Ribocation lipids

Abstract

The present invention discloses cationic lipids, which are compounds of formula (I'). The cationic lipids provided herein can be used to deliver and express mRNA and the encoded protein, for example as a component of a liposome delivery vehicle, and thus can be used to treat various diseases, disorders and conditions, such as those associated with the deficiency of one or more proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No. 62 / 672,194 filed on May 16, 2018, U.S. Patent Application No. 62 / 740,095 filed on October 2, 2018, and U.S. Patent Application No. 62 / 749,442 filed on October 23, 2018, the entirety of each of which is incorporated herein by reference. Background Art

[0003] The delivery of nucleic acids has been widely explored as a potential treatment option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for treating a variety of diseases, including those associated with the deficiency of one or more proteins. Summary of the invention

[0004] The present invention particularly provides cationic lipids that can be used for delivering mRNA. The delivery of mRNA provided by cationic lipids as described herein can lead to targeted delivery, reduce dosing frequency, improve patient tolerance, and provide more effective and less toxic mRNA therapy for treating multiple diseases (including but not limited to cancer, cardiovascular disease, cystic fibrosis, infectious diseases and nervous system diseases).

[0005] In one aspect, the present invention provides a cationic lipid of formula (I'):

[0006]

[0007] in:

[0008] R X Independently -H, -L 1 -R 1 or –L 5A -L 5B -B';

[0009] L 1 , L 2 and L 3 Each of the above is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NR L -;

[0010] Each L 4A and L 5A are independently -C(O)-, -C(O)O- or -C(O)NR L -;

[0011] Each L 4B and L 5BIndependently C1-C 20 Alkylene, C2-C 20 Alkenylene or C2-C 20 Alkynylidene;

[0012] Each B and B' is NR 4 R 5 , a 5- to 10-membered nitrogen-containing heterocyclic group or a 5- to 10-membered nitrogen-containing heteroaryl group;

[0013] Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl;

[0014] Each R 4 and R 5 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 Alkynyl; or R 4 and R 5 are combined to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and

[0015] Each R L are independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl.

[0016] In an embodiment, the present invention provides a cationic lipid of formula (I):

[0017]

[0018] In an embodiment, the present invention provides a cationic lipid of formula (Ia):

[0019]

[0020] Where L 1 , L 2 , L 3 , R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0021] In an embodiment, the present invention provides a cationic lipid of formula (Ib):

[0022]

[0023] Where: R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0024] In an embodiment, the present invention provides a cationic lipid of formula (Ic):

[0025]

[0026] Where R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0027] In an embodiment, the present invention provides a cationic lipid of formula (Id):

[0028]

[0029] Where R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0030] In an embodiment, the present invention provides a cationic lipid of formula (Ie):

[0031]

[0032] Where R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0033] In an embodiment, each L 3 are independently -C(O)- or a covalent bond.

[0034] In an embodiment, L 4A It is -C(O)-.

[0035] In an embodiment, L 4B is unsubstituted C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene. 4B is -CH2-. In the embodiment, L 4Bis -CH2CH2-. In the embodiment, L 4B is -CH2CH2CH2-. In the embodiment, L 4B It is -CH2CH2CH2CH2-.

[0036] In an embodiment, B is NR 4 R 5 , and each R 4 and R 5 is independently hydrogen or unsubstituted C1-C6 alkyl. In an embodiment, B is N(CH3)2.

[0037] In an embodiment, B is NR 4 R 5 , and R 4 and R 5 Combined to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl. In an embodiment, B is piperidinyl or piperazinyl. In an embodiment, B is 4-methylpiperazinyl.

[0038] In another aspect, the present invention provides a cationic lipid of formula (II):

[0039]

[0040] in:

[0041] R A is hydrogen or -L 1 -R 1 ;

[0042] R B is hydrogen or -L 2 -R 2 ;

[0043] R C is hydrogen or -L 3 -R 3 ;

[0044] L 1 , L 2 and L 3 Each of the above is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NR L -;

[0045] L 4 Independent for C1-C 10 Alkylene;

[0046] Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C30 Alkenyl or C6-C 30 Alkynyl;

[0047] Each R 4 and R 5 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 Alkynyl; or R 4 and R 5 are combined to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and

[0048] Each R L are independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl.

[0049] In an embodiment, R A For -L 1 -R 1 ; R B For -L 2 -R 2 ; and R C For -L 3 -R 3 .

[0050] In an embodiment, the present invention provides a cationic lipid of formula (IIa):

[0051]

[0052] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0053] In an embodiment, the present invention provides a cationic lipid of formula (IIb):

[0054]

[0055] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0056] In an embodiment, the present invention provides a cationic lipid of formula (IIc):

[0057]

[0058] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0059] In an embodiment, the present invention provides a cationic lipid of formula (IId):

[0060]

[0061] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0062] In an embodiment, the present invention provides a cationic lipid of formula (IIe):

[0063]

[0064] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0065] In an embodiment, L 4 is an unsubstituted C1-C6 alkylene group. 4B is -CH2-. In the embodiment, L 4B is -CH2CH2-. In the embodiment, L 4B It is -CH2CH2CH2-.

[0066] In an embodiment, each R 4 and R 5 are independently hydrogen or unsubstituted C1-C6 alkyl, or R 4 and R 5 Combined to form a 5- to 6-membered heterocyclic group containing one or two ring nitrogens. In an embodiment, each R 4 and R 5 For CH3.

[0067] In an embodiment, R 4 and R 5are combined to form a 5- to 10-membered heterocyclic group. 4 and R 5 In one embodiment, R 4 and R 5 The combination forms 4-methylpiperazinyl.

[0068] In an embodiment, each R 1 , R 2 and R 3 Independently C6-C 22 Alkyl, C6-C 22 Alkenyl or C6-C 22 In one embodiment, each R 1 , R 2 and R 3 For unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 Alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl, unsubstituted branched C6-C 22 In one embodiment, each R 1 , R 2 and R 3 For unsubstituted linear C6-C 22 In an embodiment, each R 1 , R 2 and R 3 For unsubstituted linear C6-C 22 In an embodiment, each R 1 , R 2 and R 3 For unsubstituted linear C6-C 22 In one embodiment, each R 1 , R 2 and R 3 For unsubstituted branched C6-C 22 In an embodiment, each R 1 , R 2 and R 3 For unsubstituted branched C6-C 22 In an embodiment, each R 1 , R 2 and R 3 For unsubstituted branched C6-C 22 Alkynyl.

[0069] In an embodiment, each R 1 , R2 and R 3 For unsubstituted C6-C 22 In an embodiment, R 6 For unsubstituted linear C6-C 14 In an embodiment, R 6 For unsubstituted branched C6-C 14 In an embodiment, each R 1 , R 2 and R 3 (CH2)7CH3, (CH2)9CH3, (CH2) 11 CH3, (CH2) 13 CH3, (CH2) 15 CH3 or (CH2) 17 CH3. In an embodiment, each R 1 , R 2 and R 3 (CH2)6CH3, (CH2)8CH3, (CH2) 10 CH3, (CH2) 12 CH3, (CH2) 14 CH3 or (CH2) 16 CH3.

[0070] In an embodiment, each R 1 , R 2 and R 3 To be –O(CO)R 6 or-C(O)OR 6 Substituted C6-C 12 Alkyl, where R 6 For unsubstituted C6-C 14 In an embodiment, each R 1 , R 2 and R 3 It is (CH2)8OC(O)(CH2)6CH3, (CH2)9OC(O)(CH2)6CH3, (CH2)7C(O)O(CH2)2CH(C5H 11 )2 or (CH2)8C(O)O(CH2)2CH(C5H 11 )2.

[0071] In an embodiment, each R 1 , R 2 and R 3 For unsubstituted C6-C 22 Alkenyl (eg, monoalkenyl, dienyl, or trienyl).

[0072] In an embodiment, each R 1, R 2 and R 3 for:

[0073] or

[0074] In an embodiment, each R 1 , R 2 and R 3 for:

[0075] or

[0076] In an embodiment, each R 1 , R 2 and R 3 for

[0077] In another aspect, the present invention provides a cationic lipid selected from the group consisting of:

[0078]

[0079]

[0080]

[0081] In another aspect, the present invention provides a cationic lipid selected from the group consisting of:

[0082]

[0083]

[0084] In an embodiment, the cationic lipid has a structure according to one of the following formulas,

[0085] In an embodiment, the cationic lipid has a structure according to one of the following formulas,

[0086]

[0087] In an embodiment, the cationic lipid is selected from the group consisting of:

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] In an embodiment, the cationic lipid is selected from the group consisting of:

[0094]

[0095]

[0096]

[0097] In embodiments, the cationic lipid has a structure according to the formula,

[0098]

[0099] In embodiments, the cationic lipid has a structure according to the formula,

[0100]

[0101] In embodiments, the cationic lipid has a structure according to the formula,

[0102]

[0103] In embodiments, the cationic lipid has a structure according to the formula,

[0104]

[0105] In an embodiment, L 5B It is -CH2-, -CH2CH2-, -CH2CH2CH2- or -CH2CH2CH2CH2-.

[0106] In an embodiment, B' is NR 4 R 5 , and each R 4 and R 5 are independently hydrogen or unsubstituted C1-C6 alkyl, or R 4 and R 5 Combined to form a 5- to 6-membered heterocyclyl containing one or two ring nitrogens. In an embodiment, B' is N(CH3)2.

[0107] In an embodiment, L 4B is -CH2-. In the embodiment, L 4B is -CH2CH2-. In the embodiment, L 4B is -CH2CH2CH2-. In the embodiment, L 4B It is -CH2CH2CH2CH2-.

[0108] In an embodiment, B' is NR 4 R 5 , and each R 4 and R 5 are independently hydrogen or unsubstituted C1-C6 alkyl, or R 4 and R 5 Combined to form a 5- to 6-membered heterocyclyl containing one or two ring nitrogens. In an embodiment, B' is N(CH3)2.

[0109] In an embodiment, L 2 and L 3 Each of the is a covalent bond.

[0110] In an embodiment, L 2 and L 3 Each of is -C(O)-.

[0111] In an embodiment, R 2 and R 3 Each of the 22 Alkyl, C6-C 22 Alkenyl or C6-C 22 Alkynyl.

[0112] In an embodiment, each R 2 and R 3 For unsubstituted linear C6-C 22 Alkyl, unsubstituted linear C6-C 22 Alkenyl, unsubstituted linear C6-C 22 Alkynyl, unsubstituted straight chain C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl, unsubstituted branched C6-C 22 In one embodiment, each R 2 and R 3 For unsubstituted linear C6-C 22 In an embodiment, each R 2 and R 3 For unsubstituted linear C6-C 22 In an embodiment, each R 2 and R 3 For unsubstituted linear C6-C 22 In one embodiment, each R 2 and R 3 For unsubstituted branched C6-C 22 In an embodiment, each R 2 and R 3 For unsubstituted branched C6-C 22In an embodiment, each R 2 and R 3 For unsubstituted branched C6-C 22 Alkynyl.

[0113] In an embodiment, R 2 and R 3 Each of the following is an unsubstituted C6-C 22 In an embodiment, R 2 and R 3 Each of (CH2)7CH3, (CH2)9CH3, (CH2) 11 CH3, (CH2) 13 CH3, (CH2) 15 CH3 or (CH2) 17 CH3. In an embodiment, R 2 and R 3 Each of (CH2)6CH3, (CH2)8CH3, (CH2) 10 CH3, (CH2) 12 CH3, (CH2) 14 CH3 or (CH2) 16 CH3. In an embodiment, R 2 and R 3 Each of -O(CO)R 6 or -C(CO)OR 6 Substituted C6-C 12 Alkyl, where R 6 For unsubstituted C6-C 14 In an embodiment, R 6 For unsubstituted linear C6-C 14 In an embodiment, R 6 For unsubstituted branched C6-C 14 In an embodiment, R 2 and R 3 Each of (CH2)8OC(O)(CH2)6CH3, (CH2)9OC(O)(CH2)6CH3, (CH2)7C(O)O(CH2)2CH(C5H 11 )2 or (CH2)8C(O)O(CH2)2CH(C5H 11 )2.

[0114] In an embodiment, R 2 and R 3 Each of the following is an unsubstituted C6-C 22 In one embodiment, C6-C 22 Alkenyl is monoalkenyl, dienyl or trienyl.

[0115] In an embodiment, R 2 and R 3 Each of

[0116] or

[0117] In an embodiment, R 2 and R 3 Each of

[0118] or

[0119] In an embodiment, the cationic lipid is selected from the group consisting of cationic lipids (22)-(462).

[0120] In another aspect, the invention features a compound having the structure,

[0121] or a salt thereof.

[0122] In another aspect, the invention features a compound having the structure,

[0123] or a salt thereof.

[0124] In some aspects, the invention provides methods for preparing the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)).

[0125] In some aspects, the invention provides a composition, such as a pharmaceutical composition comprising a cationic lipid of the invention and one or more polynucleotides.

[0126] In some embodiments, the composition (e.g., pharmaceutical composition) comprises an mRNA encoding a protein encapsulated in a liposome. In embodiments, the liposome comprises one or more cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)), one or more non-cationic lipids, one or more cholesterol-based lipids and / or one or more PEG-modified lipids. In embodiments, the liposome comprises one or more cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)), one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. In embodiments, the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In embodiments, the mRNA encodes an ornithine carbamoyltransferase (OTC) protein.In embodiments, the mRNA encodes an antigen from an infectious agent.

[0127] In embodiments, the composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated in a liposome, wherein the liposome comprises a cationic lipid as described herein (e.g., a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)). In embodiments, the composition further comprises one or more lipids selected from the group consisting of: one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids. In embodiments, the composition further comprises one or more lipids selected from the group consisting of: one or more non-cationic lipids and one or more PEG-modified lipids. In embodiments, the nucleic acid is an mRNA encoding a peptide or polypeptide. In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or treatment of a subject's lung or lung cells (e.g., the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein). In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or treatment of a subject's liver or hepatocytes (e.g., the mRNA encodes an ornithine carbamoyltransferase (OTC) protein). In an embodiment, the mRNA encodes an antigen from an infectious agent.

[0128] In some aspects, the invention provides methods of treating a disease in a subject, comprising administering to the subject a composition (eg, a pharmaceutical composition) as described herein.

[0129] In embodiments, the composition (eg, pharmaceutical composition) is formulated for intravenous (IV) administration.

[0130] In embodiments, the composition (eg, pharmaceutical composition) is formulated for intramuscular (IM) administration.

[0131] In embodiments, the composition (eg, pharmaceutical composition) is formulated for administration by inhalation. In embodiments, the composition (eg, pharmaceutical composition) is formulated for nebulization. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 hEPO protein expression following intravenous (IV) administration of lipid nanoparticle formulations comprising a cationic lipid described herein and mRNA encoding hEPO is shown. Protein expression was determined using ELISA. DETAILED DESCRIPTION

[0133] definition

[0134] In order to make the present invention more easily understood, some terms are first defined below. The following terms and other definitions of terms are set forth throughout this specification. The publications and other reference materials cited herein that describe the background technology of the present invention and provide additional details about its implementation are incorporated herein by reference.

[0135] Amino Acids As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a d-amino acid; in some embodiments, the amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and / or substitutions. Amino acids, including carboxyl and / or amino terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. The amino acid may contain one or more post-translational modifications, for example, conjugation to one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, and a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be apparent from the context in which the term is used.

[0136] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a person at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0137] Approximately or approximately: As used herein, when applied to one or more target values, the term "approximately" or "approximately" refers to a value similar to the reference value. In certain embodiments, the term "approximately" or "approximately" refers to a series of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the value, unless otherwise stated or otherwise apparent from the context (unless the number exceeds 100% of the possible value).

[0138] Bioactive: As used herein, the term "bioactive" refers to the characteristic of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered bioactive.

[0139] Delivery: As used herein, the term "delivery" encompasses local delivery and systemic delivery. For example, delivery of mRNA includes situations where the mRNA is delivered to the target tissue and the encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and situations where the mRNA is delivered to the target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), and systemically distributed and absorbed by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0140] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides into complete proteins (e.g., enzymes), and / or post-translational modifications of polypeptides or fully assembled proteins (e.g., enzymes). In this application, the terms "expression" and "production" and grammatically equivalent terms are used interchangeably.

[0141] Functional: As used herein, a "functional" biomolecule is one that is in a form that exhibits the properties and / or activities that characterize it.

[0142] Half-life: As used herein, the term "half-life" is the time required for the concentration or activity of an agent, such as an amino acid or a protein, to fall to half of its value measured at the beginning of a time period.

[0143] Improvement, increase or decrease: As used herein, the terms "improvement," "increase," or "decrease," or grammatical equivalents, refer to values ​​relative to a baseline measurement, such as the measurement of the same individual before the start of a treatment described herein, or the measurement of a control subject (or multiple control subjects) in the absence of a treatment described herein. A "control subject" is a subject having the same form of disease as the subject being treated and who is about the same age as the subject being treated.

[0144] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0145] 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).

[0146] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some of the components with which it was originally associated when it was produced (whether naturally and / or in an experimental setting), and / or (2) produced, prepared and / or manufactured by the hand of man. An isolated substance and / or entity can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were originally associated. In some embodiments, the purity of the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of the purity percentage of isolated substances and / or entities should not include excipients (e.g., buffers, solvents, water, etc.).

[0147] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA includes modified RNA and unmodified RNA. The term "modified mRNA" relates to an mRNA comprising at least one chemically modified nucleotide. The mRNA may comprise one or more coding and non-coding regions. The mRNA may be purified from a natural source, produced using a recombinant expression system, and optionally purified, chemically synthesized, and the like. Where appropriate, for example, in the case of a chemically synthesized molecule, the mRNA may comprise nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, and the like. Unless otherwise indicated, the display direction of the mRNA sequence is 5' to 3'. In some embodiments, the mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).

[0148] Nucleic acid: As used herein, the term "nucleic acid" refers to any compound and / or substance that can be incorporated into a polynucleotide chain in its broadest sense. In some embodiments, nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain by a phosphodiester bond. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" covers RNA and single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, "nucleic acid" covers ribonucleic acid (RNA), including but not limited to interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), poly-encoding nucleic acid (MCNA), polymer-encoding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA) Any one or more. 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 RNA and DNA. In embodiments, DNA can be in the form of antisense DNA, plasmid DNA, part of plasmid DNA, pre-condensed DNA, polymerase chain reaction (PCR) product, vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA or derivatives of these groups.In embodiments, RNA can be in the form of messenger RNA (mRNA), ribosomal RNA (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), splicing leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), microRNA (miRNA), RNA interacting with piwi (piRNA), small interfering RNA (siRNA), transactional 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 encoding a protein such as an enzyme.

[0149] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition can be administered, for example, for experimental purposes, diagnostic purposes, preventive purposes, cosmetic purposes, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include prenatal and postnatal.

[0150] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0151] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe 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 are salts of amino groups formed with inorganic or organic acids or by using other methods used in the art, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, 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, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate heptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C 1-4 alkyl)4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed when appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and arylsulfonates. Other pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using appropriate electrophiles such as alkyl halides to form quaternized alkylated amino salts.

[0152] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or method that affects the entire body or entire organism. Typically, systemic distribution or delivery is accomplished via the body's circulatory system (e.g., blood). This is in contrast to the definition of "local distribution or delivery."

[0153] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person who presents to a medical provider for disease diagnosis or treatment. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may suffer from a disease or condition or be susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0154] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a characteristic or property of interest in full or near full extent or degree. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0155] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some embodiments, target tissues include those tissues that exhibit disease-associated pathology, symptoms, or characteristics.

[0156] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount sufficient to treat, diagnose, prevent and / or delay the onset of symptoms of a disease, disorder and / or condition when administered to a subject suffering from or susceptible to the disease, disorder and / or condition. One of ordinary skill in the art will recognize that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0157] Treatment: As used herein, the term "treatment" refers to any method used to partially or completely relieve, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. In order to reduce the risk of developing pathology associated with a disease, treatment can be administered to subjects who do not show signs of the disease and / or show only early signs of the disease.

