in International Patent Publication WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of one of the following formulas or a pharmaceutically acceptable salt thereof:

[0107]

[0108] In the formula, R1 and R2 are each independently hydrogen, optionally substituted variable saturated or unsaturated C1-C 20 Alkyl, and optionally substituted variablely saturated or unsaturated C6-C 20 Selected from the group consisting of acyl, where L1 and L2 are each independently hydrogen, arbitrarily substituted C1-C 30 Alkyl, optionally substituted variable unsaturated C1-C 30 alkenyl, and optionally substituted C1-C 30Selected from the group consisting of alkynoyls, m and o are each independently selected from the group consisting of 0 and any positive integer (e.g., m is 3), and n is 0 or any positive integer (e.g., n is 1). In certain embodiments, the composition and method of the present invention comprise a cationic lipid (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-15,18-dien-1-amine ("HGT5000") having the following compound structure and a pharmaceutically acceptable salt thereof:

[0109]

[0110] (HGT-5000).

[0111] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely (15Z, 18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl) tetracosa-4,15,18-triene-l-amine ("HGT5001") having the following compound structure, and a pharmaceutically acceptable salt thereof:

[0112]

[0113] (HGT-5001).

[0114] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-diene-1-yl) tetracosa-5,15,18-triene-1-amine ("HGT5002") having the following compound structure, and a pharmaceutically acceptable salt thereof:

[0115]

[0116] (HGT-5002).

[0117] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids described as amino alcohol lipids in International Patent Publication WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0118] .

[0119] In some embodiments, the lipids used in the compositions and methods of the present invention are synthesized by reacting commercially available amines with lipophilic acrylates, acrylamides, or epoxides. In some embodiments, the lipids are derived from amine 86 (N,N-bis(2-hydroxyethyl)ethylenediamine) and amine 87 (N-(3-aminopropyl)diethanolamine). Lipids have several advantages as a potential new class of nucleic acid delivery reagents: (i) the chemicals used to synthesize lipidoids are simple and economical, (ii) a library of structural diversity has already been developed, and (iii) the correlation between the structure and function of the delivery system can be constructed from a large dataset accumulated by screening the lipidoid library. Since these reactions are simple, it has been possible to construct a structurally diverse lipidoid library by varying the type of amine and the length and type of the tail (or carbon-arm chain) (acrylamide / acrylate / epoxide).

[0120] In some embodiments, the lipidoid comprises about 2 to 20 carbon-arm chains. In some embodiments, the lipidoid comprises about 5 to 18 carbon-arm chains. In some embodiments, the lipidoid comprises about 10 to 16 carbon-arm chains. In some embodiments, the lipidoid comprises about 10 to 14 carbon-arm chains. In some embodiments, the lipidoid comprises about 10 to 12 carbon-arm chains. In some embodiments, the lipidoid comprises about 10 carbon-arm chains. In some embodiments, the lipidoid comprises about 12 carbon-arm chains. In some embodiments, the lipidoid comprises about 14 carbon-arm chains. In some embodiments, the lipidoid comprises about 16 carbon-arm chains.

[0121] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0122] .

[0123]

[0124] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0125] .

[0126] ​Other cationic ions suitable for use in the compositions and methods of the present invention include cationic lipids having the chemical formula 14,25-detridecyl 15,18,21,24-tetraza-octatriacontan and pharmaceutically acceptable salts thereof.

[0127] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids described in International Patent Publications WO 2013 / 063468 and WO 2016 / 205691, both of which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula or a pharmaceutically acceptable salt thereof:

[0128]

[0129] During the meal, R L Each instance of is independently a randomly substituted C6-C 40 It is an alkenyl. In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0130] .

[0131] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0132] .

[0133] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0134] .

[0135] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0136] .

[0137] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula or a pharmaceutically acceptable salt thereof:

[0138]

[0139] In the formula, each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and each R A is independently hydrogen, an optionally substituted C1-50 alkyl, an optionally substituted C2-50 alkenyl, an optionally substituted C2-50 alkynyl, an optionally substituted C3-10 carbocyclyl, an optionally substituted 3-14 heterocyclyl, an optionally substituted C6-14 aryl, an optionally substituted 5-14 heteroaryl, or a halogen, and each R B 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 heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 heteroaryl, or halogen. In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely "Target 23" having the following compound structure, and a pharmaceutically acceptable salt thereof:

[0140]

[0141] (Target 23).

[0142]

[0143] ​Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures or pharmaceutically acceptable salts thereof:

[0144] .

[0145] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0146] .

[0147] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0148] .

[0149] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formulas or pharmaceutically acceptable salts thereof:

[0150]

[0151] Each R in the equation 1 and R 2 is independently H or C1-C6 aliphatic; each m is an integer having a value from 1 to 4 independently; each A is independently a covalent bond or an arylene; and each L 1 is independently an ester group, a thioester group, a disulfide group, or an anhydride group; and each L 2 is independently C2-C 10Aliphatic and; each X 1 is independently H or OH; and each R 3 is independently C6-C 20 The composition and method of the present invention are. In some embodiments, the composition and method of the present invention comprise a cationic lipid of the following formula or a pharmaceutically acceptable salt thereof:

[0152]

[0153] (Compound 1).

[0154] In some embodiments, the composition and method of the present invention comprise a cationic lipid of the following formula or a pharmaceutically acceptable salt thereof:

[0155]

[0156] (Compound 2).

[0157] In some embodiments, the composition and method of the present invention comprise a cationic lipid of the following formula or a pharmaceutically acceptable salt thereof:

[0158]

[0159] (Compound 3).

[0160] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids such as those described in the literature of J. McClellan, MC King [Cell 2010, 141, 210-217] and the literature of Whitehead et al. [Nature Communications (2014) 5:4277], which are incorporated herein by reference. In certain embodiments, the cationic lipids of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0161] .

[0162] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0163] .

[0164] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0165] .

[0166] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0167] .

[0168] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0169] .

[0170] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0171] .

[0172] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0173] .

[0174] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0175] .

[0176] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0177] .

[0178] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0179] .

[0180] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0181] .

[0182] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0183] .

[0184] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0185] .

[0186] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0187] .

[0188] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0189] .

[0190] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0191] .

[0192] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0193] .

[0194] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0195] .

[0196] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0197] .

[0198] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0199] .

[0200] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0201] .

[0202] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0203] .

[0204] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0205] .

[0206] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0207] .

[0208] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0209] .

[0210] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0211] .

[0212] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0213] .

[0214] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0215] .

[0216] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0217] .

[0218] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0219] .

[0220] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0221] .

[0222] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula or a pharmaceutically acceptable salt thereof:

[0223]

[0224] During the meal, 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 one 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, and G 1 and G 2 Each is an independently unsubstituted C1-C 12 Alkylene or C1-C 12 It is alkenylene, and G 3 is C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, and R a is H or C1-C 12 It is alkyl, and R 1 and R 2 Each independently C6-C 24 alkyl or C6-C 24 It is alkenyl, and R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 It is alkyl, and R 5 is H or C1-C6 alkyl, and x is 0, 1, or 2.

[0225] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0226] .

[0227] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0228] .

[0229] In some embodiments, the composition and method of the present invention comprises a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0230] .

[0231] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the compositions and methods of the present invention include a compound of one of the following formulas and a pharmaceutically acceptable salt thereof:

[0232]

[0233] and

[0234]

[0235] In any one of these four chemical formulas, R4 is -(CH2) n Q and -(CH2) n Independently selected from CHQR, where Q is -OR, -OH, -O(CH2) nSelected from the group consisting of 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 heterocycles, where n is 1, 2, or 3. In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0236] .

[0237] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0238] .

[0239] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0240] .

[0241] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0242] .

[0243] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids described in International Patent Publications WO 2017 / 173054 and WO 2015 / 095340, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structures and pharmaceutically acceptable salts thereof:

[0244] .

[0245] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0246] .

[0247] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0248] .

[0249] In a given embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure and a pharmaceutically acceptable salt thereof:

[0250] .

[0251] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids described in International Patent Publication WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formulas or pharmaceutically acceptable salts thereof:

[0252]

[0253] In the formula, R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkyl amino (e.g., alkyl amino such as dimethylamino), and pyridyl, and R2 is selected from the group consisting of one of the following two formulas, and

[0254] and

[0255] In the formula, R3 and R4 are each independently arbitrarily substituted, variablely saturated or unsaturated C6-C 20Alkyl, and optionally substituted, variablely saturated or unsaturated C6-C 20 Asil, and n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or higher).

[0256] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely "HGT4001" having the following compound structure and a pharmaceutically acceptable salt thereof:

[0257]

[0258] (HGT4001).

[0259] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely "HGT4002" having the following compound structure and a pharmaceutically acceptable salt thereof:

[0260]

[0261] (HGT4002).

[0262] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely "HGT4003" having the following compound structure and a pharmaceutically acceptable salt thereof:

[0263]

[0264] (HGT4003).

[0265] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely "HGT4004" having the following compound structure and a pharmaceutically acceptable salt thereof:

[0266]

[0267] (HGT4004).

[0268] In a given embodiment, the composition and method of the present invention comprise a cationic lipid, namely “HGT4005” having the following compound structure and a pharmaceutically acceptable salt thereof:

[0269]

[0270] (HGT4005).

