Thioester cationic lipids

Abstract

The present invention discloses cationic lipids, which are compounds of formula (I), (II), (III), (IV), (V) or (VI). Cationic lipids provided herein can be used to deliver and express mRNA and encoded proteins, for example as components of liposome delivery vehicles, and therefore can be used to treat various diseases, disorders and conditions, such as those associated with the deficiency of one or more proteins.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201980048008.6, application date May 23, 2019, and invention name “Thioester cationic lipid”. The Chinese invention patent application with application number 201980048008.6 is a Chinese national phase application with international application number PCT / US2019 / 033806, which claims priority to U.S. provisional patent application number 62 / 676,147 filed on May 24, 2018, U.S. provisional patent application number 62 / 748,097 filed on October 19, 2018, and U.S. provisional patent application number 62 / 750,013 filed on October 24, 2018, the full contents of which are hereby incorporated by reference into this document. Background Art

[0002] 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

[0003] The present invention particularly provides cationic lipids that can be used for delivering mRNA.The delivery of the mRNA provided by cationic lipids as herein described can result in targeted delivery, reduce administration frequency, improve patient tolerance, and provide more effective and less toxic mRNA therapy for the treatment of various diseases (including but not limited to cancer, cardiovascular disease, cystic fibrosis, infectious diseases and nervous system diseases).

[0004] In one aspect, the present invention provides a cationic lipid of formula (I):

[0005]

[0006] in

[0007] R 1 It is hydrogen, C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl, or substructure Y;

[0008] Each a and b is an integer from 0 to 6;

[0009] Each X A1 and X B1 independently O or S;

[0010] Each L A and L B Independently C1-C 10Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene; X A2 Independently NH, NR A , CH2 or CHR A ;

[0011] X B2 Independently NH, NR B , CH2 or CHR B ;

[0012] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl; and substructure Y is In another aspect, the present invention provides a cationic lipid of formula (II):

[0013]

[0014] in

[0015] R 1 It is hydrogen, C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl, or substructure Z;

[0016] Each a and b is an integer from 0 to 6;

[0017] Each X A1 and X B1 independently O or S;

[0018] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene; X A2 Independently NH, NR A , CH2 or CHR A ;

[0019] X B2 Independently NH, NR B , CH2 or CHR B ;

[0020] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30Alkenyl or C6-C 30 Alkynyl; and substructure Z is In yet another aspect, the present invention provides a cationic lipid of formula (III):

[0021]

[0022] in

[0023] Each a is an integer from 0 to 6;

[0024] Each X A1 independently O or S;

[0025] Each L A Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0026] X A2 Independently NH, NR A , CH2 or CHR A ;as well as

[0027] Each R A Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0028] In yet another aspect, the present invention provides a cationic lipid having a structure according to formula (IV),

[0029]

[0030] in

[0031] R 1 is hydrogen, C1-C 30 Alkyl, C2-C 30 Alkenyl or C2-C 30 Alkynyl;

[0032] Each a and b is an integer from 0 to 6;

[0033] Each X A1 and X B1 independently O or S;

[0034] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene; L Care independently –C(O)– or –(CH2) b –

[0035] X A2 Independently NH, NR A , CH2 or CHR A ;

[0036] X B2 Independently NH, NR B , CH2 or CHR B ;as well as

[0037] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0038] In an embodiment, L C is -C(O)-. In an embodiment, L C Yes – (CH2) b –.

[0039] In embodiments, the cationic lipid has a structure according to Formula (IA),

[0040]

[0041] In embodiments, the cationic lipid has a structure according to Formula (I-A'),

[0042]

[0043] In embodiments, the cationic lipid has a structure according to Formula (IV-A):

[0044]

[0045] In an embodiment, each X A1 and X B1 It is O, or every X A1 and X B1 is S. In an embodiment, each X A1 and X B1 Is O. In an embodiment, each X A1 and X B1 It’s S.

[0046] In an embodiment, each a and b is independently 0, 1 or 2.

[0047] In an embodiment, each X A2 It is NR A or CHRA .

[0048] In an embodiment, each X B2 It is NR B or CHR B .

[0049] In embodiments, the cationic lipid has a structure according to Formula (IB),

[0050]

[0051] In embodiments, the cationic lipid has a structure according to Formula (I-B'),

[0052]

[0053] In an embodiment, the cationic lipid has a structure according to Formula (IB"),

[0054] wherein d is independently an integer from 0 to 5.

[0055] In embodiments, the cationic lipid has a structure according to formula (II-A),

[0056]

[0057] In embodiments, the cationic lipid has a structure according to formula (III-A),

[0058]

[0059] In embodiments, the cationic lipid has a structure according to formula (IV-B),

[0060]

[0061] In embodiments, the cationic lipid has a structure according to Formula (IV-B'),

[0062]

[0063] In embodiments, the cationic lipid has a structure according to Formula (IV-B"),

[0064]

[0065] In another aspect, the present invention provides a cationic lipid having a structure according to formula (V),

[0066]

[0067] in

[0068] Each LA and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylene; and

[0069] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 Alkynyl or C1-C 15 Alkylene-C(O)2-C1-C 15 alkyl.

[0070] In an embodiment, each L A It is C1-C 10 Alkylene.

[0071] In an embodiment, each L B It is C1-C 10 Alkylene.

[0072] In an embodiment, each L A and L B is an unsubstituted C1-C 10 Alkylene.

[0073] In embodiments, the cationic lipid has a structure according to formula (IC),

[0074]

[0075] wherein each c is independently an integer from 2 to 10.

[0076] In embodiments, the cationic lipid has a structure according to Formula (I-C'),

[0077]

[0078] wherein each c is independently an integer from 2 to 10.

[0079] In embodiments, the cationic lipid has a structure according to formula (IC"),

[0080] wherein each c is independently an integer from 2 to 10, and d is independently an integer from 0 to 5. In embodiments, d is 0, 1, 2, 3 or 4.

[0081] In embodiments, the cationic lipid has a structure according to formula (II-B),

[0082]

[0083] wherein each c is independently an integer from 2 to 10.

[0084] In embodiments, the cationic lipid has a structure according to formula (III-B),

[0085]

[0086] wherein each c is independently an integer from 2 to 10.

[0087] In embodiments, the cationic lipid has a structure according to formula (III-C),

[0088]

[0089] wherein each c is independently an integer from 2 to 10.

[0090] In embodiments, the cationic lipid has a structure according to Formula (III-C'),

[0091]

[0092] wherein each c is independently an integer from 2 to 10.

[0093] In embodiments, the cationic lipid has a structure according to Formula (IV-C"),

[0094] wherein each c is independently an integer from 2 to 10.

[0095] In another aspect, the present invention provides a cationic lipid having a structure according to formula (VI),

[0096]

[0097] in

[0098] Each R A and R B Independently C1-C 30 Alkyl, C2-C 30 Alkenyl, C2-C 30 Alkynyl or C1-C 15 Alkylene-C(O)2-C1-C 15 Alkyl; and

[0099] Each c is independently an integer from 2 to 10.

[0100] In an embodiment, each c is 2, 3, or 4. In an embodiment, each c is 4, 5, 6, 7, 8, 9, or 10. In an embodiment, each c is 4.

[0101] In an embodiment, each R A It is C6-C 20 Alkyl or C6-C 20 In an embodiment, each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0102] In an embodiment, each R B It is C6-C 20 Alkyl or C6-C 20 In an embodiment, each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0103] In an embodiment, each R A and R B It is C6-C 20 Hydroxyalkyl, or each R A and R B It is C6-C 20 In an embodiment, each R A and R B It is -CH2CH(OH)C 10 H 21 , or each R A and R B Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 In an embodiment, each R A and R B It is -CH2CH(OH)C 10 H 21 .

[0104] In an embodiment, each R A and R B It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0105]

[0106] The present invention also provides a cationic lipid: the cationic lipid is any one of Compounds 1-156.

[0107] In embodiments, the cationic lipid is

[0108]

[0109]

[0110] The present invention also provides such cationic lipids, which are:

[0111]

[0112]

[0113]

[0114] In another aspect, the invention features a composition comprising an mRNA encoding a peptide or polypeptide encapsulated within 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 as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (1)-(156)).

[0115] In an embodiment, the composition comprises mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0116] In an embodiment, the composition comprises mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0117] In embodiments, the composition comprises mRNA encoding an antigen (eg, an antigen from an infectious agent).

[0118] In another aspect, the invention features a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid as described herein (e.g., a cationic lipid of any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of compounds (I)-(156)).

[0119] In an embodiment, 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.

[0120] In an embodiment, the nucleic acid is an mRNA encoding a peptide or polypeptide.

[0121] In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or treatment of the lung or lung cells of a subject.In embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0122] In embodiments, the mRNA encodes a peptide or polypeptide for delivery to or treatment of the liver or hepatocytes of a subject. In embodiments, the mRNA encodes an ornithine transcarbamylase (OTC) protein.

[0123] In embodiments, the mRNA encodes a peptide or polypeptide for use in a vaccine. In embodiments, the mRNA encodes an antigen (e.g., an antigen from an infectious agent).

[0124] In embodiments, the composition is formulated for intravenous (IV) administration. In embodiments, the composition is formulated for intramuscular (IM) administration. In embodiments, the composition is formulated for administration by inhalation (e.g., the composition is formulated for nebulization).

[0125] In some aspects, the present invention provides a method of treating a disease in a subject, comprising administering to the subject a composition (e.g., a pharmaceutical composition) as described herein (e.g., a composition comprising a cationic lipid of any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of compounds (I)-(156)). BRIEF DESCRIPTION OF THE DRAWINGS

[0126] 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

[0127] definition

[0128] To make the present invention more easily understood, certain terms are first defined below. Additional definitions of the following and other terms are set forth throughout this specification. The publications and other reference materials cited herein to describe the background technology of the present invention and to provide additional details about its implementation are incorporated herein by reference.

[0129] 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 synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and / or substitutions. Amino acids, including the 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. An amino acid may comprise 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.

[0130] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0131] 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 range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of 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).

[0132] Bioactive: As used herein, the term "bioactive" refers to the characteristic of any agent that is active in a biological system, particularly an organism. For example, an agent that has a biological effect on an organism when administered to that organism is considered bioactive.

[0133] Delivery: As used herein, the term "delivery" encompasses both local 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 taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0134] 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," as well as grammatically equivalent terms, are used interchangeably.

[0135] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form that exhibits the properties and / or activities that characterize it.

[0136] 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 protein, to fall to half its value measured at the beginning of a time period.

[0137] Improvement, increase, or decrease: As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to values ​​relative to a baseline measurement, such as that measured in the same individual before initiation of a treatment described herein, or that measured in a control subject (or 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.

[0138] 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.

[0139] 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).

[0140] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some components with which it was originally associated when produced (whether in nature 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 other components with which it was 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. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of the purity percentage of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0141] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA includes modified RNA and unmodified RNA. The term "modified mRNA" relates to an mRNA that comprises at least one chemically modified nucleotide. An mRNA may comprise one or more coding and non-coding regions. An 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, mRNA sequences are presented in a 5' to 3' direction. 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 linkages).

[0142] Nucleic acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polynucleotide chain. In some embodiments, a 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" encompasses RNA and single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acid (RNA), including but not limited to any one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), poly-encoding nucleic acid (MCNA), polymer-encoding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses RNA and DNA. In embodiments, the DNA can be in the form of antisense DNA, plasmid DNA, a portion of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or a derivative of these groups.In embodiments, the 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), piwi-interacting RNA (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.

[0143] 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, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. This includes both prenatal and postnatal humans.

[0144] 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.

[0145] 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, 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, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconoheptate, 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) salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, 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 using appropriate electrophiles such as alkyl halides to form quaternized alkylated amino salts.

[0146] 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."

[0147] 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 can be a patient, which refers to a person who presents to a medical provider for disease diagnosis or treatment. The term "subject" can be used interchangeably with "individual" or "patient" herein. A subject can have a disease or condition or be susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0148] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand 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.

[0149] 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.

[0150] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of 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.

[0151] Treatment: As used herein, the term "treatment" refers to any method used to partially or completely alleviate, 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. Treatment can be administered to subjects who do not exhibit signs of disease and / or who exhibit only early signs of disease in order to reduce the risk of developing pathology associated with the disease.

[0152] Aliphatic: As used herein, the term aliphatic refers to C 1- C 40 Hydrocarbons, and include saturated hydrocarbons and unsaturated hydrocarbons. Aliphatic groups can be straight chain, branched chain or cyclic. For example, C1-C 20 Aliphatic groups 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 Dienyl, linear 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 comprise 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. 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', -C0H, -C0R', -CN, -OH, -OR', -OCOR', -OC0R', -NH2, -NHR', -N(R')2, -SR, or -S0R', 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 one embodiment, R' is independently an unsubstituted C1-C3 alkyl. In one embodiment, the aliphatic group is unsubstituted. In one embodiment, the aliphatic group does not include any heteroatoms.

[0153] Alkyl: As used herein, the term "alkyl" refers to acyclic straight-chain and branched hydrocarbon groups, for example, "C1-C 20"Alkyl" refers to an alkyl group having 1 to 20 carbons. The alkyl group can be straight or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like. Other alkyl groups will be apparent to those skilled in the art given the benefit of this disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R', wherein each instance of R' is independently C1-C 20 Aliphatic 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 alkyl group is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In some embodiments, the alkyl group is substituted with an -OH group and may also be referred to herein as a "hydroxyalkyl," where the prefix indicates an -OH group and "alkyl" is as described herein.

[0154] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight or branched hydrocarbon radical, and is exemplified by methylene, ethylene, isopropylene, and the like. Similarly, as used herein, the term "alkenylene" refers to an unsaturated divalent straight or branched hydrocarbon radical 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 radical 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 radical 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', -OCOR', -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 group is unsubstituted. In some embodiments, the alkylene, alkenylene, or alkynylene group does not include any heteroatoms.

[0155] 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 carbon atoms. 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, and the like. In embodiments, the alkenyl group contains 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl group contains a single carbon-carbon double bond. In embodiments, 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', -OCOR', -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, alkenyl is unsubstituted. In some embodiments, alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In some embodiments, alkenyl groups are substituted with an —OH group and may also be referred to herein as “hydroxyalkenyl,” where the prefix represents an —OH group and “alkenyl” is as described herein.

[0156] 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, for example, "C2-C 20"Alkynyl" refers to an alkynyl group having 2-20 carbon atoms. 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 an embodiment, 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-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 one embodiment, R' is independently unsubstituted C1-C3 alkyl. In one embodiment, alkynyl is unsubstituted. In one embodiment, alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0157] 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 group is monocyclic. In embodiments, the cycloalkyl group is polycyclic (e.g., bicyclic or tricyclic). In polycyclic cycloalkyl groups, the individual rings may be fused, bridged, or spirocyclic. Examples of cycloalkyl groups 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 group may be unsubstituted or substituted with one or more substituents as described herein. For example, the cycloalkyl group may be substituted with one or more of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCOR', -NH2, -NHR', -N(R')2, -SR', or -S02R' (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-C10 In an embodiment, R' is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In one embodiment, R' is independently unsubstituted C1-C3 alkyl. In one embodiment, cycloalkyl is unsubstituted. In one embodiment, cycloalkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0158] Halogen: As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0159] Cationic lipids

[0160] Liposome-based vehicles are considered to be attractive carriers for therapeutic agents and are still under development with ongoing effort. Although liposome-based vehicles 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, a significant shortcoming of liposome delivery systems relates 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 into such target cells.

