Tricine and citric acid lipids

Abstract

To provide cationic lipids which are useful for delivery and expression of mRNA and encoded protein as a component of a liposomal delivery vehicle, and useful for treating diseases, disorders and conditions associated with deficiency of proteins.SOLUTION: The invention provides a cationic lipid represented by Formula (A) or a pharmaceutically acceptable salt thereof. (In the formula: X1A is O or NR1A; X1B is a bond, C(O), CH2CO2, CH2C(O); one of X2A and X2B is O and the other is a bond; one of X3A and X3B is O and the other is a bond; one of X4A and X4B is O and the other is a covalent bond; and the cationic lipid comprises at least one ionizable nitrogen-containing group.)SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 864,818, filed June 21, 2019, the entire disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] The delivery of nucleic acids has been extensively investigated as a potential therapeutic option for certain medical conditions. Specifically, messenger RNA (mRNA) therapy has become an increasingly important option for the treatment of various diseases, including those associated with deficiencies of one or more proteins. Summary of the Invention

[0003] The present invention provides, among other things, the cationic lipid that is useful for the delivery of mRNA.The mRNA delivery provided by cationic lipid described herein can provide targeted delivery, reduce the frequency of administration, improve patient tolerance, and provide more effective and less toxic mRNA therapy for the treatment of various diseases, including but not limited to cancer, cardiovascular disease, cystic fibrosis, infectious disease and neurological disease.

[0004] In a first aspect, the present invention provides new cationic lipids.

[0005] In a second aspect, the present invention provides a liposome encapsulating an mRNA encoding a protein, the liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is a cationic lipid described herein.

[0006] In a third aspect, the present invention provides a nucleic acid encapsulated in a liposome, wherein the liposome comprises a cationic lipid as described herein.

[0007] In embodiments, the cationic lipid has a structure according to formula (A): [ka] During the ceremony, n is independently 0 or 1 at each occurrence; X 1A are independently O or NR 1A and R 1A is H or C1-C6 alkyl; X 1B is a covalent bond, C(O), CH2CO2, or CH2C(O), X 2A and X 2B One of the groups is O and the other is a covalent bond, X 3A and X 3B One of the groups is O and the other is a covalent bond, X 4A and X 4B One of the groups is O and the other is a covalent bond, R 1 independently, L 1 -B 1 , C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, R 2 independently, L 2 -B 2 , C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, R 3 independently, L 3 -B 3 , C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, R 4 independently, L 4 -B 4 , C6-C 30Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 , L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 1 , B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; The cationic lipid contains at least one ionizable nitrogen-containing group, or a pharmaceutically acceptable salt thereof.

[0008] In embodiments, the cationic lipid has a structure according to formula (I): [ka] During the ceremony, R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0009] In embodiments, the cationic lipid has a structure according to formula (I): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 2 , R 3 , and R 4 are each independently, C6-C 30Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0010] In embodiments, the cationic lipid has a structure according to formula (II): [ka] During the ceremony, R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0011] In embodiments, the cationic lipid has a structure according to formula (II): [ka] During the ceremony, R 1A is H or C1-C6 alkyl; B 1 is an ionic nitrogen-containing group, R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0012] In embodiments, the cationic lipid has a structure according to formula (AI): [ka] During the ceremony, L 1 But C1-C 10 is alkylene, R 6A are each independently H or C1-C6 alkyl; R 6B are each independently H or C1-C6 alkyl, or a pharmaceutically acceptable salt thereof.

[0013] In embodiments, the cationic lipid has a structure according to formula (AII): [ka] or a pharmaceutically acceptable salt thereof.

[0014] In embodiments, the cationic lipid has a structure according to formula (AIII): [ka] During the ceremony, R 1A is H, or a pharmaceutically acceptable salt thereof.

[0015] In embodiments, the cationic lipid has a structure according to formula (IIa): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 1A is H or C(O)-R 7 and R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0016] In embodiments, the cationic lipid has a structure according to formula (IIb): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 1A is H or C(O)-R 7 and R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0017] In embodiments, the cationic lipid has a structure according to formula (IIc): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0018] In embodiments, the cationic lipid has a structure according to formula (IId): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl or a pharmaceutically acceptable salt thereof be.

[0019] In some embodiments, R 1A is H.

[0020] In some embodiments, L 1 is unsubstituted C1-C 10 It is alkylene.

[0021] In some embodiments, L 1 is (CH2)2, (CH2)3, (CH2)4, or (CH2)5.

[0022] In some embodiments, B 1 are independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0023] In some embodiments, B 1 is independent, [ka] is.

[0024] In some embodiments, B 1 is independent, [ka] is.

[0025] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently CH 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0026] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl, C6-C 22 Alkenyl, or C6-C 22 It is alkynyl.

[0027] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl or C6-C 22 It is alkenyl.

[0028] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl or unsubstituted linear C6-C 22 It is alkenyl.

[0029] In some embodiments, R 1 is independent, C8H 17 , C 10 H 21 , C 12 H 25 , C14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0030] In some embodiments, R 1 are independently unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 Alkenyl or unsubstituted linear C6-C 22 It is alkynyl.

[0031] In some embodiments, R 2 , R 3 , and R 4 are each independently an unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0032] In some embodiments, R 2 , R 3 , and R 4 are unsubstituted C6-C 22 It is alkyl.

[0033] In some embodiments, R 2 , R 3 , and R 4 are each independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0034] In some embodiments, R2 , R 3 , and R 4 are unsubstituted C6-C 22 In some embodiments, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0035] In some embodiments, R 2 , R 3 , and R 4 are respectively as follows: [Table 1]

[0036] In some embodiments, R 2 , R 3 , and R 4 are respectively as follows: [Table 2]

[0037] In some embodiments (e.g., embodiments of Formula (II)), R 2 , R 3 , and R 4 are respectively as follows: [Table 3]

[0038] In some embodiments (e.g., embodiments of Formula (II)), R 2 , R 3 , and R 4 are respectively as follows: [Table 4]

[0039] In embodiments, the cationic lipid has a structure according to formula (III): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0040] In embodiments, the cationic lipid has a structure according to formula (III): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0041] In embodiments, the cationic lipid has a structure according to formula (IV): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0042] In embodiments, the cationic lipid has a structure according to formula (IV): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1A is H or C1-C6 alkyl; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments, R 1A is H.

[0044] In embodiments, the cationic lipid has a structure according to formula (V): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0045] In embodiments, the cationic lipid has a structure according to formula (V): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0046] In embodiments, the cationic lipid has a structure according to formula (VI): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0047] In embodiments, the cationic lipid has a structure according to formula (VI): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1A is H or C1-C6 alkyl; R 1 became independent, C6-C30 Alkyl, C6-C 30 Alkenyl, or C6-C3 0 alkynyl, or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, R 1A is H.

[0049] In some embodiments, R 1 are independently unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0050] In some embodiments, R 1 are independently unsubstituted C6-C 22 It is alkyl.

[0051] In some embodiments, R 1 are independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0052] In some embodiments, R 1 are independently unsubstituted C6-C 22 It is alkenyl.

[0053] In some embodiments, C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0054] In some embodiments (e.g., embodiments of Formula (III) or (IV)), R 1 are independently: [Table 5]

[0055] In some embodiments (e.g., embodiments of Formula (III) or (IV)), R 1 are independently: [Table 6]

[0056] In some embodiments (e.g., embodiments of Formula (V) or (VI)), R 1 are independently: [Table 7]

[0057] In some embodiments (e.g., embodiments of Formula (V) or (VI)), R 1 are independently: [Table 8]

[0058] In some embodiments, L 2 , L 3 , and L 4 are unsubstituted C1-C 10 It is alkylene.

[0059] In some embodiments, L 2 , L 3 , and L 4 are (CH2)2, (CH2)3, (CH2)4, or (CH2)5, respectively.

[0060] In some embodiments, B 2 , B 3 , and B 4 are each independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0061] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0062] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0063] In embodiments, the cationic lipid is any of compounds 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd, or a pharmaceutically acceptable salt thereof.

[0064] In embodiments, the cationic lipid is any of the compounds set forth in any of Tables AF, or a pharmaceutically acceptable salt thereof.

[0065] In another aspect, the invention features a composition including any of the liposomes described herein (eg, liposomes encapsulating mRNA encoding a protein).

[0066] In embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0067] In embodiments, the mRNA encodes an ornithine transcarbamylase (OTC) protein.

[0068] In another aspect, the invention features a composition including a nucleic acid encapsulated in a liposome described herein.

[0069] In some embodiments, the composition further comprises another lipid selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

[0070] In embodiments, the nucleic acid is an mRNA that encodes a peptide or polypeptide.

[0071] In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject.

[0072] In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject.

[0073] In embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0074] In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject.

[0075] In embodiments, the mRNA encodes an ornithine transcarbamylase (OTC) protein.

[0076] In embodiments, the mRNA encodes a peptide or polypeptide for use in a vaccine.

[0077] In embodiments, the mRNA encodes an antigen.

[0078] In some aspects, the present invention provides a method of treating a disease in a subject, comprising administering to the subject a composition described herein. [Brief explanation of the drawings]

[0079] [Figure 1] Figure 1 shows in vivo protein (i.e., firefly luciferase (FFL)) production in the lung resulting from delivery of mRNA (i.e., FFL mRNA) using lipid nanoparticles comprising Compound Ia, Compound 225, Compound 16, Compound 249, Compound 29, Compound 1, Compound 6, Compound 177, Compound 4, Compound 17, Compound 40, and Compound 10 described herein. As shown in this figure, the use of these compounds enables high levels of in vivo protein (i.e., FFL) production 24 hours after administration. DETAILED DESCRIPTION OF THE INVENTION

[0080] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of these terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are incorporated herein by reference.

[0081] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. As used herein, a "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxy- and / or amino-terminal amino acids in a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. An amino acid can participate in a disulfide bond. An amino acid may include one or more post-translational modifications, such as association with one or more chemicals (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and can refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which it is used.

[0082] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0083] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of possible values), unless otherwise specified or apparent from the context.

[0084] Biologically active: As used herein, the term "biologically active" refers to the characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.

[0085] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, the delivery of mRNA encompasses the situation in which the mRNA is delivered to a target tissue, and the encoded protein is expressed in and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and the situation in which the mRNA is delivered to a target tissue, and the encoded protein is expressed in and secreted into the patient's circulatory system (e.g., serum), and distributed throughout the body and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0086] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides into an intact protein (e.g., an enzyme), and / or the post-translational modification of the polypeptides or fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0087] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0088] Half-life: As used herein, the term "half-life" is the time required for a quantity, such as a nucleic acid or protein concentration or activity, to fall to half of its value when measured at the beginning of a period of time.

[0089] Improve, increase, or decrease: As used herein, "improve," "increase," or "decrease," or grammatical equivalents, refer to a value compared to a baseline measurement, e.g., a measurement in the same individual before initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject suffering from the same form of disease as the subject being treated and who is approximately the same age as the subject being treated.

[0090] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0091] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0092] Isolated: As used herein, the term "isolated" refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) artificially produced, prepared, and / or manufactured. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. In some embodiments, isolated agents are those that are about 80%, about 85%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. , about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of percent purity of isolated substances and / or entities should not include excipients (e.g., buffers, solvents, water, etc.).

[0093] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, liposomes used herein can be formed by mixing one or more lipids or by mixing one or more lipids with a polymer. In some embodiments, liposomes suitable for the present invention comprise a cationic lipid and optionally a non-cationic lipid, optionally a cholesterol-based lipid, and / or optionally a PEG-modified lipid.

[0094] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. The term "modified mRNA" refers to mRNA that contains at least one chemically modified nucleotide. mRNA can contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, and the like. Optionally, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. The mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA is selected from natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, The amino acid sequence may be or contain a C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine), a chemically modified base, a biologically modified base (e.g., a methylated base), an intercalated base, a modified sugar (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or a modified phosphate group (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0095] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, "nucleic acid" refers to interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), and CRISP. "Nucleic acid" encompasses ribonucleic acid (RNA), including, but not limited to, any one or more of R RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, but not limited to, any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In several embodiments, the DNA can be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a polymerase chain reaction (PCR) product, a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups. In embodiments, the RNA is a messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splice leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding RNA (lncRNA), microRNA (miRNA), Piwi-binding RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 73K It may be in the form of RNA, retrotransposons, viral genomes, viroids, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is an mRNA that encodes a protein, such as an enzyme.

[0096] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include prenatal and postnatal forms.

[0097] Pharmaceutically acceptable: The term "pharmaceutically acceptable," as used herein, refers to those substances which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0098] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art.For example, S.M. Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences (1977) 66:1-19.The pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases.Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts of amino groups formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts of amino groups formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and the like. + (C 1-4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations, and amine cations, formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, for example, alkyl halides to form quaternized alkylated amino salts.

[0099] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or approach that affects the entire body or the entire organism. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery."

[0100] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, and refers to a person who sees a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0101] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or reach completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0102] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease being treated. In some embodiments, the target tissue includes tissue that exhibits pathology, symptoms, or characteristics associated with the disease.

[0103] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent symptoms of, and / or delay the onset of, the disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutically effective amount is typically administered in a dosing regimen comprising at least one unit dose.

[0104] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to administering or administering a drug or drug to a subject or individual to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or who exhibit only early signs of disease for the purpose of reducing the risk of developing conditions associated with the disease.

[0105] Aliphatic: As used herein, the term aliphatic refers to C1- C 40 Aliphatic refers to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic groups can be linear, branched, or cyclic. For example, C1-C 20 Aliphatic includes C1-C 20 Alkyl (e.g., linear or branched C1-C 20 saturated alkyl), C2-C 20 Alkenyl (e.g., linear or branched C4-C 20 Dienyl, linear or branched C6-C 20 trienyl, etc.), and C2-C 20 Alkynyl (e.g., linear or branched C-C 20 C-C alkynyl) may be included. 20 Aliphatic includes C3-C 20 Cycloaliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl, or C8-C 20 In certain embodiments, an aliphatic group can include one or more cycloaliphatic groups and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. An aliphatic group is unsubstituted or substituted with one or more substituents described herein. For example, an aliphatic group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR′, —COH, —COR′, —CN, —OH, —OR′, —OCOR′, —OCOR′, —NH, —NHR′, —N(R′)′, —SR′, or —SOR′, where each instance of R′ is independently C-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10In embodiments, R' is independently unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not contain any heteroatoms.

[0106] Alkyl: As used herein, the term "alkyl" refers to acyclic straight and branched chain hydrocarbon groups, e.g., C1-C 20 "Alkyl" refers to an alkyl group having 1 to 20 carbons. The alkyl group can be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentylhexyl, isohexyl, and the like. Other alkyl groups will be readily apparent to those of ordinary skill in the art given the benefit of this disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents described herein. For example, the alkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -COH, -COR', -CN, -OH, -OR', -OCOR', -OCOR', -NH, -NHR', -N(R')2, -SR', or -SOR', where each instance of R' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10In some embodiments, R' is independently an unsubstituted C1-C3 alkyl. In some embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein). In some embodiments, the alkyl group is substituted with an -OH group and may also be referred to herein as a "hydroxyalkyl" group, where the prefix indicates the -OH group and "alkyl" is as described herein.