[0271] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids such as those described in U.S. Provisional Patent Application No. 62 / 672,194 filed May 16, 2018, which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids corresponding to any one of the general formulas described in U.S. Provisional Patent Application No. 62 / 672,194 or any one of structures (1a)-(21a), (1b)-(21b), and (22)-(237). In certain embodiments, the compositions and methods of the present invention include cationic lipids having a structure according to formula (I') and pharmaceutically acceptable salts thereof, wherein:

[0272]

[0273] During the meal

[0274] R X is independently -H, -L 1 -R 1 , or -L 5A -L 5B -B' and;

[0275] L 1 , L 2 , and L 3 Each is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L - and;

[0276] Each L 4A and L 5A is independently -C(O)-, -C(O)O-, or -C(O)NR L - and;

[0277] Each L 4B and L 5B C1-C independently 20 Alkylene, C2-C 20 Alkenylene, or C2-C 20 It is alkynylene;

[0278] Each B and B' is NR 4 R 5 or is a 5- to 10-membered nitrogen-containing heteroaryl;

[0279] Each R 1 , R 2 , and R 3 is independently C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 It is alkinyl;

[0280] Each R 4 , and R 5 C1-C independently 10 Alkyl, C2-C 10 Alkenyl, or C2-C 10 It is alkynyl;

[0281] Each R L It independently contains hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, or C2-C 20 It is alkinil.

[0282] In a specific embodiment, the composition and method of the present invention comprise a cationic lipid having the following compound structure, namely, a compound (139) of 62 / 672,194:

[0283]

[0284] ("18:1 carbon tail-ribose lipid").

[0285] In some embodiments, the compositions and methods of the present invention comprise a cationic lipid, namely N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”) (see Feigner et al. incorporated herein by reference [Proc. Nat'l Acad. Sci. 84, 7413 (1987)]; U.S. Patent No. 4,897,355). Other cationic lipids suitable for the compositions and methods of the present invention are, for example, 5-carboxyspermylglycinedioctadecylamide (“DOGS”); It includes 2,3-dioleyloxy-N-[2(spermin-carboxamidoethyl]-N,N-dimethyl-l-propaneamin ("DOSPA") (Behr et al. [Proc. Nat.'l Acad. Sci. 86, 6982 (1989)]; U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761); l,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); l,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0286] Further examples of cationic lipids suitable for the compositions and methods of the present invention also include: 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide("DMRIE"); 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-l-(cis,cis-9,12-octadecadieneoxy)propane("CLinDMA"); 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl ll-(cis,cis-9', l-2'-octadecanedieneoxy)propane("CpLinDMA"); N,N-dimethyl-3,4-dioleyyloxybenzylamine("DMOBA"); 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine("DLinDAP"); l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane("DLincarbDAP"); l,2-dilinoleoylcarbamyl-3-dimethylaminopropane("DLinCDAP"); 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl l-3-[(9Z, 12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine("octyl-CLinDMA"); (2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl l-3-[(9Z, 12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine("octyl-CLinDMA (2R)");(2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyh3-[(9Z, 12Z)-octadeca-9, 12-diene-1-yloxy]propan-1-amine("Octyl-CLinDMA (2S)"); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,l 2-diene-1-yl)-l ,3-dioxolane-4-yl)-N,N-dimethylethanolamine ("DLin-KC2-DMA") (WO 2010 / 042877 incorporated herein by reference; see 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, one or more of the cationic lipids comprise at least one of an imidazole, a dialkylamino, or a guanidinium moiety.;

[0287] In some embodiments, one or more cationic lipids suitable for the composition and method of the present invention comprise 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-diethyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0288] In some embodiments, the composition of the present invention comprises one or more cationic lipids constituting at least about 5 wt%, 10 wt%, 20 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, or 70 wt% of the total lipid content in the composition, e.g., lipid nanoparticles. In some embodiments, the composition of the present invention comprises one or more cationic lipids constituting at least about 5 mol%, 10 mol%, 20 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, or 70 mol% of the total lipid content in the composition (e.g., lipid nanoparticles). In some embodiments, the composition of the present invention comprises one or more cationic lipids constituting the total lipid content in the composition, for example, about 30 to 70 wt% of lipid nanoparticles (e.g., about 30 to 65 wt%, about 30 to 60 wt%, about 30 to 55 wt%, about 30 to 50 wt%, about 30 to 45 wt%, about 30 to 40 wt%, about 35 to 50 wt%, about 35 to 45 wt%, or about 35 to 40 wt%). In some embodiments, the composition of the present invention comprises one or more cationic lipids constituting the total lipid content in the composition, for example, about 30 to 70 mol% of lipid nanoparticles (e.g., about 30 to 65 mol%, about 30 to 60 mol%, about 30 to 55 mol%, about 30 to 50 mol%, about 30 to 45 mol%, about 30 to 40 mol%, about 35 to 50 mol%, about 35 to 45 mol%, or about 35 to 40 mol%).

[0289] In some embodiments, sterol cationic lipids may be used instead of or in addition to the cationic lipids described herein. Suitable sterol cationic lipids are dialkylamino-, imidazole-, and guanidinium-containing sterol cationic lipids. For example, a given embodiment relates to a composition comprising one or more sterol-based cationic lipids, such as an imidazole, e.g., an imidazole cholesterol ester, i.e., “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptane-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazole-4-yl)propanoate, as indicated by structural formula (I) below. In a given embodiment, as indicated by the following structural formula, lipid nanoparticles for delivering RNA (e.g., mRNA) encoding a functional protein may comprise one or more imidazole cationic lipids, e.g., imidazole cholesterol ester, i.e., “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptane-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazole-4-yl)propanoate:

[0290] (ICE).

[0291] In some embodiments, the percentage of cationic lipids in the liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. In some embodiments, the cationic lipid(s) constitute about 30 to 50 wt% of the liposome (e.g., about 30 to 45 wt%, about 30 to 40 wt%, about 35 to 50 wt%, about 35 to 45 wt%, or about 35 to 40 wt%). In some embodiments, the cationic lipids (e.g., ICE lipids) constitute about 30%, about 35%, about 40%, about 45%, or about 50% of the liposome in molar ratio.

[0292] Non-cationic Lipids

[0293] As used herein, the term “non-cationic lipid” refers to any neutral, zwitterionic, or anionic lipid, which is also referred to herein as “helper lipid”. As used herein, the term “anionic lipid” refers to any one of a number of lipid species that possess a net negative charge at a selected pH, e.g., physiological pH.

[0294] The present invention relates to mRNA-LNPs comprising one or more non-cationic helper lipids, including 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). In some embodiments, DEPE is the only non-cationic helper lipid in the mRNA-LNP. In other embodiments, the helper lipid portion of the mRNA-LNP comprises DEPE and cholesterol.

[0295] Without being bound by any particular theory, certain DEPE derivatives having lipid chain lengths or compositions different from DEPE are also included in the present invention. For example, the inventors have found that alkyl or alkene chains having a length of 10 to 20 carbons are particularly suitable for the formation of mRNA-LNPs. In some embodiments, DEPE derivatives having alkyl or alkene chains having a length of 16 to 20 carbons are particularly preferred. Alternatively, DEPE derivatives having alkyl or alkene chains having a length of 10 to 14 carbons, for example, 10, 12, or 14 carbons, may be particularly suitable for the practice of the present invention.

[0296] Other non-cationic lipids or helper lipids that may be included in mRNA-LNP are distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), Phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof, but are not limited to these.

[0297] In some embodiments, such non-cationic lipids may be used alone, but preferably in combination with other lipids, e.g., cationic lipids. In some embodiments, the non-cationic lipids may comprise about 5% to about 90%, or about 10% to about 70%, of the total lipids present in the liposomes. In some embodiments, the non-cationic lipids are neutral lipids, i.e., lipids that do not carry a net charge under the conditions in which the composition is formulated and / or administered. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0298] Cholesterol-based Lipids

[0299] In some embodiments, the composition (e.g., liposomal composition) comprises one or more cholesterol-based lipids. For example, a cholesterol-based lipid suitable for carrying out the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al. [Biochem. Biophys. Res. Comm. 179, 280 (1991)]; Wolf et al. [BioTechniques 23, 139 (1997)]; U.S. Patent No. 5,744,335), or imidazole cholesterol esters (ICE).

[0300] In some embodiments, cholesterol-based lipids may be present in a molar ratio (mol%) 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 the lipid nanoparticles may be greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, or greater than about 40%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0301] In some embodiments, cholesterol-based lipids may be present in a weight part (wt%) of about 1% to about 30%, or about 5% to about 20%, of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be about 5 wt% or less, about 10 wt% or less, about 20 wt% or less, about 30 wt% or less, or about 40 wt% or less.

[0302] PEGylated lipids

[0303] In some embodiments, the suitable lipid solution comprises one or more PGE-modified lipids, which are also referred to herein as PEG-modified lipids. A PEG-modified lipid or PEG-modified lipid suitable for carrying out the present invention is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K). For example, the use of derived lipids, such as derived ceramides (PEG-CER) including polyethylene glycol (PEG)-modified phospholipids and N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), is also considered in the present invention. The PEG-modified lipids considered are C6-C 20 Polyethylene glycol chains up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length are covalently bonded to lipids having alkyl chain(s) of length, but are not limited thereto. In some embodiments, the PEG-modified lipid or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, particularly useful exchangeable lipids are shorter acyl chains (e.g., C 14 or C 18 It is a PEG-ceramide having ). By adding such components, complex aggregation can be prevented, and means to extend circulation life and increase the delivery of the lipid-nucleic acid composition to target tissues can be provided (see Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or these components can be selected to be rapidly exchanged from the formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful exchangeable lipids are those with shorter acyl chains (e.g., C 14 or C 18It is a PEG-ceramide having ). The PEG-modified phospholipid and derived lipid of the present invention may comprise a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome transport vehicle.