[0161] 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 various cell types and tissues with improved efficiency. Importantly, there is still a particular need for novel cationic lipids that exhibit reduced toxicity and can effectively deliver encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0162] Novel cationic lipids, compositions comprising such lipids, and methods of use thereof are described herein. 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.

[0163] 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.

[0164] In embodiments, cationic lipids as herein described can provide one or more desired characteristics or properties.That is, in certain embodiments, the feature of cationic lipids as herein described 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 property of the liposome compositions (such as lipid nanoparticles) of their components.Especially, the feature of cationic lipids disclosed herein can be the enhanced transfection efficiency and the ability causing specific biological results thereof.Such results can include the release of cellular uptake, endosome / lysosome destruction ability and / or promotion of intracellular encapsulation material (such as, polynucleotide) such as enhanced.

[0165] Cationic lipids of formula (I), (II), (III), (IV), (V) and (VI)

[0166] In one aspect, the present invention provides a cationic lipid of formula (I):

[0167]

[0168] in

[0169] R 1 It is hydrogen, C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl, substructure Y or substructure Z;

[0170] Each a and b is an integer from 0 to 6;

[0171] Each X A1 and X B1 independently O or S;

[0172] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0173] X A2 Independently NH, NR A , CH2 or CHR A ;

[0174] X B2 Independently NH, NR B , CH2 or CHR B ;

[0175] Each R A and R B Independently C6-C 30 Alkyl, C6-C30 Alkenyl or C6-C 30 alkynyl; and

[0176] Substructure Y is

[0177] In an embodiment, R 1 is hydrogen. In an embodiment, R 1 It is C6-C 30 In an embodiment, R 1 It is C6-C 30 In an embodiment, R 1 It is C6-C 30 In one embodiment, R 1 It is substructure Y.

[0178] In one aspect, the present invention provides a cationic lipid of formula (II):

[0179]

[0180] in

[0181] R 1 It is hydrogen, C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl, or substructure Z;

[0182] Each a and b is an integer from 0 to 6;

[0183] Each X A1 and X B1 independently O or S;

[0184] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0185] X A2 Independently NH, NR A , CH2 or CHR A ;

[0186] X B2 Independently NH, NR B , CH2 or CHR B ;

[0187] Each R A and R B Independently C6-C 30 Alkyl, C6-C30 Alkenyl or C6-C 30 alkynyl; and

[0188] Substructure Z is

[0189] In an embodiment, R 1 is hydrogen. In an embodiment, R 1 It is C6-C 30 In an embodiment, R 1 It is C6-C 30 In an embodiment, R 1 It is C6-C 30 In one embodiment, R 1 It is substructure Z.

[0190] In another aspect, the present invention provides a cationic lipid of formula (III):

[0191]

[0192] in

[0193] Each a is an integer from 0 to 6;

[0194] Each X A1 independently O or S;

[0195] Each L A Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0196] X A2 Independently NH, NR A , CH2 or CHR A ;as well as

[0197] Each R A Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0198] In embodiments, the cationic lipid has a structure according to formula (IV),

[0199]

[0200] in

[0201] R 1 is hydrogen, C1-C 30 Alkyl, C2-C 30 Alkenyl or C2-C30 Alkynyl;

[0202] Each a and b is an integer from 0 to 6;

[0203] Each X A1 and X B1 independently O or S;

[0204] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0205] L C are independently –C(O)– or –(CH2) b –

[0206] X A2 Independently NH, NR A , CH2 or CHR A ;

[0207] X B2 Independently NH, NR B , CH2 or CHR B ;as well as

[0208] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0209] In another aspect, the present invention provides a cationic lipid having a structure according to formula (V),

[0210]

[0211] in

[0212] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene; and

[0213] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 Alkynyl or C1-C 15Alkylene-C(O)2-C1-C 15 Alkylene.

[0214] In embodiments, the cationic lipid has a structure according to Formula (IA),

[0215]

[0216] In embodiments, the cationic lipid has a structure according to Formula (I-A'),

[0217]

[0218] In embodiments, the cationic lipid has a structure according to formula (II-A),

[0219]

[0220] In embodiments, the cationic lipid has a structure according to Formula (IV-A):

[0221]

[0222] In an embodiment, each X A1 and X B1 It is O, or every X A1 and X B1 is S. In an embodiment, each X A1 and X B1 Is O. In an embodiment, each X A1 and X B1 It’s S.

[0223] In embodiments, each a and b are independently 0, 1, or 2. In embodiments, each a is 0. In embodiments, each a is 1. In embodiments, each a is 2. In embodiments, each b is independently 0. In embodiments, each b is 1. In embodiments, each b is 2. In embodiments, a and b are the same. In embodiments, a and b are different. In embodiments, each a and b are 0. In embodiments, each a and b are 1. In embodiments, each a and b are 2. In embodiments, each a is 0 and each b is 1. In embodiments, each a is 0 and each b is 2. In embodiments, each a is 1 and each b is 0. In embodiments, each a is 1 and each b is 2. In embodiments, each a is 2 and each b is 0. In embodiments, each a is 2 and each b is 1.

[0224] In an embodiment, each X A2 It is NR A or CHR AIn an embodiment, each X A2 It is NR A In an embodiment, each X A2 It is CHR A .

[0225] In an embodiment, each X B2 It is NR B or CHR B In an embodiment, each X B2 It is NR B In an embodiment, each X B2 It is CHR B .

[0226] In an embodiment, each X A2 It is NR A , and each X B2 It is NR B In an embodiment, each X A2 It is CHR A , and each X B2 It is CHR B .

[0227] In embodiments, the cationic lipid has a structure according to Formula (IB),

[0228]

[0229] In embodiments, the cationic lipid has a structure according to Formula (I-B'),

[0230]

[0231] In an embodiment, the cationic lipid has a structure according to Formula (IB"),

[0232] wherein d is independently an integer from 0 to 5. In an embodiment, d is 0. In an embodiment, d is 1. In an embodiment, d is 2. In an embodiment, d is 3. In an embodiment, d is 4. In an embodiment, d is 5.

[0233] In embodiments, the cationic lipid has a structure according to formula (II-A),

[0234]

[0235] In embodiments, the cationic lipid has a structure according to formula (III-A),

[0236]

[0237] In embodiments, the cationic lipid has a structure according to formula (IV-B),

[0238]

[0239] In embodiments, the cationic lipid has a structure according to Formula (IV-B'),

[0240]

[0241] In embodiments, the cationic lipid has a structure according to Formula (IV-B"),

[0242]

[0243] In an embodiment, each L A It is C1-C 10 Alkylene (e.g., C2-C 10 Alkylene, C4-C 10 In an embodiment, each L A is an unsubstituted C1-C 10 Alkylene (e.g., unsubstituted C2-C 10 Alkylene, unsubstituted C4-C 10 alkylene or unsubstituted C4-C8 alkylene). In an embodiment, each L A is substituted C1-C 10 Alkylene (e.g., substituted C2-C 10 Alkylene, substituted C4-C 10 alkylene or substituted C4-C8 alkylene). In an embodiment, each L A is -(CH2)4-. In an embodiment, each L A is -(CH2)2-. In an embodiment, each L A is -(CH2)3-. In an embodiment, each L A It is -(CH2)5-.

[0244] In an embodiment, each R A and R B It is an unsubstituted C6-C 30 In an embodiment, each R A and R B Yes (unsubstituted C3-C 15 Alkylene)-C(O)2-(unsubstituted C3-C 15 alkyl).

[0245] In an embodiment, each L B It is C1-C 10Alkylene (e.g., C2-C 10 Alkylene, C4-C 10 In an embodiment, each L B is an unsubstituted C1-C 10 Alkylene (e.g., unsubstituted C2-C 10 Alkylene, unsubstituted C4-C 10 alkylene or unsubstituted C4-C8 alkylene). In an embodiment, each L B is substituted C1-C 10 Alkylene (e.g., substituted C2-C 10 Alkylene, substituted C4-C 10 alkylene or substituted C4-C8 alkylene). In an embodiment, each L B is -(CH2)4-. In an embodiment, each L B is -(CH2)2-. In an embodiment, each L B is -(CH2)3-. In an embodiment, each L B It is -(CH2)5-.

[0246] In an embodiment, each L A and L B It is C1-C 10 In an embodiment, each L A and L B is substituted C1-C 10 In an embodiment, each L A and L B is an unsubstituted C1-C 10 In an embodiment, each L A and L B is -CH2-. In an embodiment, each L A and L B is -(CH2)2-. In an embodiment, each L A and L B is -(CH2)3-. In an embodiment, each L A and L B is -(CH2)4-. In an embodiment, each L A and L B is -(CH2)5-. In an embodiment, each L A and L B is -(CH2)6-. In an embodiment, each L A and L B is -(CH2)7-. In an embodiment, each L A and L Bis -(CH2)8-. In an embodiment, each L A and L B is -(CH2)9-. In an embodiment, each L A and L B Yes - (CH2) 10 -.

[0247] In embodiments, the cationic lipid has a structure according to formula (IC),

[0248]

[0249] wherein each c is independently an integer from 2 to 10.

[0250] In embodiments, the cationic lipid has a structure according to Formula (I-C'),

[0251]

[0252] wherein each c is independently an integer from 2 to 10.

[0253] In embodiments, the cationic lipid has a structure according to formula (IC"),

[0254] wherein each c is independently an integer from 2 to 10, and d is independently an integer from 0 to 5. In embodiments, d is 0, 1, 2, 3 or 4.

[0255] In embodiments, the cationic lipid has a structure according to formula (II-B),

[0256]

[0257] wherein each c is independently an integer from 2 to 10.

[0258] In embodiments, the cationic lipid has a structure according to formula (III-B),

[0259]

[0260] wherein each c is independently an integer from 2 to 10.

[0261] In embodiments, the cationic lipid has a structure according to formula (III-C),

[0262]

[0263] wherein each c is independently an integer from 2 to 10.

[0264] In embodiments, the cationic lipid has a structure according to Formula (IV-C'),

[0265]

[0266] wherein each c is independently an integer from 2 to 10.

[0267] In embodiments, the cationic lipid has a structure according to Formula (IV-C"),

[0268] wherein each c is independently an integer from 2 to 10.

[0269] In another aspect, the present invention provides a cationic lipid having a structure according to formula (VI),

[0270]

[0271] in

[0272] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 Alkynyl or C1-C 15 Alkylene-C(O)2-C1-C 15 alkylene; and

[0273] Each c is independently an integer from 2 to 10.

[0274] In embodiments, each c is 4, 5, 6, 7, 8, 9, or 10. In embodiments, each c is 2, 3, or 4. In embodiments, each c is 2. In embodiments, each c is 3. In embodiments, each c is 4. In embodiments, each c is 5. In embodiments, each c is 6. In embodiments, each c is 7. In embodiments, each c is 8. In embodiments, each c is 9. In embodiments, each c is 10.

[0275] In an embodiment, each R A and R B It is an unsubstituted C6-C 30 In an embodiment, each R A and R B Yes (unsubstituted C3-C 15 Alkylene)-C(O)2-(unsubstituted C3-C 15 alkyl).

[0276] In an embodiment, each R A It is C6-C 30 Alkyl (e.g., C6-C 20 Alkyl) or C6-C 30Alkenyl (e.g., C6-C 20 In an embodiment, each R A It is an unsubstituted C6-C 30 Alkyl, C6-C 30 Hydroxyalkyl, unsubstituted C6-C 30 Alkenyl or C6-C 30 In an embodiment, each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0277] In an embodiment, each R A It is C6-C 30 In an embodiment, each R A It is an unsubstituted C6-C 30 In an embodiment, each R A It is substituted C6-C 30 In an embodiment, each R A It is C6-C 30 In an embodiment, each R A It is a straight chain C6-C 30 In an embodiment, each R A It is a branched C6-C 30 alkyl.

[0278] In an embodiment, each R A It is C6-C 20 In an embodiment, each R A It is an unsubstituted C6-C 20 In an embodiment, each R A It is substituted C6-C 20 In an embodiment, each R A It is C6-C 20 In an embodiment, each R A It is a straight chain C6-C 20 In an embodiment, each R A It is a branched C6-C 20 alkyl.

[0279] In an embodiment, each R A It is C6-C 30 In an embodiment, each R A It is an unsubstituted C6-C 30 In an embodiment, each RA It is substituted C6-C 30 In an embodiment, each R A It is C6-C 30 In an embodiment, each R A It is a straight chain C6-C 30 In an embodiment, each R A It is a branched C6-C 30 In an embodiment, C6-C 30 Alkenyl is a monoalkenyl, a dienyl or a trienyl.

[0280] In an embodiment, each R A It is C6-C 20 In an embodiment, each R A It is an unsubstituted C6-C 20 In an embodiment, each R A It is substituted C6-C 20 In an embodiment, each R A It is C6-C 20 In an embodiment, each R A It is a straight chain C6-C 20 In an embodiment, each R A It is a branched C6-C 20 In one embodiment, C6-C 20 Alkenyl is a monoalkenyl, a dienyl or a trienyl.

[0281] In an embodiment, each R A It is C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A It is an unsubstituted C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A It is substituted C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A It is C4-C 10 Alkylene-C(O)2-C4-C 10 Hydroxyalkyl.

[0282] In an embodiment, each R B It is C6-C 30 Alkyl (e.g., C6-C 20 Alkyl) or C6-C 30 Alkenyl (e.g., C6-C20 In an embodiment, each R B It is an unsubstituted C6-C 30 Alkyl, C6-C 30 Hydroxyalkyl, unsubstituted C6-C 30 Alkenyl or C6-C 30 In an embodiment, each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0283] In an embodiment, each R B It is C6-C 20 In an embodiment, each R B It is an unsubstituted C6-C 20 In an embodiment, each R B It is substituted C6-C 20 In an embodiment, each R B It is C6-C 20 In an embodiment, each R B It is a straight chain C6-C 20 In an embodiment, each R B It is a branched C6-C 20 alkyl.

[0284] In an embodiment, each R B It is C6-C 30 In an embodiment, each R B It is an unsubstituted C6-C 30 In an embodiment, each R B It is substituted C6-C 30 In an embodiment, each R B It is C6-C 30 In an embodiment, each R B It is a straight chain C6-C 30 In an embodiment, each R B It is a branched C6-C 30 In one embodiment, C6-C 30 Alkenyl is a monoalkenyl, a dienyl or a trienyl.

[0285] In an embodiment, each R B It is C6-C 20 In an embodiment, each R B It is an unsubstituted C6-C20 In an embodiment, each R B It is substituted C6-C 20 In an embodiment, each R B It is C6-C 20 In an embodiment, each R B It is a straight chain C6-C 20 In an embodiment, each R B It is a branched C6-C 20 In one embodiment, C6-C 20 Alkenyl is a monoalkenyl, a dienyl or a trienyl.

