Vitamin cationic lipids

Abstract

To provide methods of treating various diseases.SOLUTION: Disclosed are cationic lipids comprising a vitamin substructure and an ionizable nitrogen-containing group. Cationic lipids provided herein can be useful for delivery and expression of mRNA and encoded protein, e.g., as a component of a liposomal delivery vehicle, and accordingly can be useful for treating various diseases, disorders and conditions, such as those associated with deficiency of one or more proteins.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 677,851 filed May 30, 2018, U.S. Provisional Application No. 62 / 677,828 filed May 30, 2018, U.S. Provisional Application No. 62 / 677,855 filed May 30, 2018, U.S. Provisional Application No. 62 / 677,818 filed May 30, 2018, U.S. Provisional Application No. 62 / 807,672 filed February 19, 2019, U.S. Provisional Application No. 62 / 807,671 filed February 19, 2019, and U.S. Provisional Application No. 62 / 807,673 filed February 19, 2019, each of which is incorporated 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 [Means for solving the problem]

[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 nervous system disease.

[0004] In one aspect, the present invention provides a liposome encapsulating an mRNA encoding a protein, the liposome comprising 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, the liposome comprising the structure of vitamin A, D, E, or K, and a moiety X 1 and at least one cationic lipid, wherein X 1 is an ionic nitrogen-containing group, and liposomes encapsulating mRNA encoding a protein are provided.

[0005] In another aspect, the invention provides a nucleic acid encapsulated in a liposome, wherein the liposome comprises the structure of vitamin A, D, E, or K, and the moiety X 1 and a cationic lipid comprising: 1 is an ionic nitrogen-containing group.

[0006] In embodiments, the cationic lipid is a cationic lipid having a structure according to any of the following structures: [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But, O, CR a Rb , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0007] In embodiments, the cationic lipid has a structure according to formula (AI): [ka]

[0008] In embodiments, the cationic lipid has a structure according to formula (A-Ia): [ka]

[0009] In embodiments, the cationic lipid has a structure according to formula (E-1): [ka]

[0010] In embodiments, the cationic lipid has a structure according to formula (E-1a): [ka]

[0011] In embodiments, the cationic lipid has a structure according to formula (K-1): [ka]

[0012] In embodiments, the cationic lipid has a structure according to formula (K-1a): [ka]

[0013] In embodiments, the cationic lipid has a structure according to formula (K-1b): [ka]

[0014] In embodiments, the cationic lipid has a structure according to formula (DA): [ka] During the ceremony, [ka] represents a single or double bond, X 1 is an ionic nitrogen-containing group, X 2 is O or S, Z is O or a covalent bond; R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; R2 is H or C1-C4-alkyl.

[0015] In embodiments, the cationic lipid has a structure according to any one of the following formulas: [ka]

[0016] In embodiments, the cationic lipid has a structure according to any one of the following formulas: [ka] [ka]

[0017] In some embodiments, X 2 is O.

[0018] In some embodiments, R 1 is C6-C 30 - alkylene.

[0019] In some embodiments, R 1 is C1-C5-alkylene.

[0020] In some embodiments, R 1 is unsubstituted C6-C 30 - alkylene.

[0021] In some embodiments, R 1 is unsubstituted C1-C5-alkylene.

[0022] In some embodiments, R 1 is -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0023] In some embodiments, R 1 is -C2H4-, -C3H6-, or -C4H8-.

[0024] In some embodiments, R 1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkylene.

[0025] In some embodiments, R 1 is C6-C 30 -Alkenylene or C8-C 20 -alkenylene.

[0026] In some embodiments, R 1 is C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14-Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 -alkenylene.

[0027] In some embodiments, R 1 represents unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0028] In some embodiments, R 1 -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2C H=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH 2-, -(CH2) 11 CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0029] In some embodiments, X 1 is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0030] In some embodiments, X 1 is a 5-6 membered nitrogen-containing heterocycloalkyl.

[0031] In some embodiments, X 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl.

[0032] In some embodiments, X 1 is a dialkylamine.

[0033] In some embodiments, X 1 is N(Me)2.

[0034] In some embodiments, X 1 teeth, [ka] where: R 3a and R 3b are each independently, C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; Each n is independently an integer having a value of about 1 to about 6.

[0035] In some embodiments, X 1 teeth, [ka] is.

[0036] In some embodiments, X 1 teeth, [ka] is.

[0037] In some embodiments, X 1 teeth, [ka] is.

[0038] In embodiments, the cationic lipid has the following structure: [ka]

[0039] In embodiments, the cationic lipid has the following structure: [ka]

[0040] In embodiments, the cationic lipid has the following structure: [ka]

[0041] In embodiments, the cationic lipid has the following structure: [ka]

[0042] In embodiments, the cationic lipid has the following structure: [ka]

[0043] In embodiments, the cationic lipid has the following structure: [ka]

[0044] In embodiments, the cationic lipid has the following structure: [ka] [ka]

[0045] In embodiments, the cationic lipid has the following structure: [ka] [ka]

[0046] In embodiments, the cationic lipid has the following structure: [ka] [ka]

[0047] In embodiments, the cationic lipid has the following structure: [ka] [ka]

[0048] In another aspect, the present invention provides a composition comprising mRNA encoding a peptide or polypeptide 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 described herein (e.g., Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA) , (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), for example, a cationic lipid of any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4).

[0049] In another aspect, the present invention provides a composition comprising mRNA encoding a peptide or polypeptide encapsulated in a liposome, the liposome comprising 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 described herein (e.g., a cationic lipid represented by Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and ( The present invention features a cationic lipid selected from the group consisting of cationic lipids of any of formulas E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4).

[0050] In embodiments, the composition comprises an mRNA encoding a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject.

[0051] In embodiments, the composition comprises mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0052] In embodiments, the composition comprises an mRNA encoding a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject.

[0053] In embodiments, the composition comprises mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0054] In embodiments, the composition comprises mRNA encoding a peptide or polypeptide for use in a vaccine.

[0055] In embodiments, the composition comprises mRNA encoding an antigen (eg, an antigen from an infectious pathogen).

[0056] In another aspect, the invention features a composition including a nucleic acid encapsulated in a liposome, wherein the liposome includes a cationic lipid described herein (e.g., a cationic lipid of any of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)).

[0057] In another aspect, the invention features a composition including a nucleic acid encapsulated in a liposome, wherein the liposome includes a cationic lipid described herein (e.g., a cationic lipid of any of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)).

[0058] In some embodiments, the composition comprises one or more cationic lipids, one or more non-cationic lipids, The composition further comprises one or more lipids selected from the group consisting of a lipid, and one or more PEG-modified lipids.

[0059] In embodiments, the nucleic acid is an mRNA that encodes a peptide or polypeptide.

[0060] In some embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject. In some embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0061] In some embodiments, the mRNA encodes a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject. In some embodiments, the mRNA encodes an ornithine transcarbamylase (OTC) protein.

[0062] In embodiments, the mRNA encodes a peptide or polypeptide for use in a vaccine. In embodiments, the mRNA encodes an antigen (e.g., an antigen from an infectious agent).

[0063] In some aspects, the present invention provides a method of treating a disease in a subject, the method comprising administering to the subject a composition (e.g., a pharmaceutical composition) described herein (e.g., a cationic lipid of any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)).

[0064] In some other aspects, the present invention provides a method of treating a disease in a subject, the method comprising administering to the subject a composition (e.g., a pharmaceutical composition) described herein (e.g., a cationic lipid of any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)).

[0065] In embodiments, the compositions are formulated for intravenous (IV) administration.

[0066] In embodiments, the compositions are formulated for intramuscular (IM) administration.

[0067] In embodiments, the composition is formulated for administration by inhalation (eg, the composition is formulated for nebulization). DETAILED DESCRIPTION OF THE INVENTION

[0068] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of these and other terms are set forth throughout the specification. Publications referenced herein to describe the background of the invention and to provide further details regarding its practice are set forth in the appended claims. and other references are incorporated herein by reference.

[0069] 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 prepared synthetically 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 substituents. 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 substitution 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, e.g., association with one or more chemicals (e.g., a methyl group, a hydrochloride 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.

[0070] 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.

[0071] 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 reference value provided. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls 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 of the reference value provided, unless otherwise provided or clear from the context (except when such number exceeds 100% of possible values).

[0072] 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.

[0073] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, delivery of mRNA can refer to a situation in which the mRNA is delivered to a target tissue, the encoded protein is expressed, and the mRNA is retained in the target tissue (also referred to as "local distribution" or "local delivery"), and a situation in which the mRNA is delivered to a target tissue, the encoded protein is expressed, and the mRNA is retained in the target tissue (also referred to as "local distribution" or "local delivery"). This includes situations where the compound is secreted into the patient's circulation (eg, serum), distributed throughout the body, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0074] 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.

[0075] 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.

[0076] 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 initially measured value over a period of time.

[0077] 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.

[0078] 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.

[0079] 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).

[0080] 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 more than about 99% of the other components with which they were originally associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 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.).

[0081] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids or by mixing one or more lipids with a polymer(s). In some embodiments, liposomes suitable for the present invention include: The liposomes comprise one or more cationic lipids, and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and / or optionally PEG-modified lipid(s).

[0082] 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, etc. 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, C5 propynyl-cytidine, C5 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), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0083] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are 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" encompasses ribonucleic acid (RNA), including, but not limited to, any one or more of 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 CRISPR 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 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 several 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 can 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.

[0084] 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.

[0085] 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.

[0086] 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. 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 include amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Salts derived from appropriate bases include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-methyl-N ... + (C 1-4 Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include, where appropriate, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, Included are non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as sulfonates and arylsulfonates. Additional pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, for example, alkyl halides, to form quaternized alkylated amino salts.

[0087] 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."

[0088] 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 can be a patient. It 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.

[0089] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a desired characteristic or property. Those skilled in the biological arts 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.

[0090] 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.

[0091] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of a symptom(s) 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.

[0092] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or who exhibit only early signs of disease, for the purpose of reducing the risk of developing conditions associated with the disease.

[0093] 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 20Alkynyl (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, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. For example, an aliphatic group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected) 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., an unsubstituted C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R' is independently unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not contain any heteroatoms.

[0094] 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., an unsubstituted C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently an unsubstituted C1-C3 alkyl. In some embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents 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.

[0095] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight- or branched-chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, and the like. Similarly, as used herein, the term "alkenylene" refers to an unsaturated divalent straight- 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" refers to an unsaturated divalent straight- 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′)′, —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., an unsubstituted C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, 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.

[0096] 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., an unsubstituted C1-C3 alkyl, or C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R' is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein). In embodiments, the alkenyl group is substituted with an -OH group and may also be referred to herein as a "hydroxyalkenyl" group, where the prefix indicates the -OH group and "alkenyl" is as described herein.

[0097] 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 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, an alkynyl contains one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more substituents described herein. For example, an alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of a 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 Alkyl, or C1- In embodiments, R' is independently an unsubstituted alkyl (e.g., an unsubstituted C-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently unsubstituted C1-C3 alkyl. In some embodiments, alkynyl is unsubstituted. In some embodiments, alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein).

[0098] Amine: The terms "amine" or "amino", used interchangeably throughout this specification, refer to the group -NZ 1 Z 2 is used herein to refer to1 and Z 2 Each Z is independently hydrogen, or alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy, amino, silyl, and combinations thereof. 1 and Z 2 may each independently be unsubstituted or substituted with one or more substituents described herein. For example, a dialkylamine group refers to the group -N(alkyl)2, where each alkyl group may be unsubstituted or substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide.

[0099] 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."

[0100] 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., an unsubstituted 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, cycloalkyl is unsubstituted. In some embodiments, cycloalkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein).

[0101] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine.

[0102] 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. Heteroalkenyls can optionally include monocyclic, bicyclic, or tricyclic rings, each of which desirably has 3 to 6 members. Heteroalkenyl groups can be substituted or unsubstituted.

[0103] 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.

[0104] 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.

[0105] 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 arrangement, 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, it also includes its 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" may be 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.

[0106] 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 1-4, 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. By way of example, in a saturated or partially unsaturated ring having 0-3 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).

[0107] A heterocyclic ring can be bonded 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 with a heterocyclyl, and 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.

[0108] 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 improvements in 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.

[0109] Specifically, there remains a need for improved cationic lipids that exhibit 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 also remains a particular need for novel cationic lipids that are characterized as having reduced toxicity and can efficiently deliver encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0110] 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.

[0111] 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 neutral or charged form.

[0112] 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 as having 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.

[0113] Exemplary vitamin-based cationic lipids are described herein.Such exemplary cationic lipids can be used in any of the compositions and methods described herein.For example, any of the vitamin-based cationic lipids can be used in any of the liposomes described herein and any of the nucleic acids encapsulated in liposomes described herein, and their compositions and methods of use.

[0114] cationic lipids Vitamin A Cationic Lipid In one aspect, the present invention provides cationic lipids derived from vitamin A.

[0115] Cationic lipids of formula (AI) In one aspect, the present invention provides a cationic lipid of formula (AI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0116] In some embodiments, X 2 is S.

[0117] In some embodiments, X 2 is C=O.

[0118] In some embodiments, X 2 is C=S.

[0119] In some embodiments, X 3 is S.

[0120] In some embodiments, X 3 is O.

[0121] In some embodiments, X 3 is CR a R b where R a and R b are each independently H, C-C-alkyl, C-C-alkoxy, C-C-cycloalkyl, C-C-alkenyl, or C-C-alkynyl; 3 is CR a R b where R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring.

