WO2019222424A1 - Ribose cationic lipids - Google Patents

Ribose cationic lipids Download PDF

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Publication number
WO2019222424A1
WO2019222424A1 PCT/US2019/032522 US2019032522W WO2019222424A1 WO 2019222424 A1 WO2019222424 A1 WO 2019222424A1 US 2019032522 W US2019032522 W US 2019032522W WO 2019222424 A1 WO2019222424 A1 WO 2019222424A1
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Prior art keywords
cationic lipid
cationic
alkyl
unsubstituted
lipid
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PCT/US2019/032522
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French (fr)
Inventor
Frank Derosa
Shrirang KARVE
Yi Zhang
Michael Heartlein
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Translate Bio, Inc.
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Publication date
Application filed by Translate Bio, Inc. filed Critical Translate Bio, Inc.
Priority to CN201980044653.0A priority Critical patent/CN112384523A/en
Priority to AU2019271215A priority patent/AU2019271215A1/en
Priority to JP2020563906A priority patent/JP7384832B2/en
Priority to US17/054,886 priority patent/US11964051B2/en
Priority to CA3100218A priority patent/CA3100218A1/en
Priority to EP19728235.3A priority patent/EP3794008A1/en
Publication of WO2019222424A1 publication Critical patent/WO2019222424A1/en
Priority to JP2023191456A priority patent/JP2023184713A/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/12—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by acids having the group -X-C(=X)-X-, or halides thereof, in which each X means nitrogen, oxygen, sulfur, selenium or tellurium, e.g. carbonic acid, carbamic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • mRNA messenger RNA
  • the present invention provides, among other things, cationic lipids useful in for delivery of mRNA. Delivery of mRNA provided by cationic lipids described herein can result in targeted delivery, reduce administration frequency, improve patient tolerability, and provide more potent and less toxic mRNA therapy for the treatment of a variety of diseases, including but not limited to cancer, cardiovascular, cystic fibrosis, infectious, and neurological diseases.
  • the present invention provides a cationic lipid of Formula (G):
  • R x is independently -H, -L ⁇ R 1 , or -L 5A -L 5B -B';
  • each of L 1 , L 2 , and L 3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NR L -; each L 4A and L 5A is independently -C(O)-, -C(0)0-, or -C(0)NR L -;
  • each L 4B and L 5B is independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene;
  • each B and B' is NR 4 R 5 , a 5- to 10-membered nitrogen-containing heterocyclyl, or a 5- to 10- membered nitrogen-containing heteroaryl;
  • each R 1 , R 2 , and R 3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
  • each R 4 and R 5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and each R L is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
  • the present invention provides a cationic lipid of Formula
  • the present invention provides a cationic lipid of Formula (la):
  • L 1 , L 2 , L 3 , R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • the present invention provides a cationic lipid of Formula (lb):
  • each of R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • the present invention provides a cationic lipid of Formula (lc):
  • the present invention provides a cationic lipid of Formula (Id):
  • R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • the present invention provides a cationic lipid of Formula (le):
  • R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • each L 3 is independently -C(O)- or a covalent bond.
  • L 4A is -C(O)-.
  • L 4B is unsubstituted C1-C6 alkylene; C2-C6 alkenylene; or C2-C6 alkynylene.
  • L 4B is -CH2-. ln embodiments, L 4B is -CH2CH2-. In embodiments, L 4B is
  • L 4B is -CH2CH2CH2CH2-
  • B is NR 4 R 5 , and each R 4 and R 5 is independently hydrogen or unsubstituted Ci- C 6 alkyl. In embodiments, B is N(CHB)2.
  • B is NR 4 R 5 , and R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl.
  • B is piperdinyl or piperazinyl.
  • B is 4-methylpiperazinyl.
  • the present invention provides a cationic lipid of Formula (I I):
  • R A is hydrogen or -lAR 1 ;
  • R B is hydrogen or -L 2 -R 2 ;
  • R c is hydrogen or -L 3 -R 3 ;
  • each of L 1 , L 2 , and L 3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NR L -;
  • L 4 is independently Ci-Cio alkylene
  • each R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl, C 6 -C 30 alkenyl, or C 6 -C 30 alkynyl;
  • each R 4 and R 5 is independently hydrogen, C 1 -C 10 alkyl; C 2 -C 10 alkenyl; or C 2 -C 10 alkynyl; or R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and
  • each R L is independently hydrogen, C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 2 -C 20 alkynyl.
  • R A is -lAR 1 ;
  • R B is -L 2 -R 2 ; and
  • the present invention provides a cationic lipid of Formula (lla):
  • R A , R B , R c , L 4 , R 4 and R 5 is as independently as defined herein.
  • the present invention provides a cationic lipid of Formula (Mb):
  • the present invention provides a cationic lipid of Formula (lie):
  • the present invention provides a cationic lipid of Formula (lid):
  • R A , R B , R c , L 4 , R 4 and R 5 is independently as defined herein.
  • the present invention provides a cationic lipid of Formula (lie):
  • R A , R B , R c , L 4 , R 4 and R 5 is independently as defined herein.
  • L 4 is unsubstituted C1-C6 alkylene.
  • L 4B is -CH2- ,ln embodiments, L 4B is -CH2CH2-. In embodiments, L 4B is -CH2CH2CH2-.
  • each R 4 and R 5 is independently hydrogen or unsubstituted C1-C6 alkyl, or R 4 and R 5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens. In embodiments, each R 4 and R 5 is CH3.
  • R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl.
  • R 4 and R 5 combine to form a piperdinyl or piperazinyl. In embodiments, R 4 and R 5 combine to form 4-methylpiperazinyl.
  • each R 1 , R 2 , and R 3 is independently C6-C22 alkyl, C6-C22 alkenyl, or C6-C22
  • each R 1 , R 2 , and R 3 is unsubstituted linear C6-C22 alkyl, unsubstituted linear C6-C22 alkenyl, unsubstituted linear C6-C22 alkynyl, unsubstituted branched C6-C22 alkyl, unsubstituted branched C6-C22 alkenyl, or unsubstituted branched C6-C22 alkynyl.
  • each R 1 , R 2 , and R 3 is unsubstituted linear C6-C22 alkyl.