[0286] In an embodiment, each R B It is C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R B It is an unsubstituted C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R B It is substituted C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R B It is C4-C 10 Alkylene-C(O)2-C4-C 10 Hydroxyalkyl.

[0287] In an embodiment, R A With R B In an embodiment, R A With R B different.

[0288] In an embodiment, each R A and R B It is C6-C 30 In an embodiment, each R A and R B It is an unsubstituted C6-C 30 In an embodiment, each R A and R B It is substituted C6-C 30 In an embodiment, each R A and R B It is C6-C 30 In an embodiment, each R A and R B It is a straight chain C6-C 30In an embodiment, each R A and R B It is a branched C6-C 30 alkyl.

[0289] In an embodiment, each R A and R B It is C6-C 20 In an embodiment, each R A and R B It is an unsubstituted C6-C 20 In an embodiment, each R A and R B It is substituted C6-C 20 In an embodiment, each R A and R B It is C6-C 20 In an embodiment, each R A and R B It is a straight chain C6-C 20 In an embodiment, each R A and R B It is a branched C6-C 20 alkyl.

[0290] In an embodiment, each R A and R B It is C6-C 30 In an embodiment, each R A and R B It is an unsubstituted C6-C 30 In an embodiment, each R A and R B It is substituted C6-C 30 In an embodiment, each R A and R B It is C6-C 30 In an embodiment, each R A and R B It is a straight chain C6-C 30 In an embodiment, each R A and R B It is a branched C6-C 30 In one embodiment, C6-C 30 Alkenyl is a monoalkenyl, a dienyl or a trienyl.

[0291] In an embodiment, each R A and R B It is C6-C 20 In an embodiment, each R A and RB It is an unsubstituted C6-C 20 In an embodiment, each R A and R B It is substituted C6-C 20 In an embodiment, each R A and R B It is C6-C 20 In an embodiment, each R A and R B It is a straight chain C6-C 20 In an embodiment, each R A and R B It is a branched C6-C 20 In one embodiment, C6-C 20 Alkenyl is a monoalkenyl, a dienyl or a trienyl.

[0292] In an embodiment, each R A and R B It is C6-C 30 Hydroxyalkyl, or each R A and R B It is C6-C 30 In an embodiment, each R A and R B It is C6-C 20 Hydroxyalkyl, or each R A and R B It is C6-C 20 Hydroxyalkenyl.

[0293] In an embodiment, each R A and R B It is C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A and R B It is an unsubstituted C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A and R B It is substituted C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A and R B It is C4-C 10 Alkylene-C(O)2-C4-C 10 Hydroxyalkyl.

[0294] In an embodiment, each RA and R B It is C6-C 30 In an embodiment, each R A and R B It is C6-C 20 In an embodiment, each R A and R B It is -CH2CH(OH)C 10 H 21 In an embodiment, each R A and R B It is -CH2CH(OH)C8H 17 In an embodiment, each R A and R B It is -CH2CH(OH)C 12 H 25 In an embodiment, each R A and R B It is -CH2CH(OH)C 14 H 29 In an embodiment, each R A and R B It is -CH2CH(OH)C 16 H 33 In an embodiment, each R A and R B It is -CH2CH(OH)C 18 H 37 .

[0295] In an embodiment, each R A and R B It is C6-C 30 In an embodiment, each R A and R B It is C6-C 20 In an embodiment, each R A and R B Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 In an embodiment, each R A and R B It is -CH2CH(OH)(CH2)6(CH=CH)C8H 17 In an embodiment, each R A and R B It is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)CH2(CH=CH)C2H5.

[0296] In an embodiment, each R A and R B It is C4-C 10 Alkylene-C(O)2-C4-C 10 In an embodiment, each R A and R B It is -CH2CH(OH)(CH2)7C(O)2C7H 15 .

[0297] In an embodiment, each R A and R B It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0298]

[0299] In an embodiment, C6-C 30 Alkyl groups (e.g., each R A and / or each R B ) is C 8-26 In an embodiment, C6-C 30 Alkyl groups (e.g., each R A and / or each R B ) is a straight chain C 8-26 alkyl.

[0300] In an embodiment, C6-C 30 Alkyl groups (e.g., each R A and / or each R B ) 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) 22CH2-, CH3(CH2) 23 CH2- or CH3(CH2) 24 CH2-.

[0301] In an embodiment, C6-C 30 Alkyl groups (e.g., each R A and / or each R B ) 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-.

[0302] In an embodiment, C6-C 30 Alkyl groups (e.g., each R A and / or each R B ) is CH3(CH2) 14 CH2-, CH3(CH2) 15 CH2- or CH3(CH2) 16 CH2-.

[0303] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) is a C with one or two carbon-carbon double bonds 8-26 Alkenyl.

[0304] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) 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-, 11CH2-, cis-CH3(CH2)7CH=CH(CH2) 13 CH2-, 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-.

[0305] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) 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-.

[0306] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) 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-.

[0307] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) is cis-CH3(CH2)7CH=CH(CH2)7CH2- or cis, cis-CH3(CH2)4CH=CH-CH2CH=CH(CH2)7CH2-.

[0308] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B) is a C having three, four, five or six carbon-carbon double bonds 8-26 aliphatic.

[0309] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B) 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=CHCH2CH2CH=CH(CH2)3CH2-, trans, trans, trans-CH3(CH2)7CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CH(CH 2) 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 Formula -CH3(CH2)4CH=CHCH=CHCH=CHCH=CHCH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-, cis, cis, cis, cis, cis-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2-, cis, cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2 CH=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=CHCH2CH=CH(CH2)4CH2-.

[0310] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) 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-.

[0311] In an embodiment, C6-C 30 Alkenyl (e.g., each R A and / or each R B ) is cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2- or cis, cis, cis, cis-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-.

[0312] In some embodiments, each R A and / or each R B is independently an aliphatic chain of a saturated or unsaturated fatty acid, ie, R'-(CH2)- for a fatty acid R'-C(O)-. In some embodiments, each R A and / or each R B 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. A and / or each R B is an aliphatic chain of octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, or heptadecanoic acid. A and / or each R B is an aliphatic chain of lauric acid, tridecyl, myristic acid, or pentadecanoic acid. In some embodiments, each R A and / or each R Bis an aliphatic chain of lauric acid or myristic acid. A and / or each R B 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. A and / or each R B is an aliphatic chain of tetracosanoic acid, oleic acid, linoleic acid, pentacosanoic acid, or cerotic acid. A and / or each R B is an aliphatic chain of oleic acid, linoleic acid, or pentacosanoic acid. In some embodiments, each R A and / or each R B is an aliphatic chain of oleic acid or linoleic acid. A and / or each R B is an aliphatic chain of oleic acid. In some embodiments, each R A and / or each R B It is the aliphatic chain of linoleic acid.

[0313] Exemplary cationic lipids

[0314] Exemplary cationic lipids include cationic lipids such as cationic lipids (1), (2), (3), (4), (5), and (6),

[0315]

[0316]

[0317] In an embodiment, the cationic lipid is compound (1). In an embodiment, the cationic lipid is compound (2). In an embodiment, the cationic lipid is compound (3). In an embodiment, the cationic lipid is compound (4). In an embodiment, the cationic lipid is compound (5). In an embodiment, the cationic lipid is compound (6).

[0318] The present invention also provides such cationic lipids, which are:

[0319]

[0320] In embodiments, the cationic lipid is compound (7). In embodiments, the cationic lipid is compound (8). In embodiments, the cationic lipid is compound (9). In embodiments, the cationic lipid is compound (10). In embodiments, the cationic lipid is compound (11). In embodiments, the cationic lipid is compound (12). In embodiments, the cationic lipid is compound (13). In embodiments, the cationic lipid is compound (14). In embodiments, the cationic lipid is compound (15). In embodiments, the cationic lipid is compound (16).

[0321] In embodiments, the cationic lipids described herein can be derived from a combination of any one of the diacids in Table A and the thiols of Table B, including the following compounds as described herein.

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347] In an embodiment, the cationic lipid is compound (17). In an embodiment, the cationic lipid is compound (18). In an embodiment, the cationic lipid is compound (19). In an embodiment, the cationic lipid is compound (20). In an embodiment, the cationic lipid is compound (21). In an embodiment, the cationic lipid is compound (22). In an embodiment, the cationic lipid is compound (23). In an embodiment, the cationic lipid is compound (24). In an embodiment, the cationic lipid is compound (25). In an embodiment, the cationic lipid is compound (26). In an embodiment, the cationic lipid is compound (27). In an embodiment, the cationic lipid is compound (28). In an embodiment, the cationic lipid is compound (29). In an embodiment, the cationic lipid is compound (30). In an embodiment, the cationic lipid is compound (31). In an embodiment, the cationic lipid is compound (32). In an embodiment, the cationic lipid is compound (33). In an embodiment, the cationic lipid is compound (34). In an embodiment, the cationic lipid is compound (35). In an embodiment, the cationic lipid is compound (36). In an embodiment, the cationic lipid is compound (37). In an embodiment, the cationic lipid is compound (38). In an embodiment, the cationic lipid is compound (39). In an embodiment, the cationic lipid is compound (40). In an embodiment, the cationic lipid is compound (41). In an embodiment, the cationic lipid is compound (42). In an embodiment, the cationic lipid is compound (43). In an embodiment, the cationic lipid is compound (44). In an embodiment, the cationic lipid is compound (45). In an embodiment, the cationic lipid is compound (46).

[0348] In embodiments, the cationic lipid is compound (7). In embodiments, the cationic lipid is compound (8). In embodiments, the cationic lipid is compound (9). In embodiments, the cationic lipid is compound (10). In embodiments, the cationic lipid is compound (11). In embodiments, the cationic lipid is compound (12). In embodiments, the cationic lipid is compound (13). In embodiments, the cationic lipid is compound (14). In embodiments, the cationic lipid is compound (15). In embodiments, the cationic lipid is compound (16). In embodiments, the cationic lipid is compound (47). In embodiments, the cationic lipid is compound (48). In embodiments, the cationic lipid is compound (49). In embodiments, the cationic lipid is compound (50). In embodiments, the cationic lipid is compound (51). In embodiments, the cationic lipid is compound (52). In embodiments, the cationic lipid is compound (53). In embodiments, the cationic lipid is compound (54). In embodiments, the cationic lipid is compound (55). In embodiments, the cationic lipid is compound (56). In embodiments, the cationic lipid is compound (57). In embodiments, the cationic lipid is compound (58). In an embodiment, the cationic lipid is compound (59). In an embodiment, the cationic lipid is compound (60). In an embodiment, the cationic lipid is compound (61). In an embodiment, the cationic lipid is compound (62). In an embodiment, the cationic lipid is compound (63). In an embodiment, the cationic lipid is compound (64). In an embodiment, the cationic lipid is compound (65). In an embodiment, the cationic lipid is compound (66).

[0349] In an embodiment, the cationic lipid is compound (67). In an embodiment, the cationic lipid is compound (68). In an embodiment, the cationic lipid is compound (69). In an embodiment, the cationic lipid is compound (70). In an embodiment, the cationic lipid is compound (71). In an embodiment, the cationic lipid is compound (72). In an embodiment, the cationic lipid is compound (73). In an embodiment, the cationic lipid is compound (74). In an embodiment, the cationic lipid is compound (75). In an embodiment, the cationic lipid is compound (76). In an embodiment, the cationic lipid is compound (77). In an embodiment, the cationic lipid is compound (78). In an embodiment, the cationic lipid is compound (79). In an embodiment, the cationic lipid is compound (80). In an embodiment, the cationic lipid is compound (81). In an embodiment, the cationic lipid is compound (82). In an embodiment, the cationic lipid is compound (83). In an embodiment, the cationic lipid is compound (84). In an embodiment, the cationic lipid is compound (85). In an embodiment, the cationic lipid is compound (86). In an embodiment, the cationic lipid is compound (87). In an embodiment, the cationic lipid is compound (88). In an embodiment, the cationic lipid is compound (89). In an embodiment, the cationic lipid is compound (90). In an embodiment, the cationic lipid is compound (91). In an embodiment, the cationic lipid is compound (92). In an embodiment, the cationic lipid is compound (93). In an embodiment, the cationic lipid is compound (94). In an embodiment, the cationic lipid is compound (95). In an embodiment, the cationic lipid is compound (96).

[0350] In an embodiment, the cationic lipid is compound (97). In an embodiment, the cationic lipid is compound (98). In an embodiment, the cationic lipid is compound (99). In an embodiment, the cationic lipid is compound (100). In an embodiment, the cationic lipid is compound (101). In an embodiment, the cationic lipid is compound (102). In an embodiment, the cationic lipid is compound (103). In an embodiment, the cationic lipid is compound (104). In an embodiment, the cationic lipid is compound (105). In an embodiment, the cationic lipid is compound (106). In an embodiment, the cationic lipid is compound (107). In an embodiment, the cationic lipid is compound (108). In an embodiment, the cationic lipid is compound (109). In an embodiment, the cationic lipid is compound (110). In an embodiment, the cationic lipid is compound (111). In an embodiment, the cationic lipid is compound (112). In an embodiment, the cationic lipid is compound (113). In an embodiment, the cationic lipid is compound (114). In an embodiment, the cationic lipid is compound (115). In an embodiment, the cationic lipid is compound (116). In an embodiment, the cationic lipid is compound (117). In an embodiment, the cationic lipid is compound (118). In an embodiment, the cationic lipid is compound (119). In an embodiment, the cationic lipid is compound (120). In an embodiment, the cationic lipid is compound (121). In an embodiment, the cationic lipid is compound (122). In an embodiment, the cationic lipid is compound (123). In an embodiment, the cationic lipid is compound (124). In an embodiment, the cationic lipid is compound (125). In an embodiment, the cationic lipid is compound (126).

[0351] In an embodiment, the cationic lipid is compound (127). In an embodiment, the cationic lipid is compound (128). In an embodiment, the cationic lipid is compound (129). In an embodiment, the cationic lipid is compound (130). In an embodiment, the cationic lipid is compound (131). In an embodiment, the cationic lipid is compound (132). In an embodiment, the cationic lipid is compound (133). In an embodiment, the cationic lipid is compound (134). In an embodiment, the cationic lipid is compound (135). In an embodiment, the cationic lipid is compound (136). In an embodiment, the cationic lipid is compound (137). In an embodiment, the cationic lipid is compound (138). In an embodiment, the cationic lipid is compound (139). In an embodiment, the cationic lipid is compound (140). In an embodiment, the cationic lipid is compound (141). In an embodiment, the cationic lipid is compound (142). In an embodiment, the cationic lipid is compound (143). In an embodiment, the cationic lipid is compound (144). In an embodiment, the cationic lipid is compound (145). In an embodiment, the cationic lipid is compound (146). In an embodiment, the cationic lipid is compound (147). In an embodiment, the cationic lipid is compound (148). In an embodiment, the cationic lipid is compound (149). In an embodiment, the cationic lipid is compound (150). In an embodiment, the cationic lipid is compound (151). In an embodiment, the cationic lipid is compound (152). In an embodiment, the cationic lipid is compound (153). In an embodiment, the cationic lipid is compound (154). In an embodiment, the cationic lipid is compound (155). In an embodiment, the cationic lipid is compound (156).