[0122] In some embodiments, X 3 is NR c where R c is independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0123] In embodiments, the cationic lipid has a structure according to formula (A-Ia): [ka]

[0124] In some embodiments, R 1 is C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl.

[0125] In some embodiments, R 1 is C6-C 30 - alkyl.

[0126] In some embodiments, R 1 is C1-C5-alkyl.

[0127] In some embodiments, R 1 is unsubstituted C6-C 30 - alkyl.

[0128] In some embodiments, R 1 is unsubstituted C1-C5-alkyl.

[0129] In some embodiments, R 1 -C1H2-, -C2H4-, -C3H6-, C4H8-, -C5H 10 -, C6H 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C25 H 50 -It is.

[0130] In some embodiments, R 1 is -CH 13 -, -C7H 25 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H2 6-, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0131] In some embodiments, R 1 is -C2H4-, -C3H6-, or -C4H8-.

[0132] In some embodiments, R 1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkylene.

[0133] In some embodiments, R 1 is C6-C 30-Alkenylene or C8-C 20 -alkenylene.

[0134] In some embodiments, R 1 is C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 -alkenylene.

[0135] In some embodiments, R 1 represents unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0136] In some embodiments, R 1 -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH2, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 CH=CH(CH2)7CH2-3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0137] An ionizable nitrogen-containing group may 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 NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0138] In some embodiments, X 1is a 5-6 membered nitrogen-containing heterocycloalkyl. Suitable 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolyl, pyrrolinyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and oxazinyl. In some embodiments, X 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl.

[0139] In some embodiments, X 1 is a dialkylamine. In some embodiments, X 1 is an unsubstituted dialkylamine. In some embodiments, X 1 is a substituted dialkylamine.

[0140] In some embodiments, X 1 is N(Me)2.

[0141] In some embodiments, X 1 teeth, [ka] where: R 3a and R 3b are each independently, C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; Each n is independently an integer having a value of about 1 to about 6.

[0142] In some embodiments, R3a and R 3b are each independently C6-C 30 - alkyl.

[0143] In some embodiments, R 3a and R 3b are each independently an unsubstituted C-C 30 - alkyl.

[0144] In some embodiments, R 3a and R 3b are each 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 , -C 23 H 47 , -C 24 H 49 , or -C 25 H 51 is.

[0145] In some embodiments, R 3a and R 3b each independently a C-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkyl.

[0146] In some embodiments, R 3a and R 3b are each independently C6-C 30 -Alkenyl or C8-C 20 -alkenyl.

[0147] In some embodiments, R 3a and R 3b are each independently C8-alkenyl, C9- Alkenylene, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 -alkenyl.

[0148] In some embodiments, R 3a and R 3b are each independently an unsubstituted C8-alkenyl, an unsubstituted C9-alkenyl, an unsubstituted C 10 -alkenyl, unsubstituted C 11 -alkenyl, unsubstituted C 12 -alkenyl, unsubstituted C 13 -alkenyl, unsubstituted C 14 -alkenyl, unsubstituted C 15 -alkenyl, unsubstituted C 16 -alkenyl, unsubstituted C 17 -alkenyl, unsubstituted C 18 -alkenyl, unsubstituted C 19 -alkenyl, and unsubstituted C 20 -alkenyl.

[0149] In some embodiments, R 3a and R 3bare each 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, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0150] In some embodiments, X 1 teeth, [ka] is.

[0151] In embodiments, the ionic nitrogen-containing group is [ka] is.

[0152] In some embodiments, X 1 teeth, [ka] is.

[0153] Cationic lipids of formula (A-II) In one aspect, the present invention provides a cationic lipid of formula (A-II): [ka] During the ceremony, X 4 is an ionic nitrogen-containing group, X 5 are each independently S, C=O, or C=S; X 6 are each independently S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0154] In some embodiments, X 5 are each S.

[0155] In some embodiments, X 5 are each C=O.

[0156] In some embodiments, X 5 where C=S.

[0157] In some embodiments, X 6 are each S.

[0158] In some embodiments, X 6 are each O.

[0159] In some embodiments, X 6 are CR a R b where R a and R b are each independently H, C-C-alkyl, C-C-alkoxy, C-C-cycloalkyl, C-C-alkenyl, or C-C-alkynyl; 6 are CR a R b where R a and R b and the carbon atoms to which they are attached. Together they form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring.

[0160] In some embodiments, X 6 are respectively, NR c where R c is independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0161] In embodiments, the cationic lipid of formula (A-II) has a structure according to formula (A-IIa): [ka]

[0162] In some embodiments, X 4is NH, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. For example, in some embodiments, X 4 teeth, [ka] is.

[0163] In some embodiments, X 4 is any ionic nitrogen-containing group described herein (e.g., X 4 is X in formula (I) or (Ia) 1 (The group may be any of the groups listed for

[0164] Vitamin D Cationic Lipid In one aspect, the present invention provides cationic lipids derived from vitamin D.

[0165] Cationic lipids of formula (DA) In one aspect, the present invention provides cationic lipids having a structure according to formula (DA): [ka] During the ceremony, [ka] represents a single or double bond, X 1 is an ionic nitrogen-containing group, X 2 is O or S, Z is O or a covalent bond; R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30-alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; R 2 is H or C1-C4-alkyl.

[0166] In some embodiments, [ka] represents a single bond.

[0167] In some embodiments, [ka] represents a double bond.

[0168] In embodiments, Z is a covalent bond (eg, a compound of formula (DI)).

[0169] In embodiments, Z is O (eg, a compound of formula (D-III)).

[0170] In some embodiments, X 2 is O.

[0171] In some embodiments, X 2 is S.

[0172] In some embodiments, R 1 is C1-C5-alkylene.

[0173] In some embodiments, R 1 is C6-C 30 - alkylene.

[0174] In some embodiments, R 1 is unsubstituted C1-C5-alkylene.

[0175] In some embodiments, R 1 is unsubstituted C6-C30 - alkylene.

[0176] In some embodiments, R 1 -C1H2-, -C2H4-, -C3H6-, C4H8-, -C5H 10 -, C6H 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0177] In some embodiments, R 1 is -C2H4-, -C3H6-, or C4H8-.

[0178] In some embodiments, R 1 is -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22-, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0179] In some embodiments, R 1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkyl.

[0180] In some embodiments, R 1 is C6-C 30 -Alkenylene or C8-C 20 -alkenylene.

[0181] In some embodiments, R 1 is C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C19 -alkenylene, and C 20 -alkenylene.

[0182] In some embodiments, R 1 represents unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0183] In some embodiments, R 1 -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH= CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CH CH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0184] In some embodiments, R 2 is H.

[0185] In some embodiments, R 2 is C1-C4-alkyl. In some embodiments, R 2 is unsubstituted C1-C4-alkyl. In some embodiments, R 2 is substituted C1-C4-alkyl. In some embodiments, R 2 is CH3. In some embodiments, R 2 is CH2CH3.

[0186] In some embodiments, X 1 is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0187] In some embodiments, X 1 is a 5-6 membered nitrogen-containing heterocycloalkyl. Suitable 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolyl, pyrrolinyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and oxazinyl. In some embodiments, X 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl.

[0188] In some embodiments, X 1 is a dialkylamine. In some embodiments, X 1 is an unsubstituted dialkylamine. In some embodiments, X 1 is a substituted dialkylamine.

[0189] In some embodiments, X 1 is N(Me)2.

[0190] In some embodiments, X 1 teeth, [ka] where: R 3a and R 3b are each independently, C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; Each n is independently an integer having a value of about 1 to about 6.

[0191] In some embodiments, R 3a and R 3b are each independently C6-C 30 - alkylene.

[0192] In some embodiments, R 3a and R 3b are each independently an unsubstituted C-C 30 - alkylene.

[0193] In some embodiments, R 3a and R 3bare each independently -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0194] In some embodiments, R 3a and R 3b each independently a C-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkylene.

[0195] In some embodiments, R 3a and R 3b are each independently C6-C 30 -Alkenylene or C8-C 20 -alkenylene.

[0196] In some embodiments, R 3a and R 3bare each independently C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 -alkenylene.

[0197] In some embodiments, R 3a and R 3b are each independently an unsubstituted C8-alkenylene, an unsubstituted C9-alkenylene, an unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0198] In some embodiments, R 3a and R 3b are each independently -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0199] In some embodiments, X 1 teeth, [ka] is.

[0200] In some embodiments, X 1 teeth, [ka] is.

[0201] In some embodiments, X 1 teeth, [ka] and suitable 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolyl, pyrrolinyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and oxazinyl. 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl, or [ka] is.

[0202] Cationic lipids of formula (DI) In one aspect, the present invention provides a cationic lipid of formula (DA) having a structure according to formula (DI): [ka] During the ceremony, [ka] represents a single or double bond, X 1 is an ionic nitrogen-containing group, X 2 is O or S, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; R 2 is H or C1-C4-alkyl. In some embodiments, R 2is H. Alternatively, in some embodiments, R 2 is C1-C4-alkyl, such as methyl, ethyl, propyl, isopropyl, or butyl. In a preferred embodiment, R 2 is H, methyl, or ethyl.

[0203] In embodiments, the cationic lipid has a structure according to formula (D-Ia): [ka]

[0204] In embodiments, the cationic lipid has a structure according to formula (D-Ib): [ka]

[0205] In embodiments, the cationic lipid has a structure according to formula (D-Ic): [ka]

[0206] In embodiments, the cationic lipid has a structure according to formula (D-Id): [ka]

[0207] In some embodiments, X 2 is O.

[0208] In some embodiments, X 2 is S.

[0209] In some embodiments, R 1 is C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30-Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene.

[0210] In some embodiments, R 1 is C6-C 30 - alkylene.

[0211] In some embodiments, R 1 is unsubstituted C6-C 30 - alkylene.

[0212] In some embodiments, R 1 is -CH 12 , -CH 14 , -CH 16 , -CH 18 , -C 10 H 20 , -C 11 H 22 , -C 12 H 24 , -C 13 H 26 , -C 14 H 28 , -C 15 H 30 , -C 16 H 32 , -C 17 H 34 , -C 18 H 36 , -C 19 H 38 , -C 20 H 40 , -C 21 H 42 , -C 22 H 44 , -C 23 H 46 , -C 24 H 48 , or -C 25 H 50 is.

[0213] In some embodiments, R1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkyl.

[0214] In some embodiments, R 1 is C6-C 30 -Alkenylene or C8-C 20 -alkenylene.

[0215] In some embodiments, R 1 is C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 -alkenylene.

[0216] In some embodiments, R 1 represents unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0217] In some embodiments, R 1-(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0218] As used herein, an ionizable nitrogen-containing group may 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.

[0219] Thus, in some embodiments, X 1is NH, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. For example, in some embodiments, X 1 teeth, [ka] is.

[0220] In some embodiments, X 1 is a 5-6 membered nitrogen-containing heterocycloalkyl. Suitable 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolyl, pyrrolinyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and oxazinyl. In some embodiments, X 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl.

[0221] In some embodiments, X 1 is a dialkylamine. In some embodiments, X 1 is an unsubstituted dialkylamine. In some embodiments, X 1 is a substituted dialkylamine.

[0222] In some embodiments, X 1 is N(Me)2.

[0223] In some embodiments, X 1 teeth, [ka] where: R 3a and R 3b are each independently, C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30-Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; Each n is independently an integer having a value of about 1 to about 6.

[0224] In some embodiments, X 1 teeth, [ka] is.

[0225] In some embodiments, X 1 teeth, [ka] is.

[0226] Cationic lipids of formula (D-II) In one aspect, the present invention provides a cationic lipid of formula (D-II): [ka] During the ceremony, X 3 is an ionic nitrogen-containing group, X 4 are each independently S or O.

[0227] In some embodiments, X 4 are each S.

[0228] In some embodiments, X 4 are each O.

[0229] In embodiments, the cationic lipid of formula (D-II) has a structure according to formula (D-IIa): [ka]

[0230] In some embodiments, X 3 is any ionic nitrogen-containing group described herein (e.g., X 3 is X in formula (DA), (DI), (D-III), (D-Ia), (D-Ib), (D-Ic), (D-Id), (D-IIIa), (D-IIIb), (D-IIIc), or (D-IIId). 1 (The group may be any of the groups listed for

[0231] In some embodiments, X 3 is NH2, guanidine, amidine, mono- or di-alkyl For example, in some embodiments, X is a 5- to 6-membered heterocycloalkyl, a 5- to 6-membered nitrogen-containing heteroaryl, or a 5- to 6-membered nitrogen-containing heteroaryl. 3 teeth, [ka] is.

[0232] Cationic lipids of formula (D-III) In one aspect, the present invention provides a cationic lipid of formula (D-III): [ka] During the ceremony, [ka] represents a single or double bond, X 1 is an ionic nitrogen-containing group, X 2 is O or S, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30-Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; R 2 is H or C1-C4-alkyl.

[0233] In some embodiments, R 2 is H. Alternatively, in some embodiments, R 2 is C1-C4-alkyl, such as methyl, ethyl, propyl, isopropyl, or butyl. In a preferred embodiment, R 2 is H, methyl, or ethyl.