  • each R 1 , R 2 , and R 3 is unsubstituted linear C 6 - C22 alkenyl. In embodiments, each R 1 , R 2 , and R 3 is unsubstituted linear C6-C22 alkynyl. In embodiments, each R 1 , R 2 , and R 3 is unsubstituted branched C6-C22 alkyl. In embodiments, each R 1 , R 2 , and R 3 is unsubstituted branched C 6 -C 22 alkenyl. In embodiments, each R 1 , R 2 , and R 3 is unsubstituted branched C 6 -C 22 alkynyl.
  • each R 1 , R 2 , and R 3 is unsubstituted C6-C22 alkyl.
  • R 6 is
  • R 6 is unsubstituted branched C6-C14 alkyl.
  • each R 1 , R 2 , and R 3 is (CH 2 ) 7 CH3, (O-hJgCHs, (CH 2 )uCH 3 , (CH 2 )i3CH 3 , (CH 2 )I 5 CH 3 , or (CH 2 )i 7 CH 3 .
  • each R 1 , R 2 , and R 3 is (CH 2 ) 6 CH 3 , (CH 2 )8CH 3 , (CH 2 )IOCH 3 , (CH 2 )i2CH 3 , (CH 2 )I 4 CH 3 , or (CH 2 )I 6 CH 3 .
  • each R 1 , R 2 , and R 3 is C 6 -C 12 alkyl substituted by -0(C0)R 6 or -C(0)OR 6 , wherein R 6 is unsubstituted C 6 -C 14 alkyl.
  • each R 1 , R 2 , and R 3 is (CH 2 ) 8 0C(0)(CH 2 ) 6 CH 3 , (CH 2 )90C(0)(CH 2 )6CH 3 , (CH 2 )7C(0)0(CH2)2CH(C 5 HII)2, or (CH 2 )8C(0)0(CH2)2CH(C 5 HII)2.
  • each R 1 , R 2 , and R 3 is unsubstituted C 6 -C 22 alkenyl (e.g., a monoalkenyl, a dienyl, or a trienyl).
  • each R 1 , R 2 , and R 3 is:
  • each R 1 , R 2 , and R 3 is:
  • the invention provides a cationic lipid selected from the group consisting of:
  • the invention provides a cationic lipid selected from the group consisting of:
  • a cationic lipid has a structure according to one of the following formulas,
  • a cationic lipid has a structure according to one of the following formulas,
  • a cationic lipid is selected from the group consisting of:
  • a cationic lipid is selected from the group consisting of:
  • a cationic lipid has a structure according to the following formula,
  • a cationic lipid has a structure according to the following formula,
  • a cationic lipid has a structure according to the following formula,
  • a cationic lipid has a structure according to the following formula,
  • L 5B is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, or -CH 2 CH 2 CH 2 CH 2 -.
  • B' is NR 4 R 5 , and each R 4 and R 5 is independently hydrogen or unsubstituted Ci- C 6 alkyl, or R 4 and R 5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
  • B' is N(CH B )2.
  • L 4B is -CH2-. In embodiments, L 4B is -CH2CH2-. In embodiments, L 4B is - CH2CH2CH2-. In embodiments, L 4B is -CH2CH2CH2CH2-.
  • B' is NR 4 R 5 , and each R 4 and R 5 is independently hydrogen or unsubstituted Ci- C 6 alkyl, or R 4 and R 5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
  • B' is N(CH B )2.
  • each of L 2 and L 3 is a covalent bond.
  • each of L 2 and L 3 is -C(O)-.
  • each of R 2 and R 3 is independently C6-C22 alkyl, C6-C22 alkenyl, or C6-C22 alkynyl.
  • each R 2 and R 3 is unsubstituted linear C6-C22 alkyl, unsubstituted linear C6-C22 alkenyl, unsubstituted linear C6-C22 alkynyl, unsubstituted branched C6-C22 alkyl, unsubstituted branched C6-C22 alkenyl, or unsubstituted branched C6-C22 alkynyl.
  • each R 2 and R 3 is unsubstituted linear C6-C22 alkyl.
  • each R 2 and R 3 is unsubstituted linear C6-C22 alkenyl.
  • each R 2 and R 3 is unsubstituted linear C6-C22 alkynyl. In embodiments, each R 2 and R 3 is unsubstituted branched C6-C22 alkyl. In embodiments, each R 2 and R 3 is unsubstituted branched C6-C22 alkenyl. In embodiments, each R 2 and R 3 is unsubstituted branched C6-C22 alkynyl.
  • each of R 2 and R 3 is unsubstituted C6-C22 alkyl.
  • each of R 2 and R 3 is (CH 2 ) 7 CH3, (CH 2 )gCH3, (CH 2 )nCH 3 , (CH 2 ) IB CH 3 , (CH 2 ) I5 CH 3 , or (CH 2 )i7CH 3 .
  • each of R 2 and R 3 is (CH 2 ) 6 CH 3 , (CH 2 )8CH 3 , (CH 2 ) IO CH 3 , (CH 2 )i2CH 3 , (CH 2 ) I4 CH 3 , or (CH 2 ) I6 CH 3 .
  • each of R 2 and R 3 is C6-C12 alkyl substituted by -0(CO)R 6 or -C(0)OR 6 , wherein R 6 is unsubstituted C 6 - C14 alkyl. In embodiments, R 6 is unsubstituted linear C6-C14 alkyl. In embodiments, R 6 is
  • each of R 2 and R 3 is (CH 2 ) 8 0C(0)(CH 2 ) 6 CH 3 , (CH 2 )90C(0)(CH 2 )6CH 3 , (CH 2 )7C(0)0(CH 2 ) 2 CH(C 5 H II ) 2 , or (CH 2 )8C(0)0(CH 2 ) 2 CH(C 5 H II ) 2 .
  • each of R 2 and R 3 is unsubstituted C6-C22 alkenyl.
  • a C6-C22 alkenyl is a monoalkenyl, a dienyl, or a trienyl.
  • each of R 2 and R 3 is
  • each of R 2 and R 3 is
  • a cationic lipid selected from the group consisting of cationic lipids (22)-(462).