[0107] Alkylene: As used herein, the term “alkylene” refers to a saturated divalent straight chain or represents a branched-chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, and the like. Similarly, the term "alkenylene," as used herein, represents an unsaturated divalent straight-chain or branched-chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, and the term "alkynylene," as used herein, represents an unsaturated divalent straight-chain or branched-chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur at any stable point along the chain. In certain embodiments, alkylene, alkenylene, or alkynylene groups can contain one or more cyclic aliphatic and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. For example, the alkylene, alkenylene, or alkynylene can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR′, —COH, —COR′, —CN, —OH, —OR′, —OCOR′, —OCOR′, —NH, —NHR′, —N(R′)2, —SR′, or —SOR′, where each instance of R′ is independently C-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., C1-C3 alkyl, or C1-C3 alkyl). 20Alkyl, C1-C 15 Alkyl, C1-C 10 In certain embodiments, R' is independently unsubstituted C1-C3 alkyl. In certain embodiments, the alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene does not contain any heteroatoms.

[0108] Alkenyl: As used herein, "alkenyl" means any straight or branched hydrocarbon chain with one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, e.g., "C2-C 20 "Alkenyl" refers to an alkenyl group having 2 to 20 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, an alkenyl contains one, two, or three carbon-carbon double bonds. In embodiments, an alkenyl contains a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., two or three) are conjugated. An alkenyl group can be unsubstituted or substituted with one or more substituents described herein. For example, an alkenyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, COR′, —COH, —COR′, —CN, —OH, —OR′, —OCOR′, —OCOR′, —NH, —NHR′, —N(R′)2, —SR′, or —SOR′, where each instance of R′ is independently C-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10In embodiments, R' is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein). In embodiments, the alkenyl group is substituted with an -OH group and may also be referred to herein as a "hydroxyalkenyl" group, where the prefix indicates the -OH group and "alkenyl" is as described herein.

[0109] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain in either a straight or branched configuration with one or more carbon-carbon triple bonds occurring at any stable point along the chain, e.g., "C-C 20 "Alkynyl" refers to an alkynyl group having 2 to 20 carbon atoms. Examples of alkynyl groups include prop-2-ynyl, butyl ... Examples of alkynyl include but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, the alkynyl group contains one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more substituents described herein. For example, the alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR′, —COH, —COR′, —CN, —OH, —OR′, —OCOR′, —OCOR′, —NH, —NHR′, —N(R′)′, —SR′, or —SOR′, where each instance of R′ is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10In some embodiments, R' is independently unsubstituted C1-C3 alkyl. In some embodiments, the alkynyl is unsubstituted. In some embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein).

[0110] Aryl: The terms "aryl" and "ar-", used alone or as part of a larger moiety, e.g., "aralkyl", "aralkoxy", or "aryloxyalkyl", refer to an optionally substituted C alkyl group containing one to three aromatic rings. 6-14 Refers to an aromatic hydrocarbon moiety. For example, an aryl group is a C 6-10 Aryl groups (i.e., phenyl and naphthyl). Aryl groups include, but are not limited to, optionally substituted phenyl, naphthyl, or anthracenyl. As used herein, the terms "aryl" and "ar-" also include groups in which an aryl ring is fused to one or more alicyclic rings to form an optionally substituted ring structure, such as a tetrahydronaphthyl ring, an indenyl ring, or an indanyl ring. The term "aryl" can be used interchangeably with the terms "aryl group," "aryl ring," and "aromatic ring."

[0111] Cycloalkyl: As used herein, the term "cycloalkyl" refers to a non-aromatic saturated cyclic group, e.g., C3-C 10"cycloalkyl" means "cycloalkyl." In embodiments, a cycloalkyl is monocyclic. In embodiments, a cycloalkyl is polycyclic (e.g., bicyclic or tricyclic). In polycyclic cycloalkyl groups, the individual rings can be fused, bridged, or spirocyclic. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornanyl, bicyclo[3.2.1]octanyl, octahydro-pentalenyl, spiro[4.5]decanyl, and the like. The term "cycloalkyl" can be used interchangeably with the term "carbocycle." Cycloalkyl groups can be unsubstituted or substituted with one or more substituents described herein. For example, a cycloalkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR′, —COH, —COR′, —CN, —OH, —OR′, —OCOR′, —OCOR′, —NH, —NHR′, —N(R′)2, —SR′, or —SOR′, where each instance of R′ is independently C-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted alkyl (e.g., C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently unsubstituted C1-C3 alkyl. In some embodiments, the cycloalkyl is unsubstituted. In some embodiments, the cycloalkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein).

[0112] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine.

[0113] Heteroalkenyl. The term "heteroalkenyl" means a branched or unbranched alkenyl group having 2 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. A heteroalkenyl can optionally include monocyclic, bicyclic, or tricyclic rings, each of which desirably has 3 to 6 members. A heteroalkenyl group can be substituted or unsubstituted.

[0114] Heteroalkynyl. The term "heteroalkynyl" means a branched or unbranched alkynyl group having 2 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkynyl can optionally include monocyclic, bicyclic, or tricyclic rings, each of which desirably has 3 to 6 members. Heteroalkynyl groups can be substituted or unsubstituted.

[0115] Heteroalkyl. The term "heteroalkyl" refers to a branched or unbranched alkyl group having 1 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include, but are not limited to, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphate diesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyl groups can optionally contain monocyclic, bicyclic, or tricyclic rings, each of which desirably has 3 to 6 members. Heteroalkyl groups can be substituted or unsubstituted. Examples of heteroalkyls include, but are not limited to, polyethers such as methoxymethyl and ethoxyethyl.

[0116] Heteroaryl: The terms "heteroaryl" and "heteroar-," e.g., "heteroaralkyl," or "heteroaralkoxy," used alone or as part of a larger moiety, refer to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms, 6, 10, or 14 π electrons shared in the cyclic array, and having 1 to 5 heteroatoms in addition to carbon atoms. Heteroaryl groups can be monocyclic, bicyclic, tricyclic, or polycyclic, e.g., monocyclic, bicyclic, or tricyclic (e.g., monocyclic or bicyclic). The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of a basic nitrogen. For example, the nitrogen atom of a heteroaryl can be a basic nitrogen atom and can be further optionally oxidized to the corresponding N-oxide. When a heteroaryl is substituted with a hydroxy group, this also includes the corresponding tautomer. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heteroalicyclic rings. Non-limiting examples of heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, The term "heteroaryl" includes dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic." and all of these terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently are optionally substituted.

[0117] Heterocyclyl: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 8-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and has one or more, such as 1 to 4, heteroatoms in addition to carbon atoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (similar to N-substituted pyrrolidinyl).

[0118] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of these ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuran, tetrahydrofuran, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, more preferably monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by heterocyclyl, where the alkyl and heterocyclyl portions are independently optionally substituted. In addition, heterocyclic rings also include groups in which a heterocyclic ring is fused to one or more aryl rings.

[0119] cationic lipids Liposome-based vehicles are considered attractive carriers for therapeutic agents and remain the subject of ongoing development efforts. Although liposome-based vehicles containing cationic lipid components have shown promising results in terms of encapsulation, stability, and site localization, there remains a great need for improved liposome-based delivery systems. For example, significant drawbacks of liposome delivery systems relate to the construction of liposomes with sufficient cell culture or in vivo stability to reach desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to efficiently release their encapsulated materials into such target cells.

[0120] Specifically, there is still a need for improved cationic lipids, which show improved pharmacokinetic properties and can deliver macromolecules such as nucleic acids to a wide variety of cell types and tissues with enhanced efficiency.Importantly, there is also still a particular need for novel cationic lipids, which are characterized by reduced toxicity and can efficiently deliver encapsulated nucleic acids and polynucleotides to target cells, tissues and organs.

[0121] Described herein are novel cationic lipids, compositions comprising such lipids, and related methods of using them.In some embodiments, the compounds described herein are useful as liposome compositions or components of liposome compositions to facilitate delivery to one or more target cells and subsequent transfection thereof.

[0122] The cationic lipids disclosed herein contain a basic ionizable functional group (e.g., an amine or a nitrogen-containing heteroaryl as described herein) that exists in a natural or charged form.

[0123] For example, a basic ionizable functional group can refer to a nitrogen functional group (e.g., NH, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl) that can be converted to a charged group by protonation with an acid or deprotonation with a base. Thus, in some embodiments, X 1 is NH, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. For example, in some embodiments, the ionizable nitrogen-containing group is [ka] is.

[0124] In some embodiments, the cationic lipids described herein may provide one or more desirable features or characteristics. That is, in certain embodiments, the cationic lipids described herein may be characterized by one or more properties that provide advantages of such compounds compared to other similarly classified lipids. For example, the cationic lipids disclosed herein may allow for control and adjustment of the properties of their component liposome compositions (e.g., lipid nanoparticles). Specifically, the cationic lipids disclosed herein may be characterized by their enhanced transfection efficiency and their ability to produce specific biological results. Such results may include, for example, enhanced cellular uptake, endosome / lysosome disruption ability, and / or facilitated release of encapsulated materials (e.g., polynucleotides) within cells.

[0125] In embodiments, the cationic lipid has a structure according to formula (A): [ka] During the ceremony, n is independently 0 or 1 at each occurrence; X 1A are independently O or NR 1A and R 1Ais H or C1-C6 alkyl; X 1B is a covalent bond, C(O), CH2CO2, or CH2C(O), X 2A and X 2B One of the groups is O and the other is a covalent bond, X 3A and X 3B One of the groups is O and the other is a covalent bond, X 4A and X 4B One of the groups is O and the other is a covalent bond, R 1 independently, L 1 -B 1 , C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, R 2 independently, L 2 -B 2 , C6-C 30 Alkyl, C6-C 30 Alkenyl, also is C6-C 30 is alkynyl, R 3 independently, L 3 -B 3 , C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, R 4 independently, L 4 -B 4 , C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 , L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 1 , B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; The cationic lipid contains at least one ionizable nitrogen-containing group, or a pharmaceutically acceptable salt thereof.

[0126] In some embodiments, each n is 1. In some embodiments, two n's are 2 and one n is 1.

[0127] In some embodiments, X 1A is O. In some embodiments, X 1A is NR 1Aである In some embodiments, X 1A is NH.

[0128] In some embodiments, X 1B is a covalent bond. In some embodiments, X 1B is C(O). In some embodiments, X 1B is CH2CO2. In some embodiments, X 1B is CH2C(O).

[0129] In some embodiments, X 2A is a covalent bond, and X 2B is O. In an embodiment, X 3A is a covalent bond, and X 3B is O. In an embodiment, X 4A is a covalent bond, and X 4B is O.

[0130] In some embodiments, X 2A is O and X 2B is a covalent bond. In some embodiments, X 3A is O and X 3B is a covalent bond. In some embodiments, X 4A is O and X 4B is a covalent bond.

[0131] In some embodiments, X 2A , X 3A , and X 4A are each covalent bonds, and X 2B , X 3B , and X 4B are each O.

[0132] In some embodiments, X 2A , X 3A , and X 4A are O and X 2B , X 3B , and X 4B are each covalent bonds.

[0133] In embodiments, the cationic lipid comprises one ionizable nitrogen-containing group.

[0134] In embodiments, the cationic lipid comprises two ionizable nitrogen-containing groups.

[0135] In embodiments, the cationic lipid comprises three ionizable nitrogen-containing groups.

[0136] In embodiments, the cationic lipid comprises four ionizable nitrogen-containing groups.

[0137] In embodiments, the cationic lipid has a structure according to formula (I): [ka] During the ceremony, R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0138] In embodiments, the cationic lipid has a structure according to formula (I): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0139] In embodiments, the cationic lipid has a structure according to formula (AI): [ka] During the ceremony, L 1 But C1-C 10 is alkylene, R 6A are each independently H or C1-C6 alkyl; R 6B are each independently H or C1-C6 alkyl, or a pharmaceutically acceptable salt thereof.

[0140] In embodiments, the cationic lipid has a structure according to formula (AII): [ka] or a pharmaceutically acceptable salt thereof.

[0141] In embodiments, the cationic lipid has a structure according to formula (AIII): [ka] During the ceremony, R1A is H, or a pharmaceutically acceptable salt thereof.

[0142] In embodiments, the cationic lipid has a structure according to formula (II): [ka] During the ceremony, R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0143] In embodiments, the cationic lipid has a structure according to formula (II): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 1A is H or C1-C6 alkyl; R 2 , R 3 , and R 4 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0144] In some embodiments, R 1A is H.

[0145] In some embodiments, L 1 is unsubstituted C1-C 10It is alkylene.

[0146] In some embodiments, L 1 is (CH2)2, (CH2)3, (CH2)4, or (CH2)5.

[0147] In some embodiments, L 1 is (CH2), (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2) 10 is.

[0148] In some embodiments, B 1 are independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0149] In some embodiments, B 1 is independent, [ka] is.

[0150] In some embodiments, B 1 is independent, [ka] is.

[0151] In some embodiments, B 1 is independent, [ka] is.

[0152] In some embodiments, B 1 is independent, [ka] is.

[0153] In some embodiments, B 1is independent, [ka] is.

[0154] In some embodiments, B 1 is independent, [ka] is.

[0155] In some embodiments, R 2 , R 3 , and R 4 are each independently CH 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0156] In some embodiments, R 2 , R 3 , and R 4 are each independently C6-C 22 Alkyl, C6-C 22 Alkenyl, or C6-C 22 It is alkynyl.

[0157] In some embodiments, R 2 , R 3 , and R 4 are each independently C6-C 22 Alkyl or C6-C 22 It is alkenyl.

[0158] In some embodiments, R 2 , R 3 , and R 4 are each independently an unsubstituted linear C6-C22 Alkyl or unsubstituted linear C6-C 22 It is alkenyl.

[0159] In some embodiments, R 2 , R 3 , and R 4 are each independently an unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0160] In some embodiments, R 2 , R 3 , and R 4 are unsubstituted C6-C 22 It is alkyl.

[0161] In some embodiments, R 2 , R 3 , and R 4 are -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 , -C 19 H 39 , -C 20 H 41 , -C 21H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49 ,or -C 25 H 51 is.

[0162] In some embodiments, R 2 , R 3 , and R 4 are each independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0163] In some embodiments, R 2 , R 3 , and R 4 are unsubstituted C6-C 22 In some embodiments, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0164] In some embodiments, R 2 , R 3 , and R 4 are respectively -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18 CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0165] In some embodiments, R 2 , R 3 , and R 4 are respectively as follows: [Table 9]

[0166] In some embodiments, R 2 , R 3 , and R 4 are respectively as follows: [Table 10]

[0167] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively as follows: [Table 11]

[0168] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively as follows: [Table 12]

[0169] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0170] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0171] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0172] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0173] In some embodiments, R 2, R 3 , and R 4 teeth, [ka] is.

[0174] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0175] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0176] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0177] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0178] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0179] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0180] In some embodiments, R 2 , R 3 , and R 4 teeth, [ka] is.

[0181] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0182] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0183] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0184] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0185] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0186] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0187] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0188] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0189] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0190] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0191] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0192] In some embodiments (e.g., embodiments of Formula (AI), (AII), (AIII), or (II)), R 2 , R 3 , and R 4 are respectively, [ka] is.

[0193] In embodiments, the cationic lipid has a structure according to formula (IIa): [ka] During the ceremony, B1 is an ionic nitrogen-containing group, R 1A is H or C(O)-R 7 and R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl, L 1 But C1-C 10 alkylene, or a pharmaceutically acceptable salt thereof.

[0194] In an embodiment of Formula (IIa), R 1A is H.

[0195] In an embodiment of Formula (IIa), R 1A is C(O)-R 7 In an embodiment of Formula (IIa), L 1 is unsubstituted C1-C 10 It is alkylene.

[0196] In some embodiments of Formula (IIa), L 1 is (CH2)2, (CH2)3, (CH2)4, or (CH2)5.