[0158] Aliphatic: As used herein, the term aliphatic refers to C 1- C 40 hydrocarbons, and include saturated hydrocarbons and unsaturated hydrocarbons. Aliphatic groups may be straight chain, branched chain or cyclic. For example, C1-C 20 The aliphatic group may include C1-C 20 Alkyl (e.g., straight or branched C1-C 20 Saturated alkyl), C2-C 20 Alkenyl (e.g., straight or branched C4-C20 Diene, straight or branched C6-C 20 triene, etc.) and C2-C 20 Alkynyl (e.g., straight-chain or branched C2-C 20 C1-C 20 Aliphatic may include C3-C 20 Cyclic aliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl or C8-C 20 In some embodiments, the aliphatic group may include one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester or amide. The aliphatic group is unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group may be substituted with one or more (e.g., 1, 2, 3, 4, 5 or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic groups (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently an unsubstituted C1-C3 alkyl. In an embodiment, the aliphatic group is unsubstituted. In an embodiment, the aliphatic group does not include any heteroatoms.

[0159] Alkyl: As used herein, the term "alkyl" refers to acyclic straight-chain and branched hydrocarbon groups, for example, "C1-C 20"Alkyl" refers to an alkyl group having 1-20 carbons. The alkyl group may be straight or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like. Other alkyl groups will be apparent to those skilled in the art in view of the benefits of this disclosure. The alkyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic groups (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted C1-C3 alkyl. In an embodiment, alkyl is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0160] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight or branched hydrocarbon group, and is exemplified by methylene, ethylene, isopropylene, and the like. Likewise, as used herein, the term "alkenylene" refers to an unsaturated divalent straight or branched hydrocarbon group having one or more unsaturated carbon-carbon double bonds, which may be present at any stable point along the chain, and the term "alkynylene" refers herein to an unsaturated divalent straight or branched hydrocarbon group having one or more unsaturated carbon-carbon triple bonds, which may be present at any stable point along the chain. In certain embodiments, an alkylene, alkenylene, or alkynylene group may contain one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. For example, the alkylene, alkenylene, or alkynylene group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic groups (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R' is independently unsubstituted C1-C3 alkyl. In some embodiments, the alkylene, alkenylene or alkynylene is unsubstituted. In some embodiments, the alkylene, alkenylene or alkynylene does not include any heteroatoms.

[0161] Alkenyl: As used herein, "alkenyl" refers to any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds, which may be present at any stable point along the chain, for example, "C2-C 20"Alkenyl" refers to an alkenyl group having 2-20 carbons. For example, alkenyl includes prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In an embodiment, the alkenyl group contains 1, 2 or 3 carbon-carbon double bonds. In an embodiment, the alkenyl group contains a single carbon-carbon double bond. In an embodiment, multiple double bonds (e.g., 2 or 3) are conjugated. The alkenyl group may be unsubstituted or substituted with one or more substituents described herein. For example, alkenyl may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic groups (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted C1-C3 alkyl. In an embodiment, alkenyl is unsubstituted. In an embodiment, alkenyl is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0162] Alkynyl: As used herein, "alkynyl" refers to any hydrocarbon chain in a straight or branched configuration having one or more carbon-carbon triple bonds at any stable point along the chain, e.g., "C2-C 20 "Alkynyl" refers to an alkynyl group having 2-20 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, the alkynyl group contains one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic groups (e.g., C1-C 20 Alkyl, C1-C15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted C1-C3 alkyl. In an embodiment, alkynyl is unsubstituted. In an embodiment, alkynyl is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0163] Cycloalkyl: As used herein, the term "cycloalkyl" refers to a non-aromatic saturated cyclic group, for example, "C3-C 10 Cycloalkyl. In embodiments, the cycloalkyl is monocyclic. In embodiments, the cycloalkyl is polycyclic (e.g., bicyclic or tricyclic). In polycyclic cycloalkyls, the individual rings may be fused, bridged, or spirocyclic. Examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[3.2.1]octyl, octahydropentalenyl, and spiro[4.5]decyl, among others. The term "cycloalkyl" may be used interchangeably with the term "carbocycle". The cycloalkyl may be unsubstituted or substituted with one or more substituents as described herein. For example, the cycloalkyl may be substituted with one or more of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each instance of R' is independently C1-C 20 Aliphatic groups (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R' is independently unsubstituted C1-C3 alkyl. In an embodiment, cycloalkyl is unsubstituted. In an embodiment, cycloalkyl is substituted (e.g., substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0164] Heterocyclyl: As used herein, "heterocyclyl" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, and phosphorus ("3-14 membered heterocyclyl"). In heterocyclyls containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, where valence permits. A heterocyclyl may be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged, or spirocyclic system, such as a bicyclic system ("bicyclic heterocyclyl") or a tricyclic system ("tricyclic heterocyclyl")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. A heterocyclic polycyclic ring system may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes a ring system in which a heterocyclyl ring as defined above is fused to one or more carbocyclyls, wherein the point of attachment is on the carbocyclyl or heterocyclyl ring, or a ring system in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in this case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Unless otherwise indicated, each instance of a heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted ("substituted heterocyclyl") with one or more substituents. In certain embodiments, a heterocyclyl is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, a heterocyclyl is a substituted 3-14 membered heterocyclyl.

[0165] In some embodiments, heterocyclyl is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heterocyclyl”). In some embodiments, heterocyclyl is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heterocyclyl”). In some embodiments, heterocyclyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0166] Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridine, oxirane, and thioalkenyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetane, and thietidinyl. Exemplary 5-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, dioxolane, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thiacyclohexane. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiolanyl, dioxanyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepane and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azocanyl, oxepane and thiepanyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthopyridinyl, decahydro-1,8-naphthopyridinyl, decahydropyrrolo[3,2-b]pyrrole, indolyl, phthalimidyl, naphthoimidyl, chromanyl, benzothiophenyl, 1H-benzo[e][1,4]diazapyridinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl , 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

[0167] Halogen: As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0168] Cationic lipids

[0169] Liposome-based carriers are considered attractive carriers for therapeutic agents, and ongoing development efforts are still required. Although liposome-based carriers comprising cationic lipid components have shown encouraging results in terms of encapsulation, stability and site localization, there is still a great need for improved liposome-based delivery systems. For example, significant drawbacks of liposome delivery systems relate to the construction of liposomes with sufficient cell culture or in vivo stability to reach the desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to effectively release their encapsulated substances to such target cells.

[0170] In particular, there is still a need for improved cationic lipids that exhibit improved pharmacokinetic properties and can deliver macromolecules such as nucleic acids to a variety of cell types and tissues with improved efficiency. Importantly, there is still a special need for novel cationic lipids that are characterized in that they have reduced toxicity and can effectively deliver encapsulated nucleic acids and polynucleotides to target cells, tissues and organs.

[0171] Described herein are novel cationic lipids, compositions comprising such lipids, and related methods of use. In embodiments, the compounds described herein can be used as liposome compositions or components of liposome compositions to facilitate delivery and subsequent transfection to one or more target cells.

[0172] The cationic lipids disclosed herein contain a basic, ionizable functional group (eg, an amine or nitrogen-containing heteroaryl group as described herein) that exists in either a neutral or charged form.

[0173] In embodiments, cationic lipids as described herein can provide one or more desired characteristics or properties.That is, in certain embodiments, the feature of cationic lipids as described herein can be to have one or more properties, and these properties provide such compound advantages relative to the lipids of other similar classifications.For example, cationic lipids disclosed herein can allow control and customize the properties of the liposome compositions (such as lipid nanoparticles) of their components.Especially, the feature of cationic lipids disclosed herein can be to enhance transfection efficiency and the ability thereof to cause specific biological results.Such results can include, for example, enhanced cellular uptake, endosome / lysosome destruction ability and / or promote the release of intracellular encapsulation materials (such as, polynucleotides).

[0174] Cationic lipids of formula (I'), (I), (II) and (III)

[0175] In one aspect, the present invention provides a cationic lipid of formula (I'):

[0176]

[0177] in:

[0178] RX Independently -H, -L 1 -R 1 or –L 5A -L 5B -B';

[0179] L 1 , L 2 and L 3 Each of the above is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NR L -;

[0180] Each L 4A and L 5A are independently -C(O)-, -C(O)O- or -C(O)NR L -;

[0181] Each L 4B and L 5B Independently C1-C 20 Alkylene, C2-C 20 Alkenylene or C2-C 20 Alkyne;

[0182] Each B and B' is NR 4 R 5 , a 5- to 10-membered nitrogen-containing heterocyclic group or a 5- to 10-membered nitrogen-containing heteroaryl group;

[0183] Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl;

[0184] Each R 4 and R 5 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 Alkynyl; or R 4 and R 5 are combined to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and

[0185] Each R L are independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl.

[0186] In an embodiment, R X is -H. In an embodiment, RX For -L 1 -R 1 In an embodiment, R X For -L 5A- L 5B- B'.

[0187] In some embodiments, the present invention provides a cationic lipid of formula (I):

[0188]

[0189] In an embodiment, L 1 is a covalent bond. In an embodiment, L 1 is -C(O)-. In an embodiment, L 1 is -C(O)O-. In an embodiment, L 1 is -C(O)S-. In an embodiment, L 1 -C(O)NR L In an embodiment, L 1 It is a covalent bond or -C(O)-.

[0190] In an embodiment, R 1 It is C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 1 It is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 It is substituted C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 1 It is C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 1 It is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 It is substituted C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 1 It is C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 1 It is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22In one embodiment, R 1 It is substituted C6-C 30 Alkynyl (e.g., substituted C6-C 22 In one embodiment, R 1 A group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl. In an embodiment, R 1 A group (eg, as described herein) is a straight chain group; for example, a straight chain alkyl, straight chain alkenyl, or straight chain alkynyl.

[0191] In an embodiment, L 2 is a covalent bond. In some embodiments, L 2 is -C(O)-. In an embodiment, L 2 is -C(O)O-. In an embodiment, L 2 is -C(O)S-. In some embodiments, L 2 -C(O)NR L In an embodiment, L 2 It is a covalent bond or -C(O)-.

[0192] In an embodiment, R 2 It is C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 2 It is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 2 It is substituted C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 2 It is C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 2 It is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 2 It is substituted C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 2 It is C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 2 It is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22In one embodiment, R 2 It is substituted C6-C 30 Alkynyl (e.g., substituted C6-C 22 In one embodiment, R 2 A group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl. In an embodiment, R 2 A group (eg, as described herein) is a straight chain group; for example, a straight chain alkyl, straight chain alkenyl, or straight chain alkynyl.

[0193] In an embodiment, L 3 is a covalent bond. In some embodiments, L 3 is -C(O)-. In an embodiment, L 3 is -C(O)O-. In an embodiment, L 3 In some embodiments, L 3 -C(O)NR L In an embodiment, L 3 It is a covalent bond or -C(O)-.

[0194] In an embodiment, -L 4A -L 4B -B is Where in is an integer from 0 to 6.

[0195] In an embodiment, -L 4A -L 4B -B is Where in is an integer from 0 to 6.

[0196] In an embodiment, -L 4A -L 4B -B is

[0197] In an embodiment, R 3 It is C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 3 It is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 3 It is substituted C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 3 It is C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R3 It is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 3 It is substituted C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 3 It is C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 3 It is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22 In one embodiment, R 3 It is substituted C6-C 30 Alkynyl (e.g., substituted C6-C 22 In one embodiment, R 3 A group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl. In an embodiment, R 3 A group (eg, as described herein) is a straight chain group; for example, a straight chain alkyl, straight chain alkenyl, or straight chain alkynyl.

[0198] In an embodiment, L 1 , L 2 and L 3 In the embodiment, L 1 , L 2 and L 3 Each of L is a covalent bond. 1 , L 2 and L 3 In the embodiment, each of L is -C(O)-. 1 , L 2 and L 3 Each of L is -C(O)O-. In an embodiment, L 1 , L 2 and L 3 In some embodiments, each of L is -C(O)S-. 1 , L 2 and L 3 Each of -C(O)NR L In an embodiment, L 1 , L 2 and L 3 Each of L is a covalent bond, or L 1 , L 2 and L 3 Each of is -C(O)-.

[0199] In an embodiment, R 1 , R 2 and R 3 In the embodiment, R 1 , R 2 and R 3 Each of C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the following is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the following is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 1 , R 2 and R 3 Each of the following is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22 In one embodiment, R 1 , R 2 and R 3 Each of the C6-C 30Alkynyl (e.g., substituted C6-C 22 In one embodiment, each R 1 , R 2 and R 3 (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl. In an embodiment, each R 1 , R 2 and R 3 (eg, as described herein) is a straight chain group; for example, a straight chain alkyl, a straight chain alkenyl, or a straight chain alkynyl.

[0200] In an embodiment, L 4A is -C(O)-. In an embodiment, L 4A is -C(O)O-. In an embodiment, L 4A -C(O)NR L -.

[0201] In an embodiment, L 4B C1-C 20 In the embodiment, L 4B is an unsubstituted C1-C 20 In the embodiment, L 4B It is a substituted C1-C 20 In an embodiment, L 4B It is C2-C 20 In an embodiment, L 4B is an unsubstituted C2-C 20 In the embodiment, L 4B It is a substituted C2-C 20 In an embodiment, L 4B It is C2-C 20 In the embodiment, L 4B is an unsubstituted C2-C 20 In the embodiment, L 4B It is a substituted C2-C 20 In one embodiment, L 4B is -CH2-. In the embodiment, L 4B is -CH2CH2-. In the embodiment, L 4B It is -CH2CH2CH2-.

[0202] In an embodiment, B is NR 4 R 5 In an embodiment, each R 4 and R 5 are independently hydrogen or unsubstituted C1-C 10 alkyl (e.g., unsubstituted C1-C6 alkyl, such as CH3), or R 4 and R 5 Combined to form a 5- to 6-membered heterocyclic group containing one or two ring nitrogens.

[0203] In an embodiment, B is NR 4 R 5 , and R 4 and R 5 are combined to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl. 4 and R 5 are combined to form a 5- to 10-membered heterocyclic group. 4 and R 5 Combined to form a 5- to 10-membered heterocyclyl containing one or two nitrogen atoms in one or more rings. 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 The combination forms 4-methylpiperazinyl.

[0204] In an embodiment, L 5A is -C(O)-. In an embodiment, L 5A is -C(O)O-. In an embodiment, L 5A -C(O)NR L -. In an embodiment, L 5A and L 4A In the embodiment, L 5A and L 4A are different.

[0205] In an embodiment, L 5B C1-C 20In the embodiment, L 5B is an unsubstituted C1-C 20 In the embodiment, L 5B It is a substituted C1-C 20 In an embodiment, L 5B It is C2-C 20 In an embodiment, L 5B is an unsubstituted C2-C 20 In the embodiment, L 5B It is a substituted C2-C 20 In an embodiment, L 5B It is C2-C 20 In the embodiment, L 5B is an unsubstituted C2-C 20 In the embodiment, L 5B It is a substituted C2-C 20 In one embodiment, L 5B is -CH2-. In the embodiment, L 5B is -CH2CH2-. In the embodiment, L 5B is -CH2CH2CH2-. In the embodiment, L 5B and L 4B In the embodiment, L 5B and L 4B are different.

[0206] In an embodiment, B' is NR 4 R 5 In an embodiment, each R 4 and R 5 are independently hydrogen or unsubstituted C1-C 10 Alkyl (eg, unsubstituted C1-C6 alkyl, such as CH3). In an embodiment, B' and B are the same. In an embodiment, B' and B are different.

[0207] In an embodiment, R 4 is hydrogen or C1-C 10 In an embodiment, R 4 is hydrogen. In an embodiment, R 4 It is C1-C10 In an embodiment, R 4 is an unsubstituted C1-C 10 In one embodiment, R 4 It is a substituted C1-C 10 In one embodiment, R 4 It is C2-C 10 In an embodiment, R 4 is an unsubstituted C2-C 10 In one embodiment, R 4 is an unsubstituted C2-C 10 In one embodiment, R 4 It is C2-C 10 In one embodiment, R 4 is an unsubstituted C2-C 10 In one embodiment, R 4 It is a substituted C2-C 10 In one embodiment, R 4 It is CH3.

[0208] In an embodiment, R 5 is hydrogen or C1-C 10 In an embodiment, R 5 is hydrogen. In an embodiment, R 5 It is C1-C 10 In an embodiment, R 5 is an unsubstituted C1-C 10 In one embodiment, R 5 It is a substituted C1-C 10 In one embodiment, R 5 It is C2-C 10 In an embodiment, R 5 is an unsubstituted C2-C 10 In one embodiment, R 5 is an unsubstituted C2-C 10 In one embodiment, R5 It is C2-C 10 In one embodiment, R 5 is an unsubstituted C2-C 10 In one embodiment, R 5 It is a substituted C2-C 10 In one embodiment, R 5 For CH3.

[0209] In an embodiment, B is N(CH3)2.

[0210] In an embodiment, B is a 5-10 membered nitrogen-containing heteroaryl. In an embodiment, B is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl or isoquinolyl. In an embodiment, B is an unsubstituted heteroaryl, which is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl or isoquinolyl. In an embodiment, B is a substituted heteroaryl group, which is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl or isoquinolyl. In an embodiment, B is imidazolyl (e.g., unsubstituted imidazolyl or substituted imidazolyl). In an embodiment, B is pyridyl (e.g., unsubstituted pyridyl or substituted pyridyl).

[0211] In an embodiment, B is a 5-10 membered nitrogen-containing heterocyclic group. In an embodiment, B is a pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydro-1,3,5-triazinyl, azepanyl, diazepanyl, azooctanyl, azacyclononanyl, etc. In an embodiment, B is a piperidinyl (e.g., unsubstituted piperidinyl or substituted piperidinyl). In an embodiment, B is a piperazinyl (e.g., unsubstituted piperazinyl or substituted piperazinyl).

[0212] In an embodiment, R L are independently hydrogen. In an embodiment, R L Independently C1-C 20 In an embodiment, R L is independently unsubstituted C1-C 20 In one embodiment, R L are independently substituted C1-C 20In one embodiment, R L Independently C2-C 20 In an embodiment, R L is independently unsubstituted C2-C 20 In one embodiment, R L are independently substituted C2-C 20 In one embodiment, R L Independently C2-C 20 In one embodiment, R L is independently unsubstituted C2-C 20 In one embodiment, R L is independently substituted C2-C 20 Alkynyl (eg, substituted C2-C6 alkynyl).

[0213] In an embodiment, the cationic lipid of formula (I) has a structure according to formula (la):

[0214]

[0215] Where L 1 , L 2 , L 3 , R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0216] In an embodiment, the cationic lipid of formula (I) has a structure according to formula (Ib):

[0217]

[0218] Where R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0219] In an embodiment, the cationic lipid of formula (I) has a structure according to formula (Ic):

[0220]

[0221] in:

[0222] R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0223] In an embodiment, the cationic lipid of formula (I) has a structure according to formula (Id):

[0224]

[0225] Where R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0226] In an embodiment, the present invention provides a cationic lipid of formula (Ie):

[0227]

[0228] Where R 1 , R 2 , R 3 , B, L 4A and L 4B Each of is independently as defined herein.

[0229] In an embodiment, the cationic lipid of formula (I) has a structure according to formula (II):

[0230]

[0231] in:

[0232] R A is hydrogen or -L 1 -R 1 ;

[0233] R B is hydrogen or -L 2 -R 2 ;

[0234] R C is hydrogen or -L 3 -R 3 ;

[0235] L 1 , L 2 and L 3 Each of the above is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NR L -;

[0236] L 4 Independent for C1-C 10 Alkylene;

[0237] Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl;

[0238] Each R 4 and R 5 are independently hydrogen, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 Alkynyl; or R 4 and R 5 Combine to form a 5- to 10-membered heterocyclic group or a 5- to 10-membered heteroaryl group;

[0239] Each R L are independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl.

[0240] In an embodiment, R A For -L 1 -R 1 ; R B For -L 2 -R 2 ; and R C For -L 3 -R 3 .

[0241] In an embodiment, R A is hydrogen. In an embodiment, R A Yes-L 1 -R 1 .

[0242] In an embodiment, L 1 is a covalent bond or -C(O)-. In an embodiment, L 1 is a covalent bond. In an embodiment, L 1 is -C(O)-. In an embodiment, L 1 is -C(O)O-. In an embodiment, L 1 is -C(O)S-. In an embodiment, L 1 -C(O)NR L -.

[0243] In an embodiment, R 1 It is C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 1 It is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 It is substituted C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 1 It is C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 1 It is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 It is substituted C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 1 It is C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 1 It is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22 In one embodiment, R 1 It is substituted C6-C 30 Alkynyl (e.g., substituted C6-C 22 alkynyl).

[0244] In an embodiment, R B is hydrogen. In an embodiment, R B Yes-L 2 -R 2 .