  • the invention features a compound having the following structure,
  • the invention features a compound having the following structure,
  • the present invention provides methods of preparing cationic lipids described herein (e.g., a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • the present invention provides a composition such as a pharmaceutical
  • composition comprising a cationic lipid of the present invention and one or more polynucleotides.
  • a composition (e.g., a pharmaceutical composition) comprises an mRNA encoding a protein, encapsulated within a liposome.
  • the liposome comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
  • a cationic lipids described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • non-cationic lipids e.g.,
  • the liposome comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), one or more non- cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
  • an mRNA encodes for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
  • an mRNA encodes for ornithine transcarbamylase (OTC) protein.
  • an mRNA encodes for an antigen from an infectious agent.
  • a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid as described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)).
  • a composition further comprises one 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.
  • a composition further comprises one more lipids selected from the group consisting of one or more non-cationic lipids and one or more PEG- modified lipids.
  • a nucleic acid is an mRNA encoding a peptide or polypeptide.
  • an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell (e.g., an mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell (e.g., an mRNA encodes ornithine transcarbamylase (OTC) protein).
  • an mRNA encodes for an antigen from an infectious agent.
  • the present invention provides methods of treating a disease in a subject comprising administering to the subject a composition (e.g., a pharmaceutical composition) as described herein.
  • a composition e.g., a pharmaceutical composition
  • a composition e.g., a pharmaceutical composition
  • a pharmaceutical composition is formulated for
  • IV intravenous
  • a composition e.g., a pharmaceutical composition
  • a pharmaceutical composition is formulated for
  • a composition e.g., a pharmaceutical composition
  • a composition is formulated for administration by inhalation.
  • a composition is formulated for nebulization.
  • FIG. 1 shows hEPO protein expression following intravenous (IV) administration of lipid
  • nanoparticle formulations comprising a cationic lipid described herein and mRNA encoding hEPO. Protein expression was determined using ELISA.
  • amino acid As used herein, the term “amino acid,” in its broadest sense, refers to any amino acid
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally occurring amino acid.
  • an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some
  • an 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.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • synthetic amino acid encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions.
  • Amino acids including carboxy- 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 change the peptide's circulating half-life without adversely affecting their activity.
  • Amino acids may participate in a disulfide bond.
  • Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g ., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.).
  • amino acid is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
  • Animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some
  • animal refers to non-human animals, at any stage of development.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
  • an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • delivery encompasses both local and systemic delivery.
  • delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery”).
  • patient's circulation system e.g., serum
  • expression refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme).
  • intact protein e.g., enzyme
  • post-translational modification e.g., enzyme
  • a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Half-life As used herein, the term "half-life" is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • improve As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • in vitro refers to events that occur in an artificial
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is "pure” if it is substantially free of other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients (e.g ., buffer, solvent, water, etc.).
  • messenger RNA As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. The term “modified mRNA” related to mRNA comprising at least one chemically modified nucleotide. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc.
  • mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc.
  • An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.
  • an mRNA is or comprises natural nucleosides [e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylgu
  • nucleic acid As used herein, the term “nucleic acid,” in its broadest sense, refers to any one
  • nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
  • nucleic acid encompasses ribonucleic acids (RNA), including but not limited to any one or more of interference RNAs (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (IncRNA), micro-RNA (miRNA) multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA).
  • RNAi interference RNAs
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • aRNA antisense RNA
  • mRNA messenger RNA
  • mmRNA modified messenger RNA
  • IncRNA micro-RNA
  • miRNA multimeric coding nucleic acid
  • PCNA polymeric coding nucleic acid
  • gRNA guide RNA
  • crRNA CRISPR RNA
  • DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • PCR polymerase chain reaction
  • RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), micro- RNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA),
  • Patient refers to any organism to which a
  • composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes.
  • Typical patients include animals ⁇ e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans).
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans.
  • a patient is a human.
  • a human includes pre- and post-natal forms.
  • compositions that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • suitable inorganic and organic acids and bases examples include those derived from suitable inorganic and organic acids and bases.
  • pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C I-4 alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.
  • Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
  • Systemic distribution or delivery As used herein, the terms “systemic distribution,” “systemic delivery,” or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of "local distribution or delivery.”
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term "subject” is used herein interchangeably with “individual” or "patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total 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 completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Target tissues refers to any tissue that is affected by a disease to be treated.
  • target tissues include those tissues that display disease-associated pathology, symptom, or feature.
  • therapeutically effective amount As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
  • Aliphatic refers to C1-C40 hydrocarbons and includes both saturated and unsaturated hydrocarbons.
  • An aliphatic may be linear, branched, or cyclic.
  • C1-C20 aliphatics can include C1-C20 alkyls (e.g., linear or branched C1-C20 saturated alkyls), C2-C20 alkenyls (e.g., linear or branched C4-C20 dienyls, linear or branched C6-C20 trienyls, and the like), and C2-C20 alkynyls (e.g., linear or branched C2-C20 alkynyls).
  • C1-C20 aliphatics can include C3-C20 cyclic aliphatics (e.g., C3-C20 cycloalkyls, C4-C20 cycloalkenyls, or C8-C20 cycloalkynyls).
  • the aliphatic may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide.
  • An aliphatic group is unsubstituted or substituted with one or more substituent groups as described herein.
  • an aliphatic may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2FI, -CO2R', -CN, -OFI, -OR', -OCOR', -OCO2R', -NFI2,
  • R' independently is C1-C20 aliphatic (e.g., Ci- C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • R' independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • R' independently is unsubstituted C 1 -C 3 alkyl.
  • the aliphatic is unsubstituted.
  • the aliphatic does not include any heteroatoms.
  • alkyl means acyclic linear and branched hydrocarbon groups, e.g. "C 1 -C 20 alkyl” refers to alkyl groups having 1-20 carbons.
  • An alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentylhexyl, Isohexyletc.
  • Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure.
  • An alkyl group may be unsubstituted or substituted with one or more substituent groups as described herein.
  • an alkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO 2 H, -CO 2 R', -CN, -OH, -OR', -OCOR', -OCO 2 R', -IMH 2 , -NHR', -N(R') 2 , -SR' or-S0 2 R', wherein each instance of R' independently is Ci- C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl).