[0197] In some embodiments of Formula (IIa), L 1 is (CH2), (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2) 10 is.

[0198] In some embodiments of Formula (IIa), L 1 is (CH2)2.

[0199] In some embodiments of Formula (IIa), B 1are independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0200] In some embodiments of Formula (IIa), B 1 is independent, [ka] is.

[0201] In some embodiments of Formula (IIa), B 1 is independent, [ka] is.

[0202] In some embodiments of Formula (IIa), B 1 is independent, [ka] is.

[0203] In some embodiments of Formula (IIa), B 1 is independent, [ka] is.

[0204] In some embodiments of Formula (IIa), B 1 is independent, [ka] is.

[0205] In some embodiments of Formula (IIa), B 1 is independent, [ka] is.

[0206] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently CH 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0207] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl, C6-C 22 Alkenyl, or C6-C 22 It is alkynyl.

[0208] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl or C6-C 22 It is alkenyl.

[0209] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl or unsubstituted linear C6-C 22 It is alkenyl.

[0210] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0211] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 It is alkyl.

[0212] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 ,-C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49 ,or -C 25 H51 is.

[0213] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are each independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0214] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 In some embodiments, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0215] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are respectively -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0216] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 are respectively as follows: [Table 13]

[0217] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0218] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0219] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0220] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0221] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0222] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0223] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0224] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0225] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0226] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0227] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0228] In some embodiments of Formula (IIa), R 2 , R 3 , R 4 , and R 7 teeth, [ka] is.

[0229] In embodiments, the cationic lipid has a structure according to formula (IIb): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 1A is H or C(O)-R 7 and R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0230] In embodiments of Formula (IIb), R 1A is H.

[0231] In embodiments of Formula (IIb), R 1A is C(O)-R 7 is.

[0232] In some embodiments of formula (IIb), B 1 are independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0233] In some embodiments of formula (IIb), B 1 is independent, [ka] is.

[0234] In some embodiments of formula (IIb), B 1 is independent, [ka] is.

[0235] In some embodiments of formula (IIb), B 1 is independent, [ka] is.

[0236] In some embodiments of formula (IIb), B 1 is independent, [ka] is.

[0237] In some embodiments of formula (IIb), B 1 is independent, [ka] is.

[0238] In some embodiments of formula (IIb), B 1 is independent, [ka] is.

[0239] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently CH 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0240] In some embodiments, R 2 , R 3, R 4 , and R 7 are each independently C6-C 22 Alkyl, C6-C 22 Alkenyl, or C6-C 22 It is alkynyl.

[0241] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl or C6-C 22 It is alkenyl.

[0242] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl or unsubstituted linear C6-C 22 It is alkenyl.

[0243] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0244] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 It is alkyl.

[0245] In some embodiments of Formula (IIb), R 2 , R3 , R 4 , and R 7 are -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 ,-C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49 ,or -C 25 H 51 is.

[0246] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are each independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0247] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C22 In some embodiments, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0248] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively -(CH 2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18 CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0249] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively as follows: [Table 14]

[0250] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0251] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0252] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0253] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0254] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0255] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0256] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0257] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0258] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0259] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0260] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0261] In some embodiments of Formula (IIb), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0262] In embodiments, the cationic lipid has a structure according to formula (IIc): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0263] In some embodiments of Formula (IIc), B 1 are independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0264] In some embodiments of Formula (IIc), B 1 is independent, [ka] is.

[0265] In some embodiments of Formula (IIc), B 1 is independent, [ka] is.

[0266] In some embodiments of Formula (IIc), B 1 is independent, [ka] is.

[0267] In some embodiments of Formula (IIc), B 1 is independent, [ka] is.

[0268] In some embodiments of Formula (IIc), B 1 is independent, [ka] is.

[0269] In some embodiments of Formula (IIc), B 1 is independent, [ka] is.

[0270] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently CH 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0271] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl, C6-C 22 Alkenyl, or C6-C 22 It is alkynyl.

[0272] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl or C6-C 22 It is alkenyl.

[0273] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl or unsubstituted linear C6-C 22 It is alkenyl.

[0274] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R7 are each independently an unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0275] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 It is alkyl.

[0276] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 ,-C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49,or -C 25 H 51 is.

[0277] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are each independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0278] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 In some embodiments, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0279] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0280] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively as follows: [Table 15]

[0281] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0282] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0283] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0284] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0285] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0286] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0287] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0288] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0289] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0290] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0291] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0292] In some embodiments of Formula (IIc), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0293] In embodiments, the cationic lipid has a structure according to formula (IId): [ka] During the ceremony, B 1 is an ionic nitrogen-containing group, R 2 , R 3 , R 4 , and R 7 are each independently, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0294] In some embodiments of formula (IId), B 1 are independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0295] In some embodiments of formula (IId), B 1 is independent, [ka] is.

[0296] In some embodiments of formula (IId), B 1 is independent, [ka] is.

[0297] In some embodiments of formula (IId), B 1 is independent, [ka] is.

[0298] In some embodiments of formula (IId), B 1 is independent, [ka] is.

[0299] In some embodiments of formula (IId), B 1 is independent, [ka] is.

[0300] In some embodiments of formula (IId), B 1 is independent, [ka] is.

[0301] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0302] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 It is alkyl.

[0303] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently CH 17 , C10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0304] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl, C6-C 22 Alkenyl, or C6-C 22 It is alkynyl.

[0305] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently C6-C 22 Alkyl or C6-C 22 It is alkenyl.

[0306] In some embodiments, R 2 , R 3 , R 4 , and R 7 are each independently an unsubstituted linear C6-C 22 Alkyl or unsubstituted linear C6-C 22 It is alkenyl.

[0307] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23, -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 ,-C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49 ,or -C 25 H 51 is.

[0308] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are each independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0309] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are unsubstituted C6-C 22 In some embodiments, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0310] In some embodiments of Formula (IId), R 2 , R 3 , R 4, and R 7 are respectively -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18 CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0311] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively as follows: [Table 16]

[0312] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0313] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0314] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0315] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0316] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0317] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0318] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0319] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0320] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0321] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0322] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0323] In some embodiments of Formula (IId), R 2 , R 3 , R 4 , and R 7 are respectively, [ka] is.

[0324] In embodiments, the cationic lipid has a structure according to formula (III): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0325] In embodiments, the cationic lipid has a structure according to formula (III): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0326] In embodiments, the cationic lipid has a structure according to formula (IV): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C3 0 alkynyl, or a pharmaceutically acceptable salt thereof.

[0327] In embodiments, the cationic lipid has a structure according to formula (IV): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1A is H or C1-C6 alkyl; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0328] In some embodiments, R 1Ais H.

[0329] In embodiments, the cationic lipid has a structure according to formula (V): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0330] In embodiments, the cationic lipid has a structure according to formula (V): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0331] In embodiments, the cationic lipid has a structure according to formula (VI): [ka] During the ceremony, R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0332] In embodiments, the cationic lipid has a structure according to formula (VI): [ka] During the ceremony, L 2 , L 3 , and L 4 are each independently, C1-C 30 Alkylene, C2-C 30 Alkenylene, or C2-C 30 is alkynylene, B 2 , B 3 , and B 4 are each independently an ionic nitrogen-containing group; R 1A is H or C1-C6 alkyl; R 1 became independent, C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl, or a pharmaceutically acceptable salt thereof.

[0333] In some embodiments, R 1A is H.

[0334] In some embodiments, R 1 is independent, C8H 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 32 is.

[0335] In some embodiments, R1 are independently unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 Alkenyl or unsubstituted linear C6-C 22 It is alkynyl.

[0336] In some embodiments, R 1 are independently unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 It is alkynyl.

[0337] In some embodiments, R 1 are independently unsubstituted C6-C 22 It is alkyl.

[0338] In some embodiments, R 1 are independently -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 , -C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C23 H 47 , -C 24 H 49 ,or -C 25 H 51 is.

[0339] In some embodiments, R 1 are independently -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 It is alkyl.

[0340] In some embodiments, R 1 are independently unsubstituted C6-C 22 It is alkenyl.

[0341] In some embodiments, R 1 are independently -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18 CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0342] In some embodiments, C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl.

[0343] In some embodiments (e.g., embodiments of Formula (III) or (IV)), R 1 are independently: [Table 17]

[0344] In some embodiments (e.g., embodiments of Formula (III) or (IV)), R 1 are independently: [Table 18]

[0345] In some embodiments, R 1 is independent, [ka] is.

[0346] In some embodiments, R 1 is independent, [ka] is.

[0347] In some embodiments, R 1 is independent, [ka] is.

[0348] In some embodiments, R 1 is independent, [ka] is.

[0349] In some embodiments, R 1 is independent, [ka] is.

[0350] In some embodiments, R 1 is independent, [ka] is.

[0351] In some embodiments, R 1 is independent, [ka] is.

[0352] In some embodiments, R 1 is independent, [ka] is.

[0353] In some embodiments, R 1 is independent, [ka] is.

[0354] In some embodiments, R 1 is independent, [ka] is.

[0355] In some embodiments, R 1 is independent, [ka] is.

[0356] In some embodiments, R 1 is independent, [ka] is.

[0357] In some embodiments (e.g., embodiments of Formula (V) or (VI)), R 1 are independently: [Table 19]

[0358] In some embodiments (e.g., embodiments of Formula (V) or (VI)), R 1 are independently: [Table 20]

[0359] In some embodiments, R 1 is independent, [ka] is.

[0360] In some embodiments, R 1 is independent, [ka] is.

[0361] In some embodiments, R 1 is independent, [ka] is.

[0362] In some embodiments, R 1 is independent, [ka] is.

[0363] In some embodiments, R 1 is independent, [ka] is.

[0364] In some embodiments, R 1 is independent, [ka] is.

[0365] In some embodiments, R 1 is independent, [ka] is.

[0366] In some embodiments, R 1 is independent, [ka] is.

[0367] In some embodiments, R 1 is independent, [ka] is.

[0368] In some embodiments, R 1 is independent, [ka] is.

[0369] In some embodiments, R 1 is independent, [ka] is.

[0370] In some embodiments, R 1 is independent, [ka] is.

[0371] In some embodiments, L 2 , L 3 , and L 4 are unsubstituted C1-C 10 It is alkylene.

[0372] In some embodiments, L 2 , L 3 , and L 4 are (CH2)2, (CH2)3, (CH2)4, or (CH2)5, respectively.

[0373] In some embodiments, L 2 , L 3 , and L 4 are (CH2), (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2) 10 is.

[0374] In some embodiments, B 2 , B 3 , and B 4are each independently NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0375] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0376] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0377] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0378] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0379] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0380] In some embodiments, B 2 , B 3 , and B 4 are each independently [ka] is.

[0381] Exemplary Cationic Lipids In embodiments, the cationic lipid is any of the compounds listed in Table A. [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4]

[0382] In embodiments, the cationic lipid is Compound 1. In embodiments, the cationic lipid is Compound 2. In embodiments, the cationic lipid is Compound 3. In embodiments, the cationic lipid is Compound 4.

[0383] In embodiments, the cationic lipid is Compound 5. In embodiments, the cationic lipid is Compound 6. In embodiments, the cationic lipid is Compound 7. In embodiments, the cationic lipid is Compound 8.

[0384] In embodiments, the cationic lipid is Compound 9. In embodiments, the cationic lipid is Compound 10. In embodiments, the cationic lipid is Compound 11. In embodiments, the cationic lipid is Compound 12.

[0385] In embodiments, the cationic lipid is Compound 13. In embodiments, the cationic lipid is Compound 14. In embodiments, the cationic lipid is Compound 15. In embodiments, the cationic lipid is Compound 16.

[0386] In embodiments, the cationic lipid is Compound 17. In embodiments, the cationic lipid is Compound 18. In embodiments, the cationic lipid is Compound 19. In embodiments, the cationic lipid is Compound 20.

[0387] In embodiments, the cationic lipid is Compound 21. In embodiments, the cationic lipid is Compound 22. In embodiments, the cationic lipid is Compound 23. In embodiments, the cationic lipid is Compound 24.

[0388] In embodiments, the cationic lipid is Compound 25. In embodiments, the cationic lipid is Compound 26. In embodiments, the cationic lipid is Compound 27. In embodiments, the cationic lipid is Compound 28.

[0389] In embodiments, the cationic lipid is Compound 29. In embodiments, the cationic lipid is Compound 30. In embodiments, the cationic lipid is Compound 31. In embodiments, the cationic lipid is Compound 32.

[0390] In some embodiments, the cationic lipid is Compound 33. In some embodiments, the cationic lipid is Compound 34. In some embodiments, the cationic lipid is Compound 3 5. In some embodiments, the cationic lipid is compound 36.

[0391] In embodiments, the cationic lipid is Compound 37. In embodiments, the cationic lipid is Compound 38. In embodiments, the cationic lipid is Compound 39. In embodiments, the cationic lipid is Compound 40.

[0392] In embodiments, the cationic lipid is Compound 41. In embodiments, the cationic lipid is Compound 42. In embodiments, the cationic lipid is Compound 43. In embodiments, the cationic lipid is Compound 44.

[0393] In some embodiments, the cationic lipid is compound Ia. In some embodiments, the cationic lipid is compound Ib. In some embodiments, the cationic lipid is compound Ic. In some embodiments, the cationic lipid is compound Id.

[0394] In embodiments, the cationic lipid is any of the compounds listed in Table B. [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4]

[0395] In embodiments, the cationic lipid is Compound 45. In embodiments, the cationic lipid is Compound 46. In embodiments, the cationic lipid is Compound 47. In embodiments, the cationic lipid is Compound 48.

[0396] In embodiments, the cationic lipid is Compound 49. In embodiments, the cationic lipid is Compound 50. In embodiments, the cationic lipid is Compound 51. In embodiments, the cationic lipid is Compound 52.

[0397] In embodiments, the cationic lipid is Compound 53. In embodiments, the cationic lipid is Compound 54. In embodiments, the cationic lipid is Compound 55. In embodiments, the cationic lipid is Compound 56.

[0398] In embodiments, the cationic lipid is Compound 57. In embodiments, the cationic lipid is Compound 58. In embodiments, the cationic lipid is Compound 59. In embodiments, the cationic lipid is Compound 60.

[0399] In embodiments, the cationic lipid is Compound 61. In embodiments, the cationic lipid is Compound 62. In embodiments, the cationic lipid is Compound 63. In embodiments, the cationic lipid is Compound 64.

[0400] In embodiments, the cationic lipid is Compound 65. In embodiments, the cationic lipid is Compound 66. In embodiments, the cationic lipid is Compound 67. In embodiments, the cationic lipid is Compound 68.

[0401] In embodiments, the cationic lipid is Compound 69. In embodiments, the cationic lipid is Compound 70. In embodiments, the cationic lipid is Compound 71. In embodiments, the cationic lipid is Compound 72.

[0402] In embodiments, the cationic lipid is Compound 73. In embodiments, the cationic lipid is Compound 74. In embodiments, the cationic lipid is Compound 75. In embodiments, the cationic lipid is Compound 76.

[0403] In embodiments, the cationic lipid is Compound 77. In embodiments, the cationic lipid is Compound 78. In embodiments, the cationic lipid is Compound 7 9. In some embodiments, the cationic lipid is compound 80.

[0404] In embodiments, the cationic lipid is Compound 81. In embodiments, the cationic lipid is Compound 82. In embodiments, the cationic lipid is Compound 83. In embodiments, the cationic lipid is Compound 84.

[0405] In embodiments, the cationic lipid is Compound 85. In embodiments, the cationic lipid is Compound 86. In embodiments, the cationic lipid is Compound 87. In embodiments, the cationic lipid is Compound 88.