[0304] PEG-modified phospholipids and derivatized lipids may constitute about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% or less of the total lipids in a suitable lipid solution on a weight or molar basis. In some embodiments, PEG-modified lipids may constitute about 5% or less of the total lipids in a suitable lipid solution on a weight or molar basis. In some embodiments, PEG-modified lipids may constitute about 4% or less of the total lipids in a suitable lipid solution on a weight or molar basis. In some embodiments, PEG-modified lipids generally constitute about 3% or less of the total lipids in a suitable lipid solution on a weight or molar basis. In some embodiments, PEG-modified lipids generally constitute about 2% or less of the total lipids in a suitable lipid solution on a weight or molar basis. In some embodiments, the PEG-modified lipid generally constitutes about 1% or less of the total lipid in a suitable lipid solution by weight or molar concentration. In some embodiments, the PEG-modified lipid constitutes about 1-5%, about 1-4%, about 1-3%, or about 1-2% of the total lipid in a suitable lipid solution by weight or molar concentration. In some embodiments, the PEG-modified lipid constitutes 0.01-3% (e.g., about 0.01-2.5%, 0.01-2%, 0.01-1.5%, or 0.01-1%) of the total lipid in a suitable lipid solution by weight or molar concentration.

[0305] molar geological ratio

[0306] According to various embodiments, the selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids, including lipid nanoparticles as well as the relative molar ratios of these lipids to one another, is based on the characteristics of the selected lipid(s), the properties of the intended target cell, and the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity, and tolerability of the selected lipid(s). Thus, the molar ratio can be appropriately adjusted.

[0307] Various combinations of lipids, namely cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol, which can be used to prepare pre-formed lipid nanoparticles are described in the literature and herein. For example, suitable lipid solutions include 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; or HGT5001, DPPC, cholesterol, and DMG-PEG2K; Or it may contain ICE, DEPE, and DMG-PEG2K.Additional combinations of lipids are known to the art, for example, PCT / US17 / 61100, filed November 10, 2017, published as WO 2018 / 089790, with the invention title "Novel ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA"; PCT / US18 / 21292, filed March 7, 2018, published as WO 2018 / 165257, with the invention title "PolyAnionic Delivery of Nucleic Acids"; PCT / US18 / 36920, filed June 11, 2018, with the invention title "Poly (Phosphoesters) for Delivery of Nucleic Acids"; and U.S. Provisional Patent Application No. 62 / 676,147, filed May 24, 2018, with the invention title "Thioester Cationic Lipids"; U.S. Provisional Patent Application No. 62 / 677,821, filed on May 30, 2018, with the title "Cationic Lipids Comprising a Steroidal Moiety"; U.S. Provisional Patent Application No. 62 / 677,809, filed on May 30, 2018, with the title "Macrocyclic Lipids"; U.S. Provisional Patent Application No. 62 / 677,818, filed on May 30, 2018, with the title "Vitamin K Cationic Lipids"; U.S. Provisional Patent Application No. 62 / 677,828, filed on May 30, 2018, with the title "Vitamin D Cationic Lipids"; U.S. Provisional Patent Application No. 62 / 677,851, filed on May 30, 2018, with the title "Vitamin A Cationic Lipids"; U.S. Provisional Patent Application No. 62 / 677,855, filed on May 30, 2018, with the title of the invention “Vitamin E Cationic Lipids”, the entire contents of which are incorporated herein by reference.

[0308] In various embodiments, cationic lipids (e.g., cKK-E12, compound 1, compound 2, or compound 3, C12-200, ICE, and / or HGT4003) constitute about 30–60% of the liposome on a molar basis (e.g., about 30–55%, about 30–50%, about 30–45%, about 30–40%, about 35–50%, about 35–45%, or about 35–40%). In some embodiments, the percentage of cationic lipids (e.g., cKK-E12, compound 1, compound 2, or compound 3, C12-200, ICE, and / or HGT4003) on a molar ratio basis is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of the liposome.

[0309] In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be approximately 30 to 60:25 to 35:20 to 30:1 to 15. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:20:10. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:25:5. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol lipid(s) to PEG-modified lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5. In some embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is 50:45:5. In some embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is 50:40:10. In some embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is 55:40:5. In some embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is 55:35:10. In some embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is 60:35:5. In some embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is 60:30:10.

[0310] In some embodiments, the liposome suitable for the present invention comprises ICE and DEPE, and the molar ratio of ICE:DEPE is greater than 1:1. In some embodiments, the molar ratio of ICE:DEPE is less than 2.5:1. In some embodiments, the molar ratio of ICE:DEPE is 1:1 to 2.5:1. In some embodiments, the molar ratio of ICE:DEPE is approximately 1.5:1. In some embodiments, the molar ratio of ICE:DEPE is approximately 1.7:1. In some embodiments, the molar ratio of ICE:DEPE is approximately 2:1. In some embodiments, the liposome suitable for the present invention comprises ICE and DMG-PEG-2K, and the molar ratio of ICE:DMG-PEG-2K is greater than 10:1. In some embodiments, the molar ratio of ICE:DMG-PEG-2K is less than 16:1. In some embodiments, the molar ratio of ICE:DMG-PEG-2K is approximately 12:1. In some embodiments, the molar ratio of ICE:DMG-PEG-2K is approximately 14:1. In some embodiments, the liposome suitable for the present invention comprises DEPE and DMG-PEG-2K, and the molar ratio of DEPE:DMG-PEG-2K is greater than 5:1. In some embodiments, the molar ratio of DEPE:DMG-PEG-2K is less than 11:1. In some embodiments, the molar ratio of DEPE:DMG-PEG-2K is approximately 7:1. In some embodiments, the molar ratio of DEPE:DMG-PEG-2K is approximately 10:1.

[0311] In some embodiments, the liposome suitable for the present invention comprises ICE, DEPE, and DMG-PEG-2K, and the molar ratio of ICE:DEPE:DMG-PEG-2K is 50:45:5. In some embodiments, the liposome suitable for the present invention comprises ICE, DEPE, and DMG-PEG-2K, and the molar ratio of ICE:DEPE:DMG-PEG-2K is 50:40:10. In some embodiments, the liposome suitable for the present invention comprises ICE, DEPE, and DMG-PEG-2K, and the molar ratio of ICE:DEPE:DMG-PEG-2K is 55:40:5. In some embodiments, the liposome suitable for the present invention comprises ICE, DEPE, and DMG-PEG-2K, and the molar ratio of ICE:DEPE:DMG-PEG-2K is 55:35:10. In some embodiments, the liposome suitable for the present invention comprises ICE, DEPE, and DMG-PEG-2K, and the molar ratio of ICE:DEPE:DMG-PEG-2K is 60:35:5. In some embodiments, the liposome suitable for the present invention comprises ICE, DEPE, and DMG-PEG-2K, and the molar ratio of ICE:DEPE:DMG-PEG-2K is 60:30:10.

[0312] polymer

[0313] In some embodiments, a suitable delivery vehicle is formulated by using a polymer as a carrier alone or in combination with other carriers comprising various lipids described herein. Accordingly, in some embodiments, the liposome delivery vehicle used herein also includes nanoparticles comprising a polymer. Suitable polymers may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, arginate, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethyleneimine (PEI). Where PEI is present, PEI with a molecular weight in the range of 10 to 40 kDa may be branched, for example, 25 kDa branched PEI (Sigma #408727).

[0314] Messenger RNA (mRNA)

[0315] The present invention can be used to encapsulate any mRNA. mRNA is generally considered a type of RNA that carries information from DNA to ribosomes. Generally, in eukaryotes, mRNA processing involves adding a "cap" to the 5' end and a "tail" to the 3' end. The typical cap is a 7-methylguanosine cap, which is guanosine connected to the first transcribed nucleotide via a 5'-5'-triphosphate bond. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The addition of the tail is typically a polyadenylation event, through which 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. Messenger RNA is translated by ribosomes into a series of amino acids that make up proteins.

[0316] mRNA can be synthesized according to any one of various known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is generally performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphate, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0317] In some embodiments, the in vitro synthesized mRNA may be purified before formulation and encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis.

[0318] The present invention can be used to formulate and encapsulate mRNA of various lengths. In some embodiments, the present invention can be used to formulate and encapsulate mRNA synthesized in vitro with a length of about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or greater than 20 kb. In some embodiments, the present invention may be used to formulate and encapsulate mRNA synthesized in vitro, having a length in the range of about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0319] The present invention may be used to formulate and encapsulate unmodified mRNA or mRNA comprising one or more modifications that generally improve stability. In some embodiments, the modification is selected from a modified nucleotide, a modified sugar phosphate backbone, and a 5' and / or 3' untranslated region.

[0320] In some embodiments, the modification of mRNA may include modification of nucleotides of RNA. The modified mRNA according to the present invention may include, for example, skeletal modification, sugar modification, or base modification. In some embodiments, mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) comprising, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotide analogs or derivatives of purines and pyrimidines, for example, 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, Pseudo-uracil(5-uracil), Dihydrouracil, 2-Thio-uracil, 4-Thio-uracil, 5-Carboxymethylaminomethyl-2-Thio-uracil, 5-(Carboxyhydroxymethyl)-uracil, 5-Fluoro-uracil, 5-Bromo-uracil, 5-Carboxymethylaminomethyl-uracil, 5-Methyl-2-Thio-uracil, 5-Methyl-uracil, N-uracil-5-Methyl Oxyacetate, 5-Methylaminomethyl-uracil, 5-Methoxyaminomethyl-2-Thio-uracil, 5'-Methoxycarbonylmethyl-uracil, 5-Methoxy-uracil, Uracil-5-Methyl Oxyacetate, It can be synthesized as uracil-5-oxyacetic acid (v), 1-methyl-usauracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, and phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine, and inosine, etc.The manufacture of such analogues is known to those skilled in the art in U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and U.S. Patent No. 5,700,642, the entire contents of which are incorporated herein by reference.

[0321] Generally, mRNA synthesis involves adding a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0322] Accordingly, in some embodiments, mRNA includes a 5' cap structure. The 5' cap is typically added as follows: First, RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphate groups; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyl transferase to form a 5'5'5 triphosphate bond; then, the 7' nitrogen of guanine is methylated by methyltransferase. 2'-O-methylation may also occur at the first base and / or second base following the 7-methylguanosine triphosphate residue. Examples of cap structures are not limited to but include m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where m represents a 2'-O methyl residue).