  • R' independently is Ci- C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1
  • R' independently is unsubstituted C 1 -C 3 alkyl.
  • the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
  • Alkylene represents a saturated divalent straight or
  • alkenylene represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain
  • alkynylene represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur in any stable point along the chain.
  • an alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide.
  • an alkylene, alkenylene, or alkynylene may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO 2 H, -CO 2 R', -CN, -OH, -OR', -OCOR', -OC0 2 R', -NH 2 , -NHR', -N(R') 2 , -SR' or-S0 2 R', wherein each instance of R' independently is C 1 -C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl).
  • R' independently is C 1 -C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 al
  • R' independently is an unsubstituted alkyl (e.g., unsubstituted C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl). In embodiments, R' independently is unsubstituted C 1 -C 3 alkyl. In certain embodiments, an alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, an alkylene, alkenylene, or alkynylene does not include any heteroatoms.
  • alkenyl means any linear or branched hydrocarbon chains having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, e.g. "C 2 -C 20 alkenyl” refers to an alkenyl group having 2-20 carbons.
  • an alkenyl group 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.
  • the alkenyl comprises 1, 2, or 3 carbon-carbon double bond.
  • the alkenyl comprises a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated.
  • An alkenyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkenyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6
  • R' independently selected substituents of halogen, -COR', -CO 2 FI, -CO 2 R', -CN, -OH, -OR', -OCOR', - OCO 2 R', -IM H 2 , -NH R', -N(R') 2 , -SR' or-S0 2 R', wherein each instance of R' independently is C 1 -C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl).
  • R' independently is an unsubstituted alkyl (e.g., unsubstituted C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl). In embodiments, R' independently is unsubstituted C 1 -C 3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
  • alkynyl means any hydrocarbon chain of either linear or branched configuration, having one or more carbon-carbon triple bonds occurring in any stable point along the chain, e.g. "C 2 -C 20 alkynyl” refers to an alkynyl group having 2-20 carbons. Examples of an alkynyl group include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex- 5-ynyl, etc. In embodiments, an alkynyl comprises one carbon-carbon triple bond.
  • An alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkynyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6
  • R' independently selected substituents of halogen, -COR', -CO 2 H, -CO 2 R', -CN, -OH, -OR', -OCOR', -OCO 2 R', -N H 2 , -NH R', -N(R') 2 , -SR' or-S0 2 R', wherein each instance of R' independently is C 1 -C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl).
  • R' independently is an unsubstituted alkyl (e.g., unsubstituted C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl). In embodiments, R' independently is unsubstituted C 1 -C 3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
  • Cycloalkyl means a nonaromatic, saturated, cyclic group, e.g. "C 3 -C 10 cycloalkyl.”
  • a cycloalkyl is monocyclic.
  • a cycloalkyl is polycyclic (e.g., bicyclic or tricyclic). In polycyclic cycloalkyl groups, individual rings can be fused, bridged, or spirocyclic.
  • cycloalkyl groups examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornanyl, bicyclo[3.2.1]octanyl, octahydro-pentalenyl, and spiro[4.5]decanyl, and the like.
  • the term "cycloalkyl” may be used interchangeably with the term "carbocycle”.
  • a cycloalkyl group may be unsubstituted or substituted with one or more substituent groups as described herein.
  • a cycloalkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO 2 H, -CO 2 R', -CN, -OH, -OR', -OCOR', -OCO 2 R', -IM H 2 , -NH R', -N(R') 2 , -SR' or-S0 2 R', wherein each instance of R' independently is C 1 -C 20 aliphatic (e.g., C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl).
  • substituents e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents
  • R' independently is an unsubstituted alkyl (e.g., unsubstituted C 1 -C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl). In embodiments, R' independently is unsubstituted C 1 -C 3 alkyl. In embodiments, the cycloalkyl is unsubstituted. In embodiments, the cycloalkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
  • heterocyclyl refers to a radical of a 3- to 14-membered non aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3-14 membered heterocyclyl").
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl”) with one or more substituents.
  • the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system
  • a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heterocyclyl").
  • a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heterocyclyl").
  • a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heterocyclyl").
  • the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
  • the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6
  • membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
  • Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without
  • Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
  • Exemplary 5- membered heterocyclyl groups containing 1 heteroatom include, without limitation
  • Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
  • Exemplary 5- membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7- membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-l,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyr
  • Halogen means fluorine, chlorine, bromine, or iodine.
  • Liposomal-based vehicles are considered an attractive carrier for therapeutic agents and remain subject to continued development efforts. While liposomal-based vehicles that comprise a cationic lipid component have shown promising results with regards to encapsulation, stability and site localization, there remains a great need for improvement of liposomal-based delivery systems. For example, a significant drawback of liposomal delivery systems relates to the construction of liposomes that have sufficient cell culture or in vivo stability to reach desired target cells and/or intracellular compartments, and the ability of such liposomal delivery systems to efficiently release their encapsulated materials to such target cells.
  • lipids comprising such lipids, and related methods of their use.
  • the compounds described herein are useful as liposomal compositions or as components of liposomal compositions to facilitate the delivery to, and subsequent transfection of one or more target cells.
  • Cationic lipids disclosed herein comprise a basic, ionizable functional group (e.g., an amine or a nitrogen-containing heteroaryl as described herein), which is present in neutral or charged form.
  • cationic lipids described herein can provide one or more desired characteristics or properties. That is, in certain embodiments, cationic lipids described herein can be characterized as having one or more properties that afford such compounds advantages relative to other similarly classified lipids.
  • cationic lipids disclosed herein can allow for the control and tailoring of the properties of liposomal compositions (e.g., lipid nanoparticles) of which they are a component.
  • cationic lipids disclosed herein can be characterized by enhanced transfection efficiencies and their ability to provoke specific biological outcomes.
  • outcomes can include, for example enhanced cellular uptake, endosomal/lysosomal disruption capabilities and/or promoting the release of encapsulated materials (e.g., polynucleotides) intracellularly.