[0406] In some embodiments, the cationic lipid is Compound IIIa. In some embodiments, the cationic lipid is Compound IIIb. In some embodiments, the cationic lipid is Compound IIIc. In some embodiments, the cationic lipid is Compound IIId.

[0407] In embodiments, the cationic lipid is any of the compounds listed in Table C. [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4]

[0408] In embodiments, the cationic lipid is Compound 89. In embodiments, the cationic lipid is Compound 90. In embodiments, the cationic lipid is Compound 91. In embodiments, the cationic lipid is Compound 92.

[0409] In embodiments, the cationic lipid is Compound 93. In embodiments, the cationic lipid is Compound 94. In embodiments, the cationic lipid is Compound 95. In embodiments, the cationic lipid is Compound 96.

[0410] In embodiments, the cationic lipid is Compound 97. In embodiments, the cationic lipid is Compound 98. In embodiments, the cationic lipid is Compound 99. In embodiments, the cationic lipid is Compound 100.

[0411] In embodiments, the cationic lipid is Compound 101. In embodiments, the cationic lipid is Compound 102. In embodiments, the cationic lipid is Compound 103. In embodiments, the cationic lipid is Compound 104.

[0412] In embodiments, the cationic lipid is Compound 105. In embodiments, the cationic lipid is Compound 106. In embodiments, the cationic lipid is Compound 107. In embodiments, the cationic lipid is Compound 108.

[0413] In embodiments, the cationic lipid is Compound 109. In embodiments, the cationic lipid is Compound 110. In embodiments, the cationic lipid is Compound 111. In embodiments, the cationic lipid is Compound 112.

[0414] In embodiments, the cationic lipid is Compound 113. In embodiments, the cationic lipid is Compound 114. In embodiments, the cationic lipid is Compound 115. In embodiments, the cationic lipid is Compound 116.

[0415] In some embodiments, the cationic lipid is Compound 117. In some embodiments, the cationic lipid is Compound 118. In some embodiments, the cationic lipid is Compound In some embodiments, the cationic lipid is compound 120.

[0416] In embodiments, the cationic lipid is Compound 121. In embodiments, the cationic lipid is Compound 122. In embodiments, the cationic lipid is Compound 123. In embodiments, the cationic lipid is Compound 124.

[0417] In embodiments, the cationic lipid is Compound 125. In embodiments, the cationic lipid is Compound 126. In embodiments, the cationic lipid is Compound 127. In embodiments, the cationic lipid is Compound 128.

[0418] In embodiments, the cationic lipid is Compound 129. In embodiments, the cationic lipid is Compound 130. In embodiments, the cationic lipid is Compound 131. In embodiments, the cationic lipid is Compound 132.

[0419] In embodiments, the cationic lipid is Compound Va. In embodiments, the cationic lipid is Compound Vb. In embodiments, the cationic lipid is Compound Vc. In embodiments, the cationic lipid is Compound Vd.

[0420] In embodiments, the cationic lipid is any of the compounds listed in Table D. [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4]

[0421] In embodiments, the cationic lipid is Compound 133. In embodiments, the cationic lipid is Compound 134. In embodiments, the cationic lipid is Compound 135. In embodiments, the cationic lipid is Compound 136.

[0422] In embodiments, the cationic lipid is Compound 137. In embodiments, the cationic lipid is Compound 138. In embodiments, the cationic lipid is Compound 139. In embodiments, the cationic lipid is Compound 140.

[0423] In embodiments, the cationic lipid is Compound 141. In embodiments, the cationic lipid is Compound 142. In embodiments, the cationic lipid is Compound 143. In embodiments, the cationic lipid is Compound 144.

[0424] In embodiments, the cationic lipid is Compound 145. In embodiments, the cationic lipid is Compound 146. In embodiments, the cationic lipid is Compound 147. In embodiments, the cationic lipid is Compound 148.

[0425] In embodiments, the cationic lipid is Compound 149. In embodiments, the cationic lipid is Compound 150. In embodiments, the cationic lipid is Compound 151. In embodiments, the cationic lipid is Compound 152.

[0426] In embodiments, the cationic lipid is Compound 153. In embodiments, the cationic lipid is Compound 154. In embodiments, the cationic lipid is Compound 155. In embodiments, the cationic lipid is Compound 156.

[0427] In embodiments, the cationic lipid is Compound 157. In embodiments, the cationic lipid is Compound 158. In embodiments, the cationic lipid is Compound 159. In embodiments, the cationic lipid is Compound 160.

[0428] In some embodiments, the cationic lipid is Compound 161. In some embodiments, the cationic lipid is Compound 162. In some embodiments, the cationic lipid is Compound In some embodiments, the cationic lipid is compound 164.

[0429] In embodiments, the cationic lipid is Compound 165. In embodiments, the cationic lipid is Compound 166. In embodiments, the cationic lipid is Compound 167. In embodiments, the cationic lipid is Compound 168.

[0430] In embodiments, the cationic lipid is Compound 169. In embodiments, the cationic lipid is Compound 170. In embodiments, the cationic lipid is Compound 171. In embodiments, the cationic lipid is Compound 172.

[0431] In embodiments, the cationic lipid is Compound 173. In embodiments, the cationic lipid is Compound 174. In embodiments, the cationic lipid is Compound 175. In embodiments, the cationic lipid is Compound 176.

[0432] In embodiments, the cationic lipid is compound IIb1. In embodiments, the cationic lipid is compound IIb2. In embodiments, the cationic lipid is compound IIb3. In embodiments, the cationic lipid is compound IIb4.

[0433] In embodiments, the cationic lipid is any of the compounds set forth in Table E. [Table 25-1] [Table 25-2] [Table 25-3] [Table 25-4]

[0434] In embodiments, the cationic lipid is Compound 177. In embodiments, the cationic lipid is Compound 178. In embodiments, the cationic lipid is Compound 179. In embodiments, the cationic lipid is Compound 180.

[0435] In embodiments, the cationic lipid is Compound 181. In embodiments, the cationic lipid is Compound 182. In embodiments, the cationic lipid is Compound 183. In embodiments, the cationic lipid is Compound 184.

[0436] In embodiments, the cationic lipid is Compound 185. In embodiments, the cationic lipid is Compound 186. In embodiments, the cationic lipid is Compound 187. In embodiments, the cationic lipid is Compound 188.

[0437] In embodiments, the cationic lipid is Compound 189. In embodiments, the cationic lipid is Compound 190. In embodiments, the cationic lipid is Compound 191. In embodiments, the cationic lipid is Compound 192.

[0438] In embodiments, the cationic lipid is Compound 193. In embodiments, the cationic lipid is Compound 194. In embodiments, the cationic lipid is Compound 195. In embodiments, the cationic lipid is Compound 196.

[0439] In embodiments, the cationic lipid is Compound 197. In embodiments, the cationic lipid is Compound 198. In embodiments, the cationic lipid is Compound 199. In embodiments, the cationic lipid is Compound 200.

[0440] In embodiments, the cationic lipid is compound 201. In embodiments, the cationic lipid is compound 202. In embodiments, the cationic lipid is compound 203. In embodiments, the cationic lipid is compound 204.

[0441] In embodiments, the cationic lipid is compound 205. In embodiments, the cationic lipid is compound 206. In embodiments, the cationic lipid is compound 207. In embodiments, the cationic lipid is compound 208.

[0442] In embodiments, the cationic lipid is compound 209. In embodiments, the cationic lipid is compound 210. In embodiments, the cationic lipid is compound 211. In embodiments, the cationic lipid is compound 212.

[0443] In embodiments, the cationic lipid is compound 213. In embodiments, the cationic lipid is compound 214. In embodiments, the cationic lipid is compound 215. In embodiments, the cationic lipid is compound 216.

[0444] In embodiments, the cationic lipid is compound 217. In embodiments, the cationic lipid is compound 218. In embodiments, the cationic lipid is compound 219. In embodiments, the cationic lipid is compound 220.

[0445] In embodiments, the cationic lipid is compound IIc1. In embodiments, the cationic lipid is compound IIc2. In embodiments, the cationic lipid is compound IIc3. In embodiments, the cationic lipid is compound IIc4. [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4]

[0446] In embodiments, the cationic lipid is compound 221. In embodiments, the cationic lipid is compound 222.

[0447] In embodiments, the cationic lipid is compound 223. In embodiments, the cationic lipid is compound 224.

[0448] In embodiments, the cationic lipid is Compound 225. In embodiments, the cationic lipid is Compound 226. In embodiments, the cationic lipid is Compound 227. In embodiments, the cationic lipid is Compound 228.

[0449] In embodiments, the cationic lipid is compound 229. In embodiments, the cationic lipid is compound 230. In embodiments, the cationic lipid is compound 231. In embodiments, the cationic lipid is compound 232.

[0450] In embodiments, the cationic lipid is compound 233. In embodiments, the cationic lipid is compound 234. In embodiments, the cationic lipid is compound 235. In embodiments, the cationic lipid is compound 236.

[0451] In embodiments, the cationic lipid is Compound 237. In embodiments, the cationic lipid is Compound 238. In embodiments, the cationic lipid is Compound 239. In embodiments, the cationic lipid is Compound 240.

[0452] In embodiments, the cationic lipid is compound 241. In embodiments, the cationic lipid is compound 242. In embodiments, the cationic lipid is compound 243. In embodiments, the cationic lipid is compound 244.

[0453] In embodiments, the cationic lipid is Compound 245. In embodiments, the cationic lipid is Compound 246. In embodiments, the cationic lipid is Compound 247. In embodiments, the cationic lipid is Compound 248.

[0454] In embodiments, the cationic lipid is compound 249. In embodiments, the cationic lipid is compound 250. In embodiments, the cationic lipid is compound 251. In embodiments, the cationic lipid is compound 252.

[0455] In embodiments, the cationic lipid is compound 253. In embodiments, the cationic lipid is compound 254. In embodiments, the cationic lipid is compound 255. In embodiments, the cationic lipid is compound 256.

[0456] In embodiments, the cationic lipid is compound 257. In embodiments, the cationic lipid is compound 258. In embodiments, the cationic lipid is compound 259. In embodiments, the cationic lipid is compound 260.

[0457] In embodiments, the cationic lipid is compound 261. In embodiments, the cationic lipid is compound 262. In embodiments, the cationic lipid is compound 263. In embodiments, the cationic lipid is compound 264.

[0458] In some embodiments, the cationic lipid is compound Ie. In some embodiments, the cationic lipid is compound If. In some embodiments, the cationic lipid is compound Ig. In some embodiments, the cationic lipid is compound Ih.

[0459] Synthesis of cationic lipids The cationic lipids described herein can be prepared according to methods known in the art. Exemplary methods include those described in US2012 / 0276482 and WO2012 / 027038, which are incorporated herein by reference.

[0460] For example, Scheme A provides two exemplary synthetic routes for preparing the cationic lipids herein. Citric acid A1 can be esterified using a Lewis acid (e.g., Bi(OTf)3) and an alcohol A2 to provide the tri-ester intermediate A3. Alternatively, a coupling agent (e.g., EDCI / DMAP) can be used to achieve this transformation. Intermediate A3 can then be converted to an acyl chloride A4 (where R 1 is an aliphatic group containing an ionizable nitrogen group as described herein) to provide cationic lipid A5, where R corresponds to any group described herein. Alternatively, trimethyl citrate A6 can be combined with A4 to provide tetraester intermediate A7. Intermediate A7 can be transesterified by saponification with base (e.g., LiOH), followed by treatment with alcohol A2 and a coupling agent to provide cationic lipid A5. [ka]

[0461] nucleic acid The cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) can be used to prepare compositions useful for delivery of nucleic acids.

[0462] Nucleic acid synthesis The nucleic acid according to the present invention can be synthesized according to any known method. For example, the mRNA according to the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, mutant T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0463] In some embodiments, to prepare mRNA according to the present invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, mutant T7, or SP6 promoter, followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0464] The desired mRNA sequence according to the present invention can be determined and incorporated into a DNA template using standard methods.For example, starting from the desired amino acid sequence (e.g., enzyme sequence), virtual reverse translation is carried out based on the degeneracy of the genetic code.Then, an optimization algorithm can be used to select suitable codons.Typically, the G / C content can be optimized to achieve the highest possible G / C content on the one hand, and to maximize the frequency of tRNA according to codon usage on the other hand.The optimized RNA sequence can be established, for example, displayed using a suitable display device, and compared with the original (wild-type) sequence.Secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or regions of RNA, respectively.

[0465] As mentioned above, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. The DNA can be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a polymerase chain reaction (PCR) product, a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups. RNAs include messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splice leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding RNA (lncRNA), microRNA (miRNA), piwi-binding RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 73K It may be in the form of RNA, retrotransposons, viral genomes, viroids, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is an mRNA that encodes a protein.

[0466] mRNA synthesis The mRNA of the present invention can be synthesized according to any of a variety of known methods. For example, the mRNA of the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application. The presence of these reagents is undesirable in the final product according to some embodiments and therefore may be referred to as impurities, and a preparation containing one or more of these impurities may be referred to as an impure preparation. In some embodiments, in vitro transcription occurs in a single batch.

[0467] In some embodiments, a DNA template is transcribed in vitro to prepare mRNA according to the present invention. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0468] The desired mRNA sequence according to the present invention can be determined and incorporated into a DNA template using standard methods.For example, starting from the desired amino acid sequence (e.g., enzyme sequence), virtual reverse translation is carried out based on the degeneracy of the genetic code.Then, an optimization algorithm can be used to select suitable codons.Typically, the G / C content can be optimized to achieve the highest possible G / C content on the one hand, and to maximize the frequency of tRNA according to codon usage on the other hand.The optimized RNA sequence can be established, for example, displayed using a suitable display device, and compared with the original (wild-type) sequence.Secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or regions of RNA, respectively.

[0469] modified mRNA In some embodiments, the mRNA of the present invention can be synthesized as unmodified mRNA or modified mRNA.Modified mRNA comprises nucleotide modification in RNA.Therefore, the modified mRNA of the present invention can comprise nucleotide modification, such as backbone modification, sugar modification, or base modification. In some embodiments, mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 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-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7 Chill-inosine, pseudouracil (5-uracil), dihydro-uracil, 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-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouracil, queosine, beta-D-mannosyl-queosine, vabutoxin and phosphoramidites, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine. The preparation of such analogs is known to those skilled in the art from, for example, U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0470] In some embodiments, mRNA can comprise RNA backbone modification.Typically, backbone modification is chemical modification of the backbone phosphate of the nucleotide contained in RNA.Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methyl phosphonate, methyl phosphoramidite, phosphoramidite, phosphorothioate (for example, cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium group, etc., which means replacing phosphodiester bond with other anionic group, cationic group, or neutral group.

[0471] In some embodiments, the mRNA may comprise a sugar modification. Exemplary sugar modifications include 4'-thio-ribonucleotides (see, e.g., U.S. Patent Application Publication No. US2016 / 0031928, incorporated herein by reference), 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, and the like. A chemical modification of the sugar of a nucleotide comprising a nucleotide includes, but is not limited to, a sugar modification selected from the group consisting of 2'-C-alkyl oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azidotriphosphate (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0472] In some embodiments, mRNA may contain a modification of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacyt ... -azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or xanthosine 5'-triphosphate.

[0473] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminus (5') and a "tail" to the C-terminus (3'). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0474] Thus, in some embodiments, the mRNA comprises a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates, then guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to create a 5'5'5 triphosphate linkage, and then the 7-nitrogen of guanine is methylated by a methyltransferase.