[0323] 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, for example, an iron-responsive element. In some embodiments, the 5' untranslated region may be about 50 to 500 nucleotides long.

[0324] In some embodiments, the 3' untranslated region comprises one or more binding sites for a polyadenylation signal, a protein affecting the positional stability of mRNA within the cell, or one or more binding sites for miRNA. In some embodiments, the 3' untranslated region may be 50 to 500 nucleotides long or longer.

[0325] While mRNA provided from an in vitro transcription reaction may be preferred in some embodiments, other mRNA sources within the scope of the invention, including mRNA generated from bacteria, fungi, plants and / or animals, are considered.

[0326] The present invention can be used to formulate and encapsulate mRNA encoding various proteins. Non-limiting examples of mRNA suitable for the present invention include mRNA encoding spinal motor neuron 1 (SMN), alpha-galactosidase (GLA), argininosuccinate synthase (ASS1), ornithine transcarbamylase (OTC), factor IX (FIX), phenylalanine hydroxylase (PAH), erythropoietin (EPO), cystic fibrosis transmembrane conduction receptor (CFFF), and firefly luciferase (FFL). Exemplary mRNA sequences as disclosed herein are listed below:

[0327] Formation of lipid nanoparticles (LNPs)

[0328] A method for preparing lipid nanoparticles that encapsulate mRNA is also provided, said 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 one or more helper lipids comprises 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); and (b) forming lipid nanoparticles from the mixture provided in step (a), said method further comprising the step of encapsulating mRNA into the lipid nanoparticles, wherein encapsulation may be performed before or after forming the lipid nanoparticles in step (b). The resulting lipid nanoparticles that encapsulate mRNA are stable (e.g., maintaining the same encapsulation of mRNA before and after freeze-thaw, or maintaining the same encapsulation of mRNA within 10% before and after freeze-thaw). In one embodiment, a method for preparing lipid nanoparticles according to the present invention explicitly excludes the use of one or more helper lipids selected from the following: dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and combinations thereof.

[0329] Various encapsulation methods are described in U.S. Patent Application Publication No. 2011 / 0244026, U.S. Patent Application Publication No. 2016 / 0038432, U.S. Patent Application Publication No. 2018 / 0153822, U.S. Patent Application Publication No. 2018 / 0125989, and U.S. Provisional Patent Application No. 62 / 877,597 filed on July 23, 2019, and may 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 of encapsulating mRNA by mixing mRNA with a mixture of lipids without first forming the lipids into lipid nanoparticles, as described in U.S. Patent No. 2016 / 0038432. As used herein, Method B or “Remix” refers to a method of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA, as described in U.S. Patent No. 2018 / 0153822. “Step Down Remix” or “Step Up Remix” is an improved method based on the “Remix” method, as described in U.S. Provisional Patent Application No. 63 / 021,319. “Step Down Remix” involves mixing a suspension of pre-formed empty lipid nanoparticles with batches of mRNA solution added sequentially. With each addition of the mRNA solution batch, intermediate mixtures are produced with different molar ratios of cationic lipids to mRNA (“N / P”), starting high and decreasing to a low N / P ratio in the final formulation. In "Step Up Remix," a suspension of pre-formed empty lipid nanoparticles is added to an mRNA solution in a batch manner, starting with an equimolar ratio of cationic lipids to mRNA. For example, pre-formed empty lipid nanoparticles are added in four batches until the ratio of cationic lipids to mRNA reaches 4:1.

[0330] In one embodiment, DEPE is present in the mixture at a concentration of 10 mol% to 50 mol%. More generally, DEPE in the mixture is present at a concentration of 25 mol% to 35 mol% of the total lipids in the mixture. In one embodiment, one or more PEG-modified lipids in the mixture are C6-C 20 It comprises a poly(ethylene) glycol chain up to 5 kDa in length covalently attached to a lipid having an alkyl chain of length. In one embodiment, the mixture of one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids further comprises one or more sterols, such as cholesterol-based lipids. In one embodiment, one or more cholesterol-based lipids are cholesterol and / or PEGylated cholesterol. In some embodiments, the ratio of cationic lipid(s) to helper lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be between about 30 to 60:25 to 35:20 to 30:1 to 15, respectively.

[0331] In some embodiments, pre-formed empty lipid nanoparticles are formed by mixing lipids dissolved in ethanol with an aqueous solution (lipid solution). In some embodiments, the lipids include one or more cationic lipids, one or more non-cationic lipids, and one or more PEG lipids. In some embodiments, the lipids also contain one or more cholesterol lipids. In some embodiments, the lipids are present as an ethanol mother liquor. Pre-formed lipid nanoparticles are formed by mixing these lipids. Generally, in some embodiments, a lipid solution containing dissolved lipids and an aqueous solution or buffer solution are mixed into a solution so that the lipids can form mRNA-free nanoparticles (i.e., pre-formed empty lipid nanoparticles).

[0332] lipid solution

[0333] According to the present invention, a lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, a suitable lipid solution is ethanol-based. For example, a suitable lipid solution may contain a mixture of preferred lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol-based. In another embodiment, a suitable lipid solution is dimethyl sulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[0334] Suitable lipid solutions can contain a mixture of desirable lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desirable lipids at a total concentration 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 preferred lipids at a total concentration in the range of about 0.1 to 100 mg / ml, 0.5 to 90 mg / ml, 1.0 to 80 mg / ml, 1.0 to 70 mg / ml, 1.0 to 60 mg / ml, 1.0 to 50 mg / ml, 1.0 to 40 mg / ml, 1.0 to 30 mg / ml, 1.0 to 20 mg / ml / ml, 1.0 to 15 mg / ml / ml, 1.0 to 10 mg / ml, 1.0 to 9 mg / ml / ml, 1.0 to 8 mg / ml, 1.0 to 7 mg / ml, 1.0 to 6 ml, or 1.0 to 5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of preferred 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.

[0335] Any desirable lipids may be mixed in any proportion suitable for encapsulating mRNA. In some embodiments, the suitable lipid solution contains a mixture of desirable lipids including a cationic lipid, a helper lipid (e.g., a non-cationic lipid and / or cholesterol lipid), and / or a PEGylated lipid. In some embodiments, the suitable lipid solution contains a mixture of desirable lipids including one or more cationic lipids, one or more helper lipids (e.g., a non-cationic lipid and / or cholesterol lipid), and / or one or more PEGylated lipids. In some embodiments, the suitable lipid solution contains a desired lipid mixture including one or more neutral lipids, one or more helper lipids, and one or more PEG-modified lipids.

[0336] In some embodiments, the pre-formed empty (i.e., mRNA-free) lipid nanoparticle formulations used to prepare the nanoparticle formulations of the present invention can be stably frozen in an 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 the empty lipid nanoparticles can produce a final formulation that can be stably stored in a frozen form without requiring any downstream purification or processing.

[0337] Formation of mRNA-LNP

[0338] As used herein, the method of forming mRNA-loaded lipid nanoparticles (mRNA-LNPs) is used interchangeably with the term "mRNA encapsulation" or any other grammatically correct expression thereof. In some embodiments, mRNA-LNPs are formed by mixing an mRNA solution with a lipid solution, wherein the mRNA solution and / or the lipid solution are heated to a predetermined temperature higher than ambient temperature prior to the mixing step (refer to U.S. Patent Application No. 14 / 790,562 filed July 2, 2015, in which the invention is titled "Encapsulation of messenger RNA," and its provisional U.S. Patent Application No. 62 / 020,163 filed July 2, 2014, the entirety of which is incorporated herein by reference).

[0339] Generally, any desired lipids may be mixed in any proportion suitable for the formation of mRNA-LNPs. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and / or one or more PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more neutral lipids, one or more helper lipids, and one or more PEG-modified lipids.

[0340] In some embodiments, an mRNA solution and a pre-formed lipid nanoparticle solution are mixed into a solution so that the mRNA is encapsulated in the lipid nanoparticles. Such a solution is also referred to as a formulation or encapsulation solution. A method for encapsulating mRNA by mixing pre-formed lipid nanoparticles with mRNA was previously described in the prior art filed as PCT / US17 / 61113 on November 10, 2017, under the title of "Improved Process of Preparing mRNA-Loaded Lipid Nanoparticles" and published as WO2018 / 089801; and simultaneously filed as U.S. Patent Application No. 15 / 809,68 under the same title. The entire contents of said application are incorporated herein by reference.

[0341] A suitable formulation or encapsulation solution comprises a solvent such as ethanol. For example, a suitable formulation or encapsulation solution comprises 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, a suitable formulation or encapsulation solution comprises a solvent such as isopropyl alcohol. For example, a suitable formulation or encapsulation solution comprises about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40% isopropyl alcohol.

[0342] In some embodiments, a suitable formulation or encapsulation solution comprises a solvent such as dimethyl sulfoxide. For example, a suitable formulation or encapsulation solution comprises about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40% dimethyl sulfoxide.

[0343] In some embodiments, a suitable formulation or encapsulation solution may contain a buffer or a salt. 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, a pre-formed empty lipid nanoparticle formulation used to prepare this novel nanoparticle formulation may be stably frozen in a 10% trehalose solution.

[0344] In some embodiments, ethanol, citrate buffer, and other destabilizing agents are not present during mRNA addition, and thus the formulation does not require any additional downstream processing. In some embodiments, the lipid nanoparticle formulation prepared by this novel method comprises pre-formed lipid nanoparticles in a trehalose solution. The absence of destabilizing agents and the stability of the trehalose solution allow the formulation scale of mRNA-encapsulated lipid nanoparticles to be easily scaled up and increase production.