  • the present invention provides a cationic lipid of Formula (G):
  • R x is independently -H, -l ⁇ -R 1 , or -L 5A -L 5B -B';
  • each of L 1 , L 2 , and L 3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NR L -; each L 4A and L 5A is independently -C(O)-, -C(0)0-, or -C(0)NR L -;
  • each L 4B and L 5B is independently C 1 -C 20 alkylene; C 2 -C 20 alkenylene; or C 2 -C 20 alkynylene;
  • each B and B' is NR 4 R 5 , a 5- to 10-membered nitrogen-containing heterocyclyl, or a 5- to 10- membered nitrogen-containing heteroaryl;
  • each R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl, C 6 -C 30 alkenyl, or C 6 -C 30 alkynyl;
  • each R 4 and R 5 is independently hydrogen, C 1 -C 10 alkyl; C 2 -C 10 alkenyl; or C 2 -C 10 alkynyl; or R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and
  • each R L is independently hydrogen, C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 2 -C 20 alkynyl.
  • R x is -H. In embodiments, R x is -l ⁇ -R 1 . In embodiments, R x is -L 5A -L 5B -B'. [0108] In some embodiments, the present invention provides a cationic lipid of Formula (I):
  • L 1 is a covalent bond. In embodiments, L 1 is -C(O)-. In embodiments, L 1 is - C(0)0-. In embodiments, L s -C(0)S-. In embodiments, L s -C(0)NR L . In embodiments, L s a covalent bond or -C(O)-.
  • R 1 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl). In embodiments, R 1 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl). In embodiments, R 1 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl). In embodiments, R 1 is C 6 -C 30 alkenyl (e.g., C 6 -C 22 alkenyl).
  • R 1 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, R 1 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl). In embodiments, R 1 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, R 1 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl).
  • R 1 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • a R 1 group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group.
  • a R 1 group (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
  • L 2 is a covalent bond. In some, L 2 is -C(O)-. In embodiments, L 2 is -C(0)0-. In embodiments, L 2 is -C(0)S-. In some embodiments, L 2 is -C(0)NR L . In embodiments, L 2 is a covalent bond or -C(O)-.
  • R 2 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl). In embodiments, R 2 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl). In embodiments, R 2 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl). In embodiments, R 2 is C 6 -C 30 alkenyl (e.g., C 6 -C 22 alkenyl).
  • R 2 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, R 2 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl). In embodiments, R 2 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, R 2 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl).
  • R 2 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • a R 2 group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group.
  • a R 2 group (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
  • L 3 is a covalent bond. In some, L 3 is -C(O)-.
  • L 3 is -C(0)0-. In embodiments, L 3 is -C(0)S-. In some embodiments, L 3 is -C(0)NR L . In embodiments, L 3 is a covalent bond or -C(O)-.
  • R 3 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl). In embodiments, R 3 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl). In embodiments, R 3 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl). In embodiments, R 3 is C 6 -C 30 alkenyl (e.g., C 6 -C 22 alkenyl).
  • R 3 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, R 3 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl). In embodiments, R 3 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, R 3 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl).
  • R 3 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • a R 3 group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group.
  • a R 3 group (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
  • each of L 1 , L 2 , and L 3 is the same group. In embodiments, each of L 1 , L 2 , and L 3 is a covalent bond. In some, each of L 1 , L 2 , and L 3 is -C(O)-. In embodiments, each of L 1 , L 2 , and L 3 is - C(0)0-. In embodiments, each of L 1 , L 2 , and L 3 is -C(0)S-. In some embodiments, each of L 1 , L 2 , and L 3 is -C(0)NR l . In embodiments, each of L 1 , L 2 , and L 3 is a covalent bond or each of L 1 , L 2 , and L 3 is
  • each of R 1 , R 2 , and R 3 is the same group.
  • each of R 1 , R 2 , and R 3 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl).
  • each of R 1 , R 2 , and R 3 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl).
  • each of R 1 , R 2 , and R 3 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl).
  • each of R 1 , R 2 , and R 3 is C 6 -C 30 alkenyl (e.g., C6- C 22 alkenyl). In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, each of R 1 , R 2 , and R 3 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl).
  • each of R 1 , R 2 , and R 3 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl). In embodiments, each of R 1 , R 2 , and R 3 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • each R 1 , R 2 , and R 3 is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group.
  • each R 1 , R 2 , and R 3 is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
  • L 4A is -C(O)-. In embodiments, L 4A is -C(0)0-. In embodiments, L 4A is -C(0)NR L -.
  • L 4B is C 1 -C 20 alkylene (e.g., C 1 -C 6 alkylene). In embodiments, L 4B is
  • L 4B is substituted C 1 -C 20 alkylene (e.g., substituted C 1 -C 6 alkylene).
  • L 4B is C 2 -C 20 alkenylene (e.g., C 2 -C 6 alkenylene).
  • L 4B is unsubstituted C 2 -C 20 alkenylene (e.g., unsubstituted C 2 -C 6 alkenylene).
  • L 4B is substituted C 2 -C 20 alkenylene (e.g., substituted C 2 -C 6 alkenylene).
  • L 4B is C 2 -C 20 alkynylene (e.g., C 2 -C 6 alkynylene).
  • L 4B is unsubstituted C 2 -C 20 alkynylene (e.g., unsubstituted C 2 -C 6 alkynylene).
  • L 4B is substituted C 2 -C 20 alkynylene (e.g., substituted C 2 -C 6 alkynylene).
  • L 4B is -CH 2 - ln embodiments, L 4B is -CH 2 CH 2 -. In embodiments, L 4B is -CH 2 CH 2 CH 2 -.
  • B is NR 4 R 5 .
  • each R 4 and R 5 is independently hydrogen or unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl such as CH3), or R 4 and R 5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
  • B is NR 4 R 5
  • R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl.
  • R 4 and R 5 combine to form a 5- to 10- membered heterocyclyl.
  • R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl comprising one or two nitrogens in the ring or rings.
  • R 4 and R 5 combine to form a piperdinyl or piperazinyl.
  • R 4 and R 5 combine to form an unsubstituted piperdinyl.
  • R 4 and R 5 combine to form an unsubstituted piperazinyl.
  • R 4 and R 5 combine to form a substituted piperdinyl. In embodiments, R 4 and R 5 combine to form a substituted piperazinyl. In embodiments, R 4 and R 5 combine to form 4- methylpiperazinyl.