[0234] In embodiments, the cationic lipid has a structure according to formula (D-IIIa): [ka]

[0235] In embodiments, the cationic lipid has a structure according to formula (D-IIIb): [ka]

[0236] In embodiments, the cationic lipid has a structure according to formula (D-IIIc): [ka]

[0237] In embodiments, the cationic lipid has a structure according to formula (D-IIId): [ka]

[0238] In some embodiments, X 2 is O.

[0239] In some embodiments, X 2 is S.

[0240] In some embodiments, R 1 is C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene.

[0241] In some embodiments, R 1 is C1-C5-alkylene.

[0242] In some embodiments, R 1 is C6-C 30 - alkylene.

[0243] In some embodiments, R 1 is unsubstituted C1-C5-alkylene.

[0244] In some embodiments, R 1 is unsubstituted C6-C 30 - alkylene.

[0245] In some embodiments, R 1 -C1H2-, -C2H4-, -C3H6-, C4H8-, -C5H 10 -, C6H 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0246] In some embodiments, R 1 is -C2H4-, -C3H6-, or C4H8-.

[0247] In some embodiments, R 1 is C6H 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0248] In some embodiments, R 1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkyl.

[0249] In some embodiments, R 1 is C6-C 30 -Alkenylene or C8-C 20 -alkenylene.

[0250] In some embodiments, R 1 is C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 -alkenylene.

[0251] In some embodiments, R 1 represents unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0252] In some embodiments, R 1 -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0253] As used herein, an ionizable nitrogen-containing 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 NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0254] In some embodiments, X 1 is a 5-6 membered nitrogen-containing heterocycloalkyl, such as, for example, 1 is a 5-6 membered nitrogen-containing heterocycloalkyl. In some embodiments, X 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl.

[0255] In some embodiments, X 1 is a substituted dialkylamine.

[0256] In some embodiments, X 1 teeth, [ka] where: R 3a and R 3b are each independently, C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; Each n is independently an integer having a value of about 1 to about 6.

[0257] In some embodiments, X 1 teeth, [ka] In some embodiments, X 1 teeth, [ka] In some embodiments, X 1 teeth, [ka] is.

[0258] Vitamin E Cationic Lipid In one aspect, the present invention provides cationic lipids derived from vitamin E.

[0259] Cationic lipids of formula (EI) In one aspect, the present invention provides a cationic lipid of formula (EI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NRc and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0260] In some embodiments, X 2 is S.

[0261] In some embodiments, X 2 is C=O.

[0262] In some embodiments, X 2 is C=S.

[0263] In some embodiments, X 3 is S.

[0264] In some embodiments, X 3 is O.

[0265] In some embodiments, X 3 is CR a R b where R a and R b are each independently H, C-C-alkyl, C-C-alkoxy, C-C-cycloalkyl, C-C-alkenyl, or C-C-alkynyl; 3 is CR a R b where Ra and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring.

[0266] In some embodiments, X 3 is NR c where R c is independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0267] In embodiments, the cationic lipid has a structure according to formula (E-Ia): [ka]

[0268] In some embodiments, R 1 is C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene.

[0269] In some embodiments, R 1 is C1-C5-alkylene.

[0270] In some embodiments, R 1 is C6-C 30 - alkylene.

[0271] In some embodiments, R 1 is unsubstituted C6-C 30 - alkylene.

[0272] In some embodiments, R 1 is unsubstituted C1-C5-alkylene.

[0273] In some embodiments, R 1 -C1H2-, -C2H4-, -C3H6-, C4H8-, -C5H 10 -, C6H 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0274] In some embodiments, R 1 is -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 -It is.

[0275] In some embodiments, R 1 is -C2H4-, -C3H6-, or C4H8-.

[0276] In some embodiments, R 1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkylene.

[0277] In some embodiments, R 1 is C6-C 30 -Alkenylene or C8-C 20 -alkenylene.

[0278] In some embodiments, R 1 is C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 -alkenylene.

[0279] In some embodiments, R 1 represents unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 -alkenylene.

[0280] In some embodiments, R 1 -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH2, -(CH2) 18CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-.

[0281] An ionizable nitrogen-containing group may 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 NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl.

[0282] In some embodiments, X 1 is a 5-6 membered nitrogen-containing heterocycloalkyl. Suitable 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolyl, pyrrolinyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and oxazinyl. In some embodiments, X 1 is a substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl.

[0283] In some embodiments, X 1 is a dialkylamine. In some embodiments, X 1 is an unsubstituted dialkylamine. In some embodiments, X 1 is a substituted dialkylamine.

[0284] In some embodiments, X 1 teeth, [ka] where: R 3a and R 3b are each independently, C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; Each n is independently an integer having a value of about 1 to about 6.

[0285] In some embodiments, R 3a and R 3b are each independently C6-C 30 - alkyl.

[0286] In some embodiments, R 3a and R 3b are each independently an unsubstituted C-C 30 - alkyl.

[0287] In some embodiments, R 3a and R 3b are each independently -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C10 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.

[0288] In some embodiments, R 3a and R 3b each independently a C-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkyl.

[0289] In some embodiments, R 3a and R 3b are each independently C6-C 30 -Alkenyl or C8-C 20 -alkenyl.

[0290] In some embodiments, R 3a and R 3b are each independently C8-alkenyl, C9-alkenylene, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14-Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 -alkenyl.

[0291] In some embodiments, R 3a and R 3b are each independently an unsubstituted C8-alkenyl, an unsubstituted C9-alkenyl, an unsubstituted C 10 -alkenyl, unsubstituted C 11 -alkenyl, unsubstituted C 12 -alkenyl, unsubstituted C 13 -alkenyl, unsubstituted C 14 -alkenyl, unsubstituted C 15 -alkenyl, unsubstituted C 16 -alkenyl, unsubstituted C 17 -alkenyl, unsubstituted C 18 -alkenyl, unsubstituted C 19 -alkenyl, and unsubstituted C 20 -alkenyl.

[0292] In some embodiments, R 3a and R 3b are each 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, -(C H2) 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, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0293] In some embodiments, X 1 teeth, [ka] is.

[0294] In embodiments, the ionic nitrogen-containing group is [ka] is.

[0295] In some embodiments, X 1 teeth, [ka] is.

[0296] Cationic lipids of formula (E-II) In one aspect, the present invention provides a cationic lipid of formula (E-II): [ka] During the ceremony, X 4is an ionic nitrogen-containing group, X 5 are each independently S, C=O, or C=S; X 6 are each independently S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0297] In some embodiments, X 5 are each S.

[0298] In some embodiments, X 5 are each C=O.

[0299] In some embodiments, X 5 where C=S.

[0300] In some embodiments, X 6 are each S.

[0301] In some embodiments, X 6 are each O.

[0302] In some embodiments, X 6 are CR a R b where R a and Rb are each independently H, C-C-alkyl, C-C-alkoxy, C-C-cycloalkyl, C-C-alkenyl, or C-C-alkynyl; 3 are CR a R b where R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring Form.

[0303] In some embodiments, X 6 are respectively, NR c where R c is independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0304] In embodiments, the cationic lipid of formula (E-II) has a structure according to formula (E-IIa): [ka]

[0305] In some embodiments, X 4 is any ionic nitrogen-containing group described herein (e.g., X 4 is X in formula (EI) or (E-Ia) 1 (The group may be any of the groups listed for

[0306] In some embodiments, X 4 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, X 4 teeth, [ka] is.

[0307] Vitamin K Cationic Lipid In one aspect, the present invention provides cationic lipids derived from vitamin K.

[0308] Cationic lipids of formula (KI) In one aspect, the present invention provides a cationic lipid of formula (KI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R care independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0309] In some embodiments, X 2 is S.

[0310] In some embodiments, X 2 is C=O.

[0311] In some embodiments, X 2 is C=S.

[0312] In some embodiments, X 3 is S.

[0313] In some embodiments, X 3 is O.

[0314] In some embodiments, X 3 is CR a R b where R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0315] In some embodiments, X 3 is CR a R b where R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring.

[0316] In some embodiments, X 3 is NR c where R cis independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0317] In embodiments, the cationic lipid of formula (KI) has a structure according to formula (K-Ia): [ka]

[0318] In embodiments, the cationic lipid of formula (KI) has a structure according to formula (K-Ib): [ka]

[0319] In some embodiments, R 1 is C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl.

[0320] In some embodiments, R 1 is C6-C 30 - alkyl.

[0321] In some embodiments, R 1 is unsubstituted C6-C 30 - alkyl.

[0322] In some embodiments, R 1 is -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H21 , -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.

[0323] In some embodiments, R 1 is a C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 - alkyl.

[0324] In some embodiments, R 1 is C6-C 30 -Alkenyl or C8-C 20 -Arke It's Nil.

[0325] In some embodiments, R 1 is C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C18 -Alkenyl, C 19 -alkenyl, and C 20 -alkenyl.

[0326] In some embodiments, R 1 represents unsubstituted C8-alkenyl, unsubstituted C9-alkenyl, unsubstituted C 10 -alkenyl, unsubstituted C 11 -alkenyl, unsubstituted C 12 -alkenyl, unsubstituted C 13 -alkenyl, unsubstituted C 14 -alkenyl, unsubstituted C 15 -alkenyl, unsubstituted C 16 -alkenyl, unsubstituted C 17 -alkenyl, unsubstituted C 18 -alkenyl, unsubstituted C 19 -alkenyl, and unsubstituted C 20 -alkenyl.

[0327] In some embodiments, R 1 are -(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, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0328] An ionizable nitrogen-containing group may 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.

[0329] Cationic lipids of formula (K-II) In one aspect, the present invention provides a cationic lipid of formula (K-II): [ka] During the ceremony, X 4 is an ionic nitrogen-containing group, X 5are each independently S, C=O, or C=S; X 6 are each independently S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b may each be combined together with the carbon atom to which they are attached to form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl.

[0330] In some embodiments, X 5 are each S.

[0331] In some embodiments, X 5 are each C=O.

[0332] In some embodiments, X 5 where C=S.

[0333] In some embodiments, X 6 are each S.

[0334] In some embodiments, X 6 are each O.

[0335] In embodiments, the cationic lipid of formula (K-II) has a structure according to formula (K-IIa): [ka]

[0336] In some embodiments, X 4 is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. For example, in some embodiments, X 4 teeth, [ka] is.

[0337] Exemplary Cationic Lipids One exemplary cationic lipid of the present invention is cationic lipid (A1). [ka]

[0338] Another exemplary cationic lipid of the present invention is cationic lipid (A2). [ka]

[0339] One exemplary cationic lipid of the present invention is cationic lipid (3). [ka]

[0340] One exemplary cationic lipid of the present invention is cationic lipid (A4). [ka]

[0341] One exemplary cationic lipid of the present invention is cationic lipid (D1). [ka]

[0342] Another exemplary cationic lipid of the present invention is cationic lipid (D2). [ka]

[0343] Yet another exemplary cationic lipid of the present invention is cationic lipid (D3). [ka]

[0344] Yet another exemplary cationic lipid of the present invention is cationic lipid (D4). [ka]

[0345] Another exemplary cationic lipid of the present invention is cationic lipid (D5). [ka]

[0346] One exemplary cationic lipid of the present invention is cationic lipid (D6). [ka]

[0347] One exemplary cationic lipid of the present invention is cationic lipid (D7). [ka]

[0348] One exemplary cationic lipid of the present invention is cationic lipid (E1). [ka]

[0349] Another exemplary cationic lipid of the present invention is cationic lipid (E2). [ka]

[0350] Yet another exemplary cationic lipid of the present invention is cationic lipid (E3). [ka]

[0351] Yet another exemplary cationic lipid of the present invention is cationic lipid (E4). [ka]

[0352] Another exemplary cationic lipid of the present invention is cationic lipid (E5). [ka]

[0353] One exemplary cationic lipid of the present invention is cationic lipid (K1). [ka]

[0354] Another exemplary cationic lipid of the present invention is cationic lipid (K2). [ka]

[0355] Yet another exemplary cationic lipid of the present invention is cationic lipid (K3). [ka]

[0356] Yet another exemplary cationic lipid of the present invention is cationic lipid (K4). [ka]

[0357] Synthesis of cationic lipids The cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), any of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), for example, any of the cationic lipids of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)) can be prepared according to methods known in the art.

[0358] Exemplary synthetic methods are provided in the Examples.

[0359] nucleic acid The cationic lipids described herein (e.g., any of the cationic lipids of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)) can be used to prepare compositions useful for delivery of nucleic acids.

[0360] 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 vary depending on the specific application.

[0361] 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.

[0362] The desired mRNA sequence(s) 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 performed 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, displayed, for example, 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.

[0363] 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. DNA can be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a product of polymerase chain reaction (PCR), a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups. RNA can be messenger RNA (mRNA), ribosomal RNA (RIRNA), or any other RNA. The RNA may be in the form of rRNA, signal recognition particle RNA (7SL 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 RNA, retrotransposon, viral genome, viroid, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is an mRNA that encodes a protein.

[0364] 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.

[0365] 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.

[0366] The desired mRNA sequence(s) 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 performed 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, displayed, for example, 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.

[0367] modified mRNA In some embodiments, mRNA according to the present invention can be synthesized as unmodified mRNA or modified mRNA. Modified mRNA includes nucleotide modifications in RNA. Thus, modified mRNA according to the present invention can include nucleotide modifications, such as backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, and the like. 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethyl Uracils can be synthesized as uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, and as 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.

[0368] In some embodiments, mRNA may comprise RNA backbone modification. Typically, backbone modification is a modification that chemically modifies 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 (e.g., 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.