[0345] mRNA solution

[0346] Since mRNA can be provided as a solution for mixing with a lipid solution, the mRNA can be encapsulated into lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations of less than 1 mg / ml. For example, a suitable mRNA solution may contain mRNA at a concentration 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, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml or less.

[0347] Generally, a suitable mRNA solution may contain a buffer and / or salt. Generally, the buffer may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, a suitable concentration of the buffer may be in the range of about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, an appropriate concentration of buffer is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or more.

[0348] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, a suitable concentration of salt in the mRNA solution may be in the range of about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. A suitable salt concentration in the mRNA solution is about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or more.

[0349] In some embodiments, a suitable mRNA solution may have a pH in the range of about 3.5 to 6.5, 3.5 to 6.0, 3.5 to 5.5, 3.5 to 5.0, 3.5 to 4.5, 4.0 to 5.5, 4.0 to 5.0, 4.0 to 4.9, 4.0 to 4.8, 4.0 to 4.7, 4.0 to 4.6, or 4.0 to 4.5. In some embodiments, a suitable mRNA solution may have a pH of about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5 or lower.

[0350] mRNA solutions suitable for the present invention can be prepared using various methods. In some embodiments, the mRNA may be dissolved directly in the buffer described herein. In some embodiments, the mRNA solution may be produced by mixing the mRNA mother liquor and the buffer before mixing the mRNA solution with the lipid solution for encapsulation. In some embodiments, the mRNA solution may be produced by mixing the mRNA mother liquor and the buffer immediately before mixing the mRNA solution with the lipid solution for encapsulation. For example, a suitable mRNA mother liquor may contain mRNA in water at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or higher.

[0351] In some embodiments, the mRNA mother liquor is mixed with a buffer using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0352] Generally, the buffer is mixed at a higher rate than the rate of the mRNA mother liquor. For example, the buffer may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x higher than the rate of the mRNA mother liquor. In some embodiments, the method according to the present invention comprises the step of first producing an mRNA solution by mixing a citrate buffer with the mRNA mother liquor. In a given embodiment, a suitable citrate buffer contains about 10 mM citrate and about 150 mM NaCl with a pH of about 4.5. In some embodiments, a suitable mRNA mother liquor contains mRNA at a concentration of about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml or more.

[0353] In some embodiments, the citrate buffer is mixed at a flow rate in the range of about 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, or 4800 to 6000 ml / min. 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.

[0354] In some embodiments, the mRNA mother liquor is mixed at a flow rate in the range of about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min. In some embodiments, the mRNA mother liquor 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.

[0355] In some embodiments, the buffer is mixed at a flow rate in the range of about 100 to 6000 ml / min (e.g., about 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, 4800 to 6000 ml / min, or 60 to 420 ml / min). In some embodiments, the buffer is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or more.

[0356] In some embodiments, the mRNA mother liquor is mixed at a flow rate in the range of about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In some embodiments, the mRNA mother liquor is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or more.

[0357] In some embodiments, pre-formed lipid nanoparticles and mRNA are mixed using a pump system. In some embodiments, the pump system includes a pulse-less flow pump. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the method using the pump system is performed on a large scale. For example, in some embodiments, the method comprises the step of mixing at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of mRNA solution with a pre-formed lipid nanoparticle solution using a pump such as that described herein to produce mRNA encapsulated in lipid nanoparticles. In some embodiments, a method of mixing mRNA with pre-formed lipid nanoparticles provides a composition according to the present invention, said composition containing at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

[0358] In some embodiments, a solution containing pre-formed lipid nanoparticles is mixed at a flow rate in the range of about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min. In some embodiments, a 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.

[0359] In some embodiments, mRNA is mixed into a solution at a flow rate in the range of about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min. In some embodiments, mRNA is mixed into a 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.

[0360] In some embodiments, the step of combining lipid nanoparticles encapsulating mRNA with pre-formed lipid particles is performed using a pump system. This combining step may be performed using a pump. In some embodiments, the lipid nanoparticles encapsulating mRNA are mixed with pre-formed lipid nanoparticles at a flow rate in the range of about 25 to 75 ml / min, about 75 to 200 ml / min, about 200 to 350 ml / min, about 350 to 500 ml / min, about 500 to 650 ml / min, about 650 to 850 ml / min, or about 850 to 1000 ml / min. In some embodiments, mRNA is mixed into a 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.

[0361] In some embodiments, the step of mixing lipid nanoparticles and mRNA is performed without any pump.

[0362] In some embodiments, the method according to the present invention comprises a step of heating one or more of the solutions to a temperature higher than the ambient temperature (or heating and maintaining it to a temperature higher than the ambient temperature) (i.e., applying heat to the solutions from a heat source), wherein the one or more solutions are a solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing mRNA encapsulated by lipid nanoparticles. In some embodiments, the method comprises a step of heating one or both of the mRNA solution and the pre-formed lipid nanoparticle solution before the mixing step. In some embodiments, the method comprises a step of heating one or more of the solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a solution containing mRNA encapsulated by lipid nanoparticles during the mixing step. In some embodiments, the method comprises a step of heating the mRNA encapsulated by lipid nanoparticles after the mixing step. In some embodiments, the temperature at which one or more of the solutions are heated (or the temperature at which one or more of the solutions are maintained) is approximately The temperature is 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature at which one or more of the solutions are heated is in the range of about 25 to 70°C, about 30 to 70°C, about 35 to 70°C, about 40 to 70°C, about 45 to 70°C, about 50 to 70°C, or about 60 to 70°C. In some embodiments, the temperature higher than the ambient temperature at which one or more of the solutions are heated is about 65°C.

[0363] In some embodiments, the method according to the present invention comprises the step of maintaining one or more of a solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a solution containing mRNA encapsulated by lipid nanoparticles at ambient temperature (i.e., not applying heat to the solution from a heat source). In some embodiments, the method comprises the step of maintaining one or both of the mRNA solution and the pre-formed lipid nanoparticle solution at ambient temperature before the mixing step. In some embodiments, the method comprises the step of maintaining one or more of the solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a solution containing mRNA encapsulated by lipid nanoparticles at ambient temperature during the mixing step. In some embodiments, the method comprises the step of maintaining the mRNA encapsulated by lipid nanoparticles at ambient temperature after the mixing step. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is about 35°C, 30°C, 25°C, 20°C, or 16°C or lower. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is in the range of about 15–35°C, about 15–30°C, about 15–25°C, about 15–20°C, about 20–35°C, about 25–35°C, about 30–35°C, about 20–30°C, about 25–30°C, or about 20–25°C. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is 20–25°C.

[0364] In some embodiments, the method according to the present invention comprises the step of mixing a solution containing pre-formed lipid nanoparticles with a solution containing mRNA to form lipid nanoparticles that encapsulate mRNA at ambient temperature.

[0365] In some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of the purified nanoparticles have a size of about 150 nm or less (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or less than about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a size of less than 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or less than about 50 nm). In some embodiments, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have a size in the range of 50 to 150 nm. In some embodiments, substantially all of the purified nanoparticles have a size in the range of 50 to 150 nm. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the purified nanoparticles have a size in the range of 80 to 150 nm. In some embodiments, substantially all of the purified nanoparticles have a size in the range of 80 to 150 nm.

[0366] In some embodiments, according to the method of the present invention, the encapsulation rate exceeds about 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, according to the method of the present invention, more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of mRNA is recovered.

[0367] In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or 1:20. The method of combining lipid nanoparticles is the same as described above for the step of mixing lipid nanoparticles with mRNA. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 20:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 19:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 15:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 10:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 9:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 8:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 7:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 6:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 5:1.In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 4:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 3:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 2:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:1. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:2. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:3. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:4. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:5. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:6. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:7. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:8. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:9. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:10.In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:12. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:15. In some embodiments, lipid nanoparticles encapsulating mRNA are combined with lipid particles pre-formed in step (b) of the method in a ratio of 1:20.

[0369] tablet

[0370] In some embodiments, pre-formed empty lipid nanoparticles or mRNA-LNPs are purified and / or concentrated. Various purification methods may be used. In some embodiments, pre-formed lipid nanoparticles are purified by tangential flow filtration (TFF). In some embodiments, lipid nanoparticles are purified by gravity-based normal 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 a chromatography method.

[0371] Pharmaceutical formulations and therapeutic uses

[0372] A composition containing mRNA-LNP can be formulated in a desired buffer, for example, PBS.

[0373] The method according to the present invention shifts the therapeutic index in a positive direction by enabling a reduction in dosage by producing an mRNA-LNP composition having higher efficacy and potency. In some embodiments, the method according to the present invention produces homogeneous mRNA-LNP having a small particle size (e.g., less than 150 nm).

[0374] Accordingly, the present invention provides a composition comprising the mRNA-LNP described herein. In some embodiments, most of the purified nanoparticles in the composition, i.e., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles, have a size of 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 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). Lipid nanoparticles with a size of less than 100 nm are particularly suitable because they can penetrate through liver perforations and access hepatocytes. Similarly, lipid nanoparticles with a size of about 100 nm or less are easily atomized and can penetrate deep into the lungs when administered to a subject using a nebulizer.

[0375] In addition, more homogeneous nanoparticles with a narrower particle size range are achieved by the method of the present invention. For example, more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the nanoparticles in the composition provided by the present invention have a size in the range of about 75 to 150 nm (e.g., about 75 to 145 nm, about 75 to 140 nm, about 75 to 135 nm, about 75 to 130 nm, about 75 to 125 nm, about 75 to 120 nm, about 75 to 115 nm, 75 to 110 nm, about 75 to 105 nm, about 75 to 100 nm, about 75 to 95 nm, about 75 to 90 nm, or about 75 to 85 nm). In some embodiments, substantially all of the purified nanoparticles have a size in the range of 75 to 100 nm (e.g., about 75 to 145 nm, about 75 to 140 nm, about 75 to 135 nm, about 75 to 130 nm, about 75 to 125 nm, about 75 to 120 nm, about 75 to 115 nm, 75 to 110 nm, about 75 to 105 nm, about 75 to 100 nm, about 75 to 95 nm, about 75 to 90 nm, or about 75 to 85 nm).