[0245] In an embodiment, L 2 is a covalent bond or -C(O)-. In an embodiment, L 2 is a covalent bond. In some embodiments, L 2 is -C(O)-. In an embodiment, L 2 is -C(O)O-. In an embodiment, L 2 is -C(O)S-. In some embodiments, L 2 -C(O)NR L .

[0246] In an embodiment, R2 It is C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 2 It is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 2 It is substituted C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 2 It is C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 2 It is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 2 It is substituted C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 2 It is C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 2 It is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22 In one embodiment, R 2 It is substituted C6-C 30 Alkynyl (e.g., substituted C6-C 22 alkynyl).

[0247] In an embodiment, R C is hydrogen. In an embodiment, R C Yes-L 3 -R 3 .

[0248] In an embodiment, L 3 is a covalent bond or -C(O)-. In an embodiment, L 3 is a covalent bond. In some embodiments, L 3 is -C(O)-. In an embodiment, L 3 is -C(O)O-. In an embodiment, L 3 In some embodiments, L 3 -C(O)NR L .

[0249] In an embodiment, R 3It is C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 3 It is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 3 It is substituted C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 3 It is C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 3 It is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 3 It is substituted C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 3 It is C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 3 It is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22 In one embodiment, R 3 It is substituted C6-C 30 Alkynyl (e.g., substituted C6-C 22 alkynyl).

[0250] In an embodiment, L 1 , L 2 and L 3 In the embodiment, L 1 , L 2 and L 3 Each of L is a covalent bond. 1 , L 2 and L 3 In the embodiment, each of L is -C(O)-. 1 , L 2 and L 3 Each of L is -C(O)O-. In an embodiment, L 1 , L 2 and L 3 In some embodiments, each of L is -C(O)S-. 1 , L 2 and L3 Each of -C(O)NR L In an embodiment, L 1 , L 2 and L 3 Each of L is a covalent bond, or L 1 , L 2 and L 3 Each of is -C(O)-.

[0251] In an embodiment, R 1 , R 2 and R 3 Each of the groups is the same. In an embodiment, R 1 , R 2 and R 3 Each of C6-C 30 Alkyl (e.g., C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the following is an unsubstituted C6-C 30 Alkyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the C6-C 30 Alkyl (e.g., substituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of C6-C 30 Alkenyl (e.g., C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the following is an unsubstituted C6-C 30 Alkenyl (e.g., unsubstituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of the C6-C 30 Alkenyl (e.g., substituted C6-C 22 In an embodiment, R 1 , R 2 and R 3 Each of C6-C 30 Alkynyl (e.g., C6-C 22 In one embodiment, R 1 , R2 and R 3 Each of the following is an unsubstituted C6-C 30 Alkynyl (e.g., unsubstituted C6-C 22 In one embodiment, R 1 , R 2 and R 3 Each of the C6-C 30 Alkynyl (e.g., substituted C6-C 22 alkynyl).

[0252] In an embodiment, L 4 Independently C1-C 10 In the embodiment, L 4 is independently unsubstituted C1-C 10 In the embodiment, L 4 are independently substituted C1-C 10 In an embodiment, L 4 It is -CH2CH2-.

[0253] In an embodiment, B is NR 4 R 5 In an embodiment, each R 4 and R 5 are independently hydrogen or unsubstituted C1-C 10 alkyl (e.g., unsubstituted C1-C6 alkyl, such as CH3), or R 4 and R 5 Combined to form a 5- to 6-membered heterocyclic group containing one or two ring nitrogens.

[0254] In an embodiment, R 4 is hydrogen or C1-C 10 In an embodiment, R 4 is hydrogen. In an embodiment, R 4 It is C1-C 10 In an embodiment, R 4 is an unsubstituted C1-C 10 In one embodiment, R 4 It is a substituted C1-C 10 In one embodiment, R 4 It is C2-C 10 In an embodiment, R 4is an unsubstituted C2-C 10 In one embodiment, R 4 is an unsubstituted C2-C 10 In one embodiment, R 4 It is C2-C 10 In one embodiment, R 4 is an unsubstituted C2-C 10 In one embodiment, R 4 It is a substituted C2-C 10 In one embodiment, R 4 For CH3.

[0255] In an embodiment, R 5 is hydrogen or C1-C 10 In an embodiment, R 5 is hydrogen. In an embodiment, R 5 It is C1-C 10 In an embodiment, R 5 is an unsubstituted C1-C 10 In one embodiment, R 5 It is a substituted C1-C 10 In one embodiment, R 5 It is C2-C 10 In an embodiment, R 5 is an unsubstituted C2-C 10 In one embodiment, R 5 is an unsubstituted C2-C 10 In one embodiment, R 5 It is C2-C 10 In one embodiment, R 5 is an unsubstituted C2-C 10 In one embodiment, R 5 It is a substituted C2-C 10 In one embodiment, R 5 For CH3.

[0256] In an embodiment, R4 and R 5 are combined to form a 5- to 10-membered heterocyclic group. 4 and R 5 Combined to form a 5- to 10-membered heterocyclyl containing one or two nitrogen atoms in one or more rings. 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 In one embodiment, R 4 and R 5 The combination forms 4-methylpiperazinyl.

[0257] In an embodiment, R L are independently hydrogen. In an embodiment, R L Independently C1-C 20 In an embodiment, R L is independently unsubstituted C1-C 20 In one embodiment, R L are independently substituted C1-C 20 In one embodiment, R L Independently C2-C 20 In an embodiment, R L is independently unsubstituted C2-C 20 In one embodiment, R L are independently substituted C2-C 20 In one embodiment, R L Independently C2-C 20 In one embodiment, R L is independently unsubstituted C2-C 20 In one embodiment, R L is independently substituted C2-C 20 Alkynyl (eg, substituted C2-C6 alkynyl).

[0258] In an embodiment, the cationic lipid of formula (II) has a structure according to formula (IIa):

[0259]

[0260] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0261] In an embodiment, the cationic lipid of formula (II) has a structure according to formula (IIb):

[0262]

[0263] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0264] In an embodiment, the cationic lipid of formula (II) has a structure according to formula (IIc):

[0265]

[0266] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0267] In an embodiment, the cationic lipid of formula (II) has a structure according to formula (IId):

[0268]

[0269] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0270] In an embodiment, the cationic lipid of formula (II) has a structure according to formula (IIe):

[0271]

[0272] Each R A , R B , R C , L 4 , R 4 and R 5 Independently as defined herein.

[0273] In an embodiment, C6-C 30 Alkyl (e.g., R 1 , R 2 and / or R 3 ) is C 8-26 In the embodiment, C6-C 30 Alkyl (e.g., R 1 , R 2 and / or R 3 ) is a straight chain C 8-26 alkyl.

[0274] In an embodiment, C6-C 30 Alkyl (e.g., R 1 , R 2 and / or R 3 ) is CH3(CH2)6CH2-, CH3(CH2)7CH2-, CH3(CH2)8CH2-, CH3(CH2)9CH2-, CH3(CH2) 10 CH2-、CH3(CH2) 11 CH2-、CH3(CH2) 12 CH2-、CH3(CH2) 13 CH2-、CH3(CH2) 14 CH2-、CH3(CH2) 15 CH2-、CH3(CH2) 16 CH2-、CH3(CH2) 17 CH2-、CH3(CH2) 18 CH2-、CH3(CH2) 19 CH2-、CH3(CH2) 20 CH2-、CH3(CH2) 21 CH2-、CH3(CH2) 22 CH2-、CH3(CH2) 23 CH2- or CH3(CH2) 24 CH2-.

[0275] In an embodiment, C6-C 30 Alkyl (such as R 1 , R 2 and / or R 3) is CH3(CH2) 13 CH2-、CH3(CH2) 14 CH2-、CH3(CH2) 15 CH2-、CH3(CH2) 16 CH2-、CH3(CH2) 17 CH2- or CH3(CH2) 18 CH2-.

[0276] In an embodiment, C6-C 30 Alkyl (such as R 1 , R 2 and / or R 3 ) is CH3(CH2) 14 CH2-、CH3(CH2) 15 CH2- or CH3(CH2) 16 CH2-.

[0277] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is a C with one or two carbon-carbon double bonds 8-26 Alkenyl.

[0278] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is cis-CH3(CH2)3CH=CH(CH2)7CH2-, cis-CH3(CH2)5CH=CH(CH2)7CH2-, cis-CH3(CH2)8CH=CH(CH2)4CH2-, cis-CH3(CH2)7CH=CH(CH2)7CH2-, cis-CH3(CH2)9CH=CH(CH2)7CH2-, cis-CH3(CH2)7CH=CH(CH2)9CH2-, trans-CH3(CH2)7CH=CH(CH2)7CH2-, trans-CH3(CH2)5CH=CH(CH2)9CH2-, cis-CH3(CH2)9CH=CH(CH2)7CH2-, cis-CH3(CH2)7CH=CH(CH2)9CH2-, 11 CH2-, cis-CH3(CH2)7CH=CH(CH2) 13CH2-, cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)7CH2-, cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)9CH2- or cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2) 11 CH2-.

[0279] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is cis-CH3(CH2)3CH=CH(CH2)7CH2-, cis-CH3(CH2)5CH=CH(CH2)7CH2-, cis-CH3(CH2)8CH=CH(CH2)4CH2-, cis-CH3(CH2)7CH=CH(CH2)7CH2-, cis-CH3(CH2)9CH=CH(CH2)7CH2-, trans-CH3(CH2)7CH=CH(CH2)7CH2-, cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)7CH2- or cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)9CH2-.

[0280] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is cis-CH3(CH2)7CH=CH(CH2)7CH2-, cis-CH3(CH2)9CH=CH(CH2)7CH2-, cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)7CH2- or cis, cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)9CH2-.

[0281] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is cis-CH3(CH2)7CH=CH(CH2)7CH2- or cis, cis-CH3(CH2)4CH=CH-CH2CH=CH(CH2)7CH2-.

[0282] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3) is a C having three, four, five or six carbon-carbon double bonds 8-26 Aliphatic group.

[0283] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3) is cis,cis,cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2-, cis,cis,cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, cis,cis,cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-, trans,trans,trans-CH3(CH2)7CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-, cis,cis,cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH( CH2)6CH2-, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)9CH2-, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, cis, cis, cis, cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)6CH2-, cis, cis, trans, trans , cis-CH3(CH2)4CH=CHCH=CHCH=CHCH=CHCH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis, cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2-, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH 2CH=CH(CH2)5CH2-, cis, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2-, cis, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2- or cis, cis, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-.

[0284] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2-, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-, cis, cis, trans, trans, cis-CH3(CH2)4CH=CHCH=CHCH=CHCH=CHCH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2-.

[0285] In an embodiment, C6-C 30 Alkenyl (e.g., R 1 , R 2 and / or R 3 ) is cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2- or cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-.

[0286] In some embodiments, R 1 , R 2 and R 3 Each of is independently a saturated or unsaturated fatty acid aliphatic chain, ie, R'-(CH2)- for fatty acids R'-C(O)-. In some embodiments, R 1 , R 2 and R 3 Each of R is independently octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, oleic acid, linoleic acid, pentacosanoic acid, or cerotic acid. In some embodiments, R and R 1 Each of R is an aliphatic chain of caprylic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, or heptadecanoic acid. 1 , R 2 and R 3 Each of R is an aliphatic chain of lauric acid, tridecyl acid, myristic acid, or pentadecanoic acid. 1, R 2 and R 3 Each of R is an aliphatic chain of lauric acid or myristic acid. 1 , R 2 and R 3 Each of R is an aliphatic chain of stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, oleic acid, linoleic acid, pentacosanoic acid, or cerotic acid. 1 , R 2 and R 3 Each of R is an aliphatic chain of lignoceric acid, oleic acid, linoleic acid, pentacosanoic acid, or cerotic acid. 1 , R 2 and R 3 Each of R is an aliphatic chain of oleic acid, linoleic acid, or pentacanoic acid. 1 , R 2 and R 3 Each of R is an aliphatic chain of oleic acid or linoleic acid. 1 , R 2 and R 3 Each of R is an aliphatic chain of oleic acid. 1 , R 2 and R 3 Each of the is an aliphatic chain of linoleic acid.

[0287] In some embodiments (eg, any of Formula (I'), (I) and (II) and any of Formula (IIIa)-(IIIaa)), R 1 , R 2 and R 3 Each of R is independently any of the following aliphatic chains. 1 , R 2 and R 3 Each of the chains is the same aliphatic chain.

[0288]

[0289]

[0290] Exemplary Cationic Lipids

[0291] Exemplary cationic lipids include cationic lipids (1a)-(21a) (Table A).

[0292]

[0293] Table A. Cationic lipids (1a)-(21a)

[0294]

[0295]

[0296] Exemplary cationic lipids include cationic lipids (1b)-(21b) (Table B).

[0297]

[0298] Table B. Cationic lipids (1b)-(21b)

[0299]

[0300]

[0301] Exemplary cationic lipids include cationic lipids according to formula (IIIa), such as cationic lipids (22)-(34) (Table C):

[0302] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table C.

[0303] Table C. Cationic lipids of formula (IIIa)

[0304]

[0305]

[0306]

[0307] In embodiments, the cationic lipid is cationic lipid (22). In embodiments, the cationic lipid is cationic lipid (23). In embodiments, the cationic lipid is cationic lipid (24). In embodiments, the cationic lipid is cationic lipid (25). In embodiments, the cationic lipid is cationic lipid (26). In embodiments, the cationic lipid is cationic lipid (27). In embodiments, the cationic lipid is cationic lipid (28). In embodiments, the cationic lipid is cationic lipid (29). In embodiments, the cationic lipid is cationic lipid (30). In embodiments, the cationic lipid is cationic lipid (31). In embodiments, the cationic lipid is cationic lipid (32). In embodiments, the cationic lipid is cationic lipid (33). In embodiments, the cationic lipid is cationic lipid (34).

[0308] Exemplary cationic lipids include cationic lipids according to formula (IIIb), such as cationic lipids (35)-(47) (Table D):

[0309] Among them, B, R 2 and R 3 Independently as described herein, including the exemplary groups of Table D.

[0310] Table D. Cationic lipids of formula (IIIb)

[0311]

[0312]

[0313]

[0314] In embodiments, the cationic lipid is a cationic lipid (35). In embodiments, the cationic lipid is a cationic lipid (36). In embodiments, the cationic lipid is a cationic lipid (37). In embodiments, the cationic lipid is a cationic lipid (38). In embodiments, the cationic lipid is a cationic lipid (39). In embodiments, the cationic lipid is a cationic lipid (40). In embodiments, the cationic lipid is a cationic lipid (41). In embodiments, the cationic lipid is a cationic lipid (42). In embodiments, the cationic lipid is a cationic lipid (43). In embodiments, the cationic lipid is a cationic lipid (44). In embodiments, the cationic lipid is a cationic lipid (45). In embodiments, the cationic lipid is a cationic lipid (46). In embodiments, the cationic lipid is a cationic lipid (47).

[0315] Exemplary cationic lipids include cationic lipids according to formula (IIIc), such as cationic lipids (48)-(60) (Table E):

[0316] Among them, B, B', R 2 and R 3 Independently as described herein, including the exemplary groups of Table E.

[0317] Table E. Cationic lipids of formula (IIIc)

[0318]

[0319]

[0320]

[0321] In embodiments, the cationic lipid is a cationic lipid (48). In embodiments, the cationic lipid is a cationic lipid (49). In embodiments, the cationic lipid is a cationic lipid (50). In embodiments, the cationic lipid is a cationic lipid (51). In embodiments, the cationic lipid is a cationic lipid (52). In embodiments, the cationic lipid is a cationic lipid (53). In embodiments, the cationic lipid is a cationic lipid (54). In embodiments, the cationic lipid is a cationic lipid (55). In embodiments, the cationic lipid is a cationic lipid (56). In embodiments, the cationic lipid is a cationic lipid (57). In embodiments, the cationic lipid is a cationic lipid (58). In embodiments, the cationic lipid is a cationic lipid (59). In embodiments, the cationic lipid is a cationic lipid (60).

[0322] Exemplary cationic lipids include cationic lipids according to formula (IIId), such as cationic lipids (61)-(71) (Table F):

[0323] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table F.

[0324] Table F. Cationic lipids of formula (IIId)

[0325]

[0326]

[0327] In embodiments, the cationic lipid is a cationic lipid (61). In embodiments, the cationic lipid is a cationic lipid (62). In embodiments, the cationic lipid is a cationic lipid (63). In embodiments, the cationic lipid is a cationic lipid (64). In embodiments, the cationic lipid is a cationic lipid (65). In embodiments, the cationic lipid is a cationic lipid (66). In embodiments, the cationic lipid is a cationic lipid (67). In embodiments, the cationic lipid is a cationic lipid (68). In embodiments, the cationic lipid is a cationic lipid (69). In embodiments, the cationic lipid is a cationic lipid (70). In embodiments, the cationic lipid is a cationic lipid (71).

[0328] Exemplary cationic lipids include cationic lipids according to formula (IIIe), such as cationic lipids (72)-(82) (Table F):

[0329] Among them, B, R2 and R 3 Independently as described herein, including the exemplary groups of Table F.

[0330] Table G. Cationic lipids of formula (IIIe)

[0331]

[0332]

[0333]

[0334] In embodiments, the cationic lipid is a cationic lipid (72). In embodiments, the cationic lipid is a cationic lipid (73). In embodiments, the cationic lipid is a cationic lipid (74). In embodiments, the cationic lipid is a cationic lipid (75). In embodiments, the cationic lipid is a cationic lipid (76). In embodiments, the cationic lipid is a cationic lipid (77). In embodiments, the cationic lipid is a cationic lipid (78). In embodiments, the cationic lipid is a cationic lipid (79). In embodiments, the cationic lipid is a cationic lipid (80). In embodiments, the cationic lipid is a cationic lipid (81). In embodiments, the cationic lipid is a cationic lipid (82).

[0335] Exemplary cationic lipids include cationic lipids according to formula (IIIf), such as cationic lipids (83)-(93) (Table H):

[0336] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table H.

[0337] Table H. Cationic lipids of formula (IIIf)

[0338]

[0339]

[0340] In embodiments, the cationic lipid is a cationic lipid (83). In embodiments, the cationic lipid is a cationic lipid (84). In embodiments, the cationic lipid is a cationic lipid (85). In embodiments, the cationic lipid is a cationic lipid (86). In embodiments, the cationic lipid is a cationic lipid (87). In embodiments, the cationic lipid is a cationic lipid (88). In embodiments, the cationic lipid is a cationic lipid (89). In embodiments, the cationic lipid is a cationic lipid (90). In embodiments, the cationic lipid is a cationic lipid (91). In embodiments, the cationic lipid is a cationic lipid (92). In embodiments, the cationic lipid is a cationic lipid (93).

[0341] Exemplary cationic lipids include cationic lipids according to formula (IIIg), such as cationic lipids (94)-(106) (Table I):

[0342] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table I.

[0343] Table I. Cationic lipids of formula (IIIg)

[0344]

[0345]

[0346]

[0347] In embodiments, the cationic lipid is a cationic lipid (94). In embodiments, the cationic lipid is a cationic lipid (95). In embodiments, the cationic lipid is a cationic lipid (96). In embodiments, the cationic lipid is a cationic lipid (97). In embodiments, the cationic lipid is a cationic lipid (98). In embodiments, the cationic lipid is a cationic lipid (99). In embodiments, the cationic lipid is a cationic lipid (100). In embodiments, the cationic lipid is a cationic lipid (101). In embodiments, the cationic lipid is a cationic lipid (102). In embodiments, the cationic lipid is a cationic lipid (103). In embodiments, the cationic lipid is a cationic lipid (104). In embodiments, the cationic lipid is a cationic lipid (105). In embodiments, the cationic lipid is a cationic lipid (106).

[0348] Exemplary cationic lipids include cationic lipids according to formula (IIIh), such as cationic lipids (107)-(119) (Table J):

[0349] Among them, B, R 2 and R 3 Independently as described herein, including the exemplary groups of Table J.

[0350] Table J. Cationic lipids of formula (IIIh)

[0351]

[0352]

[0353]

[0354] In embodiments, the cationic lipid is a cationic lipid (107). In embodiments, the cationic lipid is a cationic lipid (108). In embodiments, the cationic lipid is a cationic lipid (109). In embodiments, the cationic lipid is a cationic lipid (110). In embodiments, the cationic lipid is a cationic lipid (111). In embodiments, the cationic lipid is a cationic lipid (112). In embodiments, the cationic lipid is a cationic lipid (113). In embodiments, the cationic lipid is a cationic lipid (114). In embodiments, the cationic lipid is a cationic lipid (115). In embodiments, the cationic lipid is a cationic lipid (116). In embodiments, the cationic lipid is a cationic lipid (117). In embodiments, the cationic lipid is a cationic lipid (118). In embodiments, the cationic lipid is a cationic lipid (119).