[0369] 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-alkyloligoribonucleotides, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-amino ... and 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0370] In some embodiments, mRNA may contain a modification of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also called base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine ... Lysine 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-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaade These include, but are not limited to, adenosine 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.

[0371] Typically, mRNA synthesis involves the addition of a "cap" onto the N-terminus (5') and a "tail" onto 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.

[0372] 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 form a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G.

[0373] In some embodiments, the mRNA comprises a 3' poly(A) tail structure. The poly-A tail on 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 the mRNA typically comprises 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 in addition to or in place of the poly-A tail.

[0374] 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 iron-responsive elements. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0375] 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.

[0376] Cap Structure 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; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G.

[0377] 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.

[0378] A common cap on mRNA produced by in vitro transcription is m 7G(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 the form m as a transcription initiator. 7 G(5')ppp(5')G("m 7 It uses a preformed dinucleotide of the formula GpppG.

[0379] To date, the usual form of synthetic dinucleotide cap used in in vitro translation experiments is the anti-inverted 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.

[0380] 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, 9:1108-22 (2003)).

[0381] 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.

[0382] In some embodiments, the cap is a Cap 0 structure. A 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. A Cap 1 structure lacks a 2'-O-methyl residue attached to 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.

[0383] Various m 7 G-cap analogs are known in the art, and many of them 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, 9:1108-22 (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, 10:1479-87 (2004)), phosphorothioate cap analogs (as described in Grudzien-Nogalska, E., et al., RNA, 13:1745-55 (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.

[0384] Tail Structure Typically, the presence of a "tail" serves to protect mRNA from exonuclease degradation. PolyA tails are thought to stabilize natural messenger and synthetic sense RNA. Therefore, in certain embodiments, a long polyA tail can be added to an mRNA molecule, resulting in a more stable RNA. PolyA tails can be added using various techniques recognized in the art. For example, long polyA tails can be added to synthetic or in vitro transcribed RNA using polyA polymerase (Yokoe, et al. Nature Biotechnology. 1996;14:1252-56). 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)).

[0385] 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.

[0386] 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 is 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. It can be controlled.

[0387] 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 iron-responsive elements. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0388] 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.

[0389] Exemplary 3' and / or 5' untranslated sequences may 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 may 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, e.g., to improve the resistance of such polynucleotides to in vivo nuclease digestion.

[0390] Pharmaceutical Formulations of Cationic Lipids and Nucleic Acids In certain embodiments, the cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and formula K (e.g., (KI) to (K-II) and Cationic lipids such as any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)), 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) and subsequent transfection of one or more target cells. For example, in certain embodiments, the cationic lipids described herein (and compositions, such as liposomal compositions, comprising such lipids) can be characterized as 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.

[0391] According to the present invention, nucleic acids, e.g., mRNAs, encoding proteins described herein (e.g., full-length, fragments, or portions of proteins) can be prepared by combining nucleic acids with any of the cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), For example, it can be delivered by a delivery vehicle containing a cationic lipid selected from compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4).

[0392] As used herein, the terms "delivery vehicle," "implantation vehicle," "nanoparticle," or grammatical equivalents, are used interchangeably.

[0393] For example, the present invention provides compositions (e.g., pharmaceutical compositions) comprising a cationic lipid described herein (e.g., any of the cationic lipids of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)) and one or more polynucleotides. The composition (e.g., pharmaceutical composition) 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.

[0394] 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 involve transfecting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a cationic lipid described (e.g., a cationic lipid of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)) and a liposome formulation comprising a cationic lipid of formula K (e.g., any of (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)), such that one or more target cells are transfected with encapsulated material (e.g., one or more polynucleotides). As used herein, the term "transfect" or "transfection" refers to the intracellular introduction of one or more encapsulated material (e.g., nucleic acids and / or polynucleotides) into a cell, or preferably a target cell. The introduced polynucleotides may be stable or may be transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulated material (e.g., polynucleotides) taken up by, introduced into, and / or expressed by the target cell being transfected. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the 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 site of the pathology and subsequently expressed, while minimizing potential systemic side effects or toxicity associated with the compounds or their encapsulated contents.

[0395] For example, after transfection of one or more target cells with polynucleotides encapsulated in one or more lipid nanoparticles, including the pharmaceutical or liposomal compositions disclosed herein, the products encoded by such polynucleotides (e.g., polypeptides) can be produced. The production of a polypeptide or protein (e.g., a target gene or a target protein) can be preferably stimulated, enhancing the ability of such target cells to express a polynucleotide and, for example, produce a polypeptide or protein of interest. For example, transfection of target cells with one or more compounds or pharmaceutical compositions that encapsulate mRNA enhances (i.e., increases) the production of the protein or enzyme encoded by such mRNA.

[0396] 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 instances 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.

[0397] 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.

[0398] The terms "liposome delivery vehicle" and "liposome composition" are used interchangeably.

[0399] 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., improved delivery of encapsulated polynucleotides to one or more target cells and / or reduced 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.

[0400] Thus, in certain embodiments, compounds described herein (e.g., compounds of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)) (K-IIa)), e.g., the cationic lipids of any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4), are cationic lipids that can be used as components of liposome compositions to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by penetrating or fusing with the lipid membranes of such target cells).

[0401] As used herein, liposome delivery vehicles, e.g., lipid nanoparticles, are generally characterized as microscopic vesicles with an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of a liposome is typically composed of amphiphilic molecules, such as lipids of synthetic or natural origin, which contain spatially separated hydrophilic and hydrophobic domains. (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposome bilayer membrane may also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, liposome delivery vehicles typically serve to transport desired mRNA to target cells or tissues.

[0402] 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).

[0403] 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 lipid of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), a lipid of Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-III)), a lipid of Formula I (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-III)), a lipid of Formula II (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-III)), a lipid of Formula III (e.g., (DA), (DI) to (D-III), (D-IIa) to (D-III)), a lipid of Formula I (e.g., (DA), (DI) to (D-III), (D-IIa) to (D-III)), a lipid of Formula II (e.g., (DA), (DI) to (D-III), (D-IIa) to (D-III)), a lipid of Formula III (e.g., (DA), (DI) to (D-III), (D-IIIa) to (D-III)), a lipid of Formula I (e.g., (DA), (DI) to (D-III), (D-IIIa) to (D-III)), a lipid of Formula II (e.g., (DA), (DI) to (D-III), (D-IIIa) to (D-III)), a lipid of Formula III (e.g., (DA), (DI) to (D-III), (D-IIIa) to (D-III)), a lipid of Formula I (e.g., (DA), (DI) to (D The cationic lipid may be any of Formulas E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4). In several embodiments, the composition comprises an mRNA encoding a protein (e.g., any of the proteins described herein). In several embodiments, the composition comprises an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In several embodiments, the composition comprises an mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0404] In embodiments, a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated in a liposome, wherein the liposome comprises any of the cationic lipids described herein (e.g., any of the cationic lipids of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)).

[0405] 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.

[0406] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net positive charge.

[0407] In embodiments, the liposome delivery vehicle (e.g., lipid nanoparticle) has a net negative It may have an electric charge.

[0408] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net neutral charge.

[0409] In embodiments, the lipid nanoparticles encapsulating the nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., cationic lipids of any of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)).

[0410] For example, the cationic lipids described herein in the compositions (e.g., those of formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), those of formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), those of formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and those of formula K (e.g., (KI)-(K)) The amount of any of the cationic lipids (K-II and K-Ia) through (K-IIa), e.g., any of compounds (A1) through (A4), (D1) through (D7), (E1) through (E5), and (K1) through (K4)) may be described as a percentage ("wt %") of the combined dry weight of all lipids in the composition (e.g., the combined dry weight of all lipids present in a liposome composition).

[0411] In embodiments of the pharmaceutical compositions described herein, the cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., For example, the cationic lipid of any of (KI) to (K-II) and (K-Ia) to (K-IIa), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4), is present in an amount of about 0.5 wt % to about 30 wt % (e.g., about 0.5 wt % to about 20 wt %) of the combined dry weight of all lipids present in the composition (e.g., liposome composition).

[0412] In embodiments, the cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., (KI) to (K-II) and (K-Ia)) The cationic lipids of any of Formulas (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4) are present in an amount of about 1% to about 30%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 5% to about 25% by weight of the combined dry weight of all lipids present in the composition (e.g., liposome composition). In some embodiments, the cationic lipids described herein (e.g., those of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id)) , (D-IIa), and (D-IIIa)-(D-IIId)), any of Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., the cationic lipid of any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)) is present in an amount of about 0.5% to about 5% by weight, about 1% to about 10% by weight, about 5% to about 20% by weight, or about 10% to about 20% by weight of the combined molar amount of all lipids present in the composition, such as a liposome delivery vehicle.

[0413] In embodiments, the cationic lipids described herein (e.g., any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., compounds (A1) to (A4), (D1) to (D7), (E1 ) through (E5), and any of (K1) through (K4) cationic lipids) are present in an amount of at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of the total lipids in the composition (e.g., liposome composition).

[0414] In embodiments, the cationic lipids described herein (e.g., any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., compounds (A1) to (A4), (D1) to (D7), ( The amount of any of the cationic lipids E1)-(E5), and (K1)-(K4)) 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 combined dry weight of the total lipids in the composition (e.g., liposome composition).

[0415] 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 lipid having a structure represented by Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa)) , and (D-IIIa) to (D-IIId)), any of Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4). In some 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., any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), any of the cationic lipids of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), for example, any of the cationic lipids of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4). 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 lipid having a structure of Formula A (e.g., (AI) through (A-II) and (A-Ia) through (A-IIa)), a structure of Formula D (e.g., (DA), (DI) through (D-III), (D-Ia) through (D-IIa)), a structure of Formula E (e.g., (DA), (DI) through (D-III), (D-Ia) through (D-IIa)), a structure of Formula I (e.g., (DA), (DI) through (D-III), (D-Ia) through (D-IIa)), a structure of Formula I (e.g., (DA), (DI) through (D-III), (D-Ia) through (D-IIa)), a structure of Formula I (e.g., (DA), (DI) through (D-III), (D-Ia) through (D-IIa)), a structure of Formula II (e.g., (DA), (DI) through (D-III), (D-Ia) through (D-IIa)), a structure of Formula I (e.g., (DA), (DI) through (D-III), (D-IIa) through (D-IIa)), a structure of Formula II ... III (e.g., (DA), (DI) through (D-III), ( The cationic lipids may include any of the cationic lipids of formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4). In embodiments, this percentage results in improved beneficial effects (e.g., improved delivery to target tissues such as the liver or lungs).

[0416] The cationic lipids described herein in the compositions (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., (KI) to (K The amount of the cationic lipid of any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4) may also be described as a percentage ("mol %") of the combined molar amount of total lipid of the composition (e.g., the combined molar amount of all lipids present in the liposomal delivery vehicle).

[0417] In embodiments of the pharmaceutical compositions described herein, the cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., those of Formula B (e.g., those of Formula C (e.g., those of Formula D (e.g., those of Formula E ... For example, any of (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., the cationic lipid of any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4), is present in an amount of about 0.5 mol % to about 30 mol % (e.g., about 0.5 mol % to about 20 mol %) of the combined molar amount of all lipids present in a composition, such as a liposome delivery vehicle.

[0418] In some embodiments, the cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., (KI) to (K-II) and The cationic lipids (e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)) 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 %, or about 10 mol % to about 20 mol % of the combined molar amount of all lipids present in the composition, such as a liposome delivery vehicle. In several embodiments, the cationic lipids described herein (e.g., those of formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), those of formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), those of formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and those of formula K (e.g., (KI)-(K-II) and (K-Ia)) The cationic lipid of any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4) is present in an amount of 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 dry weight of all lipids present in the composition, such as a liposome delivery vehicle.

[0419] In certain embodiments, the cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., (KI) to (K-I)) The cationic lipid of any of compounds (I) and (K-Ia) to (K-IIa), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4), may comprise from about 0.1 mol % to about 50 mol %, or from 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).

[0420] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (I), e.g., cationic lipids of Formula (Ia), Compound (1), Compound (2), Compound (3), and / or Compound (4)) comprise more than about 0.1 mol%, or more than about 0.5 mol%, of the total lipid amount in the lipid nanoparticles (e.g., cationic lipids of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-IIa)), or The cationic lipids of any of Formulas E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4), may constitute more than about 1 mol%, or more than about 5 mol%.

[0421] In certain embodiments, the cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., For example, the cationic lipid of any of (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4), may constitute less than about 25 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 the composition (e.g., liposome delivery vehicle).

[0422] In some embodiments, the cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of Formula E (e.g., (EI) to (E-II) and and (E-Ia) to (E-IIa)), and any of formula K (e.g., any of (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4))) may be present in an amount that is less than or equal to the combined dry weight of the total lipids in the composition (e.g., liposome composition). It 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%.

[0423] In embodiments, the cationic lipids described herein (e.g., any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., compounds (A1) to (A4), (D1) to (D7), ( The amount of cationic lipid (any of E1)-(E5), and (K1)-(K4)) is present in an amount of less than or equal to 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 dry weight of the total lipids in the composition (e.g., liposome composition).

[0424] In embodiments, this percentage results in improved beneficial effects (eg, improved delivery to target tissues such as the liver or lungs).

[0425] 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).

[0426] 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.

[0427] In embodiments, the composition (e.g., lipid nanoparticle) encapsulating the nucleic acid (e.g., mRNA encoding a peptide or polypeptide) can comprise one or more cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of Formula E (e.g., For example, the cationic lipid may be any of the compounds of formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), for example, any of the compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)), and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids.