[0376] In some embodiments, the measure of dispersibility, or molecular size heterogeneity (PDI) of nanoparticles in the composition provided by the present invention is less than about 0.23 (e.g., 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 less than 0.08). In certain embodiments, the PDI is less than about 0.16.

[0377] In some embodiments, the composition according to the present invention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, according to the method according to the present invention, about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of the mRNA is recovered.

[0378] In some embodiments, the mRNA in the composition of the present invention has an integrity exceeding 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the mRNA has 100% integrity.

[0379] In some embodiments, the composition according to the present invention is formulated to allow administration of a specific dosage of the composition to a subject. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of about 5 mg / kg or less than 5 mg / kg of mRNA (i.e., mRNA less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, 1.0 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.3 mg / kg, 0.016 mg / kg, 0.05 mg / kg, and mRNA less than 0.016 mg / kg). In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles less than 4 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles less than 3 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 2 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 1 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.6 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.5 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.3 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.2 mg / kg.In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.1 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.08 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.06 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.05 mg / kg. In some embodiments, the composition of mRNA lipid nanoparticles as described herein is formulated at a dosage concentration of mRNA lipid nanoparticles of less than 0.01 mg / kg.

[0380] In a specific embodiment, the amount of mRNA required to exert a therapeutic effect is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or 99%. In a specific embodiment, the amount of polynucleotide required to exert a therapeutic effect is reduced by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 15 times, 20 times, or 25 times or more.

[0381] Accordingly, in a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to a human subject or treating a human subject. In some embodiments, the therapeutic composition comprising purified mRNA is used for delivery into the lungs or lung cells of a subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an endogenous protein that may be deficient or non-functional in a subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an endogenous protein that may be deficient or non-functional in a subject.

[0382] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to or treating the lungs or lung cells of a subject. In a specific embodiment, the present invention is useful for a method of producing mRNA encoding a cystic fibrosis transmembrane delivery regulator, namely CFTR. The CFTR mRNA is delivered to the lungs of a subject requiring a therapeutic composition for treating cystic fibrosis.

[0383] In a given embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide to be delivered to or used to treat the liver or liver cells of a subject. Such peptide or polypeptide may include those associated with urea cycle disorders, those associated with lysosomal storage disorders, those associated with glycogen storage disorders, those associated with amino acid metabolism disorders, those associated with lipid metabolism or fibrosis disorders, those associated with methylmalonic acidemia, or those associated with any other metabolic disorder for which delivering the abundant full-length mRNA to or using it to treat the liver or liver cells is a therapeutic benefit.

[0384] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with urea circulation disease. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an ornithine transcarbamylase (OTC) protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an arginosuccinate synthase 1 protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a carbamoyl phosphate synthase I protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an arginosuccinate lyase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an arginase protein.

[0385] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with lysosomal storage disorders. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an alpha galactosidase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glucocerebrosidase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an iduronate-2-sulfatase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an iduroninidase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an N-acetyl-alpha-D-glucosaminidase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a heparin N-sulfatase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a galactosamine-6 ​​sulfatase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a beta-galactosidase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a lysosomal lipase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an aral sulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a transcription factor EB (TFEB).

[0386] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with glycogen storage disorders. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an acid alpha-glucosidase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glucose-6-phosphatase (G6PC) protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a liver glycogen phosphorylase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a muscle phosphoglycerate mutase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glycogen debranching enzyme.

[0387] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with amino acid metabolism disorders. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a phenylalanine hydroxylase enzyme. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a glutaryl-CoA dehydrogenase enzyme. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a propionyl-CoA carboxylase enzyme. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising full-length mRNA encoding an oxalase alanine-glyoxylate aminotransferase.

[0388] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with lipid metabolism disorders or fibrosis disorders. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an mTOR inhibitor. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the ATPase phospholipid transporter 8B1 (ATP8B1) protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding one or more of one or more NF-kappa B inhibitors, such as I-kappa B alpha, interferon-associated developmental regulator 1 (IFRD1), and sirtuin 1 (SIRT1). In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the PPAR-gamma protein or an active variant.

[0389] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein associated with methylmalonic acidemia. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a methylmalone CoA mutase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a methylmalone CoA epimerase protein.

[0390] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA that may provide therapeutic benefits upon delivery to the liver or treatment of the liver. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the ATP7B protein, also known as the Wilson disease protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the phosphobilinogen deaminase enzyme. In a specific embodiment, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X. In a specific embodiment, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding the human hemochromatosis (HFE) protein.

[0391] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in treating a cardiovascular condition of a subject or delivering it to the subject's cardiovascular cells. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a vascular endothelial growth factor A protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a relaxin protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a bone morphogenetic protein-9 protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a bone morphogenetic protein-2 receptor protein.

[0392] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to or treating the muscle or muscle cells of a subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a dystrophin protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a frataxin protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to or treating the myocardium or myocardial cells of a subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein that regulates one or both of potassium channels and sodium channels in muscle tissue or muscle cells. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein that regulates Kv7.1 channels in muscle tissue or muscle cells. In a given embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a protein that regulates Nav1.5 channels in muscle tissue or muscle cells.

[0393] In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to or treating the nervous system or nervous system cells of a subject. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the vigilance motor neuron 1 protein. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the vigilance motor neuron 2 protein. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the frataxin protein. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the ATP-binding cassette subfamily D member 1 (ABCD1) protein. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the CLN3 protein.

[0394] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to the blood or bone marrow of a subject or blood cells or bone marrow cells, or for treating the blood or bone marrow of a subject or blood cells or bone marrow cells. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a beta-globin protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a Bruton tyrosine kinase protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.

[0395] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to or treating the kidney or kidney cells of a subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a type IV collagen alpha 5 chain (COL4A5) protein.

[0396] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering to or treating the eye or eye cells of a subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding the ATP-binding cassette subfamily A member 4 (ABCA4) protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an interretinal protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a retinal pigment epithelium-specific 65 kDa (RPE65) protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a 290 kDa centrosome protein (CDP290).

[0397] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in delivering a vaccine to a subject or the cells of a subject or treating the subject or the cells of a subject with a vaccine. For example, in a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of an infectious agent such as a virus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of an influenza virus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a respiratory syncytial virus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a rabies virus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a cytomegalovirus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a rotavirus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a hepatitis virus, e.g., hepatitis A virus, hepatitis B virus, or hepatitis C virus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a human papillomavirus. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a herpes simplex virus, e.g., herpes simplex virus type 1 or herpes simplex virus type 2.In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a human immunodeficiency virus, e.g., human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a human metapneumovirus. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a human parainfluenza virus, e.g., human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a malaria virus. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a Zika virus. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen of a chikungunya virus.

[0398] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen associated with the cancer of a subject or identified from the cancer cells of the subject. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen determined from the subject's own cancer cells, i.e., an antigen for providing a customized cancer vaccine. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antigen expressed from the KRAS gene.

[0399] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antibody. In a specific embodiment, the antibody may be a bispecific antibody. In a specific embodiment, the antibody may be part of a fusion protein. In some embodiments, in step (b) of the method, two separate mRNA-LNPs comprise mRNA encoding the light chain and the heavy chain of the antibody. In some embodiments, the mRNA-LNP composition of the present invention may comprise a combination of LNPs comprising different lipid compositions and LNPs encapsulating mRNA encoding the light chain or heavy chain of the antibody, wherein the LNPs are not identical. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antibody against OX40. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antibody against VEGF. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antibody against tissue necrosis factor alpha. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antibody against CD3. In a predetermined embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an antibody against CD19.

[0400] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an immunomodulatory substance. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding interleukin 12. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding interleukin 23. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding interleukin 36 gamma. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a constitutively active variant of a stimulator (STING) protein of one or more interferon genes.

[0401] In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an endonuclease. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding an RNA-guided DNA endonuclease protein, e.g., Cas 9 protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a meganuclease protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a transcription activator-like effector nuclease protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding a zinc finger protein.

[0402] In a given embodiment, the present invention provides a method for producing a therapeutic composition comprising purified mRNA encoding to treat an ocular disease. In some embodiments, the method is used to produce a therapeutic composition comprising purified mRNA encoding an interretinal protein.

[0403] Examples

[0404] Although specific compounds, compositions, and methods of the present invention have been described specifically in relation to specific embodiments, the following examples are merely illustrative of the invention and are not intended to limit it.

[0405] The mRNA-LNP test article described in the following examples contains mRNA encapsulated in a multicomponent lipid mixture using, in varying proportions, 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 (e.g., cholesterol designed to encapsulate mRNA), unless otherwise specified.

[0406] Example 1. Preparation of mRNA

[0407] In vitro transcription of mRNA

[0408] Unless otherwise described, mRNA was synthesized via in vitro transcription (IVT) using T7 polymerase or SP6 polymerase. Briefly, for the SP6 polymerase IVT reaction, for every 1 g of transcribed mRNA, a reaction mixture containing 20 mg of a linear double-stranded DNA plasmid containing an RNA polymerase-specific promoter, SP6 RNA polymerase, an RNase inhibitor, pyrophosphatase, 5 mM NTP, 10 mM DTT, and reaction buffer (10x - 250 mM Tris-HCl, pH 7.5, 20 mM spirmidine, 50 mM NaCl) was prepared as an RNase-free mixture and then incubated at 37°C for 60 minutes. Then, DNase I and DNase I buffer (10x - 100 mM Tris-HCl, 5 mM MgCl2, and 25 mM CaCl2, pH 7.6) were added to quench the reaction mixture, thereby facilitating the degradation of the double-stranded DNA template in the preparation for purification.