[0475] In some embodiments, the mRNA comprises a 3' poly(A) tail structure. The poly(A) tail at the 3' end of the mRNA typically comprises about 10 to 500 adenosine nucleotides. In some embodiments, the mRNA comprises a 3' poly(C) tail structure. In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region can be about 50 to 500 nucleotides in length.

[0476] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides in length or more.

[0477] Cap Structure In some embodiments, the mRNA comprises a 5' cap structure. In some embodiments, the nucleotide forming the cap is further methylated at the 3' position. In some embodiments, the nucleotide immediately adjacent to the cap is further methylated at the 2' position. Examples of cap structures include, but are not limited to, mG(5')ppp(5')(2'OMeG), mG(5')ppp(5')(2'OMeA), m(3'OMeG)(5')ppp(5')(2'OMeG), m(3'OMeG)(5')ppp(5')(2'OMeA), mG(5')ppp(5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. In certain embodiments, the cap structure is mG(5')ppp(5')(2'OMeG). Additional cap structures are described in U.S. Patent Application No. US2016 / 0032356, filed February 27, 2017, and U.S. Provisional Patent Application No. 62 / 464,327, both of which are incorporated by reference herein.

[0478] The naturally occurring cap structure is linked to the 5' end of the first transcribed nucleotide by a triphosphate bridge, resulting in m 7 It contains a 7-methylguanosine, resulting in a dinucleotide cap of G(5')ppp(5')N (where N is any nucleoside). In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyltransferase. Addition of the cap to the 5' end of the RNA occurs immediately after transcription initiation. The terminal nucleoside is typically guanosine, in the reverse orientation relative to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0479] A common cap on mRNA produced by in vitro transcription is m 7 The cap structure is G(5')ppp(5')G, which is used as a dinucleotide cap during in vitro transcription with T7 or SP6 RNA polymerase to obtain RNAs with a cap structure at their 5' end. A common method for in vitro synthesis of caPPEd mRNA is to use m as a transcription initiator.7 G(5')ppp(5')G("m 7 Prediction of the form of "GpppG" The dinucleotide formed is used.

[0480] To date, the usual form of synthetic dinucleotide cap used in in vitro translation experiments is the antipodal cap analog ("ARCA") or modified ARCA, which is generally a modified cap analog in which the 2' or 3' OH group is replaced with -OCH3.

[0481] Additional cap analogs include m 7 GpppG, m 7 GpppA, m 7 GpppC, a non-methylated cap analog (e.g., GpppG), a dimethylated cap analog (e.g., m 2,7 GpppG), trimethylated cap analogs (e.g., m 2,2,7 GpppG), dimethylated symmetric cap analogs (e.g., m 7 Gpppm 7 G), or inverted cap analogs (e.g., ARCA, m 7 , 2’Ome GpppG, m 72’d GpppG, m 7,3’Ome GpppG, m 7,3’d GpppG, and their tetraphosphate derivatives) (see, for example, Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties", RNA, 9:1108-1122 (2003)).

[0482] In some embodiments, a suitable cap is linked to the 5' end of the first transcribed nucleotide by a triphosphate bridge, resulting in m 7 7-methylguanylic acid ("m"), which gives G(5')ppp(5')N, where N is any nucleoside. 7 G") used in the embodiment of the present invention.7 A preferred embodiment of the G-cap is m 7 G(5')ppp(5')G.

[0483] In some embodiments, the cap is a Cap 0 structure. The Cap 0 structure lacks ribose 2'-O-methyl residues attached to bases 1 and 2. In some embodiments, the cap is a Cap 1 structure. The Cap 1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap is a Cap 2 structure. The Cap 2 structure has a 2'-O-methyl residue attached to both base 2 and base 3.

[0484] Various m 7 G-cap analogs are known in the art and many are commercially available. These include the m 7 GpppG and ARCA 3'-OCH3 and 2'-OCH3 cap analogs are included (Jemielity, J. et al., RNA, 9:1108-1122 (2003)). Additional cap analogs for use in embodiments of the present invention include N7-benzylated dinucleoside tetraphosphate analogs (as described in Grudzien, E. et al., RNA, 10:1479-1487 (2004)), phosphorothioate cap analogs (as described in Grudzien-Nogalska, E. et al., RNA, 13:1745-1755 (2007)), and cap analogs described in U.S. Patent Nos. 8,093,367 and 8,304,529, which are incorporated herein by reference, including biotinylated cap analogs.

[0485] Tail Structure Typically, the presence of a "tail" serves to protect mRNA from exonuclease degradation. Poly-A tails are thought to stabilize natural messenger and synthetic sense RNA. Thus, in certain embodiments, a long poly-A tail can be added to an mRNA molecule, resulting in a more stable RNA. Poly-A tails can be added using various techniques recognized in the art. For example, long poly-A tails can be added to synthetic or in vitro transcribed RNA using poly-A polymerase. (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Transcription vectors can also encode long polyA tails. In addition, polyA tails can be added by direct transcription from PCR products. PolyA can also be ligated to the 3' end of sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition).

[0486] In some embodiments, the mRNA comprises a 3' poly(A) tail structure. Typically, the length of the poly(A) tail can be at least about 10, 50, 100, 200, 300, 400, or at least 500 nucleotides. In some embodiments, the poly(A) tail at the 3' end of the mRNA typically comprises about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA comprises a 3' poly(C) tail structure. A suitable poly-C tail on the 3' end of an mRNA typically contains about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail can be added to or can replace the poly-A tail.

[0487] In some embodiments, the length of the poly-A or poly-C tail is adjusted to control the stability of the modified sense mRNA molecules of the present invention, and thus protein transcription. For example, because the length of the poly-A tail can affect the half-life of the sense mRNA molecule, the length of the poly-A tail can be adjusted to modify the level of resistance of the mRNA to nucleases, thereby controlling the time course of polynucleotide expression and / or polypeptide production in target cells.

[0488] 5' and 3' untranslated regions In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be approximately 50-500 nucleotides in length.

[0489] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for a protein that affects the positional stability of the mRNA in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides in length or longer.

[0490] Exemplary 3' and / or 5' untranslated sequences can be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, the 5' untranslated sequence can include a subsequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the polynucleotide. Inclusion of a sequence encoding human growth hormone (hGH) or a fragment thereof in the 3' end or untranslated region of a polynucleotide (e.g., mRNA) is also contemplated to further stabilize the polynucleotide. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to their unmodified counterparts, For example, it includes modifications made to improve the resistance of such polynucleotides to in vivo nuclease digestion.

[0491] Pharmaceutical Formulations of Cationic Lipids and Nucleic Acids In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd), as well as pharmaceutical and liposomal compositions comprising such lipids, can be used in formulations to facilitate delivery of encapsulated materials (e.g., one or more polynucleotides, such as mRNA) to one or more target cells and subsequent transfection thereof. For example, in certain embodiments, the cationic lipids described herein (and compositions, such as liposomal compositions, comprising such lipids) are characterized by providing one or more of receptor-mediated endocytosis, clathrin-mediated, and caveolae-mediated endocytosis, phagocytosis, and macropinocytosis, fusogenicity, endosomal or lysosomal disruption, and / or releasable properties that confer advantages of such compounds compared to other similarly classified lipids.

[0492] According to the present invention, a nucleic acid, e.g., mRNA, encoding a protein described herein (e.g., a full-length, fragment, or portion of a protein) can be delivered by a delivery vehicle comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd).

[0493] As used herein, the terms "delivery vehicle," "implantation vehicle," "nanoparticle," or grammatical equivalents, are used interchangeably.

[0494] For example, the present invention provides compositions (e.g., pharmaceutical compositions) comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) and one or more polynucleotides. The compositions (e.g., pharmaceutical compositions) may further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and / or one or more PEG-modified lipids.

[0495] In certain embodiments, the compositions exhibit enhanced (e.g., improved) ability to transfect one or more target cells. Accordingly, methods of transfecting one or more target cells are also provided herein. Such methods generally include contacting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) encapsulating one or more polynucleotides), thereby transfecting the one or more target cells with the encapsulated material (e.g., one or more polynucleotides). As used herein, the terms "transfect" or "transfection" refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and / or polynucleotides) into a cell, or preferably into a target cell. The introduced polynucleotide may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the efficiency of uptake by, introduction into, and / or transfection of a target cell. It refers to the relative amount of such encapsulated material (e.g., polynucleotide) expressed thereby. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by target cells after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby improving the likelihood that an appropriate dosage of the encapsulated material (e.g., one or more polynucleotides) will be delivered to the pathological site and subsequently expressed, while minimizing potential systemic side effects or toxicity associated with the compounds or their encapsulated contents.

[0496] For example, after transfection of one or more target cells with polynucleotides encapsulated in one or more lipid nanoparticles, including pharmaceutical compositions or liposome compositions disclosed herein, the production of the product (e.g., polypeptide or protein) encoded by such polynucleotides can be preferably stimulated, and the ability of such target cells to express polynucleotides and produce, for example, the target polypeptide or protein is enhanced.For example, transfection of target cells with one or more compounds or pharmaceutical compositions encapsulating mRNA enhances (i.e., increases) the production of the protein or enzyme encoded by such mRNA.

[0497] Furthermore, the delivery vehicles described herein (e.g., liposomal delivery vehicles) can be prepared to preferentially distribute to other target tissues, cells, or organs, such as the heart, lungs, kidneys, and spleen. In embodiments, the lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. For example, a polynucleotide (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical compositions and liposomal compositions described herein can be delivered to and / or transfect a target cell or tissue. In some embodiments, the encapsulated polynucleotide (e.g., mRNA) can be expressed, and a functional polypeptide product can be produced (and in some cases excreted) by the target cell, thereby conferring beneficial properties, for example, to the target cell or tissue. Such an encapsulated polynucleotide (e.g., mRNA) can encode, for example, a hormone, enzyme, receptor, polypeptide, peptide, or other protein of interest.

[0498] Liposomal Delivery Vehicles In some embodiments, the composition is a suitable delivery vehicle, hi embodiments, the composition is a liposomal delivery vehicle, for example, a lipid nanoparticle.

[0499] The terms "liposome delivery vehicle" and "liposome composition" are used interchangeably.

[0500] Concentration of liposome compositions with one or more of the cationic lipids disclosed herein can be used as a means of improving (e.g., reducing) toxicity or otherwise imparting one or more desirable properties to such concentrated liposome compositions (e.g., improving delivery of encapsulated polynucleotides to one or more target cells and / or reducing the in vivo toxicity of the liposome composition). Accordingly, pharmaceutical compositions, particularly liposome compositions, comprising one or more of the cationic lipids disclosed herein are also contemplated.

[0501] Thus, in certain embodiments, the compounds described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) can be used to facilitate the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) into one or more target cells (e.g., by penetrating the lipid membrane of such target cells). Cationic lipids are cationic lipids that can be used as components of liposome compositions to facilitate or enhance (by passing through or fusing with) liposomes.

[0502] As used herein, liposome delivery vehicle, for example, lipid nanoparticle, is generally considered to be a microscopic vesicle with an internal aqueous space separated from the external medium by one or more bilayer membranes.The bilayer membrane of liposome is typically formed by amphiphilic molecules such as synthetic or natural lipids, which contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998).The bilayer membrane of liposome can also be formed by amphiphilic polymers and surfactants (for example, polymerosomes, niosomes, etc.).In the context of the present invention, liposome delivery vehicle typically plays the role of transporting desired mRNA to target cells or tissues.

[0503] In certain embodiments, such compositions (e.g., liposomal compositions) are loaded with or encapsulate a material such as, for example, one or more biologically active polynucleotides (e.g., mRNA).

[0504] In some embodiments, the composition (e.g., pharmaceutical composition) comprises an mRNA encoding a protein encapsulated within a liposome. 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, wherein at least one cationic lipid is a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd). In some embodiments, the composition comprises an mRNA encoding a protein (e.g., any of the proteins described herein). In some embodiments, the composition comprises an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In some embodiments, the composition comprises an mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0505] In embodiments, a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated in a liposome, wherein the liposome comprises any cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd).

[0506] In some embodiments, the nucleic acid is an mRNA that encodes a peptide or polypeptide. In some embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in the treatment of a subject's lung or lung cells (for example, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein). In some embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in the treatment of a subject's liver or liver cells (for example, the mRNA encodes the ornithine transcarbamylase (OTC) protein). Still other exemplary mRNAs are described herein.

[0507] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net positive charge.

[0508] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net negative charge.

[0509] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net neutral charge.

[0510] In embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd).

[0511] For example, the amount of a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) in a composition can be described as a percentage ("wt %") of the combined total lipid dry weight of the composition (e.g., the combined total lipid dry weight present in a liposome composition).

[0512] In several embodiments of the pharmaceutical compositions described herein, the cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is present in an amount of about 0.5% to about 50% by weight (e.g., about 0.5% to about 20% by weight) of the combined total lipid dry weight present in the composition (e.g., liposome composition).

[0513] In embodiments, the cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is present in an amount of about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 5% to about 25%, about 10% to about 30%, or about 20% to about 40% by weight of the total combined lipid dry weight present in the composition (e.g., liposome composition). In embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) are present in an amount of about 0.5% to about 5%, about 1% to about 10%, about 5% to about 20%, or about 10% to about 20% by weight of the combined total molar amount of lipids present in a composition, such as a liposome delivery vehicle.

[0514] In embodiments, the amount of cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is present in an amount of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the total combined lipid dry weight in the composition (e.g., liposome composition).

[0515] In embodiments, the cationic lipids described herein (e.g., those represented by Formula (A), e.g., The amount of cationic lipid of any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd, is present in an amount of less than or equal to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the total combined lipid dry weight in the composition (e.g., liposome composition).

[0516] In embodiments, the composition (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises from about 0.1% to about 20% by weight (e.g., from about 0.1% to about 15% by weight) of a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd). In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.5%, about 1%, about 3%, about 5%, or about 10% by weight of a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd). In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5%, about 1%, about 3%, about 5%, about 10%, about 15%, or about 20% by weight of a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd). In embodiments, this percentage provides improved beneficial effects (e.g., improved delivery to target tissues such as the liver or lung).

[0517] The amount of a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) in a composition can also be described as a percentage ("mol %") of the total combined lipid molar amount of the composition (e.g., the total combined lipid molar amount present in a liposome delivery vehicle).

[0518] In several embodiments of the pharmaceutical compositions described herein, the cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is present in an amount of about 0.5 mol % to about 50 mol % (e.g., about 0.5 mol % to about 30 mol %) of the combined total molar amount of lipids present in the composition, such as a liposome delivery vehicle.

[0519] In embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) are present in an amount of about 0.5 mol % to about 5 mol %, about 1 mol % to about 10 mol %, about 5 mol % to about 20 mol %, about 10 mol % to about 20 mol %, about 20 mol % to about 30 mol %, about 30 mol % to about 40 mol %, about 40 mol % to about 50 mol %, or about 50 mol % to about 60 mol % of the total combined lipid molar amount present in a composition, such as a liposome delivery vehicle. In embodiments, the cationic lipids described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is present in an amount of about 1 mol% to about 50 mol%, about 1 mol% to about 40 mol%, about 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, or about 5 mol% to about 25 mol% of the combined total lipid dry weight present in a composition, such as a liposome delivery vehicle.

[0520] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) may comprise from about 0.1 mol% to about 50 mol%, or 0.5 mol% to about 50 mol%, or from about 1 mol% to about 25 mol%, or from about 1 mol% to about 10 mol% of the total lipid amount in the composition (e.g., liposome delivery vehicle).