[0428] In embodiments, the composition (e.g., lipid nanoparticle) encapsulating the nucleic acid (e.g., mRNA encoding a peptide or polypeptide) can comprise one or more cationic lipids described herein (e.g., lipid nanoparticles) of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (EI (E-II) and (E-Ia) to (E-IIa)), and formula K (e.g., any of (KI) to (K-II) and (K-Ia) to (K-IIa)), for example, a cationic lipid of any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)), and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids, and further comprising a cholesterol-based lipid.

[0429] In embodiments, the lipid nanoparticles encapsulating the nucleic acid (e.g., mRNA encoding a peptide or polypeptide) may be prepared by mixing one or more cationic lipids described herein (e.g., those of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of Formula E (e.g., (EI) to (E-II), ) and (E-Ia) to (E-IIa)), and formula K (e.g., any of (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., a cationic lipid of any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)), and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, PEGylated lipids, and cholesterol-based lipids.

[0430] 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.

[0431] Further cationic lipids In addition to any of the cationic lipids described herein (e.g., cationic lipids of any of Formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), Formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), Formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and Formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)), e.g., any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4)), the composition may include one or more additional cationic lipids.

[0432] 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 the literature, and many of them are commercially available.

[0433] Additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO 2010 / 144740, which is incorporated herein by reference. In certain embodiments, the present compositions comprise a cationic lipid 4-( (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl dimethylaminobutanoate, [ka] and pharmaceutically acceptable salts thereof.

[0434] 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 R and R are each independently hydrogen, an optionally substituted variably saturated or unsaturated C-C 20 Alkyl, and optionally substituted variably saturated or unsaturated C-C 20 acyl; 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, 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.

[0435] 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.

[0436] 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.

[0437] 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.

[0438] 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.

[0439] 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.

[0440] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0441] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0442] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0443] 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, or halogen. In certain embodiments, the composition comprises a cationic lipid "Target 23" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0444] 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] (In the formula, [ka] is) or a pharmaceutically acceptable salt thereof.

[0445] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0446] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0447] 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.

[0448] 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.

[0449] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0450] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0451] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0452] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0453] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0454] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0455] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0456] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0457] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0458] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0459] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0460] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0461] Other additional cationic lipids suitable for use in the present compositions include those described in International Patent Publication No. WO2017 / 004143, which is incorporated herein by reference.

[0462] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0463] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0464] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0465] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0466] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0467] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0468] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0469] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0470] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0471] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0472] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0473] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0474] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0475] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0476] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0477] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0478] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0479] 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 following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond, and G 1 and G2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene, G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, and R a is H or C1-C 12 alkyl, and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl, and R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 alkyl, and R 5 is H or C1-C6 alkyl and x is 0, 1, or 2.

[0480] 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.

[0481] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0482] In some embodiments, the composition comprises a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0483] 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.

[0484] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0485] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0486] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0487] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0488] 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.

[0489] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0490] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0491] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0492] In certain embodiments, the composition comprises a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0493] 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.

[0494] 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 variably saturated or unsaturated C-C 20 Alkyl and optionally substituted variably saturated or unsaturated C6-C 20acyl, 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).

[0495] In certain embodiments, the composition comprises a cationic lipid "HGT4001" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0496] In certain embodiments, the composition comprises a cationic lipid "HGT4002" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0497] In certain embodiments, the composition comprises a cationic lipid "HGT4003" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0498] In certain embodiments, the composition comprises a cationic lipid "HGT4004" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0499] In certain embodiments, the composition comprises a cationic lipid "HGT4005" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0500] In some embodiments, the composition comprises a cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”). Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, 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, and 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-carboxamido)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").

[0501] 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-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"), N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N,N-distearyl -N,N-dimethylammonium bromide ("DDAB"), N-(l,2-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-octadecadienooxy)propane ("CLinDMA"), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethylll-(cis,cis-9',l-2'- octadecadienoyl)propane ("CpLinDMA"), N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"), l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"), l,2-dilinoleylcarbamyl-3-dimethylaminopropane ("DLinC") DAP"), 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 ...12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA(2R)”), (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyl 3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA(2S)”), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (“DLin-K-XTC2-DMA”), and 2-(2,2-di((9Z,12Z)-octadeca-9,l Also included are (2-dien-1-yl)-l,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, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005), International Patent Publication No. WO 2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0502] In some embodiments, one or more cationic lipids suitable for the present compositions include 2,2-dilinoleyl-4-dimethylaminoethyl-[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]dioxolane ("XTC"); and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0503] 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%).

[0504] 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%).

[0505] 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 liposome 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 liposome 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.

[0506] 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, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or mixtures thereof.

[0507] In embodiments, the non-cationic or helper lipid is dioleoylphosphatidylethanolamine (DOPE).

[0508] In some embodiments, the non-cationic lipid is a neutral lipid, ie, a lipid that has no net charge under the conditions in which the composition is formulated and / or administered.

[0509] 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%. In some embodiments, the percentage of total 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%. In some embodiments, the percentage of total non-cationic lipids in the liposomes can be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in the liposomes can be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%.

[0510] In some embodiments, the non-cationic 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 non-cationic 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.

[0511] 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]

[0512] In embodiments, the cholesterol-based lipid is cholesterol.

[0513] In some embodiments, the cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0514] 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.

[0515] PEGylated lipids In some embodiments, a composition (eg, a liposomal composition) comprises one or more PEGylated lipids.

[0516] 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 liposomes, 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.

[0517] In some embodiments, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000).

[0518] Contemplated PEG-modified lipids (also referred to herein as PEGylated lipids, and this term is interchangeable with PEG-modified lipids) include C6-C 20 These include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids having 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 aggregation of the complex, increase circulation life, and increase delivery of the lipid-nucleic acid composition to target cells. Means can also be provided for the preparation of a drug substance containing a hydroxybenzoate (Klibanov et al. (1990) FEBS Letters, 268(1):235-37), or the components can be selected to rapidly exchange from the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0519] 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).

[0520] 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).

[0521] Pharmaceutical Formulations and Therapeutic Uses The cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., (KI) to (K-II) and (K-Ia) to (K-IIa)) Any of the cationic lipids, for example, any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4), can be used in the preparation of compositions (e.g., to construct liposome compositions) that facilitate 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 permeating or fusing with the lipid membranes of such target cells).

[0522] 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.

[0523] Similarly, in certain embodiments, the cationic lipids described herein (e.g., any of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)) may be used to prepare liposome vesicles characterized by 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.

[0524] Thus, the cationic lipids described herein (e.g., those of formula A (e.g., (AI) to (A-II) and (A-Ia) to (A-IIa)), those of formula D (e.g., (DA), (DI) to (D-III), (D-Ia) to (D-Id), (D-IIa), and (D-IIIa) to (D-IIId)), those of formula E (e.g., (EI) to (E-II) and (E-Ia) to (E-IIa)), and those of formula K (e.g., (KI) to (K-II) and ( Pharmaceutical preparations comprising any of the cationic lipids of compounds (K-Ia) to (K-IIa), for example, any of compounds (A1) to (A4), (D1) to (D7), (E1) to (E5), and (K1) to (K4), and a nucleic acid provided by the present invention can be used for various therapeutic purposes. To facilitate delivery of nucleic acids in vivo, the cationic lipids described herein (e.g., any of the cationic lipids of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)) and nucleic acids may 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 (I) or (II), e.g., cationic lipids of Formula (Ia), (IIa), Compound (1), and / or Compound (2)) can be formulated using a premixed lipid solution. In other embodiments, compositions comprising the cationic lipids described herein (e.g., any of the cationic lipids of Formula A (e.g., (AI)-(A-II) and (A-Ia)-(A-IIa)), Formula D (e.g., (DA), (DI)-(D-III), (D-Ia)-(D-Id), (D-IIa), and (D-IIIa)-(D-IIId)), Formula E (e.g., (EI)-(E-II) and (E-Ia)-(E-IIa)), and Formula K (e.g., (KI)-(K-II) and (K-Ia)-(K-IIa)), e.g., any of compounds (A1)-(A4), (D1)-(D7), (E1)-(E5), and (K1)-(K4)) may be formulated using post-insertion techniques into the lipid membrane of nanoparticles.Formulation techniques and drug administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. (latest edition).

[0525] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation administration, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, and 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). In some embodiments, administration is intramuscular. In some embodiments, administration is intravenous. In some embodiments, administration is intratracheal.

[0526] 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. Examples of 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, liquid drop, or even injection.

[0527] In some embodiments, administration is by pulmonary delivery. As used herein, pulmonary delivery refers to delivery to the lungs, for example, via the nasal passages, trachea, bronchi, bronchioles, and / or other pulmonary systems. In some embodiments, the compositions described herein are formulated for nebulization. In some embodiments, the delivery vehicle can be an aerosolized composition that can be inhaled. In some embodiments, pulmonary delivery involves inhalation (e.g., in the case of nasal, tracheal, or bronchial delivery). In some embodiments, the composition is nebulized before inhalation.

[0528] 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 in a human subject, or cells that are treated and delivered to a human subject.

[0529] 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.

[0530] 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.

[0531] 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) proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding argininosuccinate synthetase 1 proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding carbamoyl phosphate synthetase I proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding argininosuccinate lyase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding arginase proteins. to provide.

[0532] 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).

[0533] 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.

[0534] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding proteins involved in 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. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding propionyl-CoA carboxylase enzymes. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding oxalase alanine-glyoxylaminotransferase enzymes.

[0535] 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.

[0536] In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a methylmalonyl-CoA mutase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding a methylmalonyl-CoA epimerase protein.

[0537] 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.

[0538] 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 cardiovascular structures or cells. 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.

[0539] In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide for delivery to or use in the treatment of muscle or muscle cells in a subject. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding a dystrophin protein. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding a frataxin protein. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide for delivery to or use in the treatment of muscle or muscle cells in a subject. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a 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 a therapeutic composition having a 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 a therapeutic composition having a full-length mRNA encoding a protein that regulates Nav1.5 channels in muscle tissue or muscle cells.

[0540] 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.

[0541] 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.

[0542] 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.

[0543] 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 eye or ocular cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding 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 retinoschisin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding retinal pigment epithelium-specific 65 kDa (RPE65) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding 290 kDa centrosomal protein (CEP290).

[0544] 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.

[0545] 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.

[0546] In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In 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 methods for producing therapeutic compositions having full-length mRNA encoding antibodies against CD3. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding antibodies against CD19.

[0547] 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.

[0548] 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.

[0549] 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 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 1-46 [Table 1-47] [Table 1-48] [Table 1-49] [Table 1-50]

[0550] 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 2-1] [Table 2-2]

[0551] The Uniprot IDs listed in Tables 1 and 2 refer to the human version of the listed protein, and the sequences of each are 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, 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 methods of the invention may include preparing and / or administering compositions comprising 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. 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.

[0552] In embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a lysosomal protein selected from Table 3. In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNA encoding one or more lysosomal proteins and / or related proteins listed in Table 3, and thus, the compositions and methods of the invention The compositions may comprise mRNA encoding a protein listed in Table 3 (or a homolog thereof), together with other components described herein, and the methods of the invention may comprise preparing and / or administering a composition comprising mRNA encoding a protein selected from the proteins listed in Table 3 (or a homolog thereof), together with other components described herein. [Table 3-1] [Table 3-2] [Table 3-3]

[0553] 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. The sequences of the listed proteins are also available for a variety of animals, including various mammals and animals for veterinary or industrial purposes, as discussed above.

[0554] In some embodiments, 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. In some embodiments, compositions and methods of the invention provide for the delivery of mRNA encoding a therapeutic protein useful for the treatment of a disease or disorder (i.e., an indication) listed in Table 4; 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 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9]

[0555] 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]

[0556] Example Example 1: Synthesis of compound (A3) [ka] Vitamin A or retinol (0.7 g, 2.44 mmol) and 3-(4-methylpiperazin-1-yl)propanoic acid (0.50 g, 2.93 mmol) in DMF (20 mL) To a solution of 3 (III) was added HOBt (0.49 g, 3.66 mmol), HBTU (1.40 g, 3.66 mmol), and DMAP (0.45 g, 3.66 mmol), followed by the slow addition of DIPEA (2.13 mL, 12.2 mmol). The reaction was heated at 65 °C for 1 h and continued to stir at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure to give compound (3) (1.3 g) as a dark brown oil, which was purified by column chromatography.

[0557] Example 2: Synthesis of compound (A4) [ka] To a solution of vitamin A or retinol (0.25 g, 0.87 mmol) and 4-(dimethylamino)butyric acid hydrochloride (0.17 g, 1.05 mmol) in DMF (20 mL) was added HOBt (0.18 g, 1.31 mmol), HBTU (0.49 g, 1.31 mmol), and DMAP (0.16 g, 1.31 mmol), followed by the slow addition of DIPEA (0.76 mL, 4.36 mmol). The reaction was heated at 65 °C for 1 h and continued to stir at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure to give compound (4) (0.5 g) as a dark brown oil.