[0355] Exemplary cationic lipids include cationic lipids according to formula (IIIi), such as cationic lipids (120)-(132) (Table K):

[0356] Among them, B, B', R 2 and R 3 Independently as described herein, including the exemplary groups of Table K.

[0357] Table K. Cationic lipids of formula (IIIi)

[0358]

[0359]

[0360]

[0361] In embodiments, the cationic lipid is a cationic lipid (120). In embodiments, the cationic lipid is a cationic lipid (121). In embodiments, the cationic lipid is a cationic lipid (122). In embodiments, the cationic lipid is a cationic lipid (123). In embodiments, the cationic lipid is a cationic lipid (124). In embodiments, the cationic lipid is a cationic lipid (125). In embodiments, the cationic lipid is a cationic lipid (126). In embodiments, the cationic lipid is a cationic lipid (127). In embodiments, the cationic lipid is a cationic lipid (128). In embodiments, the cationic lipid is a cationic lipid (129). In embodiments, the cationic lipid is a cationic lipid (130). In embodiments, the cationic lipid is a cationic lipid (131). In embodiments, the cationic lipid is a cationic lipid (132).

[0362] Exemplary cationic lipids include cationic lipids according to formula (IIIj), such as cationic lipids (133)-(143) (Table L):

[0363] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table L.

[0364] Table L. Cationic lipids of formula (IIIj)

[0365]

[0366]

[0367] In an embodiment, the cationic lipid is a cationic lipid (133). In an embodiment, the cationic lipid is a cationic lipid (134). In an embodiment, the cationic lipid is a cationic lipid (135). In an embodiment, the cationic lipid is a cationic lipid (136). In an embodiment, the cationic lipid is a cationic lipid (137). In an embodiment, the cationic lipid is a cationic lipid (138). In an embodiment, the cationic lipid is a cationic lipid (139). In an embodiment, the cationic lipid is a cationic lipid (140). In an embodiment, the cationic lipid is a cationic lipid (141). In an embodiment, the cationic lipid is a cationic lipid (142). In an embodiment, the cationic lipid is a cationic lipid (143).

[0368] Exemplary cationic lipids include cationic lipids according to formula (IIIk), such as cationic lipids (144)-(154) (Table M):

[0369] Among them, B, R 2 and R 3 Independently as described herein, including the exemplary groups of Table M.

[0370] Table M. Cationic lipids of formula (IIIk)

[0371]

[0372]

[0373]

[0374] In embodiments, the cationic lipid is a cationic lipid (144). In embodiments, the cationic lipid is a cationic lipid (145). In embodiments, the cationic lipid is a cationic lipid (146). In embodiments, the cationic lipid is a cationic lipid (147). In embodiments, the cationic lipid is a cationic lipid (148). In embodiments, the cationic lipid is a cationic lipid (149). In embodiments, the cationic lipid is a cationic lipid (150). In embodiments, the cationic lipid is a cationic lipid (151). In embodiments, the cationic lipid is a cationic lipid (152). In embodiments, the cationic lipid is a cationic lipid (153). In embodiments, the cationic lipid is a cationic lipid (154).

[0375] Exemplary cationic lipids include cationic lipids according to formula (IIIm), such as cationic lipids (155)-(165) (Table N):

[0376] Among them, B, B', R 2 and R 3 Independently as described herein, including the exemplary groups of Table H.

[0377] Table N. Cationic lipids of formula (IIIm)

[0378]

[0379]

[0380] In embodiments, the cationic lipid is a cationic lipid (155). In embodiments, the cationic lipid is a cationic lipid (156). In embodiments, the cationic lipid is a cationic lipid (157). In embodiments, the cationic lipid is a cationic lipid (158). In embodiments, the cationic lipid is a cationic lipid (159). In embodiments, the cationic lipid is a cationic lipid (160). In embodiments, the cationic lipid is a cationic lipid (161). In embodiments, the cationic lipid is a cationic lipid (162). In embodiments, the cationic lipid is a cationic lipid (163). In embodiments, the cationic lipid is a cationic lipid (164). In embodiments, the cationic lipid is a cationic lipid (165).

[0381] Exemplary cationic lipids include cationic lipids according to formula (IIIn), such as cationic lipids (166)-(178) (Table O):

[0382] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table O.

[0383] In an embodiment, the cationic lipid according to formula (IIIn) has the following structure,

[0384] In an embodiment, R 1 , R 2 and R 3 Each of is any exemplary aliphatic group described herein (eg, Table O).

[0385] Table O. Cationic lipids of formula (IIIn) and (IIIn-1)

[0386]

[0387]

[0388]

[0389] In embodiments, the cationic lipid is a cationic lipid (166). In embodiments, the cationic lipid is a cationic lipid (167). In embodiments, the cationic lipid is a cationic lipid (168). In embodiments, the cationic lipid is a cationic lipid (169). In embodiments, the cationic lipid is a cationic lipid (170). In embodiments, the cationic lipid is a cationic lipid (171). In embodiments, the cationic lipid is a cationic lipid (172). In embodiments, the cationic lipid is a cationic lipid (173). In embodiments, the cationic lipid is a cationic lipid (174). In embodiments, the cationic lipid is a cationic lipid (175). In embodiments, the cationic lipid is a cationic lipid (176). In embodiments, the cationic lipid is a cationic lipid (177). In embodiments, the cationic lipid is a cationic lipid (178).

[0390] Exemplary cationic lipids include cationic lipids according to formula (IIIo), such as cationic lipids (179)-(191) (Table P):

[0391] Among them, B, R 2 and R 3 Independently as described herein, including the exemplary groups of Table P.

[0392] Table P. Cationic lipids of formula (IIIo)

[0393]

[0394]

[0395]

[0396] In an embodiment, the cationic lipid is a cationic lipid (179). In an embodiment, the cationic lipid is a cationic lipid (180). In an embodiment, the cationic lipid is a cationic lipid (181). In an embodiment, the cationic lipid is a cationic lipid (182). In an embodiment, the cationic lipid is a cationic lipid (183). In an embodiment, the cationic lipid is a cationic lipid (184). In an embodiment, the cationic lipid is a cationic lipid (185). In an embodiment, the cationic lipid is a cationic lipid (186). In an embodiment, the cationic lipid is a cationic lipid (187). In an embodiment, the cationic lipid is a cationic lipid (188). In an embodiment, the cationic lipid is a cationic lipid (189). In an embodiment, the cationic lipid is a cationic lipid (190). In an embodiment, the cationic lipid is a cationic lipid (191).

[0397] Exemplary cationic lipids include cationic lipids according to formula (IIIp), such as cationic lipids (192)-(204) (Table Q):

[0398] Among them, B, B', R 2 and R 3 Independently as described herein, including the exemplary groups of Table Q.

[0399] Table Q. Cationic lipids of formula (IIIp)

[0400]

[0401]

[0402]

[0403] In embodiments, the cationic lipid is a cationic lipid (192). In embodiments, the cationic lipid is a cationic lipid (193). In embodiments, the cationic lipid is a cationic lipid (194). In embodiments, the cationic lipid is a cationic lipid (195). In embodiments, the cationic lipid is a cationic lipid (196). In embodiments, the cationic lipid is a cationic lipid (197). In embodiments, the cationic lipid is a cationic lipid (198). In embodiments, the cationic lipid is a cationic lipid (199). In embodiments, the cationic lipid is a cationic lipid (200). In embodiments, the cationic lipid is a cationic lipid (201). In embodiments, the cationic lipid is a cationic lipid (202). In embodiments, the cationic lipid is a cationic lipid (203). In embodiments, the cationic lipid is a cationic lipid (204).

[0404] Exemplary cationic lipids include cationic lipids according to formula (IIIq), such as cationic lipids (205)-(215) (Table R):

[0405] Among them, B, R 1 , R 2 and R 3 Independently as described herein, including the exemplary groups of Table R.

[0406] In an embodiment, the cationic lipid according to formula (IIIq) has the structure,

[0407] In an embodiment, R 1 , R 2 and R 3 Each of is, for example, any of the exemplary aliphatic groups described in Table R.

[0408] Table R. Cationic lipids of formula (IIIq)

[0409]

[0410]

[0411] In embodiments, the cationic lipid is a cationic lipid (205). In embodiments, the cationic lipid is a cationic lipid (206). In embodiments, the cationic lipid is a cationic lipid (207). In embodiments, the cationic lipid is a cationic lipid (208). In embodiments, the cationic lipid is a cationic lipid (209). In embodiments, the cationic lipid is a cationic lipid (210). In embodiments, the cationic lipid is a cationic lipid (211). In embodiments, the cationic lipid is a cationic lipid (212). In embodiments, the cationic lipid is a cationic lipid (213). In embodiments, the cationic lipid is a cationic lipid (214). In embodiments, the cationic lipid is a cationic lipid (215).

[0412] Exemplary cationic lipids include cationic lipids according to formula (IIIr), such as cationic lipids (216)-(226) (Table S):

[0413] Among them, B, R 2 and R 3 Independently as described herein, including the exemplary groups of Table S.

[0414] Table S. Cationic lipids of formula (IIIr)

[0415]

[0416]

[0417]

[0418] In an embodiment, the cationic lipid is a cationic lipid (216). In an embodiment, the cationic lipid is a cationic lipid (217). In an embodiment, the cationic lipid is a cationic lipid (218). In an embodiment, the cationic lipid is a cationic lipid (219). In an embodiment, the cationic lipid is a cationic lipid (220). In an embodiment, the cationic lipid is a cationic lipid (221). In an embodiment, the cationic lipid is a cationic lipid (222). In an embodiment, the cationic lipid is a cationic lipid (223). In an embodiment, the cationic lipid is a cationic lipid (224). In an embodiment, the cationic lipid is a cationic lipid (225). In an embodiment, the cationic lipid is a cationic lipid (226).

[0419] Exemplary cationic lipids include cationic lipids according to formula (IIIs), such as cationic lipids (227)-(237) (Table T):

[0420] Among them, B, B', R 2 and R 3 Independently as described herein, including the exemplary groups of Table T.

[0421] Table T. Cationic lipids of formula (IIIs)

[0422]

[0423]

[0424] In an embodiment, the cationic lipid is a cationic lipid (227). In an embodiment, the cationic lipid is a cationic lipid (228). In an embodiment, the cationic lipid is a cationic lipid (229). In an embodiment, the cationic lipid is a cationic lipid (230). In an embodiment, the cationic lipid is a cationic lipid (231). In an embodiment, the cationic lipid is a cationic lipid (232). In an embodiment, the cationic lipid is a cationic lipid (233). In an embodiment, the cationic lipid is a cationic lipid (234). In an embodiment, the cationic lipid is a cationic lipid (235). In an embodiment, the cationic lipid is a cationic lipid (236). In an embodiment, the cationic lipid is a cationic lipid (237).

[0425] Exemplary cationic lipids include cationic lipids according to formula (IIIt), such as cationic lipids (238)-(261) (Table U):

[0426] wherein each R is independently as described herein, including the example groups in Table U.

[0427] Table U. Cationic lipids of formula (IIIt)

[0428]

[0429]

[0430] In embodiments, the cationic lipid is a cationic lipid (238). In embodiments, the cationic lipid is a cationic lipid (239). In embodiments, the cationic lipid is a cationic lipid (240). In embodiments, the cationic lipid is a cationic lipid (241). In embodiments, the cationic lipid is a cationic lipid (242). In embodiments, the cationic lipid is a cationic lipid (243). In embodiments, the cationic lipid is a cationic lipid (244). In embodiments, the cationic lipid is a cationic lipid (245). In embodiments, the cationic lipid is a cationic lipid (246). In embodiments, the cationic lipid is a cationic lipid (247). In embodiments, the cationic lipid is a cationic lipid (248). In embodiments, the cationic lipid is a cationic lipid (249). In embodiments, the cationic lipid is a cationic lipid (250). In embodiments, the cationic lipid is a cationic lipid (251). In embodiments, the cationic lipid is a cationic lipid (252). In embodiments, the cationic lipid is a cationic lipid (253). In embodiments, the cationic lipid is a cationic lipid (254). In embodiments, the cationic lipid is a cationic lipid (255). In an embodiment, the cationic lipid is a cationic lipid (256). In an embodiment, the cationic lipid is a cationic lipid (257). In an embodiment, the cationic lipid is a cationic lipid (258). In an embodiment, the cationic lipid is a cationic lipid (259). In an embodiment, the cationic lipid is a cationic lipid (260). In an embodiment, the cationic lipid is a cationic lipid (261).

[0431] Exemplary cationic lipids include cationic lipids according to formula (IIIu), such as cationic lipids (262)-(285) (Table V):

[0432] wherein each R is independently as described herein, including the exemplary groups of Table V.

[0433] Table V. Cationic lipids of formula (IIIu)

[0434]

[0435]

[0436] In embodiments, the cationic lipid is a cationic lipid (262). In embodiments, the cationic lipid is a cationic lipid (263). In embodiments, the cationic lipid is a cationic lipid (264). In embodiments, the cationic lipid is a cationic lipid (265). In embodiments, the cationic lipid is a cationic lipid (266). In embodiments, the cationic lipid is a cationic lipid (267). In embodiments, the cationic lipid is a cationic lipid (268). In embodiments, the cationic lipid is a cationic lipid (269). In embodiments, the cationic lipid is a cationic lipid (270). In embodiments, the cationic lipid is a cationic lipid (271). In embodiments, the cationic lipid is a cationic lipid (272). In embodiments, the cationic lipid is a cationic lipid (273). In embodiments, the cationic lipid is a cationic lipid (274). In embodiments, the cationic lipid is a cationic lipid (275). In embodiments, the cationic lipid is a cationic lipid (276). In embodiments, the cationic lipid is a cationic lipid (277). In embodiments, the cationic lipid is a cationic lipid (278). In embodiments, the cationic lipid is a cationic lipid (279). In an embodiment, the cationic lipid is a cationic lipid (280). In an embodiment, the cationic lipid is a cationic lipid (281). In an embodiment, the cationic lipid is a cationic lipid (282). In an embodiment, the cationic lipid is a cationic lipid (283). In an embodiment, the cationic lipid is a cationic lipid (284). In an embodiment, the cationic lipid is a cationic lipid (285).

[0437] Exemplary cationic lipids include cationic lipids according to formula (IIIv), such as cationic lipids (286)-(309) (Table W):

[0438] wherein each R is independently as described herein, including the exemplary groups in Table W.

[0439] Table W. Cationic lipids of formula (IIIv)

[0440]

[0441]

[0442] In embodiments, the cationic lipid is a cationic lipid (286). In embodiments, the cationic lipid is a cationic lipid (287). In embodiments, the cationic lipid is a cationic lipid (288). In embodiments, the cationic lipid is a cationic lipid (289). In embodiments, the cationic lipid is a cationic lipid (290). In embodiments, the cationic lipid is a cationic lipid (291). In embodiments, the cationic lipid is a cationic lipid (292). In embodiments, the cationic lipid is a cationic lipid (293). In embodiments, the cationic lipid is a cationic lipid (294). In embodiments, the cationic lipid is a cationic lipid (295). In embodiments, the cationic lipid is a cationic lipid (296). In embodiments, the cationic lipid is a cationic lipid (297). In embodiments, the cationic lipid is a cationic lipid (298). In embodiments, the cationic lipid is a cationic lipid (299). In embodiments, the cationic lipid is a cationic lipid (300). In embodiments, the cationic lipid is a cationic lipid (301). In embodiments, the cationic lipid is a cationic lipid (302). In embodiments, the cationic lipid is a cationic lipid (303). In an embodiment, the cationic lipid is a cationic lipid (304). In an embodiment, the cationic lipid is a cationic lipid (305). In an embodiment, the cationic lipid is a cationic lipid (306). In an embodiment, the cationic lipid is a cationic lipid (307). In an embodiment, the cationic lipid is a cationic lipid (308). In an embodiment, the cationic lipid is a cationic lipid (309).

[0443] Exemplary cationic lipids include cationic lipids according to formula (IIIw), such as cationic lipids (310)-(333) (Table X):

[0444] wherein each R is independently as described herein, including the exemplary groups of Table X.

[0445] Table X. Cationic lipids of formula (IIIw)

[0446]

[0447]

[0448] In embodiments, the cationic lipid is a cationic lipid (310). In embodiments, the cationic lipid is a cationic lipid (311). In embodiments, the cationic lipid is a cationic lipid (312). In embodiments, the cationic lipid is a cationic lipid (313). In embodiments, the cationic lipid is a cationic lipid (314). In embodiments, the cationic lipid is a cationic lipid (315). In embodiments, the cationic lipid is a cationic lipid (316). In embodiments, the cationic lipid is a cationic lipid (317). In embodiments, the cationic lipid is a cationic lipid (318). In embodiments, the cationic lipid is a cationic lipid (319). In embodiments, the cationic lipid is a cationic lipid (320). In embodiments, the cationic lipid is a cationic lipid (321). In embodiments, the cationic lipid is a cationic lipid (322). In embodiments, the cationic lipid is a cationic lipid (323). In embodiments, the cationic lipid is a cationic lipid (324). In embodiments, the cationic lipid is a cationic lipid (325). In embodiments, the cationic lipid is a cationic lipid (326). In embodiments, the cationic lipid is a cationic lipid (327). In an embodiment, the cationic lipid is a cationic lipid (328). In an embodiment, the cationic lipid is a cationic lipid (329). In an embodiment, the cationic lipid is a cationic lipid (330). In an embodiment, the cationic lipid is a cationic lipid (331). In an embodiment, the cationic lipid is a cationic lipid (332). In an embodiment, the cationic lipid is a cationic lipid (333).

[0449] Exemplary cationic lipids include cationic lipids according to formula (IIIx), such as cationic lipids (334)-(357) (Table Y):

[0450] wherein each R is independently as described herein, including the exemplary groups of Table Y.

[0451] Table Y. Cationic lipids of formula (IIIx)

[0452]

[0453]

[0454] In embodiments, the cationic lipid is a cationic lipid (334). In embodiments, the cationic lipid is a cationic lipid (335). In embodiments, the cationic lipid is a cationic lipid (336). In embodiments, the cationic lipid is a cationic lipid (337). In embodiments, the cationic lipid is a cationic lipid (338). In embodiments, the cationic lipid is a cationic lipid (339). In embodiments, the cationic lipid is a cationic lipid (340). In embodiments, the cationic lipid is a cationic lipid (341). In embodiments, the cationic lipid is a cationic lipid (342). In embodiments, the cationic lipid is a cationic lipid (343). In embodiments, the cationic lipid is a cationic lipid (344). In embodiments, the cationic lipid is a cationic lipid (345). In embodiments, the cationic lipid is a cationic lipid (346). In embodiments, the cationic lipid is a cationic lipid (347). In embodiments, the cationic lipid is a cationic lipid (348). In embodiments, the cationic lipid is a cationic lipid (349). In embodiments, the cationic lipid is a cationic lipid (350). In embodiments, the cationic lipid is a cationic lipid (351). In an embodiment, the cationic lipid is a cationic lipid (352). In an embodiment, the cationic lipid is a cationic lipid (353). In an embodiment, the cationic lipid is a cationic lipid (354). In an embodiment, the cationic lipid is a cationic lipid (355). In an embodiment, the cationic lipid is a cationic lipid (356). In an embodiment, the cationic lipid is a cationic lipid (357).

[0455] Exemplary cationic lipids include cationic lipids according to formula (IIIy), such as cationic lipids (358)-(381) (Table Z):

[0456] wherein each R is independently as described herein, including the exemplary groups in Table Z.