[0409] mRNA 5' capping

[0410] Unless otherwise described, IVT-transcribed mRNA was capped at its 5' end or capped in a subsequent enzymatic step by incorporating a cap structure as part of the IVT reaction. When capping as part of the IVT reaction, a cap analog may be incorporated into the initial RNA strand as the first "base". The cap analogs are cap 0, cap 1, cap 2, m6 A m , or it may be a non-natural cap. Alternatively, after IVT, for example using guanylate transferase, 5' N 7- By adding a methylguanylate cap 0 structure and generating a cap 1 structure by adding a methyl group to the 2' O position of the second-to-last nucleotide using 2' O-methyltransferase as described in the literature by Pechter, P. and Brownlee, GG ["Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249], uncapped and purified in vitro transcribed (IVT) mRNA was enzymatically converted to include a cap.

[0411] 3' tailing of mRNA

[0412] Unless otherwise described, IVT-transcribed mRNA was tailed at its 3' end by incorporating a tail template that tails mRNA as part of an IVT reaction into a linearized plasmid, or by tailing in a subsequent enzymatic step. When tailing as part of an IVT reaction, incorporating a poly-T or similar tailing feature into a pDNA template is performed so that a poly-A tail or a similar suitable tail is formed on the mRNA as part of the IVT process. Alternatively, a poly-A tail may be enzymatically added to the 3' end of the IVT-generated mRNA after the IVT reaction, for example using a poly-A polymerase.

[0413] Example 2. Preparation of lipid nanoparticles (LNPs) and encapsulation of mRNA

[0414] An LNP was prepared according to Method B, and mRNA was encapsulated in the LNP containing Compound 3 as a cationic lipid. Method B is further described in U.S. Patent Application Publication No. US2018153822, incorporated herein by reference for all purposes. As previously stated, the LNP formulation was a multicomponent lipid mixture comprising 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 one or more sterols (e.g., cholesterol designed to encapsulate the mRNA obtained as described in Example 1).

[0415] As used herein, Method A refers to a conventional method in which an LNP is formed from a multicomponent lipid mixture and mRNA is encapsulated in the LNP-forming components in a single step.

[0416] Method B refers to a method for encapsulating mRNA by mixing pre-formed lipid nanoparticles with mRNA. A pre-formed LNP was first prepared without mRNA by immediately mixing a multi-component lipid mixture dissolved in a solvent such as ethanol with citrate buffer. Empty lipid nanoparticles were formed through the mixing of the two streams, which was a self-assembly method. Empty lipid nanoparticles were obtained in an alcohol-containing citrate buffer from the resulting formulation, and empty lipid nanoparticles were obtained in a 10% w / v trehalose solution buffer by buffer exchange (e.g., by normal flow filtration (TFF)). Then, empty lipid nanoparticles and mRNA were mixed in an aqueous solution to form mRNA encapsulated in lipid nanoparticles.

[0417] Specifically, to prepare empty lipid nanoparticles, DEPE or DOPE as a helper lipid, Compound 3 as a cationic lipid, DMG-PEG2K as a PEG-modified lipid, and cholesterol together Table 2-1It was used at the ratio described in.

[0418] [Table 2-1]

[0419]

[0420] Surprisingly, it was found that these multicomponent lipid mixtures containing compound 3 as a cationic lipid could not be formulated using DOPE as a non-cationic helper lipid, but formed stable liposomes when DEPE was used instead of DOPE as a non-cationic helper lipid.

[0421] Example 3. Enhancing mRNA production in vivo from LNPs using DEPE

[0422] This example illustrates an unexpected increase in the efficacy of mRNA encapsulated in an LNP when an LNP containing DEPE as a helper lipid is used.

[0423] mRNA encoding EPO was synthesized as described in Example 1. Using Method B as described in Example 2, the mRNA was encapsulated in an LNP that is otherwise identical but contains a different helper lipid (see below). Table 3-1 (Reference). Specifically, each mRNA-encapsulated LNP contained different helper lipids but included the same cationic lipid (Compound 1), the same PEG-modified lipid (DMG-PEG2K), the same sterol compound (cholesterol), the same molar ratio of these lipids, the same mRNA (EPO mRNA), the same N / P ratio = 4 (i.e., molar ratio of cationic lipid to mRNA), and the same mRNA concentration (0.2 mg / mL), and was prepared according to the same method (Method B). The characteristics of the resulting mRNA-LNPs are Table 3-1 It is provided in.

[0424] [Table 3-1]

[0425]

[0426] Each of the four test items containing mRNA encapsulated in LNPs containing different helper lipids ( Table 3-1 The contents of (1–4) were intravenously administered to mice (n=5, CD-1 mice, 6–8 weeks old) via tail vein injection at a dose of 1 mg / kg mRNA in a volume of 5 mL / kg. At 6 hours post-administration, the tails were severed and intermediate whole blood was collected. At 24 hours post-administration, all animals were euthanized, and terminal blood was collected by thoracotomy. Human erythropoietin (hEPO) levels in serum samples were determined using an ELISA kit (R&D system Cat# DEP00) according to the manufacturer's instructions. Additionally, serum ALT and AST levels were measured by ELISA according to standard techniques. The results of EPO protein expression and ALT / AST were Table 3-2 It is provided in and , Fig. 1 and Fig. 2 They are each plotted as graphs.

[0427] [Table 3-2]

[0428]

[0429] Table 3-2 and Fig. 1As shown in [figure], mRNA LNPs containing DEPE as a helper lipid provided significantly higher in vivo protein expression compared to protein expression from mRNA LNPs containing other helper lipids but otherwise identical. Specifically, at 6 hours after administration, mRNA LNPs containing DEPE provided in vivo protein expression that exceeded 100% of, or more than twice, the protein expression from mRNA LNPs containing non-DEPE helper lipids, e.g., DOPE, DLOPE, or POPE, at the same time. Similarly, at 24 hours after administration, mRNA LNPs containing DEPE provided in vivo protein expression that exceeded 100% of, or more than twice, the protein expression from mRNA LNPs containing non-DEPE helper lipids, e.g., DOPE, DLOPE, or POPE, at the same time.

[0430] also, Table 3-2 and Fig. 2 As shown in the figure, at 24 hours after administration, ALT and AST levels were substantially similar for all mRNA LNPs regardless of the helper lipid, which indicates that mRNA LNPs containing DEPE as a helper lipid had similar safety and tolerability to mRNA LNPs containing helper lipids other than DEPE, e.g., DOPE, DLOPE, or POPE, while being significantly more potent.

[0431] Example 4. Preparing lipid nanoparticles (LNPs) using DEPE or DOPE as helper lipids

[0432] This example illustrates that using DEPE as a helper lipid in mRNA-encapsulated lipid nanoparticle (mRNA-LNP) formulations can increase protein expression from mRNA in vivo by up to twofold compared to conventional liposomes containing DOPE as a helper lipid. It was also observed that mRNA-LNPs containing DEPE as a helper lipid increase encapsulation efficiency compared to the same mRNA-LNPs containing DOPE as a helper lipid. In particular, this expression enhancement and this enhancement of encapsulation efficiency were observed across a wide variety of mRNA-LNPs containing different cationic lipids.

[0433] In these studies, DEPE or DOPE are included as helper lipids. Table 4 mRNA encoding OTC was encapsulated in LNPs containing various cationic lipids listed in [the document]. Table 4 Each cationic lipid described in is, as indicated by the last two digits in the description of each lipid, C 10 , C 12 , C 14 , or C 16 It contained four alkyl chains of length. The molar ratio of cationic lipid:DMG-PEG2K:cholesterol:helper lipid was approximately 40:3:25:32. For the in vivo portion of the study, 1 mg / kg of each formulation of mRNA-LNP was delivered to mice via tail vein injection. Mice were sacrificed at 24 hours, and in vivo expression of the mRNA encoding OTC was evaluated from liver homogenates of each mouse. Average protein expression is provided in the table below.

[0434] [Table 4]

[0435]

[0436] Table 4This shows that the encapsulation efficiency was superior when using DEPE as the helper lipid compared to when using DOPE. Furthermore, it was surprising to observe that while a multicomponent lipid mixture containing a specific lipidoid as the cationic lipid could not even be formulated using DOPE, it formed stable liposomes when using DEPE as the helper lipid. Table 4 shows that mRNA LNPs containing DEPE as the helper lipid exhibited significantly higher in vivo protein expression compared to protein expression from mRNA LNPs that contained DOPE as the helper lipid but were otherwise identical. This was the case when various cationic lipids were used in the LNPs.

[0437] Example 5. Enhanced in vivo protein expression by using DEPE as a helper lipid compared to other helper lipids

[0438] This embodiment illustrates that using DEPE as a helper lipid in mRNA-encapsulated lipid nanoparticles (mRNA-LNP) can increase mRNA expression in vivo compared to lipid nanoparticles using other types of helper lipids, across various encapsulation methods used to manufacture the mRNA-LNP.

[0439] In these studies, one of several different helper lipids including DMG-PEG-2000 as a PEG-modified lipid, cDD-TE-4-E12 as a cationic lipid, cholesterol, and DEPE Table 5-1, Table 5-2, Table 5-3, and Table 5-4 mRNA encoding an OTC was encapsulated in an LNP (N / P = 4) containing the molar lipid ratios as indicated in [figure]. Each mRNA-LNP formulation was prepared using one of the following four different encapsulation methods: conventional method, Table 5-1 A method for the formulation described in, Table 5-2 Remix method for the formulation described in, Table 5-3 A step-up remix method for the formulation described in, or Table 5-4Step-down remix method for the formulations described in. mRNA-LNPs were evaluated for LNP size, multidispersity, and encapsulation percentage, and the results for each are presented in the table below. For the in vivo portion of the study, 1 mg / kg of each formulation of mRNA-LNP was delivered to mice via tail vein injection (n=5). Mice were sacrificed at 24 hours, and in vivo expression of mRNA encoding OTC was evaluated from liver homogenates of each mouse. Mean protein expression is provided in the table below.