[0558] Example 3: Synthesis of compound (D5) [ka] To a solution of vitamin-D3 (1.0 g, 2.60 mmol) and 4-(dimethylamino)butyric acid hydrochloride (0.52 g, 3.12 mmol) in DMF (20 mL) was added HOBt (0.53 g, 3.90 mmol), HBTU (1.5 g, 3.90 mmol), and DMAP (0.48 g, 3.90 mmol), followed by the slow addition of DIPEA (2.3 mL, 13.0 mmol). The reaction was heated at 65 °C for 1 h and continued to stir at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (eluent: 0.5–1.0% MeOH in DCM) to give compound (5) (0.78 g, 60%) as a sticky brown solid.

[0559] Example 4: Synthesis of compound (D6) [ka] To a solution of vitamin-D3 (1.0 g, 2.60 mmol) and 3-(4-methylpiperazin-1-yl)propanoic acid (0.54 g, 3.12 mmol) in DMF (20 mL) was added HOBt (0.53 g, 3.90 mmol), HBTU (1.5 g, 3.90 mmol), and DMAP (0.5 g, 3.90 mmol), followed by the slow addition of DIPEA (2.3 mL, 12.9 mmol). The reaction was heated at 65 °C for 1 h and continued to stir at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (eluent: 0.5–1.0% MeOH in DCM) to give compound (6) (0.56 g, 40%) as a sticky yellow solid.

[0560] Example 5: Synthesis of compound (D7) [ka] To a solution of vitamin D3 (1.0 g, 2.60 mmol) and 3-(bis(3-((8-methylnonyl)oxy)-3-oxopropyl)amino)propanoic acid (1.5 g, 2.86 mmol) in DMF (20 mL) was added HOBt (0.53 g, 3.90 mmol), HBTU (1.48 g, 3.90 mmol), and DMAP (0.48 g, 3.90 mmol), followed by the slow addition of DIPEA (2.3 mL, 13.0 mmol). The reaction was heated at 65° C. for 1 h and continued stirring at room temperature for an additional 24 h. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3×100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure and the residue was purified by silica gel chromatography (eluent: 0.5-1.0% MeOH in DCM) to give compound (7) as a sticky yellow solid (0.80 g, 35%).

[0561] Example 6: Synthesis of compound (E3) [ka] To a solution of vitamin E or DL-α-tocopherol (1.0 g, 2.32 mmol) and 3-(4-methylpiperazin-1-yl)propanoic acid (0.48 g, 2.79 mmol) in DMF (20 mL) was added HOBt (0.47 g, 3.50 mmol), HBTU (1.32 g, 3.50 mmol), and DMAP (0.42 g, 3.50 mmol), followed by the slow addition of DIPEA (2.0 mL, 11.6 mmol). The reaction was heated at 65 °C for 1 hour and continued to stir at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure and the residue was purified by silica gel chromatography (eluent: 0.5-1.0% MeOH in DCM) to give compound (3) as a sticky yellow solid (0.45 g, 33%).

[0562] Example 7: Synthesis of compound (E4) Step 1 [ka] Beta-alanine (10.0 g, 112.2 mmol) was dissolved in DMSO / HO (100 mL, 1:1 v / v ratio). Isodecyl acrylate (68.1 mL, 280.6 mmol) was added to it, and the reaction mixture was heated at 85 °C for 3 days. The reaction was stopped after 3 days and cooled to room temperature. The organic layer was then separated, diluted with ethyl acetate (300 mL), and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (eluent: 1.0–3.0% MeOH in DCM) to give 3-(bis(3-((8-methylnonyl)oxy)-3-oxopropyl)amino)propanoic acid (12.0 g, 21%) as a colorless oil.

[0563] Step 2 [ka] To a solution of vitamin E or DL-α-tocopherol (0.6 g, 1.40 mmol) and 3-(bis(3-((8-methylnonyl)oxy)-3-oxopropyl)amino)propanoic acid (0.60 g, 1.17 mmol) in DMF (15 mL) was added HOBt (0.24 g, 1.75 mmol), HBTU (0.66 g, 1.75 mmol), and DMAP (0.19 g, 1.52 mmol), followed by the slow addition of DIPEA (1.02 mL, 5.85 mmol). The reaction was heated at 65° C. for 1 h and continued stirring at room temperature for an additional 24 h. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3×100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure and the residue was purified by silica gel chromatography (eluent: 0.5-1.0% MeOH in DCM) to give compound (4) as a light brown oil (0.50 g, 46%).

[0564] Example 8: Synthesis of compound (E5) [ka] To a solution of vitamin E or DL-α-tocopherol (1.0 g, 2.32 mmol) and 4-(dimethylamino)butyric acid hydrochloride (0.47 g, 2.78 mmol) in DMF (20 mL) was added HOBt (0.47 g, 3.48 mmol), HBTU (1.32 g, 3.48 mmol), and DMAP (0.42 g, 3.48 mmol), followed by the slow addition of DIPEA (2.0 mL, 11.6 mmol). The reaction was heated at 65 °C for 1 h. The mixture was heated for 1 hour and stirred at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (200 mL) and washed with brine solution (3 × 100 mL). After drying over anhydrous NaSO, the organic layer was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (eluent: 0.5–1.0% MeOH in DCM) to give compound (5) (0.78 g, 60%) as a sticky brown oil.

[0565] Example 9: Formulation of lipid nanoparticles using vitamin cationic lipids and in vivo expression of FFLuc in CD1 mice The cationic lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include those described in International Publication No. WO2018 / 089801, which is incorporated herein by reference in its entirety.

[0566] One exemplary process for formulating lipid nanoparticles is Process A of WO2018 / 089801 (see, e.g., Example 1 and Figure 1 of WO2018 / 089801). Process A ("A") relates to a conventional method for encapsulating mRNA by mixing the 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 solution of the lipid mixture (cationic lipids, helper lipids, zwitterionic lipids, PEG lipids, etc.) was prepared by dissolving the lipids in ethanol. An mRNA solution was prepared by dissolving the mRNA in a citrate buffer to obtain a concentration of 0.0833 mg / mL of mRNA in a pH 4.5 citrate buffer. Both mixtures were then heated to 65°C and mixed. These two solutions were then mixed using a pump system. In some examples, 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.

[0567] 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. A range of conditions, such as different temperatures (i.e., heating or not heating the mixture), 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 the two streams resulted in the formation of empty lipid nanoparticles, a self-assembly process. The resulting formulation mixture was empty lipid nanoparticles in a citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process to perform buffer exchange. The resulting suspension of preformed empty lipid nanoparticles was then mixed with 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.

[0568] The nanoparticle formulations in Table 5 were prepared by Process A described above for intratracheal administration using a MicroSprayer®. All formulations contained mRNA encoding firefly luciferase (FFLuc) protein and other components in the following molar ratios: cationic lipid:DMG-PEG2000, cholesterol:DOPE=40:5:25:30. [Table 5]

[0569] Example 10: In vivo expression of mRNA encoding firefly luciferase (FFLuc) protein Intratracheal administration of lipid nanoparticle formulations containing exemplary vitamin-containing cationic lipids and mRNA encoding FFLuc protein (Table 1) was performed to study mRNA delivery and resulting protein expression. Male CD1 mice, 6-8 weeks old, were dosed by a single intratracheal aerosol administration (50 μL / animal) while anesthetized with isoflurane (1%-4%) via a nose cone. 24 hours after dosing, mice were sacrificed, and both lungs were collected for ex vivo IVIS imaging after perfusion. FFLuc protein was detected in the lungs of animals dosed with the formulations in Table 5. These studies demonstrate that the vitamin-containing cationic lipids described herein are effective for in vivo delivery of mRNA and result in expression of the protein or polypeptide encoded by the delivered mRNA.