[0521] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) may constitute more than about 0.1 mol%, or more than about 0.5 mol%, or more than about 1 mol%, or more than about 5 mol%, or more than about 10 mol%, or more than about 15 mol%, or more than about 20 mol%, or more than 25 mol%, or more than 30 mol%, or more than 35 mol%, or more than 40 mol%, or more than 45 mol%, or more than 50 mol% of the total lipid amount in the lipid nanoparticle.

[0522] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) may constitute less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 30%, or less than about 25 mol%, or less than about 20 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total lipid amount in a composition (e.g., a liposome delivery vehicle).

[0523] In embodiments, the amount of cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is present in an amount of at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined total lipid dry weight in the composition (e.g., liposome composition).

[0524] In embodiments, the amount of cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) is about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 10 ...0 mol%, about 200 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 It is present in an amount of about 98 mole %, or greater than about 99 mole %.

[0525] In embodiments, this percentage results in improved beneficial effects (eg, improved delivery to target tissues such as the liver or lungs).

[0526] In some embodiments, the composition further comprises another lipid (e.g., another lipid selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids).

[0527] In certain embodiments, such pharmaceutical compositions (e.g., liposome compositions) comprise one or more of a PEG-modified lipid, a non-cationic lipid, and a cholesterol lipid. In several embodiments, such pharmaceutical compositions (e.g., liposome compositions) comprise one or more PEG-modified lipids, one or more non-cationic lipids, and one or more cholesterol lipids. In several embodiments, such pharmaceutical compositions (e.g., liposome compositions) comprise one or more PEG-modified lipids and one or more cholesterol lipids.

[0528] In embodiments, compositions (e.g., lipid nanoparticles) encapsulating nucleic acids (e.g., mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids.

[0529] In embodiments, the composition (e.g., lipid nanoparticle) encapsulating the nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd), and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids, and further comprises a cholesterol-based lipid.

[0530] In embodiments, lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, PEGylated lipids, and cholesterol-based lipids.

[0531] According to various embodiments, the cationic lipid, non-cationic lipid, and / or PEG-modified lipid that comprise lipid nanoparticles are selected, and the relative molar ratio of these lipids to each other is selected based on the characteristics of selected lipid(s), the nature of the target cell of interest, and the characteristics of the mRNA that is delivered.Further considerations include, for example, the saturation degree of the alkyl chain of selected lipid(s), as well as size, charge, pH, pKa, fusogenicity, and toxicity.Therefore, molar ratio can be adjusted accordingly.

[0532] Further cationic lipids Any of the cationic lipids described herein (e.g., cationic lipids of formula (A), e.g., any of formulas (I) to (VI), or cationic lipids 1 to 264, In addition to any of Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd), the composition may include one or more additional cationic lipids.

[0533] In some embodiments, liposome can comprise one or more additional cationic lipids.As used herein, the term " cationic lipid " refers to any of several lipid species that have net positive charge at selected pH, such as physiological pH.Some cationic lipids have been described in literature, and many of them are commercially available.

[0534] Additional cationic lipids suitable for use in the composition include those described in International Patent Publication No. WO2010 / 144740, which is incorporated herein by reference.In certain embodiments, the composition comprises the cationic lipid 4-(dimethylamino)butanoate (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl, having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0535] Other additional cationic lipids suitable for use in the present compositions include the ionic cationic lipids described in International Patent Publication No. WO2013 / 149140, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid of one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently hydrogen, an optionally substituted variably saturated or unsaturated C1-C 20 Alkyl, and optionally substituted variably saturated or unsaturated C-C 20 acyl, wherein L and L are each independently selected from the group consisting of hydrogen, optionally substituted C-C 30 Alkyl, optionally substituted variably unsaturated C-C 30 Alkenyl, and optionally substituted C-C 30 alkynyl, wherein m and o are each independently selected from the group consisting of zero and any positive integer (e.g., m is 3), and n is zero or any positive integer (e.g., n is 1). In certain embodiments, the composition comprises a cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the composition comprises a cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the composition comprises a cationic lipid having the following compound structure: (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002"), [ka] and pharmaceutically acceptable salts thereof.

[0536] Other additional cationic lipids suitable for use in the present compositions include those cationic lipids described as amino alcohol lipidoids in International Patent Publication No. WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the present compositions comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0537] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2016 / 118725, which is incorporated herein by reference. In certain embodiments, the present compositions comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0538] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2016 / 118724, which is incorporated herein by reference. In certain embodiments, the present compositions comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0539] Other cationic lipids suitable for use in the present compositions include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0540] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication Nos. WO2013 / 063468 and WO2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R L each instance of is independently an optionally substituted C-C 40 In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0541] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0542] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0543] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0544] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2015 / 184256, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and R A are each independently hydrogen, optionally substituted C alkyl, optionally substituted C alkenyl, optionally substituted C alkynyl, optionally substituted C carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C aryl, optionally substituted 5-14 membered heteroaryl, or halogen; R B are each independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, In certain embodiments, the composition comprises a cationic lipid "Target 23" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0545] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid having the following compound structure: [ka] During the ceremony, [ka] or a pharmaceutically acceptable salt thereof.

[0546] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0547] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0548] Other cationic lipids suitable for use in the present compositions include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In certain embodiments, the cationic lipid of the present compositions is a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0549] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0550] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0551] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0552] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0553] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0554] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0555] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0556] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0557] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0558] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka]

[0559] and pharmaceutically acceptable salts thereof.

[0560] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0561] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0562] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0563] Other additional cationic lipids suitable for use in the present compositions include those incorporated herein by reference. Cationic lipids include those described in International Patent Publication No. WO2017 / 004143, which is incorporated herein by reference.

[0564] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0565] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0566] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0567] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0568] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0569] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0570] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0571] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0572] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0573] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0574] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0575] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0576] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0577] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0578] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0579] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0580] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0581] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2017 / 075531, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, and L1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O- or a direct bond, and G 1 and G 2 are each independently unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene, G 3 But C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, and R a But H or C1-C 12 alkyl, and R 1 and R 2 are each independently, C6-C 24 Alkyl or C6-C 24 alkenyl, and R 3 But, H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and R 4 But C1-C 12 alkyl, and R 5 is H or C1-C6 alkyl and x is 0, 1, or 2.

[0582] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2017 / 117528, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0583] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0584] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0585] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the present invention is a compound of one of the following formulae: [ka] and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n CHQR, where Q is OR, -OH, -O(CH2) n R is independently selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), and heterocycle; 1-3 Alkyl, C 2-3 alkenyl, and H, where n is 1, 2, or 3.

[0586] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0587] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0588] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0589] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0590] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication Nos. WO2017 / 173054 and WO2015 / 095340, each of which is incorporated herein by reference.

[0591] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0592] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0593] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0594] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0595] Other additional cationic lipids suitable for use in the present compositions include cholesterol-based cationic lipids. In certain embodiments, the present compositions comprise an imidazole cholesterol ester or "ICE," which has the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0596] Other additional cationic lipids suitable for use in the present compositions include the cleavable cationic lipids described in International Patent Publication No. WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the present compositions comprise a cationic lipid of the following formula: [ka] wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl, and R2 is selected from the group consisting of one of the following two formulas: [ka] wherein R and R each independently represent an optionally substituted saturated or unsaturated C-C 20 Alkyl and optionally substituted variably saturated or unsaturated C-C 20 acyl, 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 more).

[0597] In certain embodiments, the composition comprises a cationic lipid "HGT4001" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0598] In certain embodiments, the composition comprises a cationic lipid "HGT4002" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0599] In certain embodiments, the composition comprises a cationic lipid "HGT4003" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0600] In certain embodiments, the composition comprises a cationic lipid "HGT4004" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0601] In certain embodiments, the composition comprises a cationic lipid "HGT4005" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0602] In some embodiments, the composition comprises the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”). See Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, each of which is incorporated herein by reference. DOTMA can be formulated alone or combined with neutral lipids (e.g., dioleoylphosphatidyl-ethanolamine or "DOPE") or yet other cationic or non-cationic lipids into liposome import vehicles or lipid nanoparticles. Such liposomes can be used to enhance delivery of nucleic acids to target cells. Other cationic lipids suitable for the present compositions include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"), 2,3-dioleyloxy-N-[2(spermine-carboxyamido)ethyl]-N,N-dimethyl-1-propanaminium ("DOSPA") (Behr et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, each of which is incorporated herein by reference. Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678, U.S. Pat. No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"), 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0603] Additional exemplary cationic lipids suitable for the present compositions 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-dioleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"). Linolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"), N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(l,2-dimyrityloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoyl)- oxy)propane ("CLinDMA"), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl ll-(cis,cis-9',l-2'-octadecadienooxy)propane ("CpLinDMA"), N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"), 1,2-N ,N'-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"), l,2-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLinCDAP"), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane ("DLin-K-DMA"), 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA") "), (2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"), (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2S)"), 2,Also included are 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA") and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see International Publication No. WO 2010 / 042877, Semple et al., Nature Biotech. 28:172-176 (2010), which are incorporated herein by reference). (Heyes, J., et al., J. Controlled Release 107:276-287 (2005); Morrissey, D.V., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); International Patent Publication No. WO2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole moiety, a dialkylamino moiety, or a guanidinium moiety.

[0604] In some embodiments, the one or more cationic lipids suitable for the present compositions include 2,2-dilinoleyl-4-dimethylaminoethyl-1-[1,3]-dioxolane ("XTC"), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100"), and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0605] In some embodiments, the percentage of total cationic lipid in a composition (e.g., a liposome composition) can be 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, or 95% or less of the total lipid, measured by molar ratio (mol%) or by weight (wt%).

[0606] In some embodiments, the percentage of total cationic lipid in a composition (e.g., a liposome composition) can be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% of the total lipid, measured by molar ratio (mol%) or by weight (wt%).

[0607] In some embodiments, the total cationic lipids comprise about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposomes by weight. In some embodiments, the cationic lipids comprise about 30%, about 35%, about 40%, about 45%, or about 50% of the composition (e.g., liposome composition) by molar ratio. In some embodiments, the total cationic lipid(s) comprise about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposomes by weight. In some embodiments, the cationic lipids comprise about 30%, about 35%, about 40%, about 45%, or about 50% of the composition (e.g., liposome composition) by weight.

[0608] Non-cationic / Helper Lipids The composition (e.g., liposome composition) may also include one or more non-cationic ("helper") lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral lipid, zwitterionic lipid, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidyl These include, but are not limited to, ethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or mixtures thereof.

[0609] In embodiments, the non-cationic or helper lipid is dioleoylphosphatidylethanolamine (DOPE).

[0610] In some embodiments, the non-cationic lipid is a neutral lipid, ie, a lipid that carries no net charge under the conditions in which the composition is formulated and / or administered.

[0611] In some embodiments, the noncationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the total noncationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes can be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. The percentage of total non-cationic lipids in the liposome can be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipids in the liposome is 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. In some embodiments, the percentage of total non-cationic lipids in the liposome can 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.

[0612] In some embodiments, the noncationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the total noncationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be less than about 5% by weight, less than about 10% by weight, less than about 20% by weight, less than about 30% by weight, or less than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be less than about 5% by weight, less than about 10% by weight, less than about 20% by weight, less than about 30% by weight, or less than about 40% by weight.

[0613] Cholesterol-based lipids In some embodiments, the composition (e.g., liposome composition) comprises one or more cholesterol-based lipids. For example, suitable cholesterol-based lipids include cholesterol and, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxyamidocholesterol), 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 ester (ICE) having the following structure: [ka]

[0614] In embodiments, the cholesterol-based lipid is cholesterol.

[0615] In some embodiments, the cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based 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.

[0616] In some embodiments, the cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be about 5 wt% or less, about 10 wt% or less, about 20 wt% or less, about 30 wt% or less, or about 40 wt% or less.

[0617] PEGylated lipids In some embodiments, a composition (eg, a liposomal composition) comprises one or more PEGylated lipids.

[0618] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER) containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), in combination with one or more of the cationic lipids and, in some embodiments, other lipids that make up the liposome, is also contemplated by the present invention. In some embodiments, particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 ) is a PEG-ceramide.

[0619] In some embodiments, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000).

[0620] Contemplated PEG-modified lipids (also referred to herein as PEGylated lipids, and this term is interchangeable with PEG-modified lipids) include C6-C 20 The PEG-modified or PEGylated lipid may include, but is not limited to, polyethylene glycol chains up to 5 kDa long, covalently attached to lipids with long alkyl chain(s). In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components can prevent the aggregation of the complex, increase the circulation life, and provide a means for increasing the delivery of lipid-nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or these components can be selected to rapidly exchange from the formulation in vivo (see U.S. Patent No. 5,885,613).

[0621] The PEG-modified phospholipids and derivatized lipids of the present invention may be present in a molar ratio (mol %) of about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipids present in the composition (e.g., liposome composition).

[0622] The PEG-modified phospholipids and derivatized lipids of the present invention may be present in a weight ratio (wt%) of about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the composition (e.g., liposome composition).

[0623] Pharmaceutical Formulations and Therapeutic Uses The cationic lipids described herein (e.g., cationic lipids of formula (A), e.g., any of formulas (I) to (VI), or cationic lipids 1 to 264, Ia to Ih, II b1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) can be used in the preparation of compositions (e.g., to construct liposomal compositions) that promote or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by penetrating or fusing with the lipid membranes of such target cells).

[0624] For example, when a liposome composition (e.g., lipid nanoparticle) comprises or is otherwise enriched with one or more of the compounds disclosed herein, a phase transition in the lipid bilayer of one or more target cells can facilitate delivery of the encapsulated material (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) to one or more target cells.

[0625] Similarly, in certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) can be used to prepare liposome vehicles characterized by their reduced toxicity in vivo. In certain embodiments, reduced toxicity is a function of the high transfection efficiency associated with the compositions disclosed herein, such that reduced amounts of such compositions can be administered to a subject to achieve a desired therapeutic response or outcome.

[0626] Thus, pharmaceutical formulations comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of Cationic Lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) and a nucleic acid provided by the present invention can be used for various therapeutic purposes. To facilitate in vivo delivery of a nucleic acid, a cationic lipid (e.g., a cationic lipid of Formula (A), e.g., any of Formulas (I)-(VI), or any of Cationic Lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) and a nucleic acid can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands, or stabilizing reagents. In some embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) can be formulated via a premixed lipid solution. In other embodiments, compositions comprising the cationic lipids described herein (e.g., cationic lipids of Formula (A), e.g., any of Formulas (I)-(VI), or any of cationic lipids 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd) can be formulated using post-insertion techniques into nanoparticle lipid membranes. Techniques for formulation and drug administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. (latest edition).

[0627] Suitable administration routes include, for example, oral administration, rectal administration, vaginal administration, transmucosal administration, pulmonary administration including intratracheal administration or inhalation administration, or intestinal administration, parenteral delivery including intradermal injection, transdermal (topical) injection, intramuscular injection, subcutaneous injection, intramedullary injection, as well as intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, or intranasal administration. In certain embodiments, intramuscular administration is into a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, this administration results in delivery of the nucleic acid to muscle cells. In some embodiments, this administration results in delivery of the nucleic acid to hepatocytes (i.e., liver cells).

[0628] Alternatively or additionally, the pharmaceutical preparations of the present invention can be administered in a local rather than systemic manner, for example, by directly injecting the pharmaceutical preparation into the targeted tissue, preferably in a sustained-release formulation. Local delivery can be achieved in various ways depending on the targeted tissue. Exemplary tissues to which the delivered mRNA can be delivered and / or expressed include, but are not limited to, the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In several embodiments, the targeted tissue is the liver. For example, an aerosol containing the composition of the present invention can be inhaled (in the case of nasal, tracheal, or bronchial delivery), the composition of the present invention can be injected, for example, at the site of injury, disease symptoms, or pain, and the composition can be provided in a lozenge for oral, tracheal, or esophageal use, or in the form of a liquid, tablet, or capsule for administration to the stomach or intestines, or in the form of a suppository for rectal or vaginal use, or can be delivered to the eye using a cream, droplet, or even an injection.