[0440] [Table 5-1]

[0441]

[0442] [Table 5-2]

[0443]

[0444] [Table 5-3]

[0445]

[0446] [Table 5-4]

[0447]

[0448] Table 5-1 , Table 5-2 , Table 5-3 , and Table 5-4 Figure 2 shows the protein expression levels of the mRNA at 24 hours after delivering the mRNA encapsulated in LNP to a group of mice. As shown, only LNPs prepared using the helper lipids DOPE or DEPE provided efficacy in terms of protein expression across different encapsulation methods. In particular, in vivo protein expression was highest when DEPE was used as the helper lipid to prepare the LNP, regardless of the encapsulation method used to prepare the mRNA-LNP.

[0449] As will be understood by those skilled in the art, such increased efficacy of mRNA LNPs containing DEPE as a helper lipid, but with similar safety and efficacy, provides significant advantages for delivering mRNA as a therapeutic agent.

[0450] Equalities

[0451] Those skilled in the art will be able to recognize or identify numerous equivalents to the specific embodiments of the invention described herein by using only routine experiments. The scope of the invention is not intended to be limited to the foregoing description, but rather as described in the appended claims.

Claims

Claim 1 A lipid nanoparticle for delivering mRNA to a target requiring mRNA delivery, comprising one or more cationic lipids encapsulating mRNA, one or more PEG-modified lipids, and one or more helper lipids, wherein one or more helper lipids comprise 1,2-dierukoyl-sn-glycero-3-phosphoethanolamine (DEPE). Claim 2 A lipid nanoparticle according to claim 1, wherein when the lipid nanoparticle is administered to a subject, the expression of mRNA is enhanced compared to the expression of the same mRNA in a second lipid nanoparticle having the same lipid components and amounts, except that it contains one or more different helper lipids and does not contain DEPE. Claim 3 In paragraph 2, the lipid nanoparticles wherein one or more different helper lipids comprise dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof. Claim 4 In paragraph 2 or 3, the lipid nanoparticle, wherein the expression is enhanced by at least 2 times compared to the second lipid nanoparticle. Claim 5 Lipid nanoparticles according to any one of claims 1 to 4, wherein DEPE within the lipid nanoparticles is present at a concentration of 10 mol% to 50 mol%. Claim 6 In any one of paragraphs 1 to 5, one or more cationic lipids each have a length C 10 -C 16 Lipid nanoparticles having 1 to 4 alkyl chains or containing the same. Claim 7 Lipid nanoparticles according to any one of claims 1 to 5, wherein one or more cationic lipids are lipidoids comprising four aliphatic chains. Claim 8 In paragraph 7, one or more cationic lipids are cationic lipids of the following chemical formula: or a pharmaceutically acceptable salt thereof or comprising the same; each R in the formula 1 and R 2 is independently H or C1-C6 aliphatic; each m is an integer having a value from 1 to 4 independently; each A is independently a covalent bond or an arylene; and each L 1 is independently an ester group, a thioester group, a disulfide group, or an anhydride group; and each L 2 is independently C2-C 10 Aliphatic and; each X 1 is independently H or OH; and each R 3 is independently C6-C 20 Phosphorus, lipid nanoparticles. Claim 9 In Paragraph 8, each R in the formula 3 is independently C8-C 16 Aliphatic lipid nanoparticles. Claim 10 In any one of paragraphs 1 to 9, one or more PEG-modified lipids are C6-C 20 Lipid nanoparticles comprising poly(ethylene) glycol chains up to 5 kDa in length covalently attached to lipids having alkyl chain(s) of length. Claim 11 Lipid nanoparticles according to any one of claims 1 to 10, further comprising one or more sterols. Claim 12 In paragraph 11, lipid nanoparticles comprising one or more sterols, wherein the sterols include cholesterol-based lipids. Claim 13 In paragraph 12, lipid nanoparticles, wherein one or more cholesterol-based lipids are cholesterol and / or PEGylated cholesterol. Claim 14 A lipid nanoparticle according to any one of claims 1 to 13, wherein the mRNA is a protein encoding mRNA that is translated into a therapeutic protein or peptide in vivo. Claim 15 In paragraph 14, the mRNA encoding a protein or peptide is delivered systemically, and the translated protein or peptide is detectable in the liver or serum at or after 24 hours of administration, lipid nanoparticles. Claim 16 In paragraph 15, the polypeptide is a lipid nanoparticle that is a therapeutic polypeptide. Claim 17 In paragraph 16, the therapeutic polypeptide is (a) an antibody light chain or an antibody heavy chain, or (b) a lipid nanoparticle that is a polypeptide that is absent or deficient in the subject. Claim 18 In paragraph 14, the mRNA encoding a protein is a lipid nanoparticle encoding a peptide. Claim 19 In paragraph 18, the peptide is a lipid nanoparticle that is an antigen. Claim 20 A method for delivering mRNA to a subject requiring delivery of mRNA, the method comprising the step of administering to the subject a lipid nanoparticle encapsulating mRNA comprising one or more cationic lipids, one or more PEG-modified lipids, and one or more helper lipids, wherein one or more helper lipids comprise 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). Claim 21 A method according to claim 20, wherein when lipid nanoparticles are administered to a subject, mRNA expression is enhanced compared to the expression of the same mRNA from a second lipid nanoparticle having the same lipid components and amounts, except that the second lipid nanoparticle contains one or more different helper lipids and does not contain DEPE. Claim 22 The method of claim 21, wherein one or more different helper lipids comprise dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof. Claim 23 In claim 21 or 22, the lipid nanoparticle, wherein the expression is enhanced by at least two times compared to the second lipid nanoparticle. Claim 24 In any one of claims 20 to 23, the lipid nanoparticles in which DEPE is present at a concentration of 10 mol% to 50 mol%. Claim 25 A method according to any one of claims 20 to 24, wherein one or more cationic lipids are cKK-E12 or include the same. Claim 26 A method according to any one of claims 20 to 24, wherein one or more cationic lipids are imidazole cholesterol esters (ICE) or include the same. Claim 27 In any one of paragraphs 20 to 24, one or more cationic lipids are cationic lipids of the following chemical formula: or a pharmaceutically acceptable salt thereof or comprising the same; each R in the formula 1 and R 2 is independently H or C1-C6 aliphatic; each m is an integer having a value from 1 to 4 independently; each A is independently a covalent bond or an arylene; and each L 1 is independently an ester group, a thioester group, a disulfide group, or an anhydride group; and each L 2 is independently C2-C 10 Aliphatic and; each X 1 is independently H or OH; and each R 3 is independently C6-C 20 Person, method. Claim 28 In paragraph 27, one or more cationic lipids are compound 1 or a method comprising the same. Claim 29 In any one of paragraphs 20 to 28, one or more PEG-modified lipids are C6-C 20 A method comprising a poly(ethylene) glycol chain of up to 5 kDa length covalently attached to a lipid having alkyl chain(s) of length. Claim 30 A method according to any one of claims 20 to 29, further comprising one or more sterols. Claim 31 In paragraph 30, a method wherein one or more sterols comprise cholesterol-based lipids. Claim 32 In paragraph 31, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol. Claim 33 A method according to any one of claims 21 to 32, wherein the mRNA is mRNA encoding a protein that is translated into a therapeutic protein in vivo. Claim 34 In paragraph 33, the mRNA encoding a protein is a method encoding a polypeptide. Claim 35 In paragraph 34, the polypeptide is a therapeutic polypeptide, method. Claim 36 In paragraph 35, 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. Claim 37 In paragraph 33, the mRNA encoding a protein is a method encoding a peptide. Claim 38 In paragraph 37, the peptide is an antigen, method. Claim 39 A method for preparing lipid nanoparticles that encapsulate 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 one or more helper lipids comprises 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); and (b) forming lipid nanoparticles from the mixture provided in step (a); wherein the method further comprises the step of encapsulating mRNA into the lipid nanoparticles, and the encapsulation is performed before or after the lipid nanoparticles are formed in step (b). Claim 40 In paragraph 39, lipid nanoparticles encapsulating mRNA are stable, method. Claim 41 A method according to claim 39 or 40, wherein one or more helper lipids do not comprise any one of dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine (DLOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and / or combinations thereof. Claim 42 A method according to any one of claims 39 to 41, wherein DEPE is present in the mixture at a concentration of 10 mol% to 50 mol%. Claim 43 In any one of paragraphs 39 to 42, one or more PEG-modified lipids in the mixture are C6-C 20 A method comprising a poly(ethylene) glycol chain of up to 5 kDa length covalently attached to a lipid having alkyl chain(s) of length. Claim 44 A method according to any one of paragraphs 39 to 43, wherein the mixture further comprises one or more sterols. Claim 45 In paragraph 44, a method wherein one or more sterols comprise cholesterol-based lipids. Claim 46 In paragraph 45, the cholesterol-based lipid is cholesterol and / or PEGylated cholesterol. Claim 47 A method according to any one of paragraphs 39 to 46, wherein the mRNA encodes a therapeutic peptide, polypeptide, or protein. Claim 48 A method according to any one of claims 39 to 47, wherein mRNA is encapsulated into pre-formed lipid nanoparticles. Claim 49 A method according to any one of claims 39 to 48, wherein the method further comprises the step of purifying lipid nanoparticles by tangential flow filtration (TFF) before and / or after encapsulation of mRNA. Claim 50 A method according to any one of claims 39 to 49, wherein the method further comprises the step of formulating lipid nanoparticles in a trehalose solution.

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