[0352] Synthesis of cationic lipids

[0353] The cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) can be prepared according to methods known in the art. For example, the cationic lipids of the present invention can be prepared by thioesterification of the starting carboxylic acid followed by deprotection.

[0354] In some embodiments, the cationic lipids described herein (eg, cationic lipid (1)) can be prepared according to Scheme 1.

[0355] Solution 1

[0356]

[0357] For example, an alkylating agent (such as compound A) can be treated with a thiol reagent (such as benzyl mercaptan) to obtain a diprotected bifunctional intermediate (such as compound B). Deprotection of the phthalimide portion of compound B provides a nucleophilic compound C, which can then be treated with another electrophilic reagent (such as an epoxide (compound D)) to provide a tertiary amine (compound E). The hydroxyl group of compound E can be protected using protecting groups and conditions known in the art (e.g., TBSCl / imidazole) to provide the corresponding hydroxyl-protected compound F. The -SH functional group can then be deprotected (e.g., using reducing conditions such as Na / NH3) to provide the nucleophilic thiol compound G.

[0358] Thiols (such as Compound G) can be coupled to polycarboxylic acids (e.g., Compound H) to form thioester lipids (e.g., Compound J). When such lipids contain protecting groups, deprotection (e.g., HF / pyridine when PG=TBS) can provide cationic lipids described herein (e.g., Cationic Lipid 1).

[0359] Nucleic Acids

[0360] The cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) can be used to prepare compositions for the delivery of nucleic acids.

[0361] Nucleic acid synthesis

[0362] Nucleic acids according to the present invention can be synthesized according to any known method. For example, mRNA according to the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, 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.

[0363] In some embodiments, to prepare mRNA according to the present invention, a DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter for in vitro transcription, such as T3, T7, a mutated T7 or SP6 promoter, followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0364] 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 G / C content as high as possible, on the other hand, use the frequency of considering tRNA according to codon 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 the zone of RNA.

[0365] 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), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transgenic 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.

[0366] mRNA synthesis

[0367] mRNA according to the present invention can be synthesized according to any of a variety of known methods. For example, mRNA according to the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system that may comprise DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, a pyrophosphatase, and / or an RNAse inhibitor. 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 in the final product is undesirable and therefore can 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.

[0368] In some embodiments, 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.

[0369] 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 G / C content as high as possible, on the other hand, use the frequency of considering tRNA according to codon 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 the zone of RNA.

[0370] Modified mRNA

[0371] 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-oxyacetic acid methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (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. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and U.S. Patent No. 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0372] In some embodiments, mRNA can 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-thiophosphate)), borate phosphates, positively charged guanidinium groups, etc., which means that phosphodiester bonds are replaced by other anions, cations, or neutral groups.

[0373] In some embodiments, the mRNA may include sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides it comprises, including but not limited to sugar modifications selected from the group consisting of: 4'-thioribonucleotides (see, e.g., U.S. Patent Application Publication No. US2016 / 0031928, incorporated herein by reference), 2'-deoxy-2'-fluoro oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate), 2'-deoxy-2'-deoxyuridine 5'-triphosphate, ... phosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-arabinocytidine 5'-triphosphate, 2'-arabinouridine 5'-triphosphate) or azidotriphosphate (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0374] In some embodiments, mRNA can include the modification (base modification) of the base of nucleotide. The modified nucleotides comprising base modification are also referred to as base modified nucleotides. The 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-iodine 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.

[0375] 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 degradation by exonucleases.

[0376] 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 one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, generating a 5'5'5 triphosphate bond; and then, a methyltransferase methylates the 7-nitrogen of guanine. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5')A and G(5')ppp(5')G.

[0377] In some embodiments, mRNA includes 3 ' poly (A) tail structure. The poly A tail at mRNA 3 ' end generally includes about 10 to 300 adenosine nucleotides (for example, 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 mRNA 3 ' end generally includes about 10 to 200 cytosine nucleotides (for example, 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.

[0378] 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.

[0379] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization and 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.

[0380] Cap structure

[0381] In some embodiments, the mRNA includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, generating a 5'5'5 triphosphate bond; and then, a methyltransferase methylates the 7-nitrogen of guanine. 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.

[0382] The naturally occurring cap structure consists of 7-methylguanosine linked to the 5'-end of the first transcribed nucleotide via a triphosphate bridge, resulting in m 7A dinucleotide cap consisting of G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added enzymatically. It is added to the nucleus of the cell and catalyzed by the enzyme guanylyltransferase. The cap is added to the 5' end of the RNA immediately after transcription begins. The terminal nucleoside is typically guanosine and is oriented in the opposite direction to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0383] The 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 mRNA. 7 G(5')ppp(5')G(“m 7 GpppG") dinucleotides serve as transcription initiators.

[0384] To date, the common form of synthetic dinucleotide cap 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.

[0385] 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 symmetrical 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, eg, Jemielity, J. et al., “Novel 'anti-reverse' cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).

[0386] In some embodiments, a suitable cap is a 7-methylguanylate ("m-guanylate") linked to the 5'-end of the first transcribed nucleotide via a triphosphate bridge. 7 G”), thus obtaining m 7 G(5')ppp(5')N, wherein N is any nucleoside. 7 The preferred embodiment of the G cap is m 7 G(5')ppp(5')G.

[0387] In some embodiments, the cap is a Cap0 structure. The Cap0 structure lacks the 2'-O-methyl residues attached to the ribose sugars at 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 Cap2 structure has 2'-O-methyl residues attached to both bases 2 and 3.

[0388] Various 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 use in 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.

[0389] Tail structure

[0390] Typically, the presence of a "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 an mRNA molecule to make RNA more stable. A variety of techniques recognized in the art can be used to add a poly A tail. For example, a long poly A tail can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe et al., Nature Biotechnology.1996; 14: 1252-1256). Transcription vectors can also encode long poly A tails. In addition, poly A tails can be added by direct transcription from a PCR product. Poly A can also be connected to the 3' end of 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)).

[0391] 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, or at least 500 nucleotides. In some embodiments, the poly A tail on the 3' end of the mRNA typically comprises about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNA includes a 3' poly (C) tail structure. Suitable poly-C tails on the 3' end of the mRNA typically 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.

[0392] 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 molecules of the present invention, thereby controlling protein transcription. For example, because the length of the poly A tail can affect the half-life of the sense mRNA molecule, the length of the poly A tail can be adjusted to change the level of resistance of the mRNA to nucleases, thereby controlling the time course of polynucleotide expression and / or polypeptide production in the target cell.

[0393] 5' and 3' untranslated regions

[0394] 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.

[0395] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization and 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.

[0396] Exemplary 3' and / or 5'UTR sequences can be derived from stable mRNA molecules (for example, globulin, actin, GAPDH, tubulin, histone or citric acid cycle enzyme) to increase the stability of sense mRNA molecules. For example, 5'UTR sequences can include a partial sequence of CMV immediate early 1 (IE1) gene or a fragment thereof, to improve nuclease resistance and / or improve the half-life of polynucleotides. It is also contemplated that 3' ends or non-translated regions of polynucleotides (for example, mRNA) comprise sequences encoding human growth hormone (hGH) or its fragments to further stabilize the polynucleotides. Typically, these modifications improve the stability and / or pharmacokinetic properties (for example half-life) of polynucleotides relative to their unmodified counterparts, and include, for example, modifications carried out to improve the resistance of such polynucleotides to nuclease digestion in vivo.

[0397] Cationic lipid and nucleic acid pharmaceutical preparations

[0398] In certain embodiments, the cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)), and pharmaceutical and liposome compositions comprising such lipids, can be used in formulations to facilitate delivery of encapsulated substances (e.g., one or more polynucleotides, such as mRNA) to one or more target cells, and subsequent transfection of one or more target cells. For example, in certain embodiments, the 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 that provide advantages for such compounds over other similarly classified lipids.

[0399] According to the present invention, a nucleic acid as described herein, e.g., an mRNA encoding a protein (e.g., full length, fragment, or portion of a protein), can be delivered via a delivery vehicle comprising a cationic lipid (e.g., any one of Formulas (I)-(VI), such as any one of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any one of compounds (I)-(156)).

[0400] As used herein, the terms "delivery vehicle," "transport vehicle," "nanoparticle," or grammatical equivalents are used interchangeably.

[0401] For example, the present invention provides compositions (e.g., pharmaceutical compositions) comprising a cationic lipid described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)). The composition (e.g., pharmaceutical composition) can 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.

[0402] In certain embodiments, the composition shows enhanced (e.g., improved) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally include contacting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising a cationic lipid as described herein (e.g., any one of formula (I)-(VI), such as formula (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B'), (IV-B"), (IV-C), (IV-C') or (IV-C"), or a cationic lipid of any one of compounds (I)-(156)) to facilitate transfection of the one or more target cells with a substance (e.g., one or more polynucleotides) encapsulated in the composition. 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 a cell, or preferably into a target cell. The introduced polynucleotides can be maintained stably or transiently in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulating materials (e.g., polynucleotides) taken up, introduced and / or expressed by the target cells undergoing transfection. In fact, the transfection efficiency can be estimated by the amount of reporter polynucleotide products produced by the target cells after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby increasing the possibility of delivering an appropriate dose of encapsulating material (e.g., one or more polynucleotides) to the pathological site and subsequently expressing it, while minimizing potential systemic adverse reactions or toxicity associated with the compound or its encapsulated contents.

[0403] After transfection of one or more target cells by, for example, a polynucleotide encapsulated in one or more lipid nanoparticles comprising a pharmaceutical composition or liposome composition disclosed herein, the production of a product (e.g., a polypeptide or protein) encoded by such polynucleotide can be preferably stimulated, and the ability of such target cells to express the polynucleotide and produce, for example, a polypeptide or protein 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 a protein or enzyme encoded by such mRNA.

[0404] In addition, delivery vehicle as herein described (for example liposome delivery vehicle) can be prepared into and preferentially be distributed to other target tissues, cells or organs, such as heart, lung, kidney, spleen.In embodiments, lipid nanoparticle of the present invention can be prepared to realize the delivery of the enhancement to target cell and tissue.For example, the polynucleotide (for example, mRNA) that is encapsulated in one or more compounds as herein described or pharmaceutical composition and liposome composition can be delivered to and / or transfected target cell or tissue.In some embodiments, the polynucleotide (for example, mRNA) of encapsulation can be expressed by target cell and produced (and in some cases secreted) functional polypeptide product by target cell, thus giving the characteristic that for example target cell or tissue are useful.The polynucleotide (for example, mRNA) of this type of encapsulation can encode for example hormone, enzyme, receptor, polypeptide, peptide or other protein of interest.

[0405] Liposomal delivery vehicles

[0406] In some embodiments, the composition is a suitable delivery vehicle. In embodiments, the composition is a liposomal delivery vehicle, such as a lipid nanoparticle.

[0407] The terms "liposomal delivery vehicle" and "liposomal composition" are used interchangeably.

[0408] Enriching liposome compositions with one or more cationic lipids disclosed herein can be used as a method to improve (e.g., reduce) toxicity or otherwise impart 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 the liposome composition). Thus, pharmaceutical compositions, particularly liposome compositions, comprising one or more cationic lipids disclosed herein are also contemplated.

[0409] Thus, in certain embodiments, a compound described herein (e.g., a cationic lipid of any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) is a cationic lipid that can be used as a component of a liposome composition to facilitate or enhance the delivery and release of an encapsulated substance (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membrane of such target cells).

[0410] As used herein, liposome delivery vehicles, 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 amphiphilic molecules, such as synthetic or naturally derived 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 amphiphilic polymers and surfactants (e.g., polymer vesicles, noids, etc.). In the context of the present invention, liposome delivery vehicles are generally used for transporting desired mRNA to target cells or tissues.

[0411] In certain embodiments, such compositions (eg, liposomal compositions) are loaded with or otherwise encapsulate a material, such as one or more biologically active polynucleotides (eg, mRNA).

[0412] In embodiments, the composition (e.g., a pharmaceutical composition) comprises an mRNA encoding a protein encapsulated within a liposome. In embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, and at least one cationic lipid is a cationic lipid as described herein (e.g., any one of formula (I)-(VI), such as formula (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any one of compounds (I)-(156)). In embodiments, the composition comprises an mRNA encoding a protein (e.g., any protein described herein). In embodiments, the composition comprises an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In embodiments, the composition comprises an mRNA encoding an ornithine transcarbamylase (OTC) protein. In embodiments, the mRNA encodes an antigen (eg, an antigen from an infectious agent).

[0413] In embodiments, a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises any cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)).

[0414] 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., mRNA encoding 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 liver cells (e.g., mRNA encoding ornithine carbamoyltransferase (OTC) protein). Other exemplary mRNAs are also described herein.

[0415] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) may have a net positive charge.

[0416] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) may have a net negative charge.

[0417] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) may have a net neutral charge.

[0418] In embodiments, lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding a peptide or polypeptide) comprise one or more cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)).

[0419] For example, the amount of a cationic lipid as described herein (e.g., a cationic lipid of any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) in a composition can be described as a percentage ("wt %") of the combined dry weight of all lipids of the composition (e.g., the combined dry weight of all lipids present in the liposome composition).

[0420] In embodiments of the pharmaceutical compositions described herein, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)) is present in an amount of about 0.5 wt % to about 30 wt % (e.g., about 0.5 wt % to about 20 wt %) of the combined dry weight of all lipids present in the composition (e.g., a liposome composition).

[0421] In embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)) is present in an amount from about 1% to about 30%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 5% to about 25% by weight of the combined dry weight of all lipids present in the composition (e.g., a liposome composition). In embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) is present in an amount of about 0.5% to about 5%, about 1% to about 10%, about 5% to about 20%, or about 10% to about 20% by weight of the combined molar amount of all lipids present in the composition (e.g., a liposomal delivery vehicle).

[0422] In embodiments, the amount of a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) is less than or equal to the amount of a cationic lipid as described herein. The lipids are 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 combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0423] In embodiments, the amount of a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) is less than or equal to the amount of a cationic lipid as described herein. is present in an amount of no more than 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 combined dry weight of the total lipids in a composition (e.g., a liposomal composition).

[0424] In embodiments, the composition (e.g., a liposomal delivery vehicle, such as a lipid nanoparticle) comprises from about 0.1% to about 20% by weight (e.g., from about 0.1% to about 15% by weight) of a cationic lipid described herein (e.g., a cationic lipid of any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)). 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 any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)). In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle, such as a lipid nanoparticle) comprises up to about 0.5%, about 1%, about 3%, about 5%, about 10%, about 15%, or about 20% by weight of a cationic lipid described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)). In embodiments, this percentage results in an improved beneficial effect (e.g., improved delivery to a target tissue such as the liver or lung).