[0629] The present invention provides methods for delivering compositions having full-length mRNA molecules encoding a peptide or polypeptide of interest for use in treating a subject, e.g., a human subject, or cells of a human subject, or cells that are treated and delivered to a human subject.

[0630] Thus, in certain embodiments, the present invention provides methods for producing therapeutic compositions comprising full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating a subject's lung or lung cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the ATP-binding cassette subfamily A member 3 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the dynein axoneme intermediate chain 1 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the dynein axoneme heavy chain 5 (DNAH5) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the alpha-1-antitrypsin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the forkhead box P3 (FOXP3) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding one or more surfactant proteins, e.g., one or more of surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.

[0631] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding peptides or polypeptides for delivery to or use in treating the liver or liver cells of a subject. Such peptides and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrotic disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery of enriched full-length mRNA to or treatment with the liver or liver cells provides a therapeutic benefit.

[0632] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding proteins associated with urea cycle disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding argininosuccinate synthetase 1 protein. The present invention provides a method for producing a therapeutic composition having a long mRNA. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding a carbamoyl phosphate synthetase I protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an argininosuccinate lyase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an arginase protein.

[0633] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding proteins associated with lysosomal storage disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding alpha-galactosidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding glucocerebrosidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding iduronate-2-sulfatase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding iduronidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding N-acetyl-alpha-D-glucosaminidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding heparan N-sulfatase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding galactosamine-6 ​​sulfatase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding a beta-galactosidase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding a lysosomal lipase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding an arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding transcription factor EB (TFEB).

[0634] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding proteins associated with glycogen storage disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding acid alpha-glucosidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding glucose-6-phosphatase (G6PC) proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding liver glycogen phosphorylase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding muscle phosphoglycerate mutase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding glycogen debranching enzymes.

[0635] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding proteins related to amino acid metabolism. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding phenylalanine hydroxylase enzymes. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding glutaryl-CoA dehydrogenase enzymes. Methods for producing compositions are provided. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a propionyl-CoA carboxylase enzyme. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an oxalase alanine-glyoxylaminotransferase enzyme.

[0636] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNAs encoding proteins related to lipid metabolism or fibrotic disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNAs encoding mTOR inhibitors. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNAs encoding ATPase phospholipid transport 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNAs encoding one or more NF-kappa B inhibitors, such as one or more of I-kappa B alpha, interferon-related developmental regulator 1 (IFRD1), and sirtuin 1 (SIRT1). In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNAs encoding PPAR-gamma proteins or active variants.

[0637] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding proteins associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding methylmalonyl-CoA mutase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding methylmalonyl-CoA epimerase proteins.

[0638] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA whose delivery to or treatment of the liver can provide therapeutic benefit. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the ATP7B protein, also known as the Wilson disease protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the porphobilinogen deaminase enzyme. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding the human hemochromatosis (HFE) protein.

[0639] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating cardiovascular structures or cells of a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a vascular endothelial growth factor A protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a relaxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a bone morphogenetic protein-9 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a bone morphogenetic protein-2 receptor protein.

[0640] In certain embodiments, the present invention provides therapeutics comprising full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating a muscle or muscle cell of a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a dystrophin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a frataxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating a subject's cardiac muscle or cardiac muscle cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a protein that regulates one or both of potassium and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a protein that regulates Kv7.1 channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a protein that regulates Nav1.5 channels in muscle tissue or muscle cells.

[0641] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating the nervous system or nervous system cells of a subject. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding survival motor neuron 2 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding frataxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding CLN3 protein.

[0642] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating a subject's blood or bone marrow or blood or bone marrow cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a beta-globin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a Bruton's tyrosine kinase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding one or more coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X.

[0643] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating a subject's kidney or kidney cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.

[0644] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for delivery to or use in treating the eye or ocular cells of a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a retinoschisin protein. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding a retinal pigment epithelium-specific 65 kDa (RPE65) protein. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding a 290 kDa centrosomal protein (CEP290).

[0645] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a peptide or polypeptide for use in delivering or treating a vaccine for a subject or cells of a subject. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from an infectious pathogen, such as a virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from influenza virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from respiratory syncytial virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from rabies virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from cytomegalovirus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from rotavirus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen derived from a human papillomavirus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen derived from a herpes simplex virus, such as herpes simplex virus type 1 or herpes simplex virus type 2. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen derived from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen derived from a human metapneumovirus.In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from a malaria virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from a Zika virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen from a Chikungunya virus.

[0646] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen associated with a subject's cancer or an antigen identified from the subject's cancer cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen determined from a subject's own cancer cells, i.e., for providing a personalized cancer vaccine. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antigen expressed from a mutant KRAS gene.

[0647] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an antibody. In certain embodiments, the antibody is a bispecific antibody. The antibody may be a fusion protein. In certain embodiments, the antibody may be part of a fusion protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antibody against CD3. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antibody against CD19.

[0648] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding an immunomodulator. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding interleukin-12. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding interleukin-23. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding interleukin-36 gamma. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding one or more constitutively active variants of the stimulator of interferon genes (STING) protein.

[0649] In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding an endonuclease. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding an RNA-guided DNA endonuclease protein, such as a Cas9 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding a meganuclease protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having a full-length mRNA encoding a zinc finger nuclease protein.

[0650] In embodiments, exemplary therapeutic uses result from the delivery of mRNA encoding a secreted protein. Thus, in embodiments, compositions and methods of the invention provide for the delivery of mRNA encoding a secreted protein. In some embodiments, compositions and methods of the invention provide for the delivery of mRNA encoding one or more secreted proteins listed in Table 1; thus, compositions of the invention may include mRNA encoding a protein listed in Table 1 (or a homolog thereof), along with other components described herein, and methods of the invention may include preparing and / or administering a composition comprising mRNA encoding a protein listed in Table 1 (or a homolog thereof), along with other components described herein. [Table 27-1] [Table 27-2] [Table 27-3] [Table 27-4]

Table 27-5

Table 27-6

Table 27-7

Table 27-8

Table 27-9

Table 27-10

Table 27-11

Table 27-12

Table 27-13

Table 27-14

Table 27-15

Table 27-16

Table 27-17

Table 27-18

Table 27-19

Table 27-20

Table 27-21

Table 27-22

Table 27-23

Table 27-24

Table 27-25

Table 27-26

Table 27-27

Table 27-28

Table 27-29

Table 27-30

Table 27-31

Table 27-32

Table 27-33

Table 27-34

Table 27-35

Table 27-36

Table 27-37

Table 27-38

[0651] In some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more additional exemplary proteins listed in Table 2; thus, compositions of the invention may include mRNAs encoding proteins listed in Table 2 (or homologs thereof), along with other components described herein, and methods of the invention may include preparing and / or administering a composition comprising mRNAs encoding proteins selected from proteins listed in Table 2 (or homologs thereof), along with other components described herein. [Table 28]

[0652] The Uniprot IDs listed in Tables 1 and 2 refer to the human version of the listed protein, and the sequence of each is available from the Uniprot database. The sequences of the listed proteins are also available for a variety of animals, including various mammals and animals of veterinary or industrial interest. Thus, in some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more proteins selected from mammalian homologs or homologs from animals of veterinary or industrial interest of the secreted proteins listed in Tables 1 and 2; thus, the compositions of the invention may include mRNAs encoding proteins selected from mammalian homologs or homologs from animals of veterinary or industrial interest of the proteins listed in Tables 1 and 2, along with other components described herein, and the methods of the invention provide for the delivery of proteins selected from mammalian homologs or homologs from animals of veterinary or industrial interest of the proteins listed in Tables 1 and 2. The present invention may include preparing and / or administering a composition comprising mRNA encoding the compound, along with other components described herein. In some embodiments, the mammalian homolog is selected from a mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homolog. In some embodiments, the animal of veterinary or industrial interest is selected from the above mammals, and / or chicken, duck, turkey, salmon, catfish, or tilapia.

[0653] In embodiments, compositions and methods of the invention provide for the delivery of mRNA encoding a lysosomal protein selected from Table 3. In some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNA encoding one or more lysosomal and / or related proteins listed in Table 3; thus, compositions of the invention may include mRNA encoding a protein listed in Table 3 (or a homolog thereof), along with other components described herein, and methods of the invention may include preparing and / or administering a composition comprising mRNA encoding a protein selected from a protein listed in Table 3 (or a homolog thereof), along with other components described herein. [Table 29-1] [Table 29-2]

[0654] Information regarding lysosomal proteins is available from Lubke et al., "Proteomics of the Lysosome," Biochim Biophys Acta. (2009) 1793:625-635. In some embodiments, the proteins listed in Table 3 and encoded by the mRNA in the compositions and methods of the invention are human proteins. Sequences of the listed proteins are also available for a variety of animals, including various mammals and animals of veterinary or industrial interest, as described above.

[0655] In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a therapeutic protein (e.g., cytoplasmic, transmembrane, or secreted), such as those listed in Table 4. ... Provides for the delivery of mRNA encoding a therapeutic protein useful in the treatment of a disease or disorder (i.e., an indication); thus, compositions of the invention may include mRNA encoding a therapeutic protein listed in Table 4 or not (or a homolog thereof as discussed below) for the treatment of a disease or disorder (i.e., an indication) listed in Table 4, along with other components described herein, and methods of the invention may include preparing and / or administering a composition comprising mRNA encoding such a protein (or a homolog thereof as discussed below) for the treatment of a disease or disorder listed in Table 4, along with other components described herein. [Table 30-1] [Table 30-2] [Table 30-3] [Table 30-4] [Table 30-5] [Table 30-6] [Table 30-7] [Table 30-8]

[0656] In some embodiments, the invention is used to prevent, treat, and / or cure a subject suffering from a disease or disorder listed in Tables 1, 2, 3, or 4, or a disease or disorder associated with a protein listed therein. In some embodiments, the mRNA encodes one or more of cystic fibrosis transmembrane conductance regulator (CFTR), argininosuccinate synthetase (ASS1), Factor IX, survival motor neuron 1 (SMN1), or phenylalanine hydroxylase (PAH). [Example]

[0657] Exemplary embodiments are described herein.

[0658] Example 1: Exemplary synthesis of compound (1) Compound (1) was prepared according to Scheme B. [ka]

[0659] Synthesis of (trioctyl 2-hydroxypropane-1,2,3-tricarboxylate) (A3-3) To a solution of citric acid A1 (2.1 g, 11.0 mmol) and 1-octanol A2-1 (9.4 g, 72.6 mmol) in dichloromethane (40 mL), DMAP (1.34 g, 11.0 mmol) and EDCI (14.3 g, 72.6 mmol) were added, and the resulting mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated under vacuum. The residue was dissolved in dichloromethane (200 mL) and washed with brine (100 mL × 3). After drying over anhydrous Na2SO4, the solvent was evaporated, and the crude material was purified by column chromatography (220 g SiO2: 0–20% ethyl acetate in hexane gradient) to give (trioctyl 2-hydroxypropane-1,2,3-tricarboxylate) as a colorless oil (5.2 g, 90%).

[0660] Synthesis of (trioctyl 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate) To a solution of trioctyl 2-hydroxypropane-1,2,3-tricarboxylate A3-1 (0.528 g, 1.0 mmol), DMAP (122 mg, 1.0 mmol), and pyridine (316 mg, 4.0 mmol) in 10 mL of dichloromethane, 3-(dimethylamino)propanoyl chloride A4-1 (271 mg, 2.0 mmol) was added at 0 °C, and the resulting mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed with brine (80 mL × 3). After drying over anhydrous NaSO, the solvent was evaporated and the crude was purified by column chromatography (80 g SiO: 0–10% methanol gradient in dichloromethane) to give trioctyl 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate as a colorless oil (210 mg, 33%).

[0661] 1 H NMR(300MHz,CDCl3)δ 4.56(s,br.,6H),4.24(t,2H),4.12(s,2H),2.55(t,2H),2.28-2.17 (m,14H),1.63-1.48(m,8H),1.25(s,br.,32H),0.86(t,12H).

[0662] APCI-MS analysis: calculated for C35H65NO8, [M+H] = 627.9, observed = 628.5.

[0663] Example 2: Exemplary synthesis of compound (2) [ka] Compound (2) can be prepared using an analogous process described in Scheme B.

[0664] 1H NMR(300MHz,CDCl3)δ 4.22-3.96(m,6H),3.33-3.16(m,4H),2.90-2.80(m,2H),2.62-2.49(m,2H),1.63-1.48(m,8H),1.25(s,br.,32H),1.06(t,6H),0.86(t,9H).

[0665] APCI-MS analysis: calculated for C37H69NO8, [M+H] = 655.9, observed = 656.5.

[0666] Example 3: Exemplary synthesis of compound (177) [ka]

[0667] (1,3-bis(octanoyloxy)-2-((octanoyloxy)methyl)propan-2-yl)glycine (3) and N-(1,3-bis(octanoyloxy)-2-((octanoyloxy)methyl)propan-2-yl)-N-octanoylglycine (3a ) and mixture synthesis To a solution of tricine 1 (3.0 g, 16.7 mmol) in dichloromethane (20 mL) was slowly added octanoyl chloride 2 (13.07 g, 80.37 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 5 h. TLC showed the disappearance of tricine, and MS showed both products. The volatiles were removed in vacuo to give a brown oil (6.0 g). The resulting material was used in the next step of the synthesis without further purification.

[0668] Synthesis of 2-(N-(2-(2-(dimethylamino)ethoxy)-2-oxoethyl)octanamido)-2-((octanoyloxy)methyl)propane-1,3-diyldioctanoate (Compound 177) [ka] To a mixture of (1,3-bis(octanoyloxy)-2-((octanoyloxy)methyl)propan-2-yl)glycine 3 and N-(1,3-bis(octanoyloxy)-2-((octanoyloxy)methyl)propan-2-yl)-N-octanoylglycine 3a (3.0 g, 8.37 mmol) in 20 mL of dichloromethane, EDCI (6.42 g, 33.5 mmol) and DMAP (1.02 g, 8.37 mmol) were added at 0 °C, and the resulting mixture was stirred at this temperature for 5 min. Dimethylaminoethanol 4 (3.0 g, 33.5 mmol) was added, and the resulting mixture was stirred at room temperature for 48 h. TLC and MS showed the formation of both products. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate and brine. After drying over sodium sulfate, the organic layer was evaporated under vacuum. The residue was purified by column chromatography (220 g SiO: 0-10% methanol gradient in dichloromethane) to give 2-(N-(2-(2-(dimethylamino)ethoxy)-2-oxoethyl)octanamido)-2-((octanoyloxy)methyl)propane-1,3-diyldioctanoate (125 mg) as a colorless oil.

[0669] 1 H NMR(300MHz,CDCl3)δ 4.56(s,br.,6H),4.24(t,2H),4.12(s,2H),2.55(t,2H),2.28-2.17(m,14H),1.63-1.48(m,8H),1.25(s,br.,32H),0.86(t,12H).

[0670] APCI-MS analysis: calculated for C42H78N2O9, [M+H] = 755.0, observed = 755.6.