  • L 5A is -C(O)-. In embodiments, L 5A is -C(0)0-. In embodiments, L 5A is -C(0)NR L -.
  • L 5A and L 4A are the same. In embodiments, L 5A and L 4A are different.
  • L 5B is C 1 -C 20 alkylene (e.g., C 1 -C 6 alkylene). In embodiments, L 5B is
  • L 5B is substituted C 1 -C 20 alkylene (e.g., substituted C 1 -C 6 alkylene).
  • L 5B is C 2 -C 20 alkenylene (e.g., C 2 -C 6 alkenylene).
  • L 5B is unsubstituted C 2 -C 20 alkenylene (e.g., unsubstituted C 2 -C 6 alkenylene).
  • L 5B is substituted C 2 -C 20 alkenylene (e.g., substituted C 2 -C 6 alkenylene).
  • L 5B is C 2 -C 20 alkynylene (e.g., C 2 -C 6 alkynylene).
  • L 5B is unsubstituted C 2 -C 20 alkynylene (e.g., unsubstituted C 2 -C 6 alkynylene).
  • L 5B is substituted C 2 -C 20 alkynylene (e.g., substituted C 2 -C 6 alkynylene).
  • L 5B is -CH 2 - ln embodiments, L 5B is -CH 2 CH 2 -. In embodiments, L 5B is -CH 2 CH 2 CH 2 -. In embodiments, L 5B and L 4B are the same. In embodiments, L 5B and L 4B are different.
  • B' is NR 4 R 5 .
  • each R 4 and R 5 is independently hydrogen or unsubstituted C 1 -C 10 alkyl (e.g., unsubstituted C 1 -C 6 alkyl such as CH 3 ).
  • B' and B are the same. In embodiments, B' and B are different.
  • R 4 is hydrogen or C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 4 is
  • R 4 is C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 4 is unsubstituted C 1 -C 10 alkyl (e.g., unsubstituted C 1 -C 6 alkyl). In embodiments, R 4 is substituted C 1 -C 10 alkyl (e.g., substitute C 1 -C 6 alkyl). In embodiments, R 4 is C 2 -C 10 alkenyl (e.g., C 2 -C 6 alkenyl). In embodiments, R 4 is unsubstituted C 2 -C 10 alkenyl (e.g., unsubstituted C 2 -C 6 alkenyl). In embodiments, R 4 is
  • R 4 is C 2 -C 10 alkynyl (e.g., C 2 -C 6 alkynyl). In embodiments, R 4 is unsubstituted C 2 -C 10 alkynyl (e.g., unsubstituted C 2 -C 6 alkynyl). In embodiments, R 4 is substituted C 2 -C 10 alkynyl (e.g., substituted C 2 -C 6 alkynyl). In embodiments, R 4 is CH 3 .
  • R 5 is hydrogen or C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 5 is
  • R 5 is C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 5 is unsubstituted C 1 -C 10 alkyl (e.g., unsubstituted C 1 -C 6 alkyl). In embodiments, R 5 is substituted C 1 -C 10 alkyl (e.g., substitute C 1 -C 6 alkyl). In embodiments, R 5 is C 2 -C 10 alkenyl (e.g., C 2 -C 6 alkenyl). In embodiments, R 5 is unsubstituted C 2 -C 10 alkenyl (e.g., unsubstituted C 2 -C 6 alkenyl). In embodiments, R 5 is C 1 -C 10 alkyl (e.g., C 1 -C 6 alkenyl). In embodiments, R 5 is
  • R 5 is C 2 -C 10 alkynyl (e.g., C2-C6 alkynyl). In embodiments, R 5 is unsubstituted C2-C10 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, R 5 is substituted C2-C10 alkynyl (e.g., substituted C2-C6 alkynyl). In embodiments, R 5 is CH3.
  • B is N(CH B ) 2 .
  • B is 5- to 10-membered nitrogen-containing heteroaryl.
  • B is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl, or isoquinolyl.
  • B is an unsubstituted heteroaryl that is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl, or isoquinolyl.
  • B is a substituted heteroaryl that is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl, or isoquinolyl.
  • B is imidazolyl (e.g., unsubstituted imidazolyl or substituted imidazolyl).
  • B is pyridyl (e.g., unsubstituted pyridyl or unsubstituted pyridyl).
  • B is 5- to 10-membered nitrogen-containing heterocyclyl.
  • B is pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydro-l,3,5-triazinyl, azepanyl, diazepanyl, azocanyl, azonanyl, and the like.
  • B is piperdinyl (e.g., unsubstituted piperdinyl or substituted piperdinyl).
  • B is piperazinyl (e.g., unsubstituted piperazinyl or substituted piperazinyl).
  • R L is independently hydrogen. In embodiments, R L is independently C 1 -C 20 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R L is independently unsubstituted C 1 -C 20 alkyl (e.g., unsubstituted C 1 -C 6 alkyl). In embodiments, R L is independently substituted C 1 -C 20 alkyl (e.g., substituted C 1 -C 6 alkyl). In embodiments, R L is independently C 2 -C 20 alkenyl (e.g., C 2 -C 6 alkenyl).
  • R L is independently unsubstituted C 2 -C 20 alkenyl (e.g., unsubstituted C 2 -C 6 alkenyl). In embodiments, R L is independently substituted C 2 -C 20 alkenyl (e.g., substituted C 2 -C 6 alkenyl). In embodiments, R L is independently C 2 -C 20 alkynyl (e.g., C 2 -C 6 alkynyl). In embodiments, R L is independently unsubstituted C 2 -C 20 alkynyl (e.g., unsubstituted C 2 -C 6 alkynyl).
  • R L is independently substituted C 2 -C 20 alkynyl (e.g., substituted C 2 -C 6 alkynyl).
  • a cationic lipid of Formula (I) has a structure according to Formula (la):
  • L 1 , L 2 , L 3 , R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • a cationic lipid of Formula (I) has a structure according to Formula (lb):
  • R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • a cationic lipid of Formula (I) has a structure according to Formula (lc):
  • each of R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • a cationic lipid of Formula (I) has a structure according to Formula (Id):
  • R 1 , R 2 , R 3 , B, L 4A and L 4B is independently as defined herein.