[0425] The amount of a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) in a composition can also be described as a percentage ("mole %") of the combined molar amount of the total lipids of the composition (e.g., the combined molar amounts of all lipids present in the liposomal delivery vehicle).

[0426] In embodiments of the pharmaceutical compositions described herein, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) is present in an amount of about 0.5 mol% to about 30 mol% (e.g., about 0.5 mol% to about 20 mol%) of the combined molar amount of all lipids present in the liposomal delivery vehicle.

[0427] In embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) 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 combined molar amount of all lipids present in the composition (such as a liposomal delivery vehicle). In embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) is present in an amount of about 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, or about 5 mol% to about 25 mol% of the combined dry weight of all lipids present in a composition (such as a liposomal delivery vehicle).

[0428] In certain embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) can 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 liposomal delivery vehicle).

[0429] In certain embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)) can comprise 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 amount of lipid in the lipid nanoparticle.

[0430] In certain embodiments, a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) can comprise less than about 25 mole%, or less than about 10 mole%, or less than about 5 mole%, or less than about 1 mole% of the total amount of lipid in a composition (e.g., a liposomal delivery vehicle).

[0431] In embodiments, the amount of a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) is less than or equal to the amount of a cationic lipid as described herein. The lipids are present in an amount of at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0432] In embodiments, the amount of a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) is less than or equal to the amount of a cationic lipid as described herein. The lipids are present in an amount of no more than about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0433] In embodiments, this percentage results in an improved beneficial effect (eg, improved delivery to a target tissue such as the liver or lung).

[0434] 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).

[0435] In certain embodiments, such pharmaceutical (e.g., liposome) compositions comprise one or more of PEG-modified lipids, non-cationic lipids, and cholesterol lipids. In embodiments, such pharmaceutical (e.g., liposome) compositions comprise: 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 comprise: one or more PEG-modified lipids and one or more cholesterol lipids.

[0436] 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 as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (1)-(156)), and the one or more lipids are selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.

[0437] 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 as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)); the one or more lipids are selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids; and further include cholesterol-based lipids.

[0438] 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 as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)), and one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, a PEGylated lipid, and a cholesterol-based lipid.

[0439] In embodiments of the lipid nanoparticles described herein, the lipid nanoparticles comprise one or more cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (I)-(156)), a non-cationic lipid (e.g., DOPE), a PEGylated lipid (e.g., DMG-PEG2000), and a cholesterol-based lipid (e.g., cholesterol).

[0440] 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 property of the expected target cell, 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 mol ratio can be adjusted accordingly.

[0441] Other cationic lipids

[0442] In addition to any of the cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of compounds (1)-(156)), the composition may also include one or more additional cationic lipids.

[0443] In some embodiments, liposomes can include one or more other cationic lipids. As used herein, phrase "cationic lipid" refers to any of a variety of lipid substances having a net positive charge at a selected pH such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

[0444] In some embodiments, liposomes can include one or more other cationic lipids. As used herein, phrase "cationic lipid" refers to any of a variety of lipid substances having a net positive charge at a selected pH such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

[0445] Suitable additional cationic lipids for compositions include cationic lipids as described in International Patent Publication WO 2010 / 144740, which is incorporated herein by reference. In certain embodiments, these compositions comprise cationic lipid (6Z, 9Z, 28Z, 31Z)-4-(dimethylamino)butyric acid-heptatriacontac-6,9,28,31-tetraene-19-yl ester having the following compound structure:

[0446]

[0447] and pharmaceutically acceptable salts thereof.

[0448] Other suitable additional cationic lipids for use in the composition include ionizable cationic lipids as described in International Patent Publication WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid of one of the following formulas:

[0449]

[0450] or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently selected from the group consisting of hydrogen, optionally substituted different saturated or unsaturated C1-C 20 alkyl and optionally substituted different saturated or unsaturated C6-C 20 Acyl; wherein L1 and L2 are each independently selected from the group consisting of hydrogen, optionally substituted C1-C 30 Alkyl, optionally substituted different unsaturated C1-C 30 Alkenyl and optionally substituted C1-C 30 Alkynyl; wherein m and o are each independently selected from the group consisting of: zero and any positive integer (e.g., wherein m is three); and wherein n is zero or any positive integer (e.g., wherein n is one). In certain embodiments, the composition comprises a cationic lipid (15Z, 18Z)-N, N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the following compound structure:

[0451]

[0452] and pharmaceutically acceptable salts thereof. In certain embodiments, the composition comprises a cationic lipid (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") having the following compound structure:

[0453]

[0454] and pharmaceutically acceptable salts thereof. In certain embodiments, the invention comprises a cationic lipid and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002"), wherein the cationic lipid has the following compound structure:

[0455]

[0456] and pharmaceutically acceptable salts thereof.

[0457] Other suitable additional cationic lipids for compositions include cationic lipids described as amino alcohol lipids in International Patent Publication WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0458]

[0459] and pharmaceutically acceptable salts thereof.

[0460] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publication WO 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0461]

[0462] and pharmaceutically acceptable salts thereof.

[0463] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publication WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0464]

[0465] and pharmaceutically acceptable salts thereof.

[0466] Other suitable cationic lipids for use in the composition include cationic lipids having the general formula 14,25-di-tridecyl-15,18,21,24-tetraaza-octatriacontane and pharmaceutically acceptable salts thereof.

[0467] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publications WO 2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid of the formula:

[0468]

[0469] or a pharmaceutically acceptable salt thereof, wherein R L Each instance of is independently optionally substituted C6-C 40 In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0470]

[0471] and pharmaceutically acceptable salts thereof.

[0472] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0473] and pharmaceutically acceptable salts thereof.

[0474] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0475] and pharmaceutically acceptable salts thereof.

[0476] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0477]

[0478] and pharmaceutically acceptable salts thereof.

[0479] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publication No. WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid of the formula:

[0480]

[0481] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R A R is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; and each R B In certain embodiments, the composition comprises a cationic lipid "target 23" having the following compound structure:

[0482]

[0483] and pharmaceutically acceptable salts thereof.

[0484] Other suitable additional cationic lipids for compositions include cationic lipids as described in International Patent Publication WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0485] in,

[0486]

[0487] or a pharmaceutically acceptable salt thereof.

[0488] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0489]

[0490] or a pharmaceutically acceptable salt thereof.

[0491] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0492]

[0493] or a pharmaceutically acceptable salt thereof.

[0494] Other suitable additional cationic lipids for compositions include those described in J.McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5: 4277, which are incorporated herein by reference. In certain embodiments, the cationic lipid of the compositions includes a cationic lipid with the following compound structure:

[0495]

[0496] and pharmaceutically acceptable salts thereof.

[0497] Other suitable additional cationic lipids for compositions include cationic lipids as described in International Patent Publication WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0498]

[0499] and pharmaceutically acceptable salts thereof.

[0500] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0501]

[0502] and pharmaceutically acceptable salts thereof.

[0503] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0504]

[0505] and pharmaceutically acceptable salts thereof.

[0506] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0507]

[0508] and pharmaceutically acceptable salts thereof.

[0509] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0510]

[0511] and pharmaceutically acceptable salts thereof.

[0512] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0513]

[0514] and pharmaceutically acceptable salts thereof.

[0515] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0516]

[0517] and pharmaceutically acceptable salts thereof.

[0518] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0519] and pharmaceutically acceptable salts thereof.

[0520] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0521] and pharmaceutically acceptable salts thereof.

[0522] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0523] and pharmaceutically acceptable salts thereof.

[0524] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0525] and pharmaceutically acceptable salts thereof.

[0526] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0527]

[0528] and pharmaceutically acceptable salts thereof.

[0529] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0530]

[0531] and pharmaceutically acceptable salts thereof.

[0532] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publication No. WO 2017 / 004143, which is incorporated herein by reference.

[0533] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0534]

[0535] and pharmaceutically acceptable salts thereof.

[0536] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0537]

[0538] and pharmaceutically acceptable salts thereof.

[0539] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0540]

[0541] and pharmaceutically acceptable salts thereof.

[0542] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0543]

[0544] and pharmaceutically acceptable salts thereof.

[0545] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0546]

[0547] and pharmaceutically acceptable salts thereof.

[0548] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0549]

[0550] and pharmaceutically acceptable salts thereof.

[0551] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0552]

[0553] and pharmaceutically acceptable salts thereof.

[0554] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0555]

[0556] and pharmaceutically acceptable salts thereof.

[0557] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0558]

[0559] and pharmaceutically acceptable salts thereof.

[0560] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0561]

[0562] and pharmaceutically acceptable salts thereof.

[0563] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0564]

[0565] and pharmaceutically acceptable salts thereof.

[0566] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0567]

[0568] and pharmaceutically acceptable salts thereof.

[0569] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0570]

[0571] and pharmaceutically acceptable salts thereof.

[0572] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0573]

[0574] and pharmaceutically acceptable salts thereof.

[0575] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0576]

[0577] and pharmaceutically acceptable salts thereof.

[0578] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0579]

[0580] and pharmaceutically acceptable salts thereof.

[0581] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0582]

[0583] and pharmaceutically acceptable salts thereof.

[0584] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publication WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid of the formula:

[0585]

[0586] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-; and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR aC(=O)O- or direct bond; G 1 and G 2 Each independently is an unsubstituted C1-C 12 Alkylene or C1-C 12 Alkenylene; G 3 It is C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a Is H or C1-C 12 Alkyl; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 It is H, OR 5 、CN、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 It is C1-C 12 Alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0587] Other suitable additional cationic lipids for use in the composition include cationic lipids as described in International Patent Publication WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0588]

[0589] and pharmaceutically acceptable salts thereof.

[0590] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0591]

[0592] and pharmaceutically acceptable salts thereof.

[0593] In some embodiments, the composition comprises a cationic lipid having the following compound structure:

[0594]

[0595] and pharmaceutically acceptable salts thereof.

[0596] Other suitable additional cationic lipids for use in the compositions include cationic lipids as described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the compositions and methods of the present invention include compounds having one of the following formulae:

[0597]

[0598] and pharmaceutically acceptable salts thereof. For any of the four general formulae, R4 is independently selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R), -N(H)C(S)N(H)(R) and heterocycle; R is independently selected from the group consisting of C 1-3 Alkyl, C 2-3 alkenyl and H; and n is 1, 2 or 3.

[0599] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0600]

[0601] and pharmaceutically acceptable salts thereof.

[0602] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0603]

[0604] and pharmaceutically acceptable salts thereof.

[0605] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0606]

[0607] and pharmaceutically acceptable salts thereof.

[0608] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0609]

[0610] and pharmaceutically acceptable salts thereof.

[0611] Other suitable additional cationic lipids for use in the compositions include cationic lipids as described in International Patent Publications WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference.

[0612] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0613]

[0614] and pharmaceutically acceptable salts thereof.

[0615] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0616]

[0617] and pharmaceutically acceptable salts thereof.

[0618] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0619]

[0620] and pharmaceutically acceptable salts thereof.

[0621] In certain embodiments, the composition comprises a cationic lipid having the following compound structure:

[0622]

[0623] and pharmaceutically acceptable salts thereof.

[0624] Other suitable additional cationic lipids for use in compositions include cholesterol-based cationic lipids. In certain embodiments, the compositions comprise imidazole cholesterol ester or "ICE" having the following compound structure:

[0625]

[0626] and pharmaceutically acceptable salts thereof.

[0627] Other suitable additional cationic lipids for use in the composition include cleavable cationic lipids as described in International Patent Publication WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the composition comprises a cationic lipid of the formula:

[0628]

[0629] wherein R1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R2 is selected from the group consisting of one of the following two general formulae:

[0630]

[0631] and wherein R3 and R4 are each independently selected from the group consisting of: optionally substituted different saturated or unsaturated C6-C 20 alkyl and optionally substituted different saturated or unsaturated C6-C 20 acyl; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more).

[0632] In certain embodiments, the composition comprises a cationic lipid "HGT4001" having the following compound structure:

[0633]

[0634] and pharmaceutically acceptable salts thereof.

[0635] In certain embodiments, the composition comprises a cationic lipid "HGT4002" having the following compound structure:

[0636]

[0637] and pharmaceutically acceptable salts thereof.

[0638] In certain embodiments, the composition comprises a cationic lipid "HGT4003" having the following compound structure:

[0639]

[0640] (HGT4003)

[0641] and pharmaceutically acceptable salts thereof.

[0642] In certain embodiments, the composition comprises a cationic lipid "HGT4004" having the following compound structure:

[0643]

[0644] and pharmaceutically acceptable salts thereof.

[0645] In certain embodiments, the composition comprises a cationic lipid "HGT4005" having the following compound structure:

[0646]

[0647] (HGT4005)

[0648] and pharmaceutically acceptable salts thereof.

[0649] In some embodiments, the composition comprises a cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"). Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, each of which is incorporated herein by reference. DOTMA can be formulated alone or combined with a neutral lipid (e.g., dioleoylphosphatidylethanolamine or "DOPE") or other cationic or non-cationic lipids into liposome transfer vehicles or lipid nanoparticles, such liposomes can be used to enhance the delivery of nucleic acids to target cells. Other cationic lipids suitable for use in these compositions include, for example, 5-carboxysperminylglycine di-octadecylamide ("DOGS"), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propylammonium ("DOSPA") (Behr et al., Proc. Nat'l Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678, U.S. Pat. No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"), 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0650] Additional exemplary cationic lipids suitable for use in these compositions also include: 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"), N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N,N-distearoyl-N,N-dimethylammonium bromide ("DDAB"), N-(1,2-dimyristyloxyprop-3-yl)-N,N- Dimethyl-N-hydroxyethylammonium bromide ("DMRIE"), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane ("CLinDMA"), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienyloxy)propane ("CpLinDMA"), N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane ("DOcarbDAP"), 2,3-dilinoleyloxy -N,N-dimethylpropylamine ("DLinDAP"), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLincarbDAP"), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLinCDAP"), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"), 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA"), (2R)-2-((8-[( (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (“octyl-CLinDMA(2R)”), (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (“octyl-CLinDMA(2S)”), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (“DLin-K-XTC2-DMA”), and 2-(2,2-di((9Z,(12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethylamine ("DLin-KC2-DMA") (see WO 2010 / 042877, which is incorporated herein by reference; Semple et al., Nature Biotech. 28: 172-176 (2010)). (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23 (8): 1003-1007 (2005); International Patent Publication WO 2005 / 121348). In some embodiments, the one or more cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0651] In some embodiments, one or more cationic lipids suitable for use in these compositions include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (“XTC”), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (“ALNY-100”), and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-di-undecyl-4,7,10,13-tetraazahexadecane-1,16-diamide (“NC98-5”).

[0652] 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 %).

[0653] 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 (mole %) or by weight (wt %).

[0654] In some embodiments, the total cationic lipids constitute about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposomes by weight. In some embodiments, the cationic lipids 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% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposomes by weight. In some embodiments, the cationic lipids constitute about 30%, about 35%, about 40%, about 45%, or about 50% of the composition (e.g., liposome composition) by weight.