[0671] Example 4: Formulation of lipid nanoparticles using compound (1) and in vivo expression of human erythropoietin (hEPO) The cationic lipids described herein can be used to prepare lipid nanoparticles according to methods known in the art. For example, suitable methods include those described in International Publication No. WO2018 / 089801, the entire contents of which are incorporated herein by reference.

[0672] One exemplary process for formulating lipid nanoparticles is described in WO2018 / 0898 This example illustrates Process A of WO2018 / 089801 (see, for example, Example 1 and Figure 1 of WO2018 / 089801). Process A ("A") relates to a conventional method for encapsulating mRNA by mixing mRNA with a lipid mixture without first preforming the lipids into lipid nanoparticles. In the exemplary process, an ethanolic lipid solution and an aqueous mRNA buffer solution were prepared separately. A lipid mixture (e.g., cationic lipid, helper lipid, zwitterionic lipid, PEG-lipid, etc.) solution was prepared by dissolving lipids in ethanol. An mRNA solution was prepared by dissolving mRNA in a citrate buffer. Both mixtures were then heated to 65°C before mixing. These two solutions were then mixed using a pump system. In some cases, these two solutions were mixed using a gear pump system. In certain embodiments, these two solutions were mixed using a "T" junction (or "Y" junction). The mixture was then purified by diafiltration using a TFF process. The resulting formulation was concentrated and stored at 2-8°C until further use.

[0673] A second exemplary process for formulating lipid nanoparticles is Process B of WO2018 / 089801 (see, for example, Example 2 and Figure 2 of WO2018 / 089801). Process B ("B") relates to a process for encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with the mRNA. Various conditions, such as different temperatures (i.e., whether the mixture is heated or not), buffers, and concentrations, can be used in Process B. In the exemplary process, lipids dissolved in ethanol and citrate buffer were mixed using a pump system. Instantaneous mixing of these two streams resulted in the formation of hollow lipid nanoparticles, a self-assembly process. The resulting formulation mixture was hollow lipid nanoparticles in a citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process, which involved buffer exchange. The resulting preformed empty lipid nanoparticle suspension was then mixed with the mRNA using a pump system. For certain cationic lipids, heating the solution after mixing resulted in a higher percentage of lipid nanoparticles containing mRNA and a higher overall yield of mRNA.

[0674] Lipid nanoparticle formulations containing exemplary cationic lipids and hEPO-encoding mRNA can be administered intravenously (IV) to mice to study mRNA delivery and resulting hEPO expression. Nanoparticle formulations of Compound (1) and hEPO mRNA were prepared by Process A above and administered intravenously in the compositions shown in Table 5 below. The formulations were prepared with the following molar ratios: cationic lipid:DMG-PEG2000; cholesterol:DPPC:DOPE=40:3:25:20:32. [Table 31]

[0675] Example 5: Delivery of firefly luciferase (FFL) mRNA by intratracheal administration A lipid nanoparticle formulation containing FFL mRNA, cationic lipid, DMG-PEG2000, cholesterol, and DOPE was administered to male CD1 mice (6-8 weeks old) under anesthesia by a single intratracheal aerosol administration (50 μL / animal) via a Microsprayer®. Approximately 24 hours after administration, animals received luciferin at 150 mg / kg (60 mg / mL) via intraperitoneal injection at 2.5 mL / kg. Five to 15 minutes later, all animals were imaged using an IVIS imaging system to visualize luciferase activity in the lungs. Figure 1 shows that lipid nanoparticles containing cationic lipids as described herein are effective for delivery of FFL mRNA in vivo based on positive luciferase activity.

[0676] While certain compounds, compositions, and methods of the present invention have been specifically described in accordance with certain embodiments, the disclosed examples serve only to illustrate the compounds of the present invention and are not intended to limit it.

Claims

1. A cationic lipid having a structure according to formula (A): 【Chemistry 255】 During the ceremony, n is independently 0 or 1 at each occurrence; X １Ａ are independently O or NR １Ａ and R １Ａ is H or C １ -C ６ is alkyl, X １Ｂ is a covalent bond, C(O), CH ２ CO ２ , or C.H. ２ C(O), X ２Ａ and X ２Ｂ one of which is O and the other is a covalent bond, X ３Ａ and X ３Ｂ one of which is O and the other is a covalent bond, X ４Ａ and X ４Ｂ one of which is O and the other is a covalent bond, R １ became independent, L １ -B １ , C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, R ２ became independent, L ２ -B ２ , C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, R ３ became independent, L ３ -B ３ , C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, R ４ became independent, L ４ -B ４ , C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, L １ , L ２ , L ３ , and L ４ are each independently C １ -C ３０ Alkylene, C ２ -C ３０ Alkenylene, or C ２ -C ３０ is alkynylene, B １ , B ２ , B ３ , and B ４ are each independently an ionic nitrogen-containing group; A cationic lipid, or a pharmaceutically acceptable salt thereof, wherein said cationic lipid comprises at least one ionizable nitrogen-containing group.

2. having a structure according to formula (I), 【256】 During the ceremony, R ２ , R ３ , and R ４ are each independently C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, L １ But C １ -C １０ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, which is alkylene.

3. having a structure according to formula (II): 【Chemistry 257】 During the ceremony, R ２ , R ３ , and R ４ are each independently C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, L １ But C １ -C １０ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, which is alkylene.

4. having a structure according to formula (AI): 【Chemical Formula 258】 During the ceremony, L １ But C １ -C １０ is alkylene, R ６Ａ are each independently H or C １ -C ６ is alkyl, R ６Ｂ are each independently H or C １ -C ６ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, wherein the cationic lipid is alkyl.

5. R ６Ａ is methyl, and R ６Ｂ The cationic lipid of claim 4, wherein is methyl.

6. 10. The cationic lipid of claim 1 or 4, having a structure according to formula (AII), or a pharmaceutically acceptable salt thereof. 【Chemical 259】

7. having a structure according to formula (AIII): 【Chemistry 260】 During the ceremony, R １Ａ The cationic lipid according to any one of claims 1 or 3 to 6, or a pharmaceutically acceptable salt thereof, wherein

8. having a structure according to formula (IIa): 【Chemical 261】 During the ceremony, B １ is an ionic nitrogen-containing group, R １Ａ is H or C(O)-R ７ and R ２ , R ３ , R ４ , and R ７ are each independently C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ is alkynyl, L １ But C １ -C １０ a cationic lipid, or a pharmaceutically acceptable salt thereof, which is alkylene;

9. 9. The cationic lipid of claim 8, having a structure according to formula (IIb): A commercially acceptable salt. 【Chemical 262】

10. 10. The cationic lipid of claim 8 or 9, having a structure according to formula (IIc), or a pharmaceutically acceptable salt thereof. 【Chemical 263】

11. 10. The cationic lipid of claim 8 or 9, having a structure according to formula (IId), or a pharmaceutically acceptable salt thereof. 【Chemical 264】

12. X １Ａ NR １Ａ The cationic lipid according to any one of claims 1 and 4 to 6,

13. R １Ａ The cationic lipid of any one of claims 1, 3 to 9, and 12, wherein is H.

14. X １Ｂ The cationic lipid according to any one of claims 1, 4 to 6, and 12 to 13, wherein is a covalent bond.

15. X １Ｂ is CH ２ CO ２ The cationic lipid according to any one of claims 1, 4 to 6, and 7 to 13,

16. L １ is unsubstituted C １ -C １０ The cationic lipid according to any one of claims 1 to 15, which is an alkylene.

17. L １ However, (CH ２ ) ２ , (CH ２ ) ３ , (CH ２ ) ４ , or (CH ２ ) ５ 17. The cationic lipid of claim 16, wherein:

18. L １ But (CH ２ ) ２ 18. The cationic lipid of claim 17, wherein

19. L １ But (CH ２ ) ３ 18. The cationic lipid of claim 17, wherein

20. B １ became independent and NH ２ 20. The cationic lipid according to any one of claims 1 to 3 and 8 to 19, which is a guanidine, an amidine, a mono- or di-alkylamine, a 5- to 6-membered nitrogen-containing heterocycloalkyl, or a 5- to 6-membered nitrogen-containing heteroaryl.

21. B １ became independent, 【Chemical 265】 21. The cationic lipid of claim 20, wherein

22. B １ became independent, 【Chemical Formula 266】 22. The cationic lipid of claim 21, wherein

23. R ２ , R ３ , and R ４ each independently represents an unsubstituted linear C ６ -C ２２ Alkyl, unsubstituted linear C ６ -C ２２ Alkenyl, unsubstituted linear C ６ -C ２２ Alkynyl, unsubstituted branched C ６ -C ２２ Alkyl, unsubstituted branched C ６ -C ２２ Alkenyl, or unsubstituted branched C ６ -C ２２ The cationic lipid according to any one of claims 1 to 22, which is alkynyl.

24. R ２ , R ３ , and R ４ are each unsubstituted C ６ -C ２２ The cationic lipid according to any one of claims 1 to 23, which is alkyl.

25. R ２ , R ３ , and R ４ each independently represents —O(CO)R ５ or -C(O)OR ５ C substituted by ６ -C １２ alkyl, wherein R ５ is unsubstituted C ６ -C １４ The cationic lipid according to any one of claims 1 to 22, which is alkyl.

26. R ２ , R ３ , and R ４ are each unsubstituted C ６ -C ２２ The cationic lipid according to any one of claims 1 to 23, which is alkenyl.

27. Said C ６ -C ２２ 27. The cationic lipid of claim 26, wherein the alkenyl is a monoalkenyl, dienyl, or trienyl.

28. R ２ , R ３ , and R ４ The cationic lipid according to any one of claims 1 to 22, wherein each of Table 32

29. R ２ , R ３ , R ４ , and R ７ The cationic lipid according to any one of claims 1 to 22, wherein each independently is: 【Table 33】

30. R ２ , R ３ , and R ４ The cationic lipid according to any one of claims 1 to 22, wherein each independently is: Table 34

31. R ２ , R ３ , R ４ , and R ７ The cationic lipid according to any one of claims 1 to 22, wherein each independently is: Table 35

32. R ２ , R ３ , R ４ , and R ７ are each independently C ８ H １７ , C １０ H ２１ , C １２ H ２５ , C １４ H ２９ , C １６ H ３３ , C １６ H ３１ , C １６ H ２９ , and C １６ H ３２ The cationic lipid according to any one of claims 1 to 22,

33. R ２ , R ３ , R ４ , and R ７ are each independently C ６ -C ２２ Alkyl, C ６ -C ２２ alkenyl, or C ６ -C ２２ The cationic lipid according to any one of claims 1 to 22, which is alkynyl.

34. R ２ , R ３ , R ４ , and R ７ are each independently C ６ -C ２２ Alkyl, or C ６ -C ２２ The cationic lipid according to any one of claims 1 to 22, which is alkenyl.

35. R ２ , R ３ , R ４ , and R ７ each independently represents an unsubstituted linear C ６ -C ２２ Alkyl, or unsubstituted linear C ６ -C ２２ The cationic lipid according to any one of claims 1 to 22, which is alkenyl.

36. R １ became independent and C ８ H １７ , C １０ H ２１ , C １２ H ２５ , C １４ H ２９ , C １６ H ３３ , C １６ H ３１ , C １６ H ２９ , and C １６ H ３２ The cationic lipid of claim 1, wherein

37. R １ are independently unsubstituted linear C ６ -C ２２ Alkyl, unsubstituted linear C ６ -C ２２ Alkenyl, or unsubstituted linear C ６ -C ２２ 2. The cationic lipid of claim 1, which is an alkynyl.

38. having a structure according to formula (III): 【Chemical 267】 During the ceremony, R １ became independent and C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, which is alkynyl.

39. having a structure according to formula (IV): 【Chemical 268】 During the ceremony, R １ became independent and C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, which is alkynyl.

40. R Ａ 40. The cationic lipid of claim 39, wherein

41. having a structure according to formula (V): 【Chemical 269】 During the ceremony, R １ became independent and C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, which is alkynyl.

42. having a structure according to formula (VI): 【Chemistry 270】 During the ceremony, R １ became independent and C ６ -C ３０ Alkyl, C ６ -C ３０ alkenyl, or C ６ -C ３０ 2. The cationic lipid of claim 1, or a pharmaceutically acceptable salt thereof, which is alkynyl.

43. R １Ａ 43. The cationic lipid of claim 42, wherein

44. R １ are independently unsubstituted linear C ６ -C ２２ Alkyl, unsubstituted linear C ６ -C ２２ Alkenyl, unsubstituted linear C ６ -C ２２ Alkynyl, unsubstituted branched C ６ -C ２２ Alkyl, unsubstituted branched C ６ -C ２２ Alkenyl, or unsubstituted branched C ６ -C ２２ The cationic lipid according to any one of claims 38 to 43, which is alkynyl.

45. R １ are independently unsubstituted C ６ -C ２２ 45. The cationic lipid of claim 44, which is alkyl.

46. R １ are independently —O(CO)R ５ or -C(O)OR ５ C substituted by ６ -C １２ alkyl, wherein R ５ is unsubstituted C ６ -C １４ The cationic lipid according to any one of claims 38 to 43, which is alkyl.

47. R １ are independently unsubstituted C ６ -C ２２ 45. The cationic lipid of claim 44, which is alkenyl.

48. Said C ６ -C ２２ 48. The cationic lipid of claim 47, wherein the alkenyl is a monoalkenyl, dienyl, or trienyl.

49. R １ became independent, Table 36 40. The cationic lipid of claim 38 or 39, wherein

50. R １ became independent, Table 37 43. The cationic lipid of claim 41 or 42, wherein

51. L ２ , L ３ , and L ４ are each unsubstituted C １ -C １０ The cationic lipid according to any one of claims 38 to 50, which is alkylene.

52. L ２ , L ３ , and L ４ are respectively (CH ２ ) ２ , (CH ２ ) ３ , (CH ２ ) ４ , or (CH ２ ) ５ 52. The cationic lipid of claim 51, wherein:

53. B ２ , B ３ , and B ４ are each independently NH ２ , guanidine, amidine, mono- or di-alkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

54. B ２ , B ３ , and B ４ are each independently, 【Chemical 271】 54. The cationic lipid of claim 53, wherein:

55. B ２ , B ３ , and B ４ are each independently, 【Chemical 272】 55. The cationic lipid of claim 54, wherein:

56. A cationic lipid which is any of compounds 1-264, Ia-Ih, IIb1-IIb4, IIc1-IIc4, IIIa-IIId, and Va-Vd, or a pharmaceutically acceptable salt thereof.

57. 57. A composition comprising an mRNA encoding a protein encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is as defined in any one of claims 1 to 56.

58. 58. The composition of claim 57, comprising mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

59. 58. The composition of claim 57, comprising mRNA encoding ornithine transcarbamylase (OTC) protein.

60. A composition comprising a nucleic acid encapsulated in a liposome, wherein the liposome comprises a cationic lipid according to any one of claims 1 to 56.

61. 61. The composition of claim 60, further comprising another lipid selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

62. 62. The composition of claim 60 or 61, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide.

63. 63. The composition of any one of claims 60-62, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject.

64. 64. The composition of claim 63, wherein the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein.

65. 63. The composition of any one of claims 60 to 62, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject.

66. 66. The composition of claim 65, wherein the mRNA encodes an ornithine transcarbamylase (OTC) protein.

67. 63. The composition of any one of claims 60 to 62, wherein the mRNA encodes a peptide or polypeptide for use in a vaccine.

68. 68. The composition of claim 67, wherein the mRNA encodes an antigen.

69. 69. The composition of claim 68, wherein the antigen is derived from an infectious pathogen.

Images