  • the present invention provides a cationic lipid of Formula (le):
  • a cationic lipid of Formula (I) has a structure according to Formula (II):
  • R A is hydrogen or -AR 1 ;
  • R B is hydrogen or -L 2 -R 2 ;
  • R c is hydrogen or -L 3 -R 3 ;
  • each of L 1 , L 2 , and L 3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NR L -;
  • L 4 is independently C 1 -C 10 alkylene
  • each R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl, C 6 -C 30 alkenyl, or C 6 -C 30 alkynyl;
  • each R 4 and R 5 is independently hydrogen, C 1 -C 10 alkyl; C 2 -C 10 alkenyl; or C 2 -C 10 alkynyl; or R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; each R L is independently hydrogen, C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 2 -C 20 alkynyl.
  • R A is -AR 1 ;
  • R B is -L 2 -R 2 ; and
  • R A is hydrogen. In embodiments, R A is -AR 1 .
  • L 1 is a covalent bond or -C(O)-. In embodiments, L 1 is a covalent bond. In embodiments, L 1 is -C(O)-. In embodiments, L 1 is -C(0)0-. In embodiments, L 1 is -C(0)S-. In embodiments, L 1 is -C(0)NR L -.
  • R 1 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl). In embodiments, R 1 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl). In embodiments, R 1 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl). In embodiments, R 1 is C 6 -C 30 alkenyl (e.g., C 6 -C 22 alkenyl).
  • R 1 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, R 1 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl). In embodiments, R 1 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, R 1 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl). In embodiments, R 1 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • R B is hydrogen. In embodiments, R B is -AR 2 . [0144] .
  • L 2 is a covalent bond or -C(O)-. In embodiments, L 2 is a covalent bond. In some, L 2 is -C(O)-. In embodiments, L 2 is -C(0)0-. In embodiments, L 2 is -C(0)S-. In some embodiments, L 2 is -C(0)NR L .
  • R 2 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl). In embodiments, R 2 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl). In embodiments, R 2 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl). In embodiments, R 2 is C 6 -C 30 alkenyl (e.g., C 6 -C 22 alkenyl).
  • R 2 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, R 2 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl). In embodiments, R 2 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, R 2 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl). In embodiments, R 2 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • R c is hydrogen. In embodiments, R c is -L 3 -R 3 .
  • L 3 is a covalent bond or -C(O)-. In embodiments, L 3 is a covalent bond.
  • L 3 is -C(O)-. In embodiments, L 3 is -C(0)0-. In embodiments, L 3 is -C(0)S-. In some embodiments, L 3 is -C(0)NR L .
  • R 3 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl). In embodiments, R 3 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl). In embodiments, R 3 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl). In embodiments, R 3 is C 6 -C 30 alkenyl (e.g., C 6 -C 22 alkenyl).
  • R 3 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, R 3 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl). In embodiments, R 3 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, R 3 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl). In embodiments, R 3 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • each of L 1 , L 2 , and L 3 is the same group. In embodiments, each of L 1 , L 2 , and L 3 is a covalent bond. In some, each of L 1 , L 2 , and L 3 is -C(O)-. In embodiments, each of L 1 , L 2 , and L 3 is - C(0)0-. In embodiments, each of L 1 , L 2 , and L 3 is -C(0)S-. In some embodiments, each of L 1 , L 2 , and L 3 is -C(0)NR l . In embodiments, each of L 1 , L 2 , and L 3 is a covalent bond or each of L 1 , L 2 , and L 3 is -C(O)-.
  • each of R 1 , R 2 , and R 3 is the same group.
  • each of R 1 , R 2 , and R 3 is C 6 -C 30 alkyl (e.g., C 6 -C 22 alkyl).
  • each of R 1 , R 2 , and R 3 is unsubstituted C 6 -C 30 alkyl (e.g., unsubstituted C 6 -C 22 alkyl).
  • each of R 1 , R 2 , and R 3 is substituted C 6 -C 30 alkyl (e.g., substituted C 6 -C 22 alkyl).
  • each of R 1 , R 2 , and R 3 is C 6 -C 30 alkenyl (e.g., C 6 - C 22 alkenyl). In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted C 6 -C 30 alkenyl (e.g., unsubstituted C 6 -C 22 alkenyl). In embodiments, each of R 1 , R 2 , and R 3 is substituted C 6 -C 30 alkenyl (e.g., substituted C 6 -C 22 alkenyl).
  • each of R 1 , R 2 , and R 3 is C 6 -C 30 alkynyl (e.g., C 6 -C 22 alkynyl). In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted C 6 -C 30 alkynyl (e.g., unsubstituted C 6 -C 22 alkynyl). In embodiments, each of R 1 , R 2 , and R 3 is substituted C 6 -C 30 alkynyl (e.g., substituted C 6 -C 22 alkynyl).
  • L 4 is independently C 1 -C 10 alkylene (e.g., C 1 -C 6 alkylene). In embodiments, L 4 is independently unsubstituted C 1 -C 10 alkylene (e.g., unsubstituted C 1 -C 6 alkylene). In embodiments, L 4 is independently substituted C 1 -C 10 alkylene (e.g., substituted C 1 -C 6 alkylene). In embodiments, L 4 is -CH 2 CH 2 -.
  • B is NR 4 R 5 .
  • each R 4 and R 5 is independently hydrogen or unsubstituted C 1 -C 10 alkyl (e.g., unsubstituted C 1 -C 6 alkyl such as CH 3 ), or R 4 and R 5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
  • R 4 is hydrogen or C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 4 is
  • R 4 is C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 4 is unsubstituted C 1 -C 10 alkyl (e.g., unsubstituted C 1 -C 6 alkyl). In embodiments, R 4 is substituted C 1 -C 10 alkyl (e.g., substitute C 1 -C 6 alkyl). In embodiments, R 4 is C 2 -C 10 alkenyl (e.g., C 2 -C 6 alkenyl). In embodiments, R 4 is unsubstituted C 2 -C 10 alkenyl (e.g., unsubstituted C 2 -C 6 alkenyl). In embodiments, R 4 is
  • R 4 is C 2 -C 10 alkynyl (e.g., C 2 -C 6 alkynyl). In embodiments, R 4 is unsubstituted C 2 -C 10 alkynyl (e.g., unsubstituted C 2 -C 6 alkynyl). In embodiments, R 4 is substituted C 2 -C 10 alkynyl (e.g., substituted C 2 -C 6 alkynyl). In embodiments, R 4 is CH 3 .