[0655] In some embodiments, the composition comprises one or more cationic lipids that comprise at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight of the total lipid content in the composition (e.g., lipid nanoparticle). In some embodiments, the composition comprises one or more cationic lipids that comprise at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by mole of the total lipid content in the composition (e.g., lipid nanoparticle). In some embodiments, the composition comprises one or more cationic lipids that comprise about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content of the composition (e.g., lipid nanoparticle) by weight. In some embodiments, the composition comprises one or more cationic lipids that comprise about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content of the composition (e.g., lipid nanoparticle) by mole%.

[0656] Non-cationic / helper lipids

[0657] 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 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, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof.

[0658] In an embodiment, the non-cationic or helper lipid is dioleoylphosphatidylethanolamine (DOPE).

[0659] 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.

[0660] In some embodiments, the non-cationic lipid can exist in the composition at 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 TL present in the non-cationic lipid. In some embodiments, the total non-cationic lipid can exist in the composition at 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%. In some embodiments, the percentage ratio of non-cationic lipid 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 lipid in the liposome can be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol% or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipid in the liposome 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 lipid in the liposome 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%.

[0661] In some embodiments, the non-cationic lipid can exist at 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 TL present in the compositions. In some embodiments, the total non-cationic lipid can exist at 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 TL present in the compositions. In some embodiments, the per-cent of non-cationic lipid 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 the liposome can be greater than about 5 % by weight, greater than about 10 % by weight, greater than about 20 % by weight, greater than about 30 % by weight or greater than about 40 % by weight. In some embodiments, the percentage of non-cationic lipid in the liposome is no more than about 5 % by weight, no more than about 10 % by weight, no more than about 20 % by weight, no more than about 30 % by weight or no more than about 40 % by weight. In some embodiments, the percentage of total non-cationic lipid in the liposome can be no more than about 5 % by weight, no more than about 10 % by weight, no more than about 20 % by weight, no more than about 30 % by weight or no more than about 40 % by weight.

[0662] Cholesterol-based lipids

[0663] 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. Patent No. 5,744,335) or imidazole cholesterol ester (ICE), which has the following structure,

[0664]

[0665] (“ICE”).

[0666] In embodiments, the cholesterol-based lipid is cholesterol.

[0667] In some embodiments, the lipid based on cholesterol can exist with a mol 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 ratio of the lipid based on cholesterol in the lipid nanoparticle 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 ratio of the lipid based on cholesterol in the lipid nanoparticle 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 %.

[0668] In some embodiments, the cholesterol-based lipid can exist with a weight ratio (wt %) of about 1% to about 30% or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage ratio of the cholesterol-based lipid in the lipid nanoparticle 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 ratio of the cholesterol-based lipid in the lipid nanoparticle 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 %.

[0669] PEGylated lipids

[0670] In some embodiments, a composition (eg, a liposomal composition) comprises one or more PEGylated lipids.

[0671] 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 ).

[0672] 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, lipids with C6-C 20In some embodiments, the PEG-modified lipid or PEGylated lipid is a PEGylated cholesterol or PEG-2K. Adding such components can prevent complex aggregation and can also provide a method for extending 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 formulation in vivo (see U.S. Patent No. 5,885,613).

[0673] In an embodiment, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000).

[0674] 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).

[0675] The PEG-modified phospholipids and derivatized lipids of the present invention can be present at 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).

[0676] Pharmaceutical preparations and therapeutic uses

[0677] The cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) can be used to prepare compositions (e.g., to construct liposome compositions) that can facilitate or enhance the delivery and release of an encapsulated substance (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membrane of such target cells).

[0678] For example, when a liposomal 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 the encapsulated material (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) to the one or more target cells.

[0679] Similarly, in certain embodiments, the cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) can be used to prepare liposomal vehicles characterized by reduced toxicity in vivo. 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.

[0680] Thus, pharmaceutical formulations comprising a cationic lipid as described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) and the nucleic acids provided herein can be used for a variety of therapeutic purposes. To facilitate in vivo delivery of nucleic acids, the cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or a cationic lipid of any of Compounds (I)-(156)) and nucleic acids can be formulated in combination with one or more additional drug carriers, targeting ligands, or stabilizers. In some embodiments, the cationic lipids described herein (e.g., any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (1)-(156)) can be formulated via a premixed lipid solution. In other embodiments, a composition comprising a cationic lipid described herein (e.g., a cationic lipid of any of Formulas (I)-(VI), such as any of Formulas (IA)-(IC), (I-A')-(I-C'), (IB"), (IC"), (II-A), (II-B), (III-A), (III-B), (IV-A), (IV-B), (IV-B'), (IV-B"), (IV-C), (IV-C'), or (IV-C"), or any of Compounds (I)-(156)) 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., most recent edition.

[0681] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation, or enteral administration; Parenteral delivery includes intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection; and intrathecal, directly intraventricular, intravenous, intraperitoneal or intranasal. In specific embodiments, intramuscular administration is to the muscle of the group selected from 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.

[0682] Alternatively or in addition, the pharmaceutical formulations of the present invention can be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical formulation 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, the 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.

[0683] 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 aerosolization. In embodiments, the delivery vehicle can be an inhalable aerosolized composition. In embodiments, pulmonary delivery involves inhalation (e.g., for nasal, tracheal, or bronchial delivery). In embodiments, compositions are aerosolized prior to inhalation.

[0684] 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 cells of a human subject or cells processed and delivered to a human subject.

[0685] 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, the therapeutic composition being 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 with 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 with 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 with 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 with 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 with a full-length mRNA encoding an alpha-1-antitrypsin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition with 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.

[0686] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding peptides or polypeptides for delivery to or treatment of the liver or hepatocytes of a subject. Such peptides and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or 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.

[0687] 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 1 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.

[0688] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding proteins associated with lysosomal storage diseases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding α-galactosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding glucocerebrosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding isocyanate-2-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding iduronidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding N-acetyl-α-D-glucosaminidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding heparan N-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding galactosamine-6-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding a β-galactosidase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding a lysosomal lipase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding an arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding a transcription factor EB (TFEB).

[0689] 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 α-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.

[0690] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins related to amino acid metabolism. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding phenylalanine hydroxylase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glutaryl-CoA dehydrogenase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding propionyl-CoA carboxylase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding oxalase alanine-glyoxylate aminotransferase.

[0691] 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 transporter 8B1 (ATP8B1) protein. 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 protein or active variants.

[0692] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding a methylmalonyl-CoA mutant enzyme protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding a methylmalonyl-CoA epimerase protein.

[0693] In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA, for which delivery to or treatment of the liver can provide therapeutic benefits. In certain embodiments, the present 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 present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding porphobilinogen deaminase. In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding one or more coagulase, such as factor VIII, factor IX, factor VII, and factor X. In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding human hemochromatosis (HFE) protein.

[0694] 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.

[0695] 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 a subject's muscle or muscle cells. 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, the therapeutic composition is used to deliver to or treat a subject's myocardium or myocardial cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein that regulates 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 that regulates Kv7.1 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 that regulates Nav1.5 channels in muscle tissue or muscle cells.

[0696] In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, the therapeutic composition being used to deliver to or treat a subject's nervous system or nervous system cells. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding survival motor neuron 2 protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the present invention provides methods 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 methods for preparing a therapeutic composition having a full-length mRNA encoding a CLN3 protein.

[0697] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding peptides or polypeptides for delivery to or treatment of the blood or bone marrow or blood cells or bone marrow cells of a subject. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding beta globin. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding Bruton's tyrosine kinase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.

[0698] 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 kidney or renal cells. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding type IV collagen α5 chain (COL4A5) protein.

[0699] 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 being 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.

[0700] In an embodiment, the mRNA encodes an antigen from an infectious agent.

[0701] In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or polypeptide, the therapeutic composition being used to deliver a vaccine to a subject or a subject's cell or to treat with a vaccine. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from an infectious source such as a virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from an influenza virus. In certain embodiments, the present invention provides methods 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 methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from a rabies virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the present invention provides methods 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 mRNAs encoding antigens from hepatitis viruses, such as hepatitis A, hepatitis B, or hepatitis C. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens from human papillomaviruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs 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 mRNAs 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 mRNAs encoding antigens from human metapneumoviruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising 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 comprising full-length mRNA encoding antigens from malaria viruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding antigens from Zika virus. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding antigens from Chikungunya virus.

[0702] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antigen associated with a subject's cancer or an antigen identified from a subject's cancer cells. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antigen identified from a subject's own cancer cells, i.e., providing a personalized cancer vaccine. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antigen expressed from a mutant KRAS gene.

[0703] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against CD3. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against CD19.

[0704] 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.

[0705] 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.

[0706] In embodiments, exemplary therapeutic uses result from the delivery of mRNA encoding a secretory protein. Thus, in embodiments, the compositions and methods of the present invention provide for the delivery of mRNA encoding a secretory protein. In some embodiments, the compositions and methods of the present invention provide for the delivery of mRNA encoding one or more secretory proteins listed in Table 1; thus, the compositions of the present invention may comprise mRNA encoding a protein listed in Table 1 (or a homolog thereof) and other components listed herein, and the methods of the present invention may comprise preparing and / or administering a composition comprising mRNA encoding a protein listed in Table 1 (or a homolog thereof) and other components listed herein.

[0707] Table 1. Secreted proteins

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763] In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more of the other exemplary proteins listed in Table 2; thus, the compositions of the invention can comprise mRNAs encoding proteins listed in Table 2 (or homologs thereof) in addition to the other components listed herein, and the methods of the invention can comprise preparing and / or administering compositions comprising mRNAs encoding proteins selected from the proteins listed in Table 2 (or homologs thereof) in addition to the other components listed herein.

[0764] Table 2. Other exemplary proteins

[0765]

[0766]

[0767] The Uniprot IDs listed in Tables 1 and 2 refer to the 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. Thus, 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 from animals of veterinary or industrial interest of the secreted proteins listed in Tables 1 and 2; thus, the compositions of the present invention may comprise mRNA encoding a protein selected from mammalian homologs or homologs from animals of veterinary or industrial interest of the proteins listed in Tables 1 and 2, as well as other components listed herein; and the methods of the present invention may comprise preparing and / or administering a composition comprising mRNA encoding a protein selected from mammalian homologs or homologs from animals of veterinary or industrial interest of the proteins listed in Tables 1 and 2, as well as other components listed herein. In some embodiments, the mammalian homolog is selected from a mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homolog. In some embodiments, the animal of veterinary or industrial interest is selected from the mammals listed above and / or chicken, duck, turkey, salmon, catfish, or tilapia.

[0768] 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) in addition to 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) in addition to other components listed herein.

[0769] Table 3. Lysosomes and related proteins

[0770]

[0771]

[0772]

[0773] Information on lysosomal proteins can be found in 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 interest as described above.

[0774] In some embodiments, the compositions and methods of the present 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 present invention provide for the delivery of mRNA encoding therapeutic proteins that are useful for treating the diseases or conditions (i.e., indications) listed in Table 4; thus, the compositions of the present invention may comprise mRNA encoding therapeutic proteins listed or not listed in Table 4 (or homologs thereof, as described below) and other components described herein for treating the diseases or conditions (i.e., indications) listed in Table 4, and the methods of the present invention may comprise preparing and / or administering compositions comprising mRNA encoding such proteins (or homologs thereof, as described below) and other components described herein for treating the diseases or conditions listed in Table 4.

[0775] Table 4. Exemplary indications and related proteins

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788] 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).

[0789] The present invention also provides the following embodiments:

[0790] Embodiment 1. A cationic lipid having a structure according to formula (I),

[0791]

[0792] in

[0793] R 1 It is hydrogen, C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl, or substructure Y;

[0794] Each a and b is an integer from 0 to 6;

[0795] Each X A1 and X B1 independently O or S;

[0796] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0797] X A2 Independently NH, NR A , CH2 or CHR A ;

[0798] X B2 Independently NH, NR B , CH2 or CHR B ;

[0799] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 alkynyl; and

[0800] Substructure Y is

[0801] Embodiment 2. The cationic lipid according to embodiment 1, having a structure according to formula (IA),

[0802]

[0803] Embodiment 3. The cationic lipid according to embodiment 1, which has a structure according to formula (I-A'):

[0804]

[0805] Embodiment 4. The cationic lipid according to any one of embodiments 1-3, wherein each X A1 and X B1 It is O, or every X A1 and X B1 It’s S.

[0806] Embodiment 5. The cationic lipid according to embodiment 4, wherein each X A1 and X B1 It's O.

[0807] Embodiment 6. The cationic lipid of any one of embodiments 1-5, wherein each a and b is independently 0, 1 or 2.

[0808] Embodiment 7. The cationic lipid according to any one of embodiments 1-6, wherein each X A2 It is NR A or CHR A .

[0809] Embodiment 8. The cationic lipid according to any one of embodiments 1-7, wherein each X B2 It is NR B or CHR B .

[0810] Embodiment 9. The cationic lipid of any one of embodiments 1-8, having a structure according to formula (IB),

[0811]

[0812] Embodiment 10. The cationic lipid of any one of embodiments 1-8, having a structure according to formula (IB"):

[0813]

[0814] wherein d is independently an integer from 0 to 5.

[0815] Embodiment 11. The cationic lipid of any one of embodiments 1-8, having a structure according to formula (I-B'):

[0816]

[0817] Embodiment 12. The cationic lipid according to any one of embodiments 1-11, wherein each L A It is C1-C 10 Alkylene.

[0818] Embodiment 13. A cationic lipid according to any one of embodiments 1-12, wherein each L B It is C1-C 10 Alkylene.

[0819] Embodiment 14. The cationic lipid according to any one of embodiments 1-13, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

[0820] Embodiment 15. The cationic lipid of any one of embodiments 12-14, having a structure according to formula (IC),

[0821]

[0822] wherein each c is independently an integer from 2 to 10.

[0823] Embodiment 16. The cationic lipid of any one of embodiments 12-14, having a structure according to formula (IC")

[0824]

[0825] wherein each c is independently an integer from 2 to 10, and d is independently an integer from 0 to 5.

[0826] Embodiment 17. The cationic lipid of embodiment 16, wherein d is 0, 1, 2, 3 or 4.

[0827] Embodiment 18. The cationic lipid of any one of embodiments 12-14, having a structure according to formula (I-C'),

[0828]

[0829] wherein each c is independently an integer from 2 to 10.

[0830] Embodiment 19. The cationic lipid of any one of embodiments 15-18, wherein each c is 2, 3 or 4.

[0831] Embodiment 20. The cationic lipid of any one of embodiments 15-18, wherein each c is 4, 5, 6, 7, 8, 9 or 10.

[0832] Embodiment 21. The cationic lipid of embodiment 20, wherein each c is 4.