[0570] While certain compounds, compositions, and methods of the present invention have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the present invention and are not intended to limit it. Illustrative Embodiments First Set of Embodiments 1. A liposome encapsulating an mRNA encoding a protein, said liposome comprising 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 a cationic lipid having a structure according to formula (AI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30-alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl. 2. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has a structure according to formula (A-Ia): [ka] 3.R 1 But C6-C 30 3. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein the carboxyl group is -alkylene. 4.R 1 A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein 5.R 1 but unsubstituted C6-C 304. A liposome encapsulating an mRNA encoding the protein of embodiment 3, wherein the carboxyl group is -alkylene. 6.R 1 is unsubstituted C1-C5-alkylene. 7.R 1 But -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 A liposome encapsulating mRNA encoding the protein of embodiment 5, wherein the mRNA is - 8.R 1 A liposome encapsulating an mRNA encoding the protein of embodiment 6, wherein -C2H4-, -C3H6-, or C4H8-. 9.R 1 is C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 306. A liposome encapsulating an mRNA encoding the protein of embodiment 5, wherein the carboxyl group is -alkylene. 10.R 1 But C6-C 30 -Alkenylene or C8-C 20 3. A liposome encapsulating an mRNA encoding the protein of embodiment 1 or 2, wherein the carboxyl group is -alkenylene. 11.R 1 However, C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 3. A liposome encapsulating mRNA encoding the protein of embodiment 1 or 2, wherein the mRNA is selected from the group consisting of methyl-, ... 12.R 1 is unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 13. A liposome encapsulating mRNA encoding the protein of embodiment 12, wherein the liposome is selected from the group consisting of -alkenylene. 13.R 1is -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 C H=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 A liposome encapsulating mRNA encoding a protein according to embodiment 1 or 2, selected from CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-. 14.X 1 A liposome encapsulating an mRNA encoding a protein according to any one of embodiments 1 to 13, wherein is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. 15.X 1 is a 5- to 6-membered nitrogen-containing heterocycloalkyl. 16.X 1 16. A liposome encapsulating an mRNA encoding the protein of embodiment 15, wherein is substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl. 17.X 1 15. A liposome encapsulating an mRNA encoding a protein according to embodiment 14, wherein is a dialkylamine. 18.X 1 but: [ka] During the ceremony, R 3a and R 3b are each independently, C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; A liposome encapsulating mRNA encoding the protein according to embodiment 17, wherein each n is independently an integer having a value of about 1 to about 6. 19.R 3a and R 3b are each independently, C6-C 30 19. A liposome encapsulating an mRNA encoding the protein of embodiment 18, wherein the liposome is -alkyl. 20.R 3a and R 3b each independently represents an unsubstituted C-C 30 20. A liposome encapsulating an mRNA encoding the protein of embodiment 19, wherein the liposome is -alkyl. 21.R 3a and R 3b are each independently, C6-C 30 21. A liposome encapsulating an mRNA encoding a protein according to embodiment 20, wherein the mRNA is selected from the group consisting of -alkyl. 22.R 3a and R 3b are each independently, C6-C 30 -Alkenyl or C8-C 20 19. A liposome encapsulating an mRNA encoding the protein of embodiment 18, wherein the carboxyl group is -alkenyl. 23.R 3a and R 3b are each independently C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C1 4-Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 23. A liposome encapsulating an mRNA encoding a protein according to embodiment 22, wherein the mRNA is selected from the group consisting of aryl, aryl- ... 24.R 3a and R 3b are each 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) 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 A liposome encapsulating mRNA encoding a protein according to embodiment 22 or 23, selected from CH=CH(CH2)7CH3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. 25.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 13, wherein: 26.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 13, wherein: 27.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 13, wherein: 28. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] 29. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] 30. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] 31. A composition comprising a liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 30. 32. mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein 32. The composition of embodiment 31, comprising NA. 33. The composition of embodiment 31, comprising mRNA encoding ornithine transcarbamylase (OTC) protein. 34. A nucleic acid encapsulated in a liposome, wherein the liposome comprises a cationic lipid having a structure according to formula (AI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl. 35. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has a structure according to formula (A-Ia): [ka] 36.R 1 But C6-C 30 36. The liposomally encapsulated nucleic acid of embodiment 34 or 35, wherein -alkylene. 37.R 1 36. The liposomally encapsulated nucleic acid of embodiment 34 or 35, wherein is C1-C5-alkylene. 38.R 1 but unsubstituted C6-C 30 37. The liposomally encapsulated nucleic acid of embodiment 36, wherein N is -alkylene. 39.R 1 38. The liposomally encapsulated nucleic acid of embodiment 37, wherein is unsubstituted C1-C5-alkylene. 40.R 1 But -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 39. The liposomally encapsulated nucleic acid of embodiment 38, selected from: 41.R 1 40. The liposomally encapsulated nucleic acid of embodiment 39, wherein is -C2H4-, -C3H6-, or C4H8-. 42.R 1 is C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 39. The liposomally encapsulated nucleic acid of embodiment 38, wherein N is -alkylene. 43.R 1 But C6-C 30 -Alkenylene or C8-C 20 36. The liposomally encapsulated nucleic acid of embodiment 34 or 35, wherein -alkenylene. 44.R 1 However, C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 36. The liposomally encapsulated nucleic acid according to embodiment 34 or 35, wherein the nucleic acid is selected from the group consisting of: -alkenylene; 45.R 1 is unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 44. The liposomally encapsulated nucleic acid of embodiment 43, wherein the nucleic acid is selected from the group consisting of: -alkenylene; 46.R 1 is -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 36. The nucleic acid encapsulated in a liposome according to embodiment 34 or 35, wherein the nucleic acid is selected from CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-. 47.X 1 47. The liposomally encapsulated nucleic acid of any one of embodiments 34 to 46, wherein is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. 48.X 1 48. The liposomally encapsulated nucleic acid of embodiment 47, wherein is a 5-6 membered nitrogen-containing heterocycloalkyl. 49.X 1 49. The liposomally encapsulated nucleic acid of embodiment 48, wherein is substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl. 50.X 1 48. The liposomally encapsulated nucleic acid of embodiment 47, wherein is a substituted dialkylamine. 51.X 1 but: [ka] During the ceremony, R 3a and R 3b are each independently, C1-C 30-Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; The liposome-encapsulated nucleic acid of embodiment 50, wherein each n is independently an integer having a value of from about 1 to about 6. 52.R 3a and R 3b are each independently, C6-C 30 52. The liposomally encapsulated nucleic acid of embodiment 51, wherein the nucleic acid is -alkyl. 53.R 3a and R 3b each independently represents an unsubstituted C-C 30 53. The liposomally encapsulated nucleic acid of embodiment 52, wherein the nucleic acid is -alkyl. 54.R 3a and R 3b are each 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 54. The liposomally encapsulated nucleic acid of embodiment 53, selected from: 55.R 3a and R 3b are each independently, C6-C 30 -Alkenyl or C8-C 20 52. The liposomally encapsulated nucleic acid of embodiment 51, wherein: 56.R 3a and R 3b are each independently C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19- Alkenyl, and C 20 56. The liposomally encapsulated nucleic acid of embodiment 55, wherein the nucleic acid is selected from the group consisting of: -alkenyl; 57.R 3a and R 3b are each 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) 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 57. The nucleic acid encapsulated in a liposome according to embodiment 55 or 56, wherein the nucleic acid is selected from CH=CH(CH2)7CH3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. 58.X 1 but, [ka] The nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 46, wherein 59.X 1 but, [ka] The nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 46, wherein 60.X 1 but, [ka] The nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 46, wherein 61. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has the following structure: [ka] 62. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has the following structure: [ka] 63. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has the following structure: [ka] 64. A composition comprising a nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 63. 65. The composition of embodiment 64, 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. 66. The composition of embodiment 64 or 65, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide. 67. The composition of any one of embodiments 64 to 66, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject. 68. The composition of embodiment 67, wherein the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. 69. The composition of any one of embodiments 64-66, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject. 70. The composition of embodiment 69, wherein the mRNA encodes an ornithine transcarbamylase (OTC) protein. 71. The composition of any one of embodiments 64 to 66, wherein the mRNA encodes a peptide or polypeptide for use in a vaccine. 72. The composition of embodiment 71, wherein the mRNA encodes an antigen. Second Set of Embodiments 1. A liposome encapsulating an mRNA encoding a protein, said liposome comprising 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 a cationic lipid having a structure according to formula (DA): [ka] During the ceremony, [ka] represents a single or double bond, X 1 is an ionic nitrogen-containing group, X 2 is O or S, Z is O or a covalent bond; R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; R 2 is H or C1-C4-alkyl. 2. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has a structure according to formula (DI): [ka] 3. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has a structure according to formula (D-III): [ka] 4. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein the cationic lipid has a structure according to formula (D-Ia): [ka] 5. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 3, wherein the cationic lipid has a structure according to formula (D-IIIa): [ka] 6. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein the cationic lipid has a structure according to formula (D-Ib): [ka] 7. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 3, wherein the cationic lipid has a structure according to formula (D-IIIb): [ka] 8. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein the cationic lipid has a structure according to formula (D-Ic): [ka] 9. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 3, wherein the cationic lipid has a structure according to formula (D-IIIc): [ka] 10. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein the cationic lipid has a structure according to formula (D-Id): [ka] 11. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 3, wherein the cationic lipid has a structure according to formula (D-IIId): [ka] 12.X 2 A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 11, wherein 13.R 1 is C1-C5-alkylene. Liposomes containing mRNA encoding the protein. 14.R 1 But C6-C 30 A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 12, wherein the aryl group is -alkylene. 15.R 1 is unsubstituted C1-C5-alkylene. 16.R 1 but unsubstituted C6-C 30 15. A liposome encapsulating an mRNA encoding the protein of embodiment 14, wherein the carboxyl group is -alkylene. 17.R 1 16. A liposome encapsulating an mRNA encoding the protein of embodiment 15, wherein is -C2H4-, -C3H6-, or C4H8-. 18.R 1 But -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 17. A liposome encapsulating mRNA encoding the protein of embodiment 16, wherein the mRNA is - 19.R 1 is C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 15. A liposome encapsulating an mRNA encoding the protein of embodiment 14, wherein the carboxyl group is -alkylene. 20.R 1 But C6-C 30 -Alkenylene or C8-C 20 A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 12, wherein the liposome is -alkenylene. 21.R 1 However, C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 13. A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 12, wherein the aryl group is selected from the group consisting of aryl, aryl- ... 22.R1 is unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 22. A liposome encapsulating mRNA encoding the protein of embodiment 21, wherein the liposome is selected from the group consisting of -alkenylene. 23.R 1 is -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 A liposome encapsulating mRNA encoding a protein according to any one of embodiments 1 to 12, wherein the protein is selected from CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-. 24.X 1 A liposome encapsulating an mRNA encoding a protein according to any one of embodiments 1 to 23, wherein is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. 25.X 1 is a 5- to 6-membered nitrogen-containing heterocycloalkyl. 26.X 1 26. A liposome encapsulating an mRNA encoding a protein according to embodiment 25, wherein is substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl. 27.X 1 25. A liposome encapsulating an mRNA encoding a protein according to embodiment 24, wherein 28.X 1 but: [ka] During the ceremony, R3a and R 3b are each independently, C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; A liposome encapsulating mRNA encoding the protein according to embodiment 27, wherein each n is independently an integer having a value of about 1 to about 6. 29.R 3a and R 3b are each independently, C6-C 30 29. A liposome encapsulating an mRNA encoding the protein of embodiment 28, wherein the liposome is -alkyl. 30.R 3a and R 3b each independently represents an unsubstituted C-C 30 30. A liposome encapsulating an mRNA encoding the protein of embodiment 29, wherein the liposome is -alkyl. 31.R 3a and R 3b are each 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 , -C 23 H 47 , -C 24 H 49 , or -C 25 H 51 A liposome encapsulating mRNA encoding the protein of embodiment 30, wherein the mRNA is 32.R 3a and R 3b are each independently, C6-C 30 -Alkenyl or C8-C 20 29. A liposome encapsulating an mRNA encoding the protein of embodiment 28, wherein the carboxyl group is -alkenyl. 33.R 3a and R 3b are each independently C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 33. A liposome encapsulating an mRNA encoding a protein according to embodiment 32, wherein the mRNA is selected from the group consisting of aryl, aryl- ... 34.R 3a and R 3b are each 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 A liposome encapsulating mRNA encoding a protein according to embodiment 32 or 33, selected from CH=CH(CH2)7CH3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. 35.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 23, wherein: 36.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 23, wherein: 37.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 23, wherein: 38. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] [ka] [ka] 39. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] [ka] 40. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] [ka] 41. A composition comprising a liposome encapsulating mRNA encoding a protein according to any one of embodiments 1 to 40. 42. The composition of embodiment 41, comprising mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. 43. The composition of embodiment 41, comprising mRNA encoding ornithine transcarbamylase (OTC) protein. 44. A nucleic acid encapsulated in a liposome, wherein the liposome comprises a cationic lipid having a structure according to formula (DA): [ka] During the ceremony, [ka] represents a single or double bond, X 1 is an ionic nitrogen-containing group, X 2 is O or S, Z is O or a covalent bond; R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; R 2 is H or C1-C4-alkyl. 45. The liposomally encapsulated nucleic acid of embodiment 44, wherein the cationic lipid has a structure according to formula (DI): [ka] 46. ​​A liposome encapsulating an mRNA encoding a protein according to embodiment 44, wherein the cationic lipid has a structure according to formula (D-III): [ka] 47. The liposomally encapsulated nucleic acid of embodiment 44 or 45, wherein the cationic lipid has a structure according to formula (D-Ia). [ka] 48. The liposomally encapsulated nucleic acid of embodiment 44 or 46, wherein the cationic lipid has a structure according to formula (D-IIIa). [ka] 49. The liposomally encapsulated nucleic acid of embodiment 44 or 45, wherein the cationic lipid has a structure according to formula (D-Ib). [ka] 50. The liposomally encapsulated nucleic acid of embodiment 44 or 46, wherein the cationic lipid has a structure according to formula (D-IIIb). [ka] 51. The liposomally encapsulated nucleic acid of embodiment 44 or 45, wherein the cationic lipid has a structure according to formula (D-Ic): [ka] 52. The method of embodiment 44 or 45, wherein the cationic lipid has a structure according to formula (D-IIIc): 47. A nucleic acid encapsulated in a liposome according to claim 46. [ka] 53. The liposomally encapsulated nucleic acid of embodiment 44 or 45, wherein the cationic lipid has a structure according to formula (D-Id): [ka] 54. The liposomally encapsulated nucleic acid of embodiment 44 or 46, wherein the cationic lipid has a structure according to formula (D-IIId). [ka] 55.X 2 The liposome-encapsulated nucleic acid according to any one of embodiments 44 to 54, wherein is O. 56.R 1 The liposome-encapsulated nucleic acid according to any one of embodiments 44 to 55, wherein is C1-C5-alkylene. 