[0457] Table Z. Cationic lipids of formula (IIIy)

[0458]

[0459]

[0460] In embodiments, the cationic lipid is a cationic lipid (358). In embodiments, the cationic lipid is a cationic lipid (359). In embodiments, the cationic lipid is a cationic lipid (360). In embodiments, the cationic lipid is a cationic lipid (361). In embodiments, the cationic lipid is a cationic lipid (362). In embodiments, the cationic lipid is a cationic lipid (363). In embodiments, the cationic lipid is a cationic lipid (364). In embodiments, the cationic lipid is a cationic lipid (365). In embodiments, the cationic lipid is a cationic lipid (366). In embodiments, the cationic lipid is a cationic lipid (367). In embodiments, the cationic lipid is a cationic lipid (368). In embodiments, the cationic lipid is a cationic lipid (369). In embodiments, the cationic lipid is a cationic lipid (370). In embodiments, the cationic lipid is a cationic lipid (371). In embodiments, the cationic lipid is a cationic lipid (372). In embodiments, the cationic lipid is a cationic lipid (373). In embodiments, the cationic lipid is a cationic lipid (374). In embodiments, the cationic lipid is a cationic lipid (375). In an embodiment, the cationic lipid is a cationic lipid (376). In an embodiment, the cationic lipid is a cationic lipid (377). In an embodiment, the cationic lipid is a cationic lipid (378). In an embodiment, the cationic lipid is a cationic lipid (379). In an embodiment, the cationic lipid is a cationic lipid (380). In an embodiment, the cationic lipid is a cationic lipid (381).

[0461] Exemplary cationic lipids include cationic lipids according to formula (IIIz), such as cationic lipids (382)-(405) (Table AA):

[0462] wherein each R is independently as described herein, including the example groups of Table AA.

[0463] Table AA. Cationic lipids of formula (IIIz)

[0464]

[0465]

[0466] In embodiments, the cationic lipid is a cationic lipid (382). In embodiments, the cationic lipid is a cationic lipid (383). In embodiments, the cationic lipid is a cationic lipid (384). In embodiments, the cationic lipid is a cationic lipid (385). In embodiments, the cationic lipid is a cationic lipid (386). In embodiments, the cationic lipid is a cationic lipid (387). In embodiments, the cationic lipid is a cationic lipid (388). In embodiments, the cationic lipid is a cationic lipid (389). In embodiments, the cationic lipid is a cationic lipid (390). In embodiments, the cationic lipid is a cationic lipid (391). In embodiments, the cationic lipid is a cationic lipid (392). In embodiments, the cationic lipid is a cationic lipid (393). In embodiments, the cationic lipid is a cationic lipid (394). In embodiments, the cationic lipid is a cationic lipid (395). In embodiments, the cationic lipid is a cationic lipid (396). In embodiments, the cationic lipid is a cationic lipid (397). In embodiments, the cationic lipid is a cationic lipid (398). In embodiments, the cationic lipid is a cationic lipid (399). In an embodiment, the cationic lipid is a cationic lipid (400). In an embodiment, the cationic lipid is a cationic lipid (401). In an embodiment, the cationic lipid is a cationic lipid (402). In an embodiment, the cationic lipid is a cationic lipid (403). In an embodiment, the cationic lipid is a cationic lipid (404). In an embodiment, the cationic lipid is a cationic lipid (405).

[0467] Exemplary cationic lipids include cationic lipids according to formula (IIIaa), such as cationic lipids (406)-(429) (Table AB):

[0468] wherein each R is independently as described herein, including the example groups of Table AB.

[0469] Table AB. Cationic lipids of formula (IIIaa)

[0470]

[0471]

[0472] In embodiments, the cationic lipid is a cationic lipid (406). In embodiments, the cationic lipid is a cationic lipid (407). In embodiments, the cationic lipid is a cationic lipid (408). In embodiments, the cationic lipid is a cationic lipid (409). In embodiments, the cationic lipid is a cationic lipid (410). In embodiments, the cationic lipid is a cationic lipid (411). In embodiments, the cationic lipid is a cationic lipid (412). In embodiments, the cationic lipid is a cationic lipid (413). In embodiments, the cationic lipid is a cationic lipid (414). In embodiments, the cationic lipid is a cationic lipid (415). In embodiments, the cationic lipid is a cationic lipid (416). In embodiments, the cationic lipid is a cationic lipid (417). In embodiments, the cationic lipid is a cationic lipid (418). In embodiments, the cationic lipid is a cationic lipid (419). In embodiments, the cationic lipid is a cationic lipid (420). In embodiments, the cationic lipid is a cationic lipid (421). In embodiments, the cationic lipid is a cationic lipid (422). In embodiments, the cationic lipid is a cationic lipid (423). In an embodiment, the cationic lipid is a cationic lipid (424). In an embodiment, the cationic lipid is a cationic lipid (425). In an embodiment, the cationic lipid is a cationic lipid (426). In an embodiment, the cationic lipid is a cationic lipid (427). In an embodiment, the cationic lipid is a cationic lipid (428). In an embodiment, the cationic lipid is a cationic lipid (429).

[0473] Exemplary cationic lipids include cationic lipids according to formula (IIIaa), such as cationic lipids (430)-(453) (Table AC):

[0474] wherein each R is independently as described herein, including the example groups in Tables AC.

[0475] Table AC. Cationic lipids of formula (IIIab)

[0476]

[0477]

[0478] In embodiments, the cationic lipid is a cationic lipid (430). In embodiments, the cationic lipid is a cationic lipid (431). In embodiments, the cationic lipid is a cationic lipid (432). In embodiments, the cationic lipid is a cationic lipid (433). In embodiments, the cationic lipid is a cationic lipid (434). In embodiments, the cationic lipid is a cationic lipid (435). In embodiments, the cationic lipid is a cationic lipid (436). In embodiments, the cationic lipid is a cationic lipid (437). In embodiments, the cationic lipid is a cationic lipid (438). In embodiments, the cationic lipid is a cationic lipid (439). In embodiments, the cationic lipid is a cationic lipid (440). In embodiments, the cationic lipid is a cationic lipid (441). In embodiments, the cationic lipid is a cationic lipid (442). In embodiments, the cationic lipid is a cationic lipid (443). In embodiments, the cationic lipid is a cationic lipid (444). In embodiments, the cationic lipid is a cationic lipid (445). In embodiments, the cationic lipid is a cationic lipid (446). In embodiments, the cationic lipid is a cationic lipid (447). In an embodiment, the cationic lipid is a cationic lipid (448). In an embodiment, the cationic lipid is a cationic lipid (449). In an embodiment, the cationic lipid is a cationic lipid (450). In an embodiment, the cationic lipid is a cationic lipid (451). In an embodiment, the cationic lipid is a cationic lipid (452). In an embodiment, the cationic lipid is a cationic lipid (453).

[0479] Exemplary cationic lipids include cationic lipids (454)-(462) of Tables AD.

[0480] Table AD. Exemplary cationic lipids

[0481]

[0482]

[0483] In an embodiment, the cationic lipid is a cationic lipid (454). In an embodiment, the cationic lipid is a cationic lipid (455). In an embodiment, the cationic lipid is a cationic lipid (456). In an embodiment, the cationic lipid is a cationic lipid (457). In an embodiment, the cationic lipid is a cationic lipid (458). In an embodiment, the cationic lipid is a cationic lipid (459). In an embodiment, the cationic lipid is a cationic lipid (460). In an embodiment, the cationic lipid is a cationic lipid (461). In an embodiment, the cationic lipid is a cationic lipid (462).

[0484] Synthesis of cationic lipids

[0485] The cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) can be prepared according to methods known in the art. Additional exemplary methods are provided in the Examples described herein.

[0486] Scheme 1 provides an exemplary synthesis of the cationic lipids described herein.

[0487] Scheme 1. Exemplary synthesis of cationic lipids

[0488]

[0489] In Scheme 1, a carbohydrate such as Compound A is combined with a cationic electrophile (e.g., Compound B, 3-(dimethylamino)propionic acid or a salt thereof) to provide Compound C. The compound can be treated with various electrophiles to provide a cationic lipid as described herein (e.g., a cationic lipid of Formula (I) and (II)). For example, Compound C can be treated with an aliphatic electrophile (e.g., an alkyl halide or alkenyl halide) such as Compound D to provide a product cationic lipid E. Alternatively, Compound C can be treated with an acylating agent (e.g., an acyl halide) such as Compound F to provide a product cationic lipid G.

[0490] Scheme 2 provides an exemplary synthesis using D-ribose and an acylating agent.

[0491] Scheme 2. Exemplary Synthesis of Ribocic Lipids

[0492]

[0493] In Scheme 2, ribose (Compound A1) can be combined with 3-(dimethylamino)propionic acid (Compound B) using iodine and tri(p-tolyl)phosphine in dichloromethane to give Compound C1. Treatment of Compound C1 with an excess of acyl halide (Compound F1) under basic conditions can give the acylated cationic lipid G1.

[0494] Nucleic Acids

[0495] Compositions useful for delivering nucleic acids can be prepared using the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II), or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b), and (22)-(462)).

[0496] Nucleic acid synthesis

[0497] 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). In short, IVT is generally performed using a linear or circular DNA template comprising 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, a mutant T7 or SP6 RNA polymerase), DNAse I, a pyrophosphatase and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0498] In some embodiments, in order to prepare mRNA according to the present invention, the DNA template is transcribed in vitro. Suitable DNA templates generally have a promoter for in vitro transcription, such as T3, T7, mutated T7 or SP6 promoters, followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0499] Can use standard method to measure according to the required mRNA sequence of the present invention and mix it in the DNA template.For example, starting from required amino acid sequence (for example enzyme sequence), carry out virtual reverse translation based on degenerate genetic code.Then can use optimization algorithm to select suitable codon.Usually, can optimize G / C content on the one hand, to realize the highest possible G / C content, on the other hand, consider the frequency of tRNA according to codon use as far as possible.Can for example set up and display optimized RNA sequence by suitable display device, and compare it with original (wild type) sequence.Can also analyze secondary structure to calculate respectively stable and destabilizing characteristic or RNA region.

[0500] As mentioned above, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. The DNA can be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, precondensed DNA, polymerase chain reaction (PCR) product, vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA, or derivatives of these groups. The RNA can be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SLRNA 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 (RNaseP), Y RNA, telomerase RNA component (TERC), splicing leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), microRNA (miRNA), RNA interacting with piwi (piRNA), small interfering RNA (siRNA), transactional 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 encoding a protein.

[0501] Synthesis of mRNA

[0502] The mRNA according to the present invention can be synthesized according to any of a variety of known methods. For example, the mRNA according to the present invention can be synthesized by in vitro transcription (IVT). In short, IVT is generally performed using a linear or circular DNA template comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase and / or RNAse inhibitors. The exact conditions will vary depending on the specific application. The exact conditions will vary depending on the specific application. According to several embodiments, the presence of these reagents is undesirable in the final product and can therefore be referred to as an impurity, and a preparation containing one or more of these impurities can be referred to as an impure preparation. In some embodiments, in vitro transcription occurs in a single batch.

[0503] In some embodiments, in order to prepare mRNA according to the present invention, the DNA template is transcribed in vitro. Suitable DNA templates generally have 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.

[0504] Can use standard method to measure according to the required mRNA sequence of the present invention and mix it in the DNA template.For example, starting from required amino acid sequence (for example enzyme sequence), carry out virtual reverse translation based on degenerate genetic code.Then can use optimization algorithm to select suitable codon.Usually, can optimize G / C content on the one hand, to realize the highest possible G / C content, on the other hand, consider the frequency of tRNA according to codon use as far as possible.Can for example set up and display optimized RNA sequence by suitable display device, and compare it with original (wild type) sequence.Can also analyze secondary structure to calculate respectively stable and destabilizing characteristic or RNA region.

[0505] Modified mRNA

[0506] In some embodiments, the mRNA according to the present invention can be synthesized as unmodified mRNA or modified mRNA. The modified mRNA includes nucleotide modifications in the RNA. Therefore, the modified mRNA according to the present invention can include nucleotide modifications, for example, 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-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyladenine, N6-methyladenine, N6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil, uracil, 5-uracil, dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate (v), 1-methylpseudouracil, quercetin, β-D-mannosylquercetin, whibutoxin and pyrophosphamide, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, the disclosures of which are incorporated by reference in their entirety.

[0507] In some embodiments, mRNA may include RNA backbone modifications. Generally, backbone modifications are modifications in which the phosphates of the nucleotide backbones contained in the RNA are chemically modified. Exemplary backbone modifications generally include, but are not limited to, modifications selected from the group consisting of: methylphosphonates, methylphosphonamides, phosphoramidates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphates)), boric acid phosphates, positively charged guanidinium groups, etc., which means replacing phosphodiester bonds with other anions, cations, or neutral groups.

[0508] In some embodiments, mRNA may include sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides it includes, including but not limited to sugar modifications selected from the group consisting of: 4'-thioribonucleotides (see, e.g., U.S. Patent Application Publication No. US 2016 / 0031928, incorporated herein by reference), 2'-deoxy-2'-fluoro oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy 2'-deamine oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleosides Acid, 2'-deoxy-2'-C-alkyl oligoribonucleotide (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotide and its isomers (2'-arabinocytidine 5'-triphosphate, 2'-arabinouridine 5'-triphosphate) or azido triphosphate (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0509] In some embodiments, mRNA may include modification of the base of the nucleotide (base modification). Modified nucleotides comprising base modification are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside, 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate , 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole nucleoside 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 xanthine riboside 5'-triphosphate.

[0510] Typically, mRNA synthesis involves the addition of a "cap" at the N(5') terminus and a "tail" at the C(3') terminus. The presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0511] Thus, in some embodiments, the mRNA includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylate transferase to produce a 5'5'5 triphosphate bond; 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.

[0512] In some embodiments, mRNA includes 3' poly (A) tail structure. The poly A tail at the 3' end of mRNA generally includes 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, mRNA includes 3' poly (C) tail structure. The suitable poly C tail on the 3' end of mRNA generally includes 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 the poly A tail, or the poly A tail can be substituted.

[0513] In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA, such as an iron response element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides.

[0514] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization stability of the mRNA in the cell, or one or more miRNA binding sites. In some embodiments, the length of the 3' untranslated region can be between 50 and 500 nucleotides or longer.

[0515] Cap structure

[0516] In some embodiments, the mRNA includes a 5' cap structure. The 5' cap is generally added as follows: first, an RNA terminal phosphatase removes a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylate transferase to produce a 5'5'5 triphosphate bond; 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.

[0517] The naturally occurring cap structure consists of 7-methylguanosine attached to the 5'-end of the first transcribed nucleotide via a triphosphate bridge, resulting in m 7A dinucleotide cap of G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added to the nucleus of the cell and is catalyzed by the enzyme guanylyltransferase. The cap is added to the 5' end of the RNA immediately after transcription begins. The terminal nucleoside is usually guanosine and is in the opposite direction to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0518] A common cap for mRNA produced by in vitro transcription is m 7 G(5')ppp(5')G has been used as a dinucleotide cap during in vitro transcription with T7 or SP6 RNA polymerase to obtain RNA with a cap structure at its 5'-end. The main method for synthesizing caPPEd mRNA in vitro is to use preformed m 7 G(5')ppp(5')G("m 7 GpppG”) as a transcription initiator.

[0519] To date, the common form of synthetic dinucleotide caps used in in vitro translation experiments is the anti-reverse cap analog ("ARCA") or modified ARCA, which is typically a modified cap analog in which the 2' or 3' OH group is replaced by -OCH3.

[0520] Other cap analogs include, but are not limited to, chemical structures selected from the group consisting of: m 7 GpppG、m 7 GpppA,m 7 GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (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 anti-reverse 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 its tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel 'anti-reverse' cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).

[0521] In some embodiments, a suitable cap is a 7-methylguanylate ("m-guanylate") attached via a triphosphate bridge to the 5'-end of the first transcribed nucleotide. 7 G”), thus obtaining m 7 G(5')ppp(5')N, wherein N is any nucleoside. 7 The preferred embodiment of G cap is m 7 G(5')ppp(5')G.

[0522] In some embodiments, the cap is a Cap0 structure. The Cap0 structure lacks the 2'-O-methyl residues attached to the ribose of bases 1 and 2. In some embodiments, the cap is a Cap1 structure. The Cap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. The 2'-O-methyl residues of the Cap2 structure are attached to both bases 2 and 3.

[0523] Multiple m 7 G cap analogs are known in the art, many of which are commercially available. These cap analogs include the m 7 GpppG, and ARCA 3'-OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for embodiments of the present invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E. et al., RNA, 13: 1745-1755 (2007)), and cap analogs described in U.S. Pat. Nos. 8,093,367 and 8,304,529 (including biotinylated cap analogs), which are incorporated herein by reference.

[0524] Tail structure

[0525] Generally, the presence of the "tail" is used to protect mRNA from exonuclease degradation. It is believed that the poly A tail can stabilize natural messengers and synthesize sense RNA. Therefore, in certain embodiments, a long poly A tail can be added to the mRNA molecule to make the RNA more stable. A variety of techniques recognized in the art can be used to add the poly A tail. For example, a long poly A tail can be added to synthetic or in vitro transcribed RNA (Yokoe et al., Nature Biotechnology.1996; 14: 1252-1256) using poly A polymerase. The transcription vector can also encode a long poly A tail. In addition, the poly A tail can be added by direct transcription from the PCR product. Poly A can also be connected to the 3' end of the sense RNA with RNA ligase (see, for example, Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0526] In some embodiments, mRNA includes 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, at least 500 nucleotides. In some embodiments, the poly A tail on the 3' end of the mRNA typically includes 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, mRNA includes a 3' poly (C) tail structure. Suitable poly-C tails on the 3' end of the mRNA generally include 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 the poly-A tail, or it can replace the poly-A tail.

[0527] In some embodiments, the length of the poly A tail or poly C tail is adjusted to control the stability of the modified sense mRNA molecule of the present invention, thereby controlling the transcription of the protein. For example, since the length of the poly A tail can affect the half-life of the sense mRNA molecule, the length of the poly A tail can be adjusted to change the resistance level of the mRNA to nucleases, thereby controlling the time course of polynucleotide expression and / or polypeptide production in the target cell.

[0528] 5' and 3' untranslated regions

[0529] In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA, such as an iron response element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides.

[0530] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization stability of the mRNA in the cell, or one or more miRNA binding sites. In some embodiments, the length of the 3' untranslated region can be between 50 and 500 nucleotides or longer.

[0531] Exemplary 3' and / or 5'UTR sequences can be derived from stable mRNA molecules (e.g., globulin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) to increase the stability of sense mRNA molecules. For example, the 5'UTR sequence can include a partial sequence or a fragment of the CMV immediate early 1 (IE1) gene to improve nuclease resistance and / or improve the half-life of the polynucleotide. It is also contemplated that the 3' end or untranslated region of the polynucleotide (e.g., mRNA) includes a sequence encoding human growth hormone (hGH) or a fragment thereof to further stabilize the polynucleotide. Typically, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to its unmodified counterpart, and include, for example, modifications made to improve the resistance of such polynucleotides to nuclease digestion in vivo.

[0532] Cationic lipid and nucleic acid pharmaceutical preparations

[0533] In certain embodiments, cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) and drugs and liposome compositions comprising such lipids can be used in formulations to facilitate delivery of encapsulated materials (e.g., one or more polynucleotides, such as mRNA) to one or more target cells and subsequent transfection. For example, in certain embodiments, cationic lipids described herein (and compositions comprising such lipids, such as liposome compositions) are characterized by causing 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, which provide advantages of such compounds over other similarly classified lipids.

[0534] According to the present invention, nucleic acids, such as mRNA encoding a protein as described herein (e.g., full length, fragment or portion of the protein) can be delivered via a delivery vector comprising a cationic lipid as described herein (e.g., a cationic lipid of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)).

[0535] As used herein, the terms "delivery vehicle," "transport vehicle," "nanoparticle," or grammatical equivalents are used interchangeably.

[0536] For example, the present invention provides compositions (e.g., pharmaceutical compositions) comprising a cationic lipid as described herein (e.g., a cationic lipid of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) and one or more polynucleotides. The composition (e.g., pharmaceutical composition) may also 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.

[0537] In certain embodiments, the composition exhibits enhanced (e.g., improved) ability to transfect one or more target cells. Therefore, a method for transfecting one or more target cells is also provided herein. Such methods generally include making one or more target cells and cationic lipids disclosed herein and / or pharmaceutical compositions (e.g., cationic lipids (e.g., cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) liposome formulations) such that the material encapsulated therein (e.g., one or more polynucleotides) transfects one or more target cells. As used herein, the term "transfection" or "transfection" refers to the intracellular introduction of one or more encapsulating materials (e.g., nucleic acids and / or polynucleotides) into cells, or preferably into target cells. The introduced polynucleotides can be stably or transiently maintained in target cells. The term "transfection efficiency" refers to the relative amount of such encapsulating materials (e.g., polynucleotides) taken up, introduced and / or expressed by target cells subjected to transfection. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by target cells after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby increasing the likelihood of delivering an appropriate dose of encapsulated material (e.g., one or more polynucleotides) to a pathological site and subsequently expressing the encapsulated material, while minimizing potential systemic adverse reactions or toxicity associated with the compound or its encapsulated contents.