[0833] Embodiment 22. A cationic lipid according to any one of embodiments 1-21, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0834] Embodiment 23. The cationic lipid according to embodiment 22, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0835] Embodiment 24. A cationic lipid according to any one of embodiments 1-23, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0836] Embodiment 25. The cationic lipid according to embodiment 24, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0837] Embodiment 26. The cationic lipid according to any one of embodiments 1-25, wherein each R A and R B It is C6-C 20 Hydroxyalkyl, or each R A and R B It is C6-C 20 Hydroxyalkenyl.

[0838] Embodiment 27. The cationic lipid according to embodiment 26, wherein each R A and R B It is -CH2CH(OH)C10 H 21 , or each R A and R B Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 .

[0839] Embodiment 28. The cationic lipid according to embodiment 27, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 .

[0840] Embodiment 29. The cationic lipid according to embodiment 26, wherein each R A and R B It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0841]

[0842]

[0843] Embodiment 30. The cationic lipid according to embodiment 1, which has the following structure,

[0844]

[0845] Embodiment 31. The cationic lipid according to embodiment 1, which has the following structure,

[0846]

[0847] Embodiment 32. A cationic lipid having a structure according to formula (II),

[0848]

[0849] in

[0850] Each a is independently an integer from 1 to 5;

[0851] b is independently an integer from 0 to 6;

[0852] Each X A1 and X B1 independently O or S;

[0853] Each L A and L B Independently C1-C 10 Alkylene, C2-C10 Alkenylene or C2-C 10 Alkynylidene;

[0854] Each X A2 Independently NH, NR A , CH2 or CHR A ;

[0855] X B2 Independently NH, NR B , CH2 or CHR B ;as well as

[0856] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0857] Embodiment 33. The cationic lipid according to embodiment 32, wherein each X A1 and X B1 It is O, or every X A1 and X B1 It’s S.

[0858] Embodiment 34. The cationic lipid according to embodiment 33, wherein each X A1 and X B1 It's O.

[0859] Embodiment 35. The cationic lipid of any one of embodiments 32-34, wherein each a is independently 1, 2, or 3; and b is independently 0, 1, or 2.

[0860] Embodiment 36. The cationic lipid of embodiment 35, wherein each a is 1 and b is 0.

[0861] Embodiment 37. A cationic lipid according to any one of embodiments 32-36, wherein each X A2 It is NR A or CHR A .

[0862] Embodiment 38. A cationic lipid according to any one of embodiments 32-37, wherein each X B2 It is NR B or CHR B .

[0863] Embodiment 39. The cationic lipid of any one of embodiments 32-38, which has a structure according to formula (II-A),

[0864]

[0865] Embodiment 40. A cationic lipid according to any one of embodiments 32-39, wherein each L A It is C1-C 10 Alkylene.

[0866] Embodiment 41. A cationic lipid according to any one of embodiments 32-40, wherein each L B It is C1-C 10 Alkylene.

[0867] Embodiment 42. A cationic lipid according to any one of embodiments 32-41, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

[0868] Embodiment 43. The cationic lipid of any one of embodiments 32-42, having a structure according to formula (II-B),

[0869]

[0870] wherein each c is independently an integer from 2 to 10.

[0871] Embodiment 44. A cationic lipid according to embodiment 43, wherein each c is 2, 3 or 4.

[0872] Embodiment 45. The cationic lipid of embodiment 43, wherein each c is 4, 5, 6, 7, 8, 9 or 10.

[0873] Embodiment 46. The cationic lipid of embodiment 45, wherein each c is 4.

[0874] Embodiment 47. A cationic lipid according to any one of embodiments 32-46, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0875] Embodiment 48. The cationic lipid of embodiment 47, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0876] Embodiment 49. The cationic lipid according to any one of embodiments 32-48, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0877] Embodiment 50. The cationic lipid of embodiment 49, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0878] Embodiment 51. A cationic lipid according to any one of embodiments 32-50, wherein each R A and R B It is C6-C 20 Hydroxyalkyl, or each R A and R B It is C6-C 20 Hydroxyalkenyl.

[0879] Embodiment 52. The cationic lipid according to embodiment 51, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 , or each R A and R B Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 .

[0880] Embodiment 53. The cationic lipid according to embodiment 52, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 .

[0881] Embodiment 54. The cationic lipid of embodiment 51, wherein each R A and R B It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0882]

[0883] Embodiment 55. The cationic lipid according to embodiment 32, which has the following structure,

[0884] Embodiment 56. A cationic lipid having a structure according to formula (III):

[0885]

[0886] in

[0887] Each a is independently an integer from 0 to 6;

[0888] Each X A1 independently O or S;

[0889] Each L A Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylidene;

[0890] X A2 Independently NH, NR A , CH2 or CHR A ;as well as

[0891] Each R A Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0892] Embodiment 57. The cationic lipid according to embodiment 56, wherein each X A1 It is O, or every X A1 It’s S.

[0893] Embodiment 58. The cationic lipid according to embodiment 57, wherein each X A1 It's O.

[0894] Embodiment 59. The cationic lipid of any one of embodiments 56-58, wherein each a is independently 0, 1 or 2.

[0895] Embodiment 60. The cationic lipid of embodiment 59, wherein each a is 1.

[0896] Embodiment 61. The cationic lipid according to any one of embodiments 56-60, wherein each X A2 It is NR A or CHR A .

[0897] Embodiment 62. The cationic lipid of any one of embodiments 56-61, having a structure according to formula (III-A),

[0898]

[0899] Embodiment 63. A cationic lipid according to any one of embodiments 56-62, wherein each L A It is C1-C 10 Alkylene.

[0900] Embodiment 64. The cationic lipid according to any one of embodiments 56-63, wherein each L A is an unsubstituted C1-C 10 Alkylene.

[0901] Embodiment 65. The cationic lipid of any one of embodiments 56-64, having a structure according to formula (III-B),

[0902]

[0903] wherein each c is independently an integer from 2 to 10.

[0904] Embodiment 66. A cationic lipid according to embodiment 65, wherein each c is 2, 3 or 4.

[0905] Embodiment 67. The cationic lipid of embodiment 65, wherein each c is 4, 5, 6, 7, 8, 9 or 10.

[0906] Embodiment 68. A cationic lipid according to embodiment 67, wherein each c is 4.

[0907] Embodiment 69. The cationic lipid according to any one of embodiments 56-68, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0908] Embodiment 70. The cationic lipid of embodiment 69, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0909] Embodiment 71. A cationic lipid according to any one of embodiments 56-70, wherein each R A It is C6-C 20 Hydroxyalkyl, or each R A It is C6-C 20 Hydroxyalkenyl.

[0910] Embodiment 72. The cationic lipid of embodiment 71, wherein each R A It is -CH2CH(OH)C 10 H 21 , or each R A Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 .

[0911] Embodiment 73. The cationic lipid according to embodiment 72, wherein each R A It is -CH2CH(OH)C 10 H 21 .

[0912] Embodiment 74. The cationic lipid of embodiment 71, wherein each R A It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0913]

[0914]

[0915] Embodiment 75. The cationic lipid according to embodiment 56, which has the following structure,

[0916] Embodiment 76. A cationic lipid having a structure according to formula (IV),

[0917]

[0918] in

[0919] R 1 is hydrogen, C1-C 30 Alkyl, C2-C 30 Alkenyl or C2-C 30 Alkynyl;

[0920] Each a and b is an integer from 0 to 6;

[0921] Each X A1 and X B1 independently O or S;

[0922] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10Alkenylene or C2-C 10 Alkynylidene;

[0923] L C are independently –C(O)– or –(CH2) b –

[0924] X A2 Independently NH, NR A , CH2 or CHR A ;

[0925] X B2 Independently NH, NR B , CH2 or CHR B ;as well as

[0926] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

[0927] Embodiment 77. The cationic lipid according to embodiment 76, wherein L C Yes – (CH2) b –.

[0928] Embodiment 78. The cationic lipid of embodiment 76, which has a structure according to formula (IV-A):

[0929]

[0930] Embodiment 79. The cationic lipid according to any one of embodiments 76-78, wherein each X A1 and X B1 It is O, or every X A1 and X B1 It’s S.

[0931] Embodiment 80. The cationic lipid of embodiment 79, wherein each X A1 and X B1 It's O.

[0932] Embodiment 81. The cationic lipid of any one of embodiments 76-80, wherein each a and b is independently 0, 1 or 2.

[0933] Embodiment 82. The cationic lipid according to any one of embodiments 76-81, wherein each X A2 It is NR A or CHR A .

[0934] Embodiment 83. The cationic lipid according to any one of embodiments 76-82, wherein each X B2 It is NR B or CHR B .

[0935] Embodiment 84. The cationic lipid of any one of embodiments 76-83, which has a structure according to formula (IV-B):

[0936]

[0937] Embodiment 85. The cationic lipid of any one of embodiments 76-83, which has a structure according to formula (IV-B'):

[0938]

[0939] Embodiment 86. The cationic lipid according to any one of embodiments 76-85, wherein each L A It is C1-C 10 Alkylene.

[0940] Embodiment 87. The cationic lipid according to any one of embodiments 76-86, wherein each L B It is C1-C 10 Alkylene.

[0941] Embodiment 88. A cationic lipid according to any one of embodiments 76-87, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

[0942] Embodiment 89. The cationic lipid of any one of embodiments 86-88, which has a structure according to formula (IV-C):

[0943]

[0944] wherein each c is independently an integer from 2 to 10.

[0945] Embodiment 90. The cationic lipid of any one of embodiments 86-88, which has a structure according to formula (IV-C'):

[0946]

[0947] wherein each c is independently an integer from 2 to 10.

[0948] Embodiment 91. The cationic lipid according to embodiment 76, wherein LC It is –C(O)–.

[0949] Embodiment 92. The cationic lipid of embodiment 91, which has a structure according to formula (IV-B")

[0950]

[0951] Embodiment 93. The cationic lipid according to embodiment 92, wherein each L A It is C1-C 10 Alkylene.

[0952] Embodiment 94. The cationic lipid according to embodiment 92 or 93, wherein each L B It is C1-C 10 Alkylene.

[0953] Embodiment 95. A cationic lipid according to any one of embodiments 92-94, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

[0954] Embodiment 96. The cationic lipid of embodiment 95, which has a structure according to formula (IV-C"):

[0955]

[0956] wherein each c is independently an integer from 2 to 10.

[0957] Embodiment 97. The cationic lipid of any one of embodiments 89, 90, and 96, wherein each c is 2, 3, or 4.

[0958] Embodiment 98. The cationic lipid of any one of embodiments 89, 90, and 96, wherein each c is 4, 5, 6, 7, 8, 9, or 10.

[0959] Embodiment 99. The cationic lipid of embodiment 98, wherein each c is 4.

[0960] Embodiment 100. A cationic lipid according to any one of embodiments 76-99, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0961] Embodiment 101. The cationic lipid according to embodiment 100, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0962] Embodiment 102. The cationic lipid according to any one of embodiments 76-101, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

[0963] Embodiment 103. The cationic lipid according to embodiment 102, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

[0964] Embodiment 104. A cationic lipid according to any one of embodiments 76-103, wherein each R A and R B It is C6-C 20 Hydroxyalkyl, or each R A and R B It is C6-C 20 Hydroxyalkenyl.

[0965] Embodiment 105. The cationic lipid of embodiment 104, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 , or each R A and R B Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 .

[0966] Embodiment 106. The cationic lipid of embodiment 105, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 .

[0967] Embodiment 107. The cationic lipid of embodiment 104, wherein each R A and R B It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0968]

[0969] Embodiment 108. The cationic lipid of embodiment 76, which has the following structure:

[0970]

[0971] Embodiment 109. A cationic lipid having a structure according to formula (V),

[0972]

[0973] in

[0974] Each L A and L B Independently C1-C 10 Alkylene, C2-C 10 Alkenylene or C2-C 10 Alkynylene; and

[0975] Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 Alkynyl or C1-C 15 Alkylene-C(O)2-C1-C 15 alkyl.

[0976] Embodiment 110. The compound according to embodiment 109, wherein each L A and L B Independently C1-C 10 Alkylene.

[0977] Embodiment 111. The compound according to embodiment 109, wherein each L A and L B Independently C2-C 10 Alkenylene or C1-C 10 Alkylene-C(O)2-C1-C 10 Alkylene.

[0978] Embodiment 112. A cationic lipid according to formula (VI),

[0979]

[0980] in

[0981] Each R A and R B Independently C1-C 30Alkyl, C2-C 30 Alkenyl, C2-C 30 Alkynyl or C1-C 15 Alkylene-C(O)2-C1-C 15 Alkyl; and

[0982] Each c is independently an integer from 2 to 10.

[0983] Embodiment 113. A cationic lipid according to embodiment 112, wherein each c is 2, 3 or 4.

[0984] Embodiment 114. The cationic lipid of embodiment 112, wherein each c is 4, 5, 6, 7, 8, 9 or 10.

[0985] Embodiment 115. A cationic lipid according to embodiment 114, wherein each c is 4.

[0986] Embodiment 116. A cationic lipid according to any one of embodiments 112-115, wherein each R A and R B It is an unsubstituted C6-C 30 Alkenyl.

[0987] Embodiment 117. A cationic lipid according to any one of embodiments 112-115, wherein each R A and R B Yes (unsubstituted C3-C 15 Alkylene)-C(O)2-(unsubstituted C3-C 15 alkyl).

[0988] Embodiment 118. A cationic lipid according to any one of embodiments 112-115, wherein each R A and R B It is -CH2CH(OH)R C , and where R C Select from the group consisting of:

[0989]

[0990]

[0991] Embodiment 119. A cationic lipid, which is any one of cationic lipids 1-156.

[0992] Embodiment 120. A cationic lipid, which is:

[0993]

[0994]

[0995] Embodiment 121. A composition comprising an mRNA encoding a peptide or polypeptide encapsulated within 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 embodiments 1-120.

[0996] Embodiment 122. The composition of embodiment 121, comprising mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0997] Embodiment 123. The composition of embodiment 121, comprising mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0998] Embodiment 124. The composition of embodiment 121, comprising mRNA encoding an antigen.

[0999] Embodiment 125. The composition of embodiment 124, wherein the antigen is from an infectious agent.

[1000] Embodiment 126. A composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid according to any one of embodiments 1-120.

[1001] Embodiment 127. The composition of embodiment 126, 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.

[1002] Embodiment 128. The composition of embodiment 126 or 127, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide.

[1003] Embodiment 129. The composition of any one of Embodiments 126-128, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of the lungs or lung cells of a subject.

[1004] Embodiment 130. The composition of embodiment 129, wherein the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[1005] Embodiment 131. The composition of any one of Embodiments 126-128, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of the liver or hepatocytes of a subject.

[1006] Embodiment 132. The composition of embodiment 131, wherein the mRNA encodes an ornithine transcarbamylase (OTC) protein.

[1007] Embodiment 133. The composition of any one of Embodiments 126-128, wherein the mRNA encodes a peptide or polypeptide for use in a vaccine.

[1008] Embodiment 134. The composition of embodiment 133, wherein the mRNA encodes an antigen.

[1009] Embodiment 135. The composition of embodiment 134, wherein the antigen is from an infectious agent.

[1010] Embodiment 136. The composition of any one of Embodiments 121-135, formulated for intravenous (IV) administration.