57.R 1 But C6-C 3056. The liposome-encapsulated nucleic acid of any one of embodiments 44 to 55, wherein the aryl group is -alkylene. 58.R 1 57. The liposomally encapsulated nucleic acid of embodiment 56, wherein is unsubstituted C1-C5-alkylene. 59.R 1 but unsubstituted C6-C 30 58. The liposomally encapsulated nucleic acid of embodiment 57, wherein: 60.R 1 59. The liposomally encapsulated nucleic acid of embodiment 58, wherein is —C2H4—, —C3H6—, or —C4H8—. 61.R 1 But -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, and -C 25 H 50 60. The liposomally encapsulated nucleic acid of embodiment 59, wherein 62.R 1is C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 58. The liposomally encapsulated nucleic acid of embodiment 57, wherein: 63.R 1 But C6-C 30 -Alkenylene or C8-C 20 56. The liposomally encapsulated nucleic acid of any one of embodiments 44 to 55, wherein the nucleic acid is -alkenylene. 64.R 1 However, C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 56. The liposomally encapsulated nucleic acid according to any one of embodiments 44 to 55, wherein the aryl group is selected from the group consisting of aryl, aryl- ... 65.R 1 is unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 65. The liposomally encapsulated nucleic acid of embodiment 64, wherein the nucleic acid is selected from the group consisting of: -alkenylene; 66.R 1is -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH( CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 56. A liposome-encapsulated nucleic acid according to any one of embodiments 44 to 55, selected from -CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-. 67.X 1 NH2, guanidine, amidine, mono- or di-alkylamine, 5-6 56. The liposomally encapsulated nucleic acid of any one of embodiments 44 to 55, wherein the aryl group is a 5- or 6-membered nitrogen-containing heterocycloalkyl, or a 5- or 6-membered nitrogen-containing heteroaryl. 68.X 1 68. The liposomally encapsulated nucleic acid of embodiment 67, wherein is a 5-6 membered nitrogen-containing heterocycloalkyl. 69.X1 69. The liposomally encapsulated nucleic acid of embodiment 68, wherein is substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl. 70.X 1 68. The liposomally encapsulated nucleic acid of embodiment 67, wherein is a dialkylamine. 71.X 1 but: [ka] During the ceremony, R 3a and R 3b are each independently, C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; The liposome-encapsulated nucleic acid of embodiment 70, wherein each n is independently an integer having a value of from about 1 to about 6. 72.R 3a and R 3b are each independently, C6-C 30 70. The liposomally encapsulated nucleic acid of embodiment 69, wherein said nucleic acid is -alkyl. 73.R 3a and R 3b each independently represents an unsubstituted C-C 30 73. The liposomally encapsulated nucleic acid of embodiment 72, wherein said nucleic acid is -alkyl. 74.R 3a and R 3b are each independently -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C12 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 74. The liposomally encapsulated nucleic acid of embodiment 73, wherein 75.R 3a and R 3b are each independently, C6-C 30 -Alkenyl or C8-C 20 70. The liposomally encapsulated nucleic acid of embodiment 69, wherein: 76.R 3a and R 3b are each independently C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 76. The liposomally encapsulated nucleic acid of embodiment 75, wherein the nucleic acid is selected from the group consisting of: -alkenyl; 77.R 3a and R 3bare each 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 77. The nucleic acid encapsulated in a liposome according to embodiment 75 or 76, wherein the nucleic acid is selected from CH=CH(CH2)7CH3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. 78.X 1 but, [ka] 56. The nucleic acid encapsulated in a liposome according to any one of embodiments 44 to 55, wherein: 79.X 1 but, [ka] 56. The nucleic acid encapsulated in a liposome according to any one of embodiments 44 to 55, wherein: 80.X 1 but, [ka] 56. The nucleic acid encapsulated in a liposome according to any one of embodiments 44 to 55, wherein: 81. The liposomally encapsulated nucleic acid of embodiment 44, wherein the cationic lipid has the following structure: [ka] [ka] [ka] [ka] 82. The liposomally encapsulated nucleic acid of embodiment 44, wherein the cationic lipid has the following structure: [ka] [ka] 83. The liposomally encapsulated nucleic acid of embodiment 44, wherein the cationic lipid has the following structure: [ka] [ka] 84. A composition comprising a nucleic acid encapsulated in a liposome according to any one of embodiments 44 to 83. 85. The composition of embodiment 84, 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. 86. The composition of embodiment 84 or 85, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide. 87. The composition of any one of embodiments 82-83, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject. 88. The composition of embodiment 87, wherein the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. 89. The composition of any one of embodiments 84-86, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject. 90. The composition of embodiment 89, wherein the mRNA encodes an ornithine transcarbamylase (OTC) protein. 91. The composition of any one of embodiments 84 to 86, wherein the mRNA encodes a peptide or polypeptide for use in a vaccine. 92. The composition of embodiment 91, wherein the mRNA encodes an antigen. Third Set of Embodiments 1. A liposome encapsulating an mRNA encoding a protein, said liposome comprising 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 a cationic lipid having a structure according to formula (EI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30-alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl. 2. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has a structure according to formula (E-Ia): [ka] 3.R 1 But C6-C 30 3. A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein the carboxyl group is -alkylene. 4.R 1 A liposome encapsulating an mRNA encoding a protein according to embodiment 1 or 2, wherein 5.R 1 but unsubstituted C6-C 304. A liposome encapsulating an mRNA encoding the protein of embodiment 3, wherein the carboxyl group is -alkylene. 6.R 1 is unsubstituted C1-C5-alkylene. 7.R 1 But -CH 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, or -C 25 H 50 A liposome encapsulating mRNA encoding the protein of embodiment 5, wherein the mRNA is - 8.R 1 A liposome encapsulating an mRNA encoding the protein of embodiment 6, wherein -C2H4-, -C3H6-, or C4H8-. 9.R 1 is C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 306. A liposome encapsulating an mRNA encoding the protein of embodiment 5, wherein the carboxyl group is -alkylene. 10.R 1 But C6-C 30 -Alkenylene or C8-C 20 3. A liposome encapsulating an mRNA encoding the protein of embodiment 1 or 2, wherein the carboxyl group is -alkenylene. 11.R 1 However, C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C 11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 3. A liposome encapsulating mRNA encoding the protein of embodiment 1 or 2, wherein the mRNA is selected from the group consisting of methyl-, ... 12.R 1 is unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 13. A liposome encapsulating mRNA encoding the protein of embodiment 12, wherein the liposome is selected from the group consisting of -alkenylene. 13.R 1is -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18 CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=C HCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 A liposome encapsulating mRNA encoding a protein according to embodiment 1 or 2, selected from CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-. 14.X 1 A liposome encapsulating an mRNA encoding a protein according to any one of embodiments 1 to 13, wherein is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. 15.X 1 is a 5- to 6-membered nitrogen-containing heterocycloalkyl. 16.X 1 16. A liposome encapsulating an mRNA encoding the protein of embodiment 15, wherein is substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl. 17.X 1 15. A liposome encapsulating an mRNA encoding a protein according to embodiment 14, wherein is a dialkylamine. 18.X 1 but: [ka] During the ceremony, R 3a and R 3b are each independently, C1-C 30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; A liposome encapsulating mRNA encoding the protein according to embodiment 17, wherein each n is independently an integer having a value of about 1 to about 6. 19.R 3a and R 3b are each independently, C6-C 30 19. A liposome encapsulating an mRNA encoding the protein of embodiment 18, wherein the liposome is -alkyl. 20.R 3a and R 3b each independently represents an unsubstituted C-C 30 20. A liposome encapsulating an mRNA encoding the protein of embodiment 19, wherein the liposome is -alkyl. 21.R 3a and R 3b are each independently, C6-C 30 21. A liposome encapsulating an mRNA encoding a protein according to embodiment 20, wherein the mRNA is selected from the group consisting of -alkyl. 22.R 3a and R 3b are each independently, C6-C 30 -Alkenyl or C8-C 20 19. A liposome encapsulating an mRNA encoding the protein of embodiment 18, wherein the carboxyl group is -alkenyl. 23.R 3a and R 3b are each independently C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 23. A liposome encapsulating an mRNA encoding a protein according to embodiment 22, wherein the mRNA is selected from the group consisting of aryl, aryl- ... 24.R 3a and R 3b are each independently -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH 2)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 A liposome encapsulating mRNA encoding a protein according to embodiment 22 or 23, selected from CH=CH(CH2)7CH3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. 25.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 13, wherein: 26.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 13, wherein: 27.X 1 but, [ka] A liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 13, wherein: 28. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] [ka] 29. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] 30. A liposome encapsulating an mRNA encoding a protein according to embodiment 1, wherein the cationic lipid has the following structure: [ka] [ka] 31. A composition comprising a liposome encapsulating mRNA encoding the protein according to any one of embodiments 1 to 30. 32. The composition of embodiment 31, comprising mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. 33. The composition of embodiment 31, comprising mRNA encoding ornithine transcarbamylase (OTC) protein. 34. A nucleic acid encapsulated in a liposome, wherein the liposome comprises a cationic lipid having a structure according to formula (EI): [ka] R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl, or R a and R b together with the carbon atoms to which they are attached form a saturated or unsaturated C5-C6-cycloalkyl or 5- to 6-membered heterocyclic ring, R c are independently H, C1-C6-alkyl, C1-C6-alkoxy, C3-C6-cycloalkyl, C2-C6-alkenyl, or C2-C6-alkynyl. 35. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has a structure according to formula (E-Ia): [ka] 36.R 1 But C6-C 30 36. The liposomally encapsulated nucleic acid of embodiment 34 or 35, wherein -alkylene. 37.R 1 36. The liposomally encapsulated nucleic acid of embodiment 34 or 35, wherein is C1-C5-alkylene. 38.R 1 but unsubstituted C6-C 30 37. The liposomally encapsulated nucleic acid of embodiment 36, wherein N is -alkylene. 39.R 1 38. The liposomally encapsulated nucleic acid of embodiment 37, wherein is unsubstituted C1-C5-alkylene. 40.R 1 But -CH 12 -, -C7H14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -C 11 H 22 -, -C 12 H 24 -, -C 13 H 26 -, -C 14 H 28 -, -C 15 H 30 -, -C 16 H 32 -, -C 17 H 34 -, -C 18 H 36 -, -C 19 H 38 -, -C 20 H 40 -, -C 21 H 42 -, -C 22 H 44 -, -C 23 H 46 -, -C 24 H 48 -, and -C 25 H 50 39. The liposome-encapsulated nucleic acid of embodiment 38, wherein 41.R 1 40. The liposomally encapsulated nucleic acid of embodiment 39, wherein is -C2H4-, -C3H6-, or C4H8-. 42.R 1 is C6-C substituted with one or more substituents selected from halogen, hydroxyl, amino, thiol, ester, and thioester; 30 39. The liposomally encapsulated nucleic acid of embodiment 38, wherein N is -alkylene. 43.R 1 But C6-C 30 -Alkenyl or C8-C 20 36. The liposomally encapsulated nucleic acid of embodiment 34 or 35, wherein -alkenylene. 44.R 1 However, C8-alkenylene, C9-alkenylene, C 10 -Alkenylene, C11 -Alkenylene, C 12 -Alkenylene, C 13 -Alkenylene, C 14 -Alkenylene, C 15 -Alkenylene, C 16 -Alkenylene, C 17 -Alkenylene, C 18 -Alkenylene, C 19 -alkenylene, and C 20 36. The liposomally encapsulated nucleic acid according to embodiment 34 or 35, wherein the nucleic acid is selected from the group consisting of: -alkenylene; 45.R 1 is unsubstituted C8-alkenylene, unsubstituted C9-alkenylene, unsubstituted C 10 -alkenylene, unsubstituted C 11 -alkenylene, unsubstituted C 12 -alkenylene, unsubstituted C 13 -alkenylene, unsubstituted C 14 -alkenylene, unsubstituted C 15 -alkenylene, unsubstituted C 16 -alkenylene, unsubstituted C 17 -alkenylene, unsubstituted C 18 -alkenylene, unsubstituted C 19 -alkenylene, and unsubstituted C 20 45. The liposomally encapsulated nucleic acid of embodiment 44, wherein the nucleic acid is selected from the group consisting of: -alkenylene. 46.R 1 is -(CH2)4CH=CH-, -(CH2)5CH=CH-, -(CH2)6CH=CH-, -(CH2)7CH=CH-, -(CH2)8CH=CH-, -(CH2)9CH=CH-, -(CH2) 10 CH=CH-, -(CH2) 11 CH=CH-, -(CH2) 12 CH=CH-, -(CH2) 13 CH=CH-, -(CH2) 14 CH=CH-, -(CH2) 15 CH=CH-, -(CH2) 16 CH=CH-, -(CH2) 17 CH=CH-, -(CH2) 18CH=CH-, -(CH2)7CH=CH(CH2)3CH2-, -(CH2)7CH=CH(CH2)5CH2-, -(CH2)4CH=CH(CH2)8CH2-, -(CH2)7CH=CH(CH2)7CH2-, -(CH2)6CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, -(CH2)3CH=CHCH2CH =CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH2-, -(CH2) 11 36. The nucleic acid encapsulated in a liposome according to embodiment 34 or 35, wherein the nucleic acid is selected from CH=CH(CH2)7CH2-, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH-. 47.X 1 47. The liposomally encapsulated nucleic acid of any one of embodiments 34 to 46, wherein is NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. 48.X 1 48. The liposomally encapsulated nucleic acid of embodiment 47, wherein is a 5-6 membered nitrogen-containing heterocycloalkyl. 49.X 1 49. The liposomally encapsulated nucleic acid of embodiment 48, wherein is substituted or unsubstituted pyrrolidinyl, piperidinyl, pyrazolidinyl, or piperazinyl. 50.X 1 48. The liposomally encapsulated nucleic acid of embodiment 47, wherein is a substituted dialkylamine. 51.X 1 but: [ka] During the ceremony, R 3a and R 3b are each independently, C1-C30 -Alkyl, C2-C 30 -Alkenyl, C2-C 30 -Alkynyl, hetero-C1-C 30 -Alkyl, Hetero-C1-C 30 -Alkenyl, hetero-C1-C 30 -alkynyl, polymeric, C5-C6-cycloalkyl, 5- to 6-membered heterocycloalkyl, C5-C6-aryl, or 5- to 6-membered heteroaryl; The liposome-encapsulated nucleic acid of embodiment 50, wherein each n is independently an integer having a value of from about 1 to about 6. 52.R 3a and R 3b are each independently, C6-C 30 52. The liposomally encapsulated nucleic acid of embodiment 51, wherein the nucleic acid is -alkyl. 53.R 3a and R 3b each independently represents an unsubstituted C-C 30 53. The liposomally encapsulated nucleic acid of embodiment 52, wherein the nucleic acid is -alkyl. 54.R 3a and R 3b are each 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 H45 , -C 23 H 47 , -C 24 H 49 , or -C 25 H 51 54. The liposomally encapsulated nucleic acid of embodiment 53, selected from: 55.R 3a and R 3b are each independently, C6-C 30 -Alkenyl or C8-C 20 52. The liposomally encapsulated nucleic acid of embodiment 51, wherein: 56.R 3a and R 3b are each independently C8-alkenyl, C9-alkenyl, C 10 -Alkenyl, C 11 -Alkenyl, C 12 -Alkenyl, C 13 -Alkenyl, C 14 -Alkenyl, C 15 -Alkenyl, C 16 -Alkenyl, C 17 -Alkenyl, C 18 -Alkenyl, C 19 -alkenyl, and C 20 56. The liposomally encapsulated nucleic acid of embodiment 55, wherein the nucleic acid is selected from the group consisting of: -alkenyl; 57.R 3a and R 3b are each 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) 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 57. The nucleic acid encapsulated in a liposome according to embodiment 55 or 56, wherein the nucleic acid is selected from CH=CH(CH2)7CH3, and -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. 58.X 1 but, [ka] The nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 46, wherein 59.X 1 but, [ka] The nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 46, wherein 60.X 1 but, [ka] The nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 46, wherein 61. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has the following structure: [ka] 62. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has the following structure: [ka] 63. The liposomally encapsulated nucleic acid of embodiment 34, wherein the cationic lipid has the following structure: [ka] 64. A composition comprising a nucleic acid encapsulated in a liposome according to any one of embodiments 34 to 63. 65. The composition of embodiment 64, 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. 66. The composition of embodiment 64 or 65, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide. 67. The composition of any one of embodiments 64 to 66, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the lung or lung cells of a subject. 68. The composition of embodiment 67, wherein the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. 69. The composition of any one of embodiments 64-66, wherein the mRNA encodes a peptide or polypeptide for delivery to or use in treating the liver or liver cells of a subject. 70. The composition of embodiment 69, wherein the mRNA encodes an ornithine transcarbamylase (OTC) protein. 71. The composition of any one of embodiments 64 to 66, wherein the mRNA encodes a peptide or polypeptide for use in a vaccine. 72. The composition of embodiment 71, wherein the mRNA encodes an antigen. Fourth Set of Embodiments 1. A cationic lipid having a structure according to formula (KI): [ka] During the ceremony, R 1 But C1-C 30 -Alkylene, C2-C 30 -Alkenylene, C2-C 30 -Alkynylene, hetero-C1-C 30 -Alkylene, Hetero-C1-C 30 -Alkenylene, hetero-C1-C 30 -alkynylene, polymer, C5-C6-cycloalkylene, 5- to 6-membered heterocycloalkylene, C5-C6-arylene, or 5- to 6-membered heteroarylene; X 1 is an ionic nitrogen-containing group, X 2 is S, C=O, or C=S, X 3 But S, O, CR a R b , or NR c and R a and R b are each independently H, C1-C6-alkyl, C...

Claims

[Claim 1] The invention described in the specification.

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