[0538] After transfection of one or more target cells by, for example, polynucleotides encapsulated in one or more lipid nanoparticles comprising a pharmaceutical composition or liposome composition disclosed herein, the production of products (e.g., polypeptides or proteins) encoded by such polynucleotides can be preferably stimulated, and the ability of such target cells to express polynucleotides and produce, for example, polypeptides or proteins of interest can be enhanced. For example, transfection of target cells by one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of proteins or enzymes encoded by such mRNAs.

[0539] In addition, delivery vectors as described herein (e.g., liposome delivery vectors) can be prepared to preferentially distribute to other target tissues, cells or organs, such as heart, lung, kidney, spleen. In embodiments, lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more compounds as described herein or pharmaceutical compositions and liposome compositions can be delivered to and / or transfected target cells or tissues. In some embodiments, the polynucleotides (e.g., mRNA) encapsulated can be expressed by target cells and produced (and in some cases secreted) functional polypeptide products by target cells, thereby giving, for example, target cells or tissues useful characteristics. Such encapsulated polynucleotides (e.g., mRNA) can encode, for example, hormones, enzymes, receptors, polypeptides, peptides or other proteins of interest.

[0540] Liposome delivery vehicle

[0541] In some embodiments, the composition is a suitable delivery vehicle. In embodiments, the composition is a liposomal delivery vehicle, such as a lipid nanoparticle.

[0542] The terms "liposomal delivery vehicle" and "liposomal composition" are used interchangeably.

[0543] Enriching liposome compositions with one or more cationic lipids disclosed herein can be used as a method of improving (e.g., reducing) toxicity or otherwise imparting one or more desired properties to such enriched liposome compositions (e.g., improving the delivery of encapsulated polynucleotides to one or more target cells and / or reducing the in vivo toxicity of liposome compositions). Therefore, pharmaceutical compositions, particularly liposome compositions, comprising one or more cationic lipids disclosed herein are also contemplated.

[0544] Thus, in certain embodiments, the compounds described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) 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 permeating or fusing with the lipid membranes of such target cells).

[0545] As used herein, liposome delivery vectors, such as lipid nanoparticles, are generally characterized as microscopic vesicles with internal water space, which are isolated from external media by one or more double-layered films. The double-layer membrane of liposomes is generally formed by amphipathic molecules, such as synthetic or natural lipids (Lasic, Trends Biotechnol., 16: 307-321, 1998) comprising spatially separated hydrophilic and hydrophobic domains. The double-layer membrane of liposomes can also be formed by amphipathic polymers and surfactants (e.g., polymer vesicles, lipoids, etc.). In the context of the present invention, liposome delivery vectors are generally used for transporting desired mRNA to target cells or tissues.

[0546] In certain embodiments, such compositions (eg, liposome compositions) load or otherwise encapsulate a material, such as one or more biologically active polynucleotides (eg, mRNA).

[0547] In embodiments, compositions (e.g., pharmaceutical compositions) include mRNAs encoding proteins encapsulated in liposomes. In embodiments, liposomes include one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, and at least one cationic lipid is a cationic lipid as described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)). In embodiments, compositions include mRNAs encoding proteins (e.g., any proteins described herein). In embodiments, compositions include mRNAs encoding cystic fibrosis transmembrane conductance regulator (CFTR) proteins. In embodiments, compositions include mRNAs encoding ornithine transcarbamylase (OTC) proteins. In embodiments, mRNAs encode antigens from infectious agents.

[0548] In an embodiment, the composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated in a liposome, wherein the liposome comprises any cationic lipid as described herein (e.g., a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)).

[0549] In embodiments, nucleic acid is an mRNA encoding a peptide or polypeptide. In embodiments, mRNA encoding is used to deliver to or treat a subject's lung or pneumonocyte peptide or polypeptide (e.g., mRNA encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein). In embodiments, mRNA encoding is used to deliver to or treat a subject's liver or hepatocyte peptide or polypeptide (e.g., mRNA encoding ornithine carbamoyltransferase (OTC) protein). In embodiments, mRNA encoding is used to deliver to or treat a subject's liver or hepatocyte peptide or polypeptide.

[0550] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) may have a net positive charge.

[0551] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) may have a net negative charge.

[0552] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) may have a net neutral charge.

[0553] In an embodiment, the lipid nanoparticles encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., a cationic lipid of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)).

[0554] For example, the amount of a cationic lipid as described herein (e.g., a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) in a composition can be described as the total dry weight of all lipids of the composition (e.g., the total dry weight of all lipids present in a liposome composition).

[0555] In embodiments of the pharmaceutical compositions described herein, a cationic lipid as described herein (e.g., a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) 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 total dry weight of all lipids present in the composition (e.g., a liposome composition).

[0556] In an embodiment, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is present in an amount of about 1 wt % to about 30 wt %, about 1 wt % to about 20 wt %, about 1 wt % to about 15 wt %, about 1 wt % to about 10 wt % or about 5 wt % to about 25 wt % of the total dry weight of all lipids present in the composition (e.g., a liposome composition). In embodiments, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is present in an amount of about 0.5 wt % to about 5 wt %, about 1 wt % to about 10 wt %, about 5 wt % to about 20 wt % or about 10 wt % to about 20 wt % of the total molar amount of all lipids present in a composition, such as a liposome delivery vehicle.

[0557] In an embodiment, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is present in an amount of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% by weight of the total dry weight of the total lipids in the composition (e.g., a liposome composition).

[0558] In an embodiment, the amount of the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is present in an amount that does not exceed about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% by weight of the total dry weight of the total lipids in the composition (e.g., a liposome composition).

[0559] In embodiments, the composition (e.g., a liposomal delivery vehicle, such as a lipid nanoparticle) comprises about 0.1% to about 20% by weight (e.g., about 0.1% to about 15% by weight) of a cationic lipid described herein (e.g., a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)). In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle, such as a lipid nanoparticle) comprises about 0.5%, about 1%, about 3%, about 5% or about 10% by weight of a cationic lipid described herein (e.g., a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)). In embodiments, the delivery vehicle (e.g., a liposome 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 as described herein (e.g., a cationic lipid of Formula (I'), (I), (II), or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b), and (22)-(462). In embodiments, this percentage results in an improved beneficial effect (e.g., improved delivery to a targeted tissue such as the liver or lung).

[0560] The amount of a cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) in a composition can also be described as a percentage ("mole %") of the total molar amount of total lipids in the composition (e.g., the total molar amount of all lipids present in the liposome delivery vehicle).

[0561] In embodiments of the pharmaceutical compositions described herein, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) 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 total molar amount of all lipids present in the composition, such as a liposome delivery vehicle.

[0562] In embodiments, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is 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 total molar amount of all lipids present in a composition, such as a liposome delivery vehicle. In embodiments, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) 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 total molar amount of all lipids present in a composition, such as a liposome delivery vehicle.

[0563] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) 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 amount of lipid in a composition (e.g., a liposome delivery vehicle).

[0564] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) may account for greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than about 1 mol%, or greater than about 5 mol% of the total lipid in the lipid nanoparticles.

[0565] In certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) may account for 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 in the composition (e.g., liposome delivery vehicle).

[0566] In embodiments, the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is present in an amount of at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol% or about 99 mol% of the total dry weight of total lipids in the composition (e.g., a liposome composition).

[0567] In an embodiment, the amount of the cationic lipid of Formula (I'), (I), (II) or (IIIa)-(IIIab) (e.g., any of the cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) is present in an amount that does not exceed 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 total dry weight of the total lipids in the composition (e.g., a liposome composition).

[0568] In embodiments, this percentage results in an improved beneficial effect (eg, improved delivery to a targeted tissue such as the liver or lung).

[0569] In embodiments, the composition further comprises one or more lipids (eg, one or more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids).

[0570] In certain embodiments, such pharmaceutical (e.g., liposome) compositions include one or more of PEG-modified lipids, non-cationic lipids, and cholesterol lipids. In embodiments, such pharmaceutical (e.g., liposome) compositions include: one or more PEG-modified lipids, one or more non-cationic lipids, and one or more cholesterol lipids. In embodiments, such pharmaceutical (e.g., liposome) compositions include: one or more PEG-modified lipids and one or more cholesterol lipids.

[0571] In an embodiment, the composition (e.g., lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids and pegylated lipids.

[0572] In an embodiment, the composition (e.g., lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)); one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids and pegylated lipids; and further comprises a cholesterol-based lipid.

[0573] In an embodiment, lipid nanoparticles encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)), and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, pegylated lipids and cholesterol-based lipids.

[0574] In embodiments of the lipid nanoparticles described herein, the lipid nanoparticles comprise one or more cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)), non-cationic lipids (e.g., DOPE), pegylated lipids (e.g., DMG-PEG2000) and cholesterol-based lipids (e.g., cholesterol).

[0575] According to various embodiments, the selection of the cationic lipid, non-cationic lipid and / or PEG-modified lipid comprising lipid nanoparticles and the relative molar ratio of such lipids to each other are based on the characteristics of the selected lipid, the properties of the expected target cells, the characteristics of the mRNA to be delivered. Other considerations include, for example, the size, charge, pH, pKa, fusogenicity and toxicity of the saturation of the alkyl chain and the selected lipid. Therefore, the molar ratio can be adjusted accordingly.

[0576] Other cationic lipids

[0577] In addition to any of the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)), the composition may include one or more additional cationic lipids.

[0578] In some embodiments, liposomes may include one or more other cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a variety of lipid substances having a net positive charge at a selected pH such as a physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

[0579] Other particularly suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication WO 2010 / 053572 (particularly C12-200 described in paragraph

[00225] ) and WO 2012 / 170930, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention employ lipid nanoparticles comprising additional cationic lipids, described in U.S. Provisional Patent Application 61 / 617,468, filed March 29, 2012 (incorporated herein by reference), such as (15Z, 18Z)-N, N-dimethyl-6-(9Z, 12Z)-octadecane-9,12-diene-1-yl)tetraconic-15,18-diene-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadec-9,12-dien-1-yl)tetracosa-4,15,18-trien-l-amine (HGT5001) and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadec-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

[0580] In some embodiments, the composition (e.g., liposome composition) comprises additional cationic lipids described in WO2013 / 063468, filed October 26, 2012, and U.S. Provisional Application No. 61 / 953,516, filed March 14, 2014, both of which are incorporated herein by reference.

[0581] In certain embodiments, the composition (e.g., liposome composition) comprises an additional cationic lipid cKK-E12 or (3,6-bis(4-(bis(2-hydroxylauryl)amino)butyl)piperazine-2,5-dione). The structure of cKK-E12 is shown below:

[0582]

[0583] In some embodiments, the additional cationic lipid can be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" (Feigner et al., Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or combined with neutral lipids (e.g., dioleoylphosphatidylethanolamine or "DOPE") or other cationic or non-cationic lipids into liposome transfer vehicles or lipid nanoparticles, and such liposomes can be used to enhance the delivery of nucleic acids to target cells. Other suitable cationic lipids include, for example, 5-carboxyspermpropylglycine dioctadecylamide or "DOGS"; 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propylammonium or "DOSPA" (Behr et al., Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP"; or 1,2-dioleoyl-3-trimethylammonium propane or "DOTAP".

[0584] Additional exemplary cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinoyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DLenDMA", "DODAC", N,N-distearyl-N,N-dimethyl bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholesterol-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane or "CLinDMA", 2-[5'-(cholesterol-5-ene-3-β-oxy)-3'-oxapentane]-1-[[[[[[[[[[[[[[[[[[[[[[ oxy)-3-dimethyl 1-1-(cis,cis-9',1-2'-octadecadienyloxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxyoxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleylcarbamoyl-3-dimethyl aminopropane or "DLincarbDAP", 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin--DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA", and 2-(2,2-di((9Z,12Z)-octadec-9,1 2-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanolamine (DLin-KC2-DMA) (see WO 2010 / 042877; Semple et al., Nature Biotech. 28:172-176 (2010)) or mixtures thereof. (Heyes, J. et al., J Controlled Release 107:276-287 (2005); Morrissey, DV. et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication WO 2005 / 121348 A1). In some embodiments, the one or more additional cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0585] In some embodiments, the one or more other cationic lipids may be selected from XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 ((6Z,9Z,28Z,31Z)-heptatriacont ... ,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012 / 170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011 / 068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61 / 617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61 / 617,468), amino alcohol lipids such as those disclosed in WO2010 / 053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. "Cationiclipid saturation influences intracellular delivery of encapsulated nucleicacids" J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, SC et al., "Rational Design of Cationic Lipids for siRNA Delivery" Nature Biotech. 2010, 28, 172-176), C12-200 (Love, KT et al., "Lipid-like materials for low-dose in vivo genesilencing” PNAS 2010,107,1864-1869).

[0586] In some embodiments, the percentage of total cationic lipids in a composition (e.g., a liposome composition) may be no more than 10%, no more than 20%, no more than 30%, no more than 40%, no more than 50%, no more than 60%, no more than 70%, no more than 80%, no more than 90%, or no more than 95% of the total lipids, as measured on a molar basis (mol %) or by weight (wt %).

[0587] In some embodiments, the percentage of total cationic lipids in a composition (e.g., a liposome composition) may 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 lipids, as measured on a molar basis (mol %) or by weight (wt %).

[0588] In some embodiments, the total cationic lipids constitute about 30-50% of the liposomes by weight (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%). In some embodiments, the cationic lipids constitute 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 lipids constitute about 30-50% of the liposomes by weight (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%). In some embodiments, the cationic lipids constitute about 30%, about 35%, about 40%, about 45%, or about 50% of the composition (e.g., liposome composition) by weight.

[0589] Non-cationic / helper lipids

[0590] Compositions (e.g., liposome compositions) can also include one or more non-cationic ("helper") lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid species that carry a net negative charge at a selected pH, such as a physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid dioleoylphosphatidylethanolamine (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof.

[0591] In an embodiment, the non-cationic or helper lipid is dioleoylphosphatidylethanolamine (DOPE).

[0592] In some embodiments, the non-cationic lipid is a neutral lipid, ie, a lipid that carries no net charge under the conditions under which the composition is formulated and / or administered.

[0593] In some embodiments, non-cationic lipids can exist in a mol 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, total non-cationic lipids can exist in a mol 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 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 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 non-cationic lipids in liposomes is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol% or no more than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in liposomes can be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol% or no more than about 40 mol%.

[0594] In some embodiments, non-cationic lipids can exist 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, total non-cationic lipids can exist 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 liposomes can be greater than about 5 wt %, greater than about 10 wt %, greater than about 20 wt %, greater than about 30 wt %, or greater than about 40 wt %. In some embodiments, the percentage of total non-cationic lipid in liposome can be greater than about 5 wt %, greater than about 10 wt %, greater than about 20 wt %, greater than about 30 wt % or greater than about 40 wt %. In some embodiments, the percentage of non-cationic lipid in liposome is no more than about 5 wt %, no more than about 10 wt %, no more than about 20 wt %, no more than about 30 wt % or no more than about 40 wt %. In some embodiments, the percentage of total non-cationic lipid in liposome can be no more than about 5 wt %, no more than about 10 wt %, no more than about 20 wt %, no more than about 30 wt % or no more than about 40 wt %.

[0595] Cholesterol-based lipids

[0596] 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-ethylcarboxamide cholesterol), 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. Pat. No. 5,744,335) or imidazole cholesterol ester (ICE), which has the following structure,

[0597]

[0598] In embodiments, the cholesterol-based lipid is cholesterol.

[0599] In some embodiments, lipids based on cholesterol can exist with a molar ratio (mol %) of about 1% to about 30% or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of lipids based on cholesterol in lipid nanoparticles can be greater than about 5 mol %, greater than about 10 mol %, greater than about 20 mol %, greater than about 30 mol % or greater than about 40 mol %. In some embodiments, the percentage of lipids based on cholesterol in lipid nanoparticles can be no more than about 5 mol %, no more than about 10 mol %, no more than about 20 mol %, no more than about 30 mol % or no more than about 40 mol %.

[0600] In some embodiments, cholesterol-based lipids can exist with a weight ratio (wt%) of about 1% to about 30% or about 5% to about 20% of the total lipids present in the liposome. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles can be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt% or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles can be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt% or no more than about 40 wt%.

[0601] PEGylated lipids

[0602] In some embodiments, a composition (eg, a liposomal composition) comprises one or more PEGylated lipids.

[0603] For example, the present invention also contemplates the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide) in combination with one or more cationic lipids and, in some embodiments, other lipids to form liposomes. In some embodiments, particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C8 PEG-2000 ceramides). 14 or C 18 ).

[0604] Contemplated PEG-modified lipids (also referred to herein as PEGylated lipids, which term is interchangeable with PEG-modified lipids) include, but are not limited to, 20In some embodiments, the lipid or PEGylated lipid modified by PEG is PEGylated cholesterol or PEG-2K. Adding such components can prevent complex aggregation, and can also provide a method for extending the circulation life and increasing the delivery of lipid-nucleic acid compositions to target cells (Klibanov et al., (1990) FEBS Letters, 268 (1): 235-237), or they can be selected to quickly exchange out of the preparation in vivo (see U.S. Patent No. 5,885,613).

[0605] In an embodiment, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000).

[0606] The PEG-modified phospholipids and derivatized lipids of the present invention can 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., a liposome composition).

[0607] The PEG-modified phospholipids and derivatized lipids of the present invention can 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 lipids present in the composition (e.g., a liposome composition).

[0608] Pharmaceutical preparations and therapeutic uses

[0609] The cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) can be used to prepare compositions (e.g., for constructing liposome compositions) that promote or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).

[0610] For example, when a liposome composition (e.g., lipid nanoparticle) contains 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 encapsulated materials (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) to the one or more target cells.

[0611] Similarly, in certain embodiments, the cationic lipids described herein (e.g., cationic lipids of Formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) can be used to prepare liposome carriers characterized by reduced in vivo toxicity. In certain embodiments, the 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.

[0612] Therefore, pharmaceutical preparations comprising cationic lipids described herein (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) and nucleic acids provided by the present invention can be used for various therapeutic purposes. In order to promote the in vivo delivery of nucleic acids, cationic lipids (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) and nucleic acids can be formulated in combination with one or more other drug carriers, targeting ligands or stabilizers. In some embodiments, cationic lipids (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) can be prepared by premixing lipid solutions. In other embodiments, compositions comprising cationic lipids (e.g., cationic lipids of formula (I'), (I), (II) or (IIIa)-(IIIab), such as cationic lipids (1a)-(21a), (1b)-(21b) and (22)-(462)) can be formulated into the lipid membrane of the nanoparticle using post-insertion techniques. Techniques for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0613] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation, or enteral administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection; and intrathecal, directly intraventricular, intravenous, intraperitoneal or intranasal. In a specific embodiment, intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments, administration causes nucleic acid delivery to muscle cells. In some embodiments, administration causes nucleic acid delivery to hepatocytes (i.e., liver cells). In embodiments, administration is intramuscular. In embodiments, administration is intravenous.

[0614] Alternatively or in addition, the pharmaceutical preparations of the present invention can be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical preparation directly into the target tissue, preferably in the form of a sustained release formulation. Depending on the tissue to be targeted, local delivery can be affected in various ways. Exemplary tissues in which the delivered mRNA can be delivered and / or expressed include, but are not limited to, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin and / or cerebrospinal fluid. In an embodiment, the tissue to be targeted is in the liver. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal or bronchial delivery); for example, the composition of the present invention can be injected into the site of injury, disease manifestation or pain; the composition can be provided in the form of a lozenge for oral, tracheal or esophageal application; it can be supplied to the stomach or intestine in the form of a liquid, tablet or capsule, or it can be supplied to the rectum or vagina in the form of a suppository; or it can even be delivered to the eye by using a cream, drops or even an injection.

[0615] In embodiments, administration is via pulmonary delivery. As used herein, pulmonary delivery refers to delivery to the lungs via, for example, the nasal cavity, trachea, bronchi, bronchioles and / or other pulmonary systems. In embodiments, compositions as described herein are formulated for atomization. In embodiments, the delivery vehicle can be an inhalable atomized composition. In embodiments, pulmonary delivery involves inhalation (e.g., for nasal, tracheal or bronchial delivery). In embodiments, compositions are atomized before inhalation.

[0616] 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, such as a human subject or a cell of a human subject or a cell processed and delivered to a human subject.