  • R 5 is hydrogen or C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 5 is
  • R 5 is C 1 -C 10 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R 5 is unsubstituted C 1 -C 10 alkyl (e.g., unsubstituted C 1 -C 6 alkyl). In embodiments, R 5 is substituted C 1 -C 10 alkyl (e.g., substitute C 1 -C 6 alkyl). In embodiments, R 5 is C 2 -C 10 alkenyl (e.g., C 2 -C 6 alkenyl). In embodiments, R 5 is unsubstituted C 2 -C 10 alkenyl (e.g., unsubstituted C 2 -C 6 alkenyl). In embodiments, R 5 is C 1 -C 10 alkyl (e.g., C 1 -C 6 alkenyl). In embodiments, R 5 is
  • R 5 is C 2 -C 10 alkynyl (e.g., C 2 -C 6 alkynyl). In embodiments, R 5 is unsubstituted C 2 -C 10 alkynyl (e.g., unsubstituted C 2 -C 6 alkynyl). In embodiments, R 5 is substituted C 2 -C 10 alkynyl (e.g., substituted C 2 -C 6 alkynyl). In embodiments, R 5 is CH 3 .
  • R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl.
  • R 4 and R 5 combine to form a 5- to 10-membered heterocyclyl comprising one or two nitrogens in the ring or rings. In embodiments, R 4 and R 5 combine to form a piperdinyl or piperazinyl. In embodiments, R 4 and R 5 combine to form an unsubstituted piperdinyl.
  • R 4 and R 5 combine to form an unsubstituted piperazinyl. In embodiments, R 4 and R 5 combine to form a substituted piperdinyl. In embodiments, R 4 and R 5 combine to form a substituted piperazinyl. In embodiments, R 4 and R 5 combine to form 4-methylpiperazinyl.
  • R L is independently hydrogen. In embodiments, R L is independently C 1 -C 20 alkyl (e.g., C 1 -C 6 alkyl). In embodiments, R L is independently unsubstituted C 1 -C 20 alkyl (e.g., unsubstituted C 1 -C 6 alkyl). In embodiments, R L is independently substituted C 1 -C 20 alkyl (e.g., substituted C 1 -C 6 alkyl). In embodiments, R L is independently C 2 -C 20 alkenyl (e.g., C 2 -C 6 alkenyl).
  • R L is independently unsubstituted C 2 -C 20 alkenyl (e.g., unsubstituted C 2 -C 6 alkenyl). In embodiments, R L is independently substituted C 2 -C 20 alkenyl (e.g., substituted C 2 -C 6 alkenyl). In embodiments, R L is independently C 2 -C 20 alkynyl (e.g., C 2 -C 6 alkynyl). In embodiments, R L is independently unsubstituted C 2 -C 20 alkynyl (e.g., unsubstituted C 2 -C 6 alkynyl). In embodiments, R L is independently substituted C 2 -C 20 alkynyl (e.g., substituted C 2 -C 6 alkynyl).
  • a cationic lipid of Formula (II) has a structure according to Formula (lla):
  • R A , R B , R c , L 4 , R 4 and R 5 is as independently as defined herein.
  • a cationic lipid of Formula (II) has a structure according to Formula (lib):
  • a cationic lipid of Formula (II) has a structure according to Formula (lie):
  • R A , R B , R c , L 4 , R 4 and R 5 is independently as defined herein.
  • a cationic lipid of Formula (II) has a structure according to Formula (lid):
  • R A , R B , R c , L 4 , R 4 and R 5 is independently as defined herein.
  • a cationic lipid of Formula (II) has a structure according to Formula (lie):
  • R A , R B , R c , L 4 , R 4 and R 5 is independently as defined herein.
  • a C6-C30 alkyl (e.g., R 1 , R 2 , and/or R 3 ) is a Cg-26 alkyl.
  • a C6-C30 alkyl (e.g., R 1 , R 2 , and/or R 3 ) is a straight-chain Cg-26 alkyl.
  • a C6-C30 alkyl (e.g., R 1 , R 2 , and/or R 3 ) is CFIsiCFhJeCFh-, CFIsiCFhJyCFh-,
  • a C6-C30 alkyl (e.g., R 1 , R 2 , and/or R 3 ) is CH3(CH2)i3CH2-, CH3(CH2)i4CH2-, CH 3 (CH 2 )i5CH 2 -, CH 3 (CH 2 )i6CH 2 -, CH 3 (CH 2 )i7CH 2 - or CH3(CH 2 )igCH 2 -.
  • a C6-C30 alkyl e.g., R 1 , R 2 , and/or R 3
  • a C6-C30 alkenyl (e.g., R 1 , R 2 , and/or R 3 ) is a Cg-26 alkenyl having one or two
  • a C6-C30 alkenyl (e.g., R 1 , R 2 , and/or R 3 ) is Cg-26 aliphatic having three, four, five or six carbon-carbon double bonds.
  • a C6-C30 alkenyl (e.g., R 1 , R 2 , and/or R 3 ) is
  • a C 6 -C 30 alkenyl (e.g., R 1 , R 2 , and/or R 3 ) is
  • a C 6 -C 30 alkenyl (e.g., R 1 , R 2 , and/or R 3 ) is
  • each of R 1 , R 2 and R 3 independently is an aliphatic chain of a saturated or unsaturated fatty acid, i.e., R'-(CH 2 )- for a fatty acid R'-C(O)-.
  • each of R 1 , R 2 and R 3 independently is the aliphatic chain of caprylic, pelargonic, capric, undecylic, lauric, tridecyclic, myristic, pentadecylic, margaric, stearic, nonadecylic, arachidic, heneicosylic, behenic, triosylic, lignoceric, oleic, linoleic, pentacosylic or cerotic acid.
  • each of R and R 1 is the aliphatic chain of caprylic, pelargonic, capric, undecylic, lauric, tridecyclic, myristic, pentadecylic, or margaric acid.
  