[1011] Embodiment 137. The composition of any one of Embodiments 121-135, formulated for intramuscular (IM) administration.

[1012] Embodiment 138. The composition of any one of Embodiments 121-135, formulated for administration by inhalation.

[1013] Embodiment 139. The composition of embodiment 138, wherein the composition is formulated for aerosolization.

[1014] While 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 limit the same.

[1015] Example

[1016] Example 1: Synthesis of cationic lipids

[1017] The cationic lipids described herein can be prepared according to the exemplary synthesis of Scheme 1.

[1018] In embodiments, the cationic lipids described herein can be prepared by conjugating a thiol to a dicarboxylic acid under suitable conditions. Exemplary dicarboxylic acids are described in Table A and exemplary thiols are described in Table B. Thus, suitable cationic lipids include those produced from any combination of the precursors described in Tables A and B.

[1019] Table A. Dicarboxylic Acids

[1020]

[1021] Table B. Thiols

[1022]

[1023]

[1024]

[1025]

[1026]

[1027] Example 2: Exemplary Synthesis of Compound 16

[1028] Cationic lipids described herein can be prepared according to the exemplary synthesis of Scheme 2. An exemplary synthesis of compound 16 is provided herein.

[1029] Option 2

[1030]

[1031] Synthesis of 2-(4-(Benzylthio)butyl)isoindoline-1,3-dione (D2)

[1032]

[1033] At 0 ° C, benzyl mercaptan (4.96g, 40mmol) was added to a mixture of sodium hydride (1.76g, 44mmol, 60% dispersion in mineral oil) dissolved in 60ml N,N-dimethylformamide. After stirring for 30min, a solution of N-(4-bromobutyl)phthalimide D1 (12g, 42.5mmol) dissolved in 20mL N,N-dimethylformamide was added, and the resulting mixture was slowly heated to room temperature and stirred overnight. The reaction mixture was quenched with methanol and saturated NH4Cl solution and then extracted with ethyl acetate. The organic layer was washed with water (300mL×3) and then dried over Na2SO4. After filtering and concentrating, the crude product was purified by flash column chromatography (330g silica gel column, 0-60% ethyl acetate gradient dissolved in hexane) to obtain 10.5g product D2 as a white solid (yield: 81%).

[1034] Synthesis of 4-(Benzylthio)butan-1-amine (D3)

[1035]

[1036] A mixture of 2-(4-(benzylthio)butyl)isoindoline-1,3-dione D2 (10.5 g, 32.5 mmol) and hydrazine hydrate (3.8 mL, 67 mmol) dissolved in methanol (300 mL) was heated to gentle reflux for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was filtered through celite and washed with methanol (30 mL). The combined filtrates were concentrated under reduced pressure to give 11.5 g (~50% purity) of crude product D3 as a white solid, which was used in the next step without purification.

[1037] Synthesis of 1,1'-((4-(Benzylthio)butyl)azanediyl)bis(dodecan-2-ol) (D5)

[1038]

[1039] A mixture of 4-(benzylthio)butan-1-amine D3 (11.5 g, ~50% purity, ~29.5 mmol), diisopropylethylamine (15 mL) and 1,2-epoxydodecane D4 (26 g, 141 mmol) dissolved in 210 mL of methanol was heated to gentle reflux for 20 h under a nitrogen atmosphere. After the reaction mixture was concentrated, the crude product was purified by flash column chromatography (330 g silica gel column, 0-40% ethyl acetate in hexanes gradient) to give 12 g of product D5 as a colorless oil (yield: 66% over 2 steps).

[1040] Synthesis of N-(4-(Benzylthio)butyl)-2-((tert-butyldimethylsilyl)oxy)-N-(2-((tert-butyldimethylsilyl)oxy)dodecyl)dodecane-1-amine (D6)

[1041]

[1042] To 1,1'-((4-(benzylthio)butyl)azanediyl)bis(dodecan-2-ol) 5 (11.5 g, 29.5 mmol) dissolved in 60 mL of anhydrous N,N-dimethylformamide was added imidazole (8.6 g, 127 mmol) and DMAP (0.5 g, 4 mmol), followed by tert-butyldimethylsilyl chloride (12.8 g, 85 mmol), and the resulting mixture was stirred at room temperature under a nitrogen atmosphere for 48 h. The reaction mixture was concentrated under reduced pressure, and the residue was partitioned between CHCl (300 mL) and water (300 mL). The organic layer was separated, and the aqueous layer was extracted with CHCl (300 mL×2). The combined organic phases were dried over NaSO. After filtration and concentration, the crude product was purified by flash column chromatography (330 g silica gel column, 0-25% ethyl acetate in hexanes gradient) to afford 13.6 g of product D6 as a colorless oil (yield: 81%).

[1043] 4-(Bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butane-1-thiol (D7)

[1044]

[1045] In a 1L three-necked round-bottom flask equipped with a magnetic stirring bar, a dry ice-acetone condenser and a nitrogen inlet, N-(4-(benzylthio)butyl)-2-((tert-butyldimethylsilyl)oxy)-N-(2-((tert-butyldimethylsilyl)oxy)dodecyl)dodecane-1-amine D6 (13.6 g, 17 mmol) was dissolved in 250 mL of anhydrous ether and then cooled to -78 ° C in a dry ice-acetone bath. Liquid ammonia (400 mL) was condensed into the reaction flask, and sodium (1 g, 43 mmol) was added portionwise under a nitrogen atmosphere to form a dark blue solution. After the addition was complete, the reaction was stirred at this temperature for one hour. The reaction was quenched by adding solid NH4Cl (20 g, 373 mmol). The dry ice-acetone bath was replaced with a water bath, and the solvent was then removed by blowing in nitrogen. The white solid residue was ground with ether (300 mL × 4). After concentration, the crude product was purified by flash column chromatography (330 g silica gel column, 0-25% ethyl acetate in hexanes gradient) to afford 8.6 g of product D7 as a colorless oil (yield: 71%).

[1046] Synthesis of S,S-bis(4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butyl)butanebis(phosphorothioate) (D9)

[1047]

[1048] DMAP (52 mg, 0.43 mmol) and EDCI (330 mg, 1.7 mmol) were added to a mixture of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butane-1-thiol 7 (600 mg, 0.86 mmol) and succinic acid D8 (50 mg, 0.43 mmol) dissolved in 15 mL of CH2Cl2. The resulting mixture was stirred at room temperature for 18 h. After the reaction mixture was concentrated, the crude product was purified by flash column chromatography (80 g silica gel column, 0-15% ethyl acetate gradient in hexane) to give 560 mg of product D9 as a colorless oil (yield: 88%).

[1049] Synthesis of S,S-bis(4-(bis(2-hydroxydodecyl)amino)butyl)butane bis(phosphorothioate) (cationic lipid 16)

[1050]

[1051] In a 100 ml Teflon flask, HF-pyridine solution (8 mL, 308 mmol, 70 wt%) was added dropwise to a solution of S,S-bis(4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butyl)butanebis(phosphorothioate)D9 (560 mg, 0.38 mmol) in 16 mL of anhydrous tetrahydrofuran at 0°C. The resulting mixture was slowly heated to room temperature and stirred overnight. The reaction mixture was diluted with CHCl (50 mL) and neutralized with aqueous NaCO. The CHCl layer was separated and the aqueous layer was extracted with CHCl (30 mL x 3). The combined organic phases were dried over NaSO and evaporated. The residue was purified by flash column chromatography (12 g silica gel column, pre-inactivated with 1% triethylamine in hexane, eluent: 0-100% ethyl acetate in hexane gradient) to give 336 mg of the product cationic lipid 16 as a colorless oil (yield: 85%).

[1052] Other cationic lipids can be prepared according to the same reaction procedure as described above, but with different tails derived from different epoxides D4.

[1053] Example 3: Lipid Nanoparticle Formulation Using Thioester Cationic Lipids

[1054] According to methods known in the art, cationic lipids as described herein can be used to prepare lipid nanoparticles. For example, suitable methods include the methods described in International Publication No. WO 2018 / 089801, which is incorporated herein by reference in its entirety.

[1055] 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 an aqueous buffered solution of mRNA are prepared separately. A solution of a lipid mixture (cationic lipids, helper 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. The two solutions are then 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 "Y" confluence). The mixture is then purified by diafiltration using a TFF method. The resulting preparation is concentrated and stored at 2-8°C until further use.

[1056] A second exemplary method for lipid nanoparticle preparation is method B of WO 2018 / 089801 (see, for example, Example 2 and Figure 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 empty lipid nanoparticles in a citrate buffer containing alcohol. The preparation is then subjected to a TFF purification process, in which buffer exchange occurs. The resulting suspension of preformed empty lipid nanoparticles is then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution after mixing results in a higher percentage of lipid nanoparticles containing mRNA and a higher total mRNA yield.

[1057] The lipid nanoparticle formulations of Table 5 were prepared as described in WO 2018 / 089801 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.

[1058] Table 5. Exemplary lipid nanoparticle formulations

[1059]

[1060] Example 4: In vivo expression of hEPO in CD1 mice using thioester lipids

[1061] Intravenous (IV) administration of lipid nanoparticle formulations (Table 5) comprising thioester cationic lipids and mRNA encoding hEPO 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 dose level of 1 mg / kg. Blood samples were collected by tail shearing at 6 hours and 24 hours after administration. The hEPO protein expression levels measured in serum samples by ELISA ( Figure 1 ). These studies show that the thioester 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 (IV-A), in Each a and b is an integer from 0 to 6; Each X A1 and X B1 independently O or S; Each L A and L B Independently C1-C 10 alkylene; X A2 It is NR A ; X B2 It is NR B ;as well as Each R A and R B Independently C6-C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 Alkynyl.

2. The cationic lipid according to claim 1, wherein each X A1 and X B1 It is O, or every X A1 and X B1 It’s S.

3. Cationic lipid according to claim 2, wherein each X A1 and X B1 It's O.

4. The cationic lipid of claim 1, wherein each a and b is independently 0, 1 or 2.

5. The cationic lipid according to any one of claims 1 to 4, having a structure according to formula (IV-B):

6. The cationic lipid according to any one of claims 1 to 4, having a structure according to formula (IV-B'):

7. according to the cationic lipid described in any one of claims 1-4, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

8. cationic lipid according to claim 5, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

9. cationic lipid according to claim 6, wherein each L A and L B is an unsubstituted C1-C 10 Alkylene.

10. The cationic lipid of claim 7, having a structure according to formula (IV-C): wherein each c is independently an integer from 2 to 10.

11. The cationic lipid according to claim 7, having a structure according to formula (IV-C'): wherein each c is independently an integer from 2 to 10.

12. The cationic lipid of claim 10, wherein each c is 2, 3 or 4.

13. The cationic lipid of claim 11, wherein each c is 2, 3 or 4.

14. The cationic lipid of claim 10, wherein each c is 4, 5, 6, 7, 8, 9 or 10.

15. The cationic lipid of claim 11, wherein each c is 4, 5, 6, 7, 8, 9 or 10.

16. The cationic lipid of claim 14, wherein each c is 4.

17. The cationic lipid of claim 15, wherein each c is 4.

18. according to the cationic lipid described in any one of claims 1-4, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

19. The cationic lipid of claim 5, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

20. The cationic lipid of claim 6, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

21. The cationic lipid of claim 10, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

22. The cationic lipid of claim 11, wherein each R A It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

23. The cationic lipid of claim 10, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

24. The cationic lipid of claim 11, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

25. The cationic lipid of claim 18, wherein each R A It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

26. according to the cationic lipid described in any one of claims 1-4, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

27. The cationic lipid of claim 5, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

28. The cationic lipid of claim 6, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

29. The cationic lipid of claim 10, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

30. The cationic lipid of claim 11, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

31. The cationic lipid of claim 18, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

32. The cationic lipid of claim 24, wherein each R B It is C6-C 20 Alkyl or C6-C 20 Alkenyl.

33. The cationic lipid of claim 26, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

34. The cationic lipid of claim 27, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

35. according to the cationic lipid of claim 28, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

36. The cationic lipid of claim 29, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

37. The cationic lipid of claim 30, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

38. according to the cationic lipid of claim 31, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

39. according to the cationic lipid of claim 32, wherein each R B It is an unsubstituted C6-C 20 Alkyl, C6-C 20 Hydroxyalkyl, unsubstituted C6-C 20 Alkenyl or C6-C 20 Hydroxyalkenyl.

40. according to the cationic lipid described in any one of claims 1-4, wherein each R A and R B It is C6-C 20 Hydroxyalkyl, or each R A and R B It is C6-C 20 Hydroxyalkenyl.

41. according to the cationic lipid of claim 40, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 , or each R A and R B Is -CH2CH(OH)(CH2)6(CH=CH)CH2(CH=CH)C5H 11 .

42. according to the cationic lipid of claim 41, wherein each R A and R B It is -CH2CH(OH)C 10 H 21 .

43. according to the cationic lipid of claim 40, wherein each R A and R B is -CH2CH(OH)R C , and where R C Select from the group consisting of:

44. A cationic lipid having the following structure:

45. A composition comprising an mRNA encoding a peptide or polypeptide encapsulated within 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 1-44.

46. ​​The composition of claim 45, comprising mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

47. The composition of claim 45, comprising mRNA encoding ornithine transcarbamylase (OTC) protein.

48. The composition of claim 45, comprising mRNA encoding an antigen.

49. The composition of claim 48, wherein the antigen is from an infectious agent.

50. A composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises the cationic lipid of any one of claims 1-44.

51. The composition of claim 50, 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.

52. The composition of claim 50, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide.

53. The composition of claim 51, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide.

54. The composition of claim 50, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of the lungs or lung cells of a subject.

55. The composition of any one of claims 51-53, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of a lung or lung cell of a subject.

56. The composition of claim 54, wherein the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein.

57. The composition of claim 55, wherein the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

58. The composition of claim 50, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of the liver or hepatocytes of a subject.

59. The composition of any one of claims 51-53, wherein the mRNA encodes a peptide or polypeptide for delivery to or treatment of a subject's liver or hepatocytes.

60. The composition of claim 58, wherein the mRNA encodes ornithine transcarbamylase (OTC) protein.

61. The composition of claim 50, wherein the mRNA encodes a peptide or polypeptide for use in a vaccine.

62. The composition of any one of claims 51-53, wherein the mRNA encodes a peptide or polypeptide for a vaccine.

63. The composition of claim 61, wherein the mRNA encodes an antigen.

64. The composition of claim 63, wherein the antigen is from an infectious agent.

65. The composition of claim 45, formulated for intravenous (IV) administration.

66. The composition of claim 50, formulated for intravenous (IV) administration.

67. The composition of claim 45, formulated for intramuscular (IM) administration.

68. The composition of claim 50, formulated for intramuscular (IM) administration.

69. The composition of claim 45, formulated for administration by inhalation.

70. The composition of claim 50, formulated for administration by inhalation.

71. The composition of claim 69, wherein the composition is formulated for aerosolization.

72. The composition of claim 70, wherein the composition is formulated for aerosolization.

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