[0617] Therefore, in certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a full-length mRNA encoding a peptide or polypeptide, which is used to deliver to or treat a subject's lung or lung cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an ATP-binding cassette subfamily A member 3 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a dynamin axon intermediate chain 1 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a dynamin axon heavy chain 5 (DNAH5) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an alpha-1-antitrypsin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a forkhead box P3 (FOXP3) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding one or more surfactant proteins, such as one or more of surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.

[0618] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide for delivery to or treatment of a subject's liver or hepatocytes. Such peptides and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder, for which delivery to or treatment of the liver or hepatocytes with enriched full-length mRNA provides a therapeutic benefit.

[0619] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with urea cycle disorders. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding ornithine transcarbamylase (OTC) proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding argininosuccinate synthetase 1 proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding carbamoyl phosphate synthetase I proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding argininosuccinate lyase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding argininosuccinate lyase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding arginase proteins.

[0620] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with lysosomal storage diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding alpha-galactosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glucocerebrosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding isocyanate-2-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding iduronidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding N-acetyl-α-D-glucosaminidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding heparan N-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding galactosamine-6 ​​sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding beta-galactosidase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding lysosomal lipase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding transcription factor EB (TFEB).

[0621] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with glycogen storage diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding acid alpha-glucosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glucose-6-phosphatase (G6PC) proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding liver glycogen phosphorylase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding muscle phosphoglycerate mutant enzyme proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glycogen debranching enzymes.

[0622] In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins related to amino acid metabolism. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding phenylalanine hydroxylase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glutaryl-CoA dehydrogenase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding propionyl-CoA carboxylase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding oxalase alanine-glyoxylate aminotransferase.

[0623] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with lipid metabolism or fibrotic diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding mTOR inhibitors. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding ATPase phospholipid transport 8B1 (ATP8B1) proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding one or more NF-κB inhibitors, such as one or more of I-κBα, interferon-related developmental regulator 1 (IFRD1), and Sirtuin 1 (SIRT1). In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding PPAR-γ proteins or active variants.

[0624] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding methylmalonyl-CoA mutant enzyme proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding methylmalonyl-CoA epimerase proteins.

[0625] In certain embodiments, the invention provides a method for preparing a therapeutic composition with full-length mRNA, for which delivery to or treatment of the liver can provide a therapeutic beneficial effect. In certain embodiments, the invention provides a method for preparing a therapeutic composition with full-length mRNA encoding ATP7B protein (also known as Wilson disease protein). In certain embodiments, the invention provides a method for preparing a therapeutic composition with full-length mRNA encoding porphobilinogen deaminase. In certain embodiments, the invention provides a method for preparing a therapeutic composition with full-length mRNA encoding one or more clotting enzymes, such as factor VIII, factor IX, factor VII and factor X. In certain embodiments, the invention provides a method for preparing a therapeutic composition with full-length mRNA encoding human hemochromatosis (HFE) protein.

[0626] In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide for delivery to or treatment of a subject's cardiovascular system or cardiovascular cells. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a vascular endothelial growth factor A protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a relaxin protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a bone morphogenetic protein 9 protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a bone morphogenetic protein 2 receptor protein.

[0627] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, which is used to deliver to or treat a subject's muscle or muscle cell. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a dystrophin. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, which is used to deliver to or treat a subject's myocardium or myocardial cell. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein regulating one or both of potassium channels and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein regulating a Kv7.1 channel in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein regulating a Nav1.5 channel in muscle tissue or muscle cells.

[0628] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, which is used to deliver to or treat the nervous system or nervous system cells of a subject. For example, in certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a survival motor neuron 2 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a CLN3 protein.

[0629] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, which is used to deliver to or treat the blood or bone marrow or blood cells or bone marrow cells of a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a beta globin. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a Bruton's tyrosine kinase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding one or more coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X.

[0630] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide for delivery to or treatment of a subject's kidney or renal cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding type IV collagen α5 chain (COL4A5) protein.

[0631] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, the therapeutic composition is used to deliver to or treat an eye or eye cell of a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding retinal chitin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding retinal pigment epithelium-specific 65kDa (RPE65) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a centrosomal protein (CEP290) of 290kDa.

[0632] In an embodiment, the mRNA encodes an antigen from an infectious agent.

[0633] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, which is used to deliver a vaccine to a subject or a subject's cell or treat with a vaccine. For example, in certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an antigen from an infectious source such as a virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an antigen from an influenza virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antigen from a respiratory syncytial virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an antigen from a rabies virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having a full-length mRNA encoding an antigen from a rotavirus. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from hepatitis viruses, such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from human papillomavirus. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from herpes simplex viruses, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from human immunodeficiency viruses, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from human metapneumonitis viruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from human parainfluenza viruses, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from malaria viruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from Zika viruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antigens from Chikungunya viruses.

[0634] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens associated with a subject's cancer or antigens identified from a subject's cancer cells. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens determined from a subject's own cancer cells, i.e., providing personalized cancer vaccines. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens expressed from a mutant KRAS gene.

[0635] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies. In certain embodiments, the antibodies may be bispecific antibodies. In certain embodiments, the antibodies may be part of a fusion protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to OX40. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to VEGF. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to tissue necrosis factor alpha. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to CD3. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to CD19.

[0636] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding immunomodulators. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding interleukin 12. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding interleukin 23. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding interleukin 36γ. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding constitutively active variants of one or more stimulators of interferon genes (STING) proteins.

[0637] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding endonucleases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding RNA-guided DNA endonucleases proteins, such as Cas 9 proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding meganuclease proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding transcription activator-like effector nuclease proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding zinc finger nuclease proteins.

[0638] In embodiments, exemplary therapeutic uses result from the delivery of mRNA encoding secretory proteins. Thus, in embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding secretory proteins. In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding one or more secretory proteins listed in Table 1; thus, the compositions of the invention may comprise mRNA encoding proteins listed in Table 1 (or homologs thereof) and other components listed herein, and the methods of the invention may comprise preparing and / or administering compositions comprising mRNA encoding proteins listed in Table 1 (or homologs thereof) and other components listed herein.

[0639] Table 1. Secreted proteins

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697] In some embodiments, the compositions and methods of the present invention provide for the delivery of one or more mRNAs encoding one or more other exemplary proteins listed in Table 2; thus, the compositions of the present invention may include mRNAs encoding proteins listed in Table 2 (or homologs thereof) and other components listed herein, and the methods of the present invention may include preparing and / or administering compositions comprising mRNAs encoding proteins selected from the proteins listed in Table 2 (or homologs thereof) and other components listed herein.

[0698] Table 2. Other exemplary proteins

[0699]

[0700]

[0701] The Uniprot IDs listed in Tables 1 and 2 refer to human versions, and the listed proteins and their respective sequences are available from the Uniprot database. The sequences of the listed proteins are also generally applicable to a variety of animals, including various mammals and animals of veterinary or industrial interest. Therefore, in some embodiments, the compositions and methods of the present invention provide for the delivery of one or more mRNAs encoding one or more proteins selected from mammalian homologs or homologs of veterinary or industrial interest animals of the secreted proteins listed in Tables 1 and 2; therefore, the compositions of the present invention may comprise mRNAs and other components listed herein, encoding proteins selected from mammalian homologs or homologs of veterinary or industrial interest animals of the proteins listed in Tables 1 and 2; and the methods of the present invention may include preparing and / or administering compositions comprising mRNAs and other components listed herein, encoding proteins of mammalian homologs or homologs of veterinary or industrial interest animals of the proteins listed in Tables 1 and 2. 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 mammals listed above and / or chicken, duck, turkey, salmon, catfish or tilapia.

[0702] 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 and / or related proteins listed in Table 3; thus, the compositions of the invention may comprise mRNA encoding a protein listed in Table 3 (or a homolog thereof) and other components listed 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) and other components listed herein.

[0703] Table 3. Lysosomes and related proteins

[0704]

[0705]

[0706]

[0707] Information about lysosomal proteins can be obtained 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 mRNA in the compositions and methods of the present invention are human proteins. The sequences of the listed proteins can also be used in a variety of animals, including a variety of mammals and animals of veterinary or industrial concern as described above.

[0708] In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding therapeutic proteins (e.g., cytosolic, transmembrane or secreted) such as those listed in Table 4. In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding therapeutic proteins that can be used to treat diseases or conditions (i.e., indications) listed in Table 4; thus, the compositions of the invention may include mRNA encoding therapeutic proteins listed or not listed in Table 4 (or homologs thereof, as described below) and other components described herein for treating diseases or conditions (i.e., indications) listed in Table 4, and the methods of the invention may include preparing and / or administering compositions comprising mRNA encoding such proteins (or homologs thereof, as described below) and other components described herein for treating diseases or conditions listed in Table 4.

[0709] Table 4. Exemplary indications and related proteins

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722] In some embodiments, the present invention is used to prevent, treat and / or cure a subject affected by a disease or condition listed in Tables 1, 2, 3 or 4 or 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).

[0723] Although certain compounds, compositions, and methods of the present invention have been specifically described according to certain embodiments, the following examples are merely illustrative of the compounds of the present invention and are not intended to be limiting thereof.

[0724] Example

[0725] Example 1: General Synthesis of Cationic Lipids

[0726] Cationic lipids (eg, cationic lipids (1a)-(21a), (1b)-(21b), and (22)-(462)) can be prepared according to Scheme 1 or Scheme 2 as described herein.

[0727] Example 2: Exemplary Synthesis of Common Synthetic Intermediates

[0728] Scheme 3 provides an exemplary synthesis of common synthetic intermediates.

[0729] Scheme 3. Synthesis of methyl ((2R,3R,4R,5S)-3,4,5-tri((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl) 3-(dimethylamino)propanoate

[0730]

[0731] Synthesis of (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol (A3):

[0732]

[0733] Trityl chloride A2 (180 g, 0.65 mole) was added to a solution of D-ribose A1 (100 g, 0.66 mole) in 300 mL of pyridine, and the resulting mixture was stirred at room temperature overnight. After evaporation of the solvent, the residue was dissolved in dichloromethane (1.5 L), washed with brine (500 mL × 3), and then dried over anhydrous Na2SO4. After filtering and concentrating, the crude product was dissolved in dichloromethane (1.5 L), and the solution was then slowly added to a mixture of hexane (4.0 L) and dichloromethane (400 mL). The mixture was stirred for 1 hour while a precipitate was formed. After filtration, the solid was washed with hexane and dried under vacuum to obtain 104 g of the desired product A3 as a white solid (yield: 40%).

[0734] Synthesis of (2S,3R,4R,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-tri(tert-butyldimethylsilane) (A4):

[0735]

[0736] To a solution of (2R, 3R, 4S, 5R) -5- ((trityloxy) methyl) tetrahydrofuran -2,3,4- triol A3 (40g, 0.102 mole) and imidazole (34.8g, 0.51 mole) in 400mL DMF was added tert-butyldimethylsilyl chloride (76.8g, 0.51mol) at 0°C, and the resulting mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum. The residue was dissolved in ethyl acetate (500mL), washed with brine (300mL × 3), and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated under vacuum. The crude product was purified by column chromatography (ethyl acetate / hexane 0-15%) to obtain 65g of the desired product A4 as a white solid (yield: 86%).

[0737] Synthesis of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methanol (A5):

[0738]

[0739] To a solution of (((2S,3R,4R,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy))tri(tert-butyldimethylsilane) A4 (65 g, 88 mmol) in 600 mL of dichloromethane was added triethylsilane (60 mL, 0.352 mole), followed by trifluoroacetic acid (13.8 mL, 0.176 mole) at 0 ° C. The resulting mixture was stirred at this temperature for 4 hours. The reaction mixture was quenched with saturated NaHCO3 solution to pH 7. The organic phase was separated and washed with saturated NaHCO3 solution (200 mL×3) and brine (200 mL), and then dried over anhydrous MgSO4. After filtration, the solvent was evaporated under vacuum to give 71 g of crude product A5, which was used in the next step without further purification.

[0740] Synthesis of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methyl 3-(dimethylamino)propanoate (A7):

[0741]

[0742] To a solution of ((2R, 3R, 4R, 5S) -3, 4, 5- tris ((tert-butyldimethylsilyl) oxy) tetrahydrofuran -2- yl) methanol A5 (71 g, 88 mmol) and 3- (dimethylamino) propionic acid B1 (13.6 g, 88 mmol) in 500 mL of dichloromethane, DMAP (10.7 g, 88 mmol) and EDCI (16.8 g, 88 mmol) were added, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was washed with brine (200 mL × 3) and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated under vacuum. The crude product was purified by column chromatography (330 g silica gel, 0-100% ethyl acetate / hexane gradient) to give 19 g of the desired product A7 as a colorless oil (yield: 36%).

[0743] Example 3: Exemplary Synthesis of Lipids Comprising Biodegradable Lipid Arms

[0744] Synthesis of (2S,3R,4R,5R)-5-(((3-(dimethylamino)propionyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl tridecanoate (2b):

[0745]

[0746] To a solution of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methyl 3-(dimethylamino)propanoate A7 (0.8 g, 1.35 mmol) in 1 mL of THF was added HF-pyridine (0.72 mL, 40.5 mmol), and the mixture was stirred at room temperature. After 3 hours, 2 mL of dichloromethane, 4 mL of pyridine and decanoyl chloride (2.3 g, 12.15 mmol) were subsequently added, and the resulting mixture was stirred at room temperature for four days. The volatiles were evaporated under vacuum, and the residue was then dissolved in dichloromethane (50 mL), washed with brine (50 mL×3), and dried over anhydrous Na2SO4. After filtration and concentration, the crude product was purified by column chromatography (80 g silica gel gold column, 0-10% methanol / dichloromethane gradient) to give 375 mg of the desired product (2b) as a clear oil (yield: 39%).

[0747] This method is applicable to the preparation of other lipids described herein, which can be obtained in similar yields by this representative method.

[0748] Example 4: Exemplary Synthesis of Lipids Comprising Biodegradable Lipid Arms

[0749] Scheme 4. Synthesis of lipids with biodegradable lipid groups

[0750]

[0751] Synthesis of (2S,3R,4R,5R)-5-((Triphenylmethoxy)methyl)tetrahydrofuran-2,3,4-triyl trioleate (A9)

[0752]

[0753] At 0°C, to a solution of (2R, 3R, 4S, 5R) -5- ((trityloxy) methyl) tetrahydrofuran -2,3,4- triol A3 (5.0 g, 1.67 mmol) in dichloromethane / pyridine (1 / 1 v / v, 100 mL), oleoyl chloride A8 (23 g, 9.4 mmol) was added, and the mixture was warmed to room temperature and stirred at room temperature for 5 days. After concentration, the residue was dissolved in dichloromethane, washed with water and brine. After drying with sodium sulfate and concentration, the crude product was purified by flash chromatography (SiO2: 0-40% dichloromethane / hexane) to give 4 g of the desired product A9.

[0754] Synthesis of (2S,3R,4R,5R)-5-(Hydroxymethyl)tetrahydrofuran-2,3,4-triyl trioleate (A10)

[0755] To a solution of (2S, 3R, 4R, 5R) -5- ((trityloxy) methyl) tetrahydrofuran-2,3,4-triyl trioleate A9 (0.8 g, 0.67 mmol) and triethylsilane (0.62 mL, 3.9 mmol) in 50 mL of dichloromethane at 0 ° C, trifluoroacetic acid (0.12 mL, 1.06 mmol) was added. After stirring at this temperature for 90 minutes, the reaction mixture was quenched with saturated sodium bicarbonate, then washed with saturated sodium bicarbonate and brine, and dried over MgSO4. After concentration, 0.81 g of crude product A10 was obtained, which was used in the next step without purification.

[0756] Synthesis of (2S,3R,4R,5R)-5-(((4-(dimethylaminodimethylamino)butyryl)oxy)methyl)tetrahydrofuran-2,3,4-triyl trioleate (211)

[0757]

[0758] A mixture of (2S, 3R, 4R, 5R) -5- (hydroxymethyl) tetrahydrofuran -2,3,4- triyl trioleate A9 (0.8 g, 0.67 mmol), EDCI (130 g, 0.67 mmol), 3- (dimethylamino) propionic acid hydrochloride (108 mg, 0.7 mmol) and DMAP (82 mg, 0.67 mmol) in 50 mL of dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and then washed with water and brine. After concentration, the crude product was purified by flash chromatography (SiO2: 0-10% MeOH / dichloromethane) to give 250 mg of the desired product (211) (yield: 36%, two steps).

[0759] As shown in Scheme 4, different acylating agents can be used in place of 3-(dimethylamino)propionic acid hydrochloride to provide other cationic lipids as described herein. Other lipids were prepared according to the representative procedure and obtained in similar yields.

[0760] Example 5: Exemplary Synthesis of Lipids Containing Aliphatic Lipid Arms—NaH Method

[0761] Scheme 5. Sodium hydride route to prepare lipids with aliphatic lipid groups

[0762]

[0763] Synthesis of (2S,3R,4R,5R)-2,3,4-tri(octyloxy)-5-((trityloxy)methyl)tetrahydrofuran (A12)

[0764]

[0765] To a solution of (2R, 3R, 4S, 5R) -5- ((trityloxy) methyl) tetrahydrofuran-2,3,4-triol A3 (3.0 g, 7.6 mmol) in 90 mL DMF was added sodium hydride (60% dispersion in oil, 2.7 g, 67.5 mmol) at 0 ° C. After 30 minutes, 1-bromooctane A11 (8.1 mL, 47 mmol) and tetrabutylammonium iodide (600 mg, 1.6 mmol) were added, and the resulting solution was stirred at room temperature overnight. The reaction mixture was quenched with ice / water and then extracted with ethyl acetate. The combined organic layers were washed with water and brine. After concentration, the crude product was purified by flash column chromatography (SiO2: 0-40% ethyl acetate / hexane) to give 290 mg of the desired product A12 as a colorless oil (yield: 5%).

[0766] Synthesis of ((2R,3R,4R,5S)-3,4,5-tri(octyloxy)tetrahydrofuran-2-yl)methanol (A13)

[0767]

[0768] To a solution of (2S, 3R, 4R, 5R)-2,3,4-tris(octyloxy)-5-((trityloxy)methyl)tetrahydrofuran A12 (290 mg, 0.4 mmol) and triethylsilane (0.25 mL, 1.6 mmol) in 2 mL of dichloromethane was added trifluoroacetic acid (0.06 mL, 0.8 mmol) at 0 °C. After stirring at this temperature for 1 hour, the reaction mixture was quenched with saturated NaHCO3, then washed with saturated NaHCO3 and brine, and dried over MgSO4. After concentration, 325 mg of crude product A13 was obtained, which was used in the next step without purification.

[0769] Synthesis of ((2R,3R,4R,5S)-3,4,5-tri(octyloxy)tetrahydrofuran-2-yl)methyl 3-(dimethylamino)propanoate (11b)

[0770]

[0771] A mixture of ((2R, 3R, 4R, 5S) -3, 4, 5- tri (octyloxy) tetrahydrofuran -2-yl) methanol A13 (325 mg, 0.4 mmol), EDCI (76 mg, 0.4 mmol), 3- (dimethylamino) propionic acid hydrochloride B1 (61 mg, 0.4 mmol) and DMAP (48 mg, 0.4 mmol) in 6 mL of dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and then washed with water and brine. After concentration, the crude product was purified by flash column chromatography (SiO2: (0-10% MeOH / dichloromethane) to give 102 mg of the desired product as a colorless oil (yield: 43%, two steps).

[0772] Example 6: Exemplary Synthesis of Lipids Containing Aliphatic Lipid Arms—Ti(O t Bu)4 Method

[0773] Scheme 6. Titanium-mediated synthesis of lipids with aliphatic arms

[0774]

[0775] Synthesis of (2R,3R,4S,5R)-2-(Decyloxy)-5-((Trityloxy)methyl)tetrahydrofuran-3,4-diol (A15)

[0776]

[0777] To a suspension of (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol A3 (3.0 g, 7.65 mmol), lithium bromide (0.66 g, 7.65 mmol) and mandelic acid (0.58 g, 3.82 mmol) in 15 mL of 1-decanol A14 was added titanium (IV) tert-butoxide (0.26 g, 0.76 mmol). The mixture was stirred at room temperature for 2 days. After removal of THF, the residue was purified by column chromatography (SiO2, 0-10% MeOH / dichloromethane) to give 1.9 g of the desired product A15 as a colorless oil (yield: 44%).

[0778] Synthesis of (2R,3R,4R,5R)-2,3,4-tri(decyloxy)-5-((trityloxy)methyl)tetrahydrofuran (A16)

[0779]

[0780] To a solution of (2R,3R,4S,5R)-2-(decyloxy)-5-((trityloxy)methyl)tetrahydrofuran-3,4-diol A15 (1.7 g, 3 mmol) in DMF / THF (100 mL / 100 mL) at 0°C was added sodium hydride (60% dispersion in oil, 0.72 g, 18.2 mmol). After 5 minutes, 1-bromodecane (3.78 g, 15.2 mmol) was added and the resulting solution was stirred at room temperature overnight. The reaction mixture was quenched with ice / water and then extracted with ethyl acetate. The combined organic layers were washed with water and brine. After concentration, the crude product was purified by flash column chromatography (SiO2: 0-100% dichloromethane / hexanes) to give 1.5 g of the desired product A16 (yield: 55%).

[0781] Synthesis of ((2R,3R,4R,5R)-3,4,5-tri(decyloxy)tetrahydrofuran-2-yl)methanol (A17)

[0782]

[0783] To a solution of (2R, 3R, 4R, 5R)-2,3,4-tris(decyloxy)-5-((trityloxy)methyl)tetrahydrofuran A16 (1.5 g, 1.67 mmol) and triethylsilane (1.5 mL, 9.4 mmol) in 35 mL of dichloromethane was added trifluoroacetic acid (0.3 mL, 3.89 mmol) at 0°C. After stirring at this temperature for 90 minutes, the reaction mixture was quenched with saturated NaHCO3, then washed with saturated NaHCO3 and brine, and dried over MgSO4. After concentration, 1.7 g of crude product A17 was obtained, which was used in the next step without purification.

[0784] Synthesis of ((2R,3R,4R,5R)-3,4,5-tri(decyloxy)tetrahydrofuran-2-yl)methyl 3-(dimethylamino)propanoate (12b)

[0785]

[0786] A mixture of ((2R,3R,4R,5R)-3,4,5-tri(decyloxy)tetrahydrofuran-2-yl)methanol 17 (1.7 g, 1.67 mmol), EDCI (320 mg, 1.67 mmol), 3-(dimethylamino)propionic acid hydrochloride 6 (260 mg, 1.9 mmol) and DMAP (200 mg, 1.67 mmol) in 100 mL of dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and then washed with water and brine. After concentration, the crude product was purified by flash column chromatography (SiO2: 0-10% MeOH / dichloromethane) to give 460 mg of the desired product 12b (yield: 36%, two steps).

[0787] Other lipids were prepared according to the representative procedure with similar yields.

[0788] Example 7: Exemplary Synthesis of Lipids Containing Aliphatic Lipid Arms - Tetraacetyl Ribose Method

[0789]

[0790] Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(dec-9-en-1-yloxy)tetrahydrofuran-3,4-diyl diacetate (A21)

[0791]

[0792] At 0 ° C, 1,2,3,5-tetra-o-acetyl-β-l-ribofuranose A19 (6.36 g, 20 mmol) and dec-9-ene-1-ol A20 (4.3 mL, 24 mmol) in 100 mL of dichloromethane were added BF3 · OEt2 (3.2 mL, 26 mmol), and the reaction mixture was kept at this temperature for 3 hours. The reaction mixture was poured into ice-cold saturated NaHCO3 and extracted with dichloromethane. The combined organic layers were dried over sodium sulfate. After concentration, the crude product was purified by flash column chromatography (SiO2: ethyl acetate / hexane 0-60%) to obtain a mixture of the desired product A21 and dec-9-ene-1-ol A20 (3.12 g) as a light yellow oil, which was used in the next step without further purification.

[0793] Synthesis of (2R,3R,4S,5R)-2-(Decan-9-en-1-yloxy)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (A22)

[0794]

[0795] A mixture of (2R, 3R, 4R, 5R) -2- (acetoxymethyl) -5- (dec-9-en-1-yloxy) tetrahydrofuran-3,4-diyl acetate A21 and dec-9-en-1-ol A20 (3.12 g) was dissolved in THF / MeOH (40 mL / 40 mL), and a solution of lithium hydroxide hydrate (3.16 g, 75 mmol) in 5 mL of water was added. The resulting mixture was stirred at room temperature overnight. After concentration to dryness, the residue was dissolved in dichloromethane and washed with brine. The organic layer was dried over sodium sulfate. After concentration, the crude product was purified by flash chromatography (SiO2: methanol / dichloromethane 0-20%) to give 2.08 g of pure product A22 as a white solid (yield: 36%, two steps).

[0796] Synthesis of (2R,3R,4S,5R)-2-(Decan-9-en-1-yloxy)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3,4-diol (A23)

[0797]

[0798] To a solution of (2R, 3R, 4S, 5R) -2- (dec-9-ene-1-yloxy) -5- (hydroxymethyl) tetrahydrofuran-3, 4-diol A22 (1.63 g, 5.55 mmol) and imidazole (755 mg, 11.1 mmol) in DMF (60 mL) was added triisopropylsilyl chloride (1.3 mL, 6.1 mmol) and stirred at room temperature overnight. The reaction mixture was concentrated to dryness. The residue was partitioned with water and ether. After separation, the organic layer was dried and concentrated. The crude product was purified by flash chromatography (SiO2: methanol / dichloromethane 0-10%) to give 1.76 g of the desired product A23 as a colorless oil (yield: 71%).

[0799] Synthesis of triisopropyl(((2R,3R,4R,5R)-3,4,5-tri(dec-9-en-1-yloxy)tetrahydrofuran-2-yl)methoxy)silane (A25)

[0800]

[0801] To a solution of (2R,3R,4S,5R)-2-(dec-9-en-1-yloxy)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3,4-diol A23 (1.76 g, 3.96 mmol) in DMF / THF (40 mL / 40 mL) at 0°C was added sodium hydride (0.95 g, 23.7 mmol). After 10 minutes, 10-bromodec-1-ene A24 (4.33 g, 19.8 mmol) was added and the resulting solution was stirred at room temperature overnight. The reaction mixture was quenched with ice / water and then extracted with ether. The combined organic layers were washed with water and brine. After concentration, the crude product was purified by flash chromatography (SiO2: ethyl acetate / hexane 0-5%) to give 2.45 g of product A25 as a colorless oil (yield: 85%).

[0802] Synthesis of 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy))trinonanal (A26)

[0803]

[0804] To a solution of triisopropyl(((2R,3R,4R,5R)-3,4,5-tris((9-(benzyloxy)nonyl)oxy)tetrahydrofuran-2-yl)methoxy)silane A25 (2.20 g, 3.05 mmol) and 2,6-lutidine (4.3 mL, 36.6 mmol) in 80 mL of dioxane was added an aqueous solution of osmium oxide (4 wt %, 0.84 mL, 0.137 mmol) followed by an aqueous solution of sodium periodate (7.83 g in 10 mL of water). After the reaction mixture was stirred at room temperature for 4 hours, saturated sodium sulfite was added. The resulting mixture was stirred for 10 minutes and extracted with dichloromethane. The organic layer was separated and washed with 2N HCl. After drying over sodium sulfate, the solvent was removed under vacuum to give 2.30 g of the desired product A26 as a yellow semisolid which was used in the next step without purification.

[0805] Synthesis of 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy))trinonanoic acid (A27)

[0806]

[0807] To a solution of 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy)trinonanal A26 (1.0 g, 1.3 mmol) in tBuOH / water (15 mL / 1 mL) were added sodium chlorite (0.54 g, 5.9 mmol) and sodium dihydrogen phosphate (0.95 g, 7.9 mmol), and the resulting mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction mixture was diluted with saturated sodium dihydrogen phosphate and extracted with ethyl acetate, and the combined organic layers were washed with brine. After concentration, 1.13 g of the desired product A27 was obtained as a colorless oil, which was used in the next step without purification.

[0808] Synthesis of tri(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy))trispelargonate (A29)

[0809]

[0810] A mixture of 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy)trinonanoic acid A27 (1.13 g, 1.46 mmol), 3-pentyloctan-1-ol A28 (3.5 g, 17.5 mmol), EDCI (3.35 g, 17.5 mmol) and DMAP (215 mg, 1.75 mmol) in 100 mL of dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with 1N HCl and extracted with dichloromethane, and the combined organic layers were washed with brine. After concentration, the crude product was purified by flash chromatography (SiO2: ethyl acetate / hexane 0-10%) to give 0.64 g of product A29 as a colorless oil (yield: 33%).

[0811] Synthesis of Tri(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4-triyl)tri(oxy))tripelanate (A30)

[0812]

[0813] To a solution of tri(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxyl))trinonanoate A29 (0.64 g, 0.48 mmol) in 8 mL of THF was added HF·pyridine (70%, 1 mL), and the resulting solution was stirred for 3 hours. The reaction mixture was diluted with dichloromethane and saturated sodium bicarbonate was added to adjust to pH 7. The organic layer was separated and dried over sodium sulfate. After concentration, the crude product was purified by flash chromatography (SiO2: ethyl acetate / hexane 0-15%) to give 0.51 g of pure product A30 as a colorless oil (yield: 91%).

[0814] Tri(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-(((3-(dimethylamino)propionyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tri(oxy)tripelanoate (21b)

[0815]

[0816] A mixture of tri(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4-triyl)tri(oxyl))trisnonanoate A30 (0.51 g, 0.437 mmol), 3-(dimethylamino)propionic acid hydrochloride B1 (67 mg, 0.437 mmol), EDCI (84 mg, 0.437 mmol) and DMAP (53 mg, 0.437 mmol) in 30 mL of dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with dichloromethane, and the combined organic layers were washed with brine. After concentration, the crude product was purified by flash chromatography (SiO2: methanol / dichloromethane 0-5%) to give 322 mg of the desired product 21b as a clear oil (yield: 58%).

[0817] According to the same procedure, compounds 19b and 20b were prepared in similar yields.

[0818]

[0819] Example 8: Lipid Nanoparticle Formulation Using Ribosyl Cationic Lipids

[0820] According to methods known in the art, cationic lipids described herein can be used to prepare lipid nanoparticles. For example, suitable methods include methods described in International Publication No. WO 2018 / 089801, which is incorporated herein by reference in its entirety.

[0821] An exemplary method of lipid nanoparticle formulation is method A of WO 2018 / 089801 (see, e.g., Examples 1 and 2 of WO 2018 / 089801). Figure 1 ). Method A ("A") relates to a conventional method for encapsulating mRNA by mixing mRNA with a lipid mixture without first preforming the lipids into lipid nanoparticles. In an exemplary method, an ethanol lipid solution and a buffered aqueous solution of mRNA are prepared separately. A solution of a lipid mixture (cationic lipids, auxiliary lipids, zwitterionic lipids, PEG lipids, etc.) is prepared by dissolving the lipids in ethanol. The mRNA solution is prepared by dissolving the mRNA in a citrate buffer, thereby producing an mRNA concentration of 0.0833 mg / ml in a citrate buffer at a pH of 4.5. The mixture is then heated to 65°C before mixing. Then, the two solutions are mixed using a pump system. In some cases, the two solutions are mixed using a gear pump system. In certain embodiments, the two solutions are mixed using a "T" confluence (or a "Y" confluence). The mixture is then purified by diafiltration by a TFF method. The resulting preparation is concentrated and stored at 2-8°C until further use.

[0822] A second exemplary method for lipid nanoparticle preparation is method B of WO 2018 / 089801 (see, e.g., Example 2 and FIG. 2 of WO 2018 / 089801). Method B ("B") refers to a process of encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with mRNA. A range of different conditions can be used in method B, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations. In an exemplary method, lipids dissolved in ethanol and citrate buffer are mixed using a pump system. The instantaneous mixing of the two streams results in the formation of empty lipid nanoparticles, which is a self-assembly process. The resulting preparation mixture is an empty lipid nanoparticle in a citrate buffer containing alcohol. The preparation is then subjected to a TFF purification process, in which buffer exchange occurs. A suspension of the resulting preformed empty lipid nanoparticles is then mixed with mRNA using a pump system. For some cationic lipids, heating the solution after mixing results in a higher percentage of lipid nanoparticles containing mRNA and a higher total mRNA yield.

[0823] As described in WO 2018 / 089801, the lipid nanoparticle formulations of Table 5 were prepared by using method A or method B. All lipid nanoparticle formulations contained hEPO mRNA and different lipids in the following molar % ratios: cationic lipid: DMG-PEG2000; cholesterol: DOPE = 40:5:25:30.

[0824] Table 5. Exemplary lipid nanoparticle formulations

[0825]

[0826]

[0827] Example 9: In vivo expression of hEPO in CD1 mice using ribolipids

[0828] Lipid nanoparticle formulations (Table 5) comprising ribose cationic lipids and mRNA encoding hEPO were administered intravenously (IV) to study mRNA delivery and resulting hEPO expression. Male CD1 mice aged 6-8 weeks were given a single intravenous injection of LNP formulations at a dosage level of 1 mg / kg. Blood samples were collected by tail clipping at 6 hours and 24 hours after administration. The hEPO protein expression level measured in serum samples by ELISA ( Figure 1 ). These studies show that the ribose cationic lipids described herein are highly effective in delivering mRNA in vivo, resulting in high expression of the protein or polypeptide encoded by the delivered mRNA.

Claims

1. A cationic lipid having a structure according to formula (I'), in R X For -L 1 -R 1 ; L 1 , L 2 and L 3 Each of is independently a covalent bond, -C(O)- or -C(O)O-; L 4A is -C(O)- or -C(O)O-; L 4B For unsubstituted C1-C 20 Alkylene; B is NR 4 R 5 or a 5- to 10-membered nitrogen-containing heterocyclic group; Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl; Each R 4 and R 5 are independently C1-C6 alkyl.

2. cationic lipid according to claim 1, wherein L 4A It is -C(O)-.

3. cationic lipid according to claim 1, wherein L 4B It is -CH2-, -CH2CH2- or -CH2CH2CH2-.

4. cationic lipid according to claim 1, wherein B is NR 4 R 5 ,and Each R 4 and R 5 are independently C1-C6 alkyl.

5. The cationic lipid according to claim 4, wherein B is N(CH3)2.

6. A cationic lipid having a structure according to formula (II) in R A For -L 1 -R 1 , R B For -L 2 -R 2 , and R C For -L 3 -R 3 ; L 1 , L 2 and L 3 Each of is independently a covalent bond or -C(O)-; L 4 C1-C 10 Alkylene; Each R 1 , R 2 and R 3 Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl; Each R 4 and R 5 are independently unsubstituted C1-C6 alkyl or R 4 and R 5 Combined to form a 5- to 10-membered nitrogen-containing heterocyclic group.

7. The cationic lipid according to claim 6, having a structure according to formula (IIa), 8. The cationic lipid according to claim 6, having a structure according to formula (IIb) or (IIc), 9. The cationic lipid according to claim 8, having a structure according to formula (IId) or (IIe), 10. according to any one of claims 6-9 cationic lipids, wherein L 4 It is an unsubstituted C1-C6 alkylene group.

11. The cationic lipid according to claim 10, wherein L 4 It is -CH2-, -CH2CH2-, -CH2CH2CH2- or -CH2CH2CH2CH2-.

12. according to any one of claims 6-9 cationic lipids, wherein each R 4 and R 5 are independently unsubstituted C1-C6 alkyl, or R 4 and R 5 Combined to form a 5- to 6-membered nitrogen-containing heterocyclic group containing one or two ring nitrogens.

13. The cationic lipid according to claim 12, wherein each R 4 and R 5 For CH3.

14. according to any one of claims 6-9 cationic lipids, wherein each R 1 , R 2 and R 3 are independently unsubstituted C6-C 22 Alkyl, unsubstituted C6-C 22 Alkenyl or unsubstituted C6-C 22 Alkynyl.

15. The cationic lipid according to claim 14, wherein each R 1 , R 2 and R 3 is an unsubstituted straight-chain C6-C 22 Alkyl, unsubstituted straight chain C6-C 22 Alkenyl or unsubstituted straight chain C6-C 22 Alkynyl.

16. The cationic lipid according to claim 14, wherein each R 1 , R 2 and R 3 For unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C6-C 22 Alkynyl.

17. The cationic lipid according to claim 14, wherein each R 1 , R 2 and R 3 For unsubstituted C6-C 22 alkyl.

18. The cationic lipid of claim 14, wherein each R 1 , R 2 and R 3 (CH2)7CH3, (CH2)9CH3, (CH2) 11 CH3, (CH2) 13 CH3, (CH2) 15 CH3 or (CH2) 17 CH3.

19. The cationic lipid of claim 14, wherein each R 1 , R 2 and R 3 (CH2)6CH3, (CH2)8CH3, (CH2) 10 CH3, (CH2) 12 CH3, (CH2) 14 CH3 or (CH2) 16 CH3.

20. A cationic lipid having a structure according to formula (II), in R A For -L 1 -R 1 , R B For -L 2 -R 2 , and R C For -L 3 -R 3 ; L 1 , L 2 and L 3 Each of is independently a covalent bond or -C(O)-; L 4 C1-C 10 Alkylene; Each R 1 , R 2 and R 3 To be –O(CO)R 6 or-C(O)OR 6 Substituted C6-C 12 Alkyl, where R 6 For unsubstituted C6-C 14 Alkyl; and Each R 4 and R 5 are independently unsubstituted C1-C6 alkyl; or R 4 and R 5 Combined to form a 5- to 10-membered nitrogen-containing heterocyclic group.

21. The cationic lipid according to claim 20, having a structure according to formula (IIa), 22. The cationic lipid of claim 20, having a structure according to formula (IIb) or (IIc), 23. The cationic lipid of claim 20, having a structure according to formula (IId) or (IIe), 24. according to the cationic lipid of claim 20, wherein R 6 is an unsubstituted straight-chain C6-C 14 alkyl.

25. according to the cationic lipid of claim 20, wherein R 6 For unsubstituted branched C6-C 14 alkyl.

26. according to the cationic lipid described in claim 24 or 25, wherein each R 1 , R 2 and R 3 It is (CH2)8OC(O)(CH2)6CH3, (CH2)9OC(O)(CH2)6CH3, (CH2)7C(O)O(CH2)2CH(C5H 11 )2 or (CH2)8C(O)O(CH2)2CH(C5H 11 )2.

27. according to the cationic lipid described in any one of claims 6-9, wherein each R 1 , R 2 and R 3 For unsubstituted C6-C 22 Alkenyl.

28. according to the cationic lipid of claim 27, wherein said C6-C 22 Alkenyl is monoalkenyl, dienyl or trienyl.

29. according to the cationic lipid of claim 27, wherein each R 1 , R 2 and R 3 for 30. according to the cationic lipid of claim 27, wherein each R 1 , R 2 and R 3 for 31. A cationic lipid selected from the group consisting of: 。 32. A cationic lipid selected from the group consisting of: 。 33. A cationic lipid selected from the group consisting of: 。 34. A cationic lipid selected from the group consisting of: 。 35. A cationic lipid selected from the group consisting of: and 。 36. A composition comprising mRNA encoding a protein encapsulated in a liposome, wherein 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 according to any one of claims 1-35.

37. The composition of claim 36 comprising mRNA encoding an antigen from an infectious agent.

38. The composition of claim 36, comprising mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

39. The composition of claim 36, comprising mRNA encoding ornithine transcarbamylase (OTC) protein.

40. A composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid according to any one of claims 1-35.

41. The composition of claim 40, further comprising one or more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

42. The composition of claim 40, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide.

43. The composition of claim 40, comprising mRNA encoding an antigen from an infectious agent.

44. The composition of claim 42, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of a lung or lung cell of a subject.

45. The composition of claim 44, wherein the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

46. ​​The composition of claim 42, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of the liver or hepatocytes of a subject.

47. The composition of claim 46, wherein the mRNA encodes ornithine transcarbamylase (OTC) protein.

48. The composition of claim 36, formulated for intravenous (IV) administration.

49. The composition of claim 36, formulated for intramuscular (IM) administration.

50. The composition of claim 36 formulated for administration by inhalation.

51. The composition of claim 50, wherein the composition is formulated for aerosolization.

52. A composition comprising mRNA encoding a protein encapsulated in a liposome, wherein 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 according to claim 6.

53. The composition of claim 52, comprising mRNA encoding an antigen from an infectious agent, a cystic fibrosis transmembrane conductance regulator (CFTR) protein, or an ornithine transcarbamylase (OTC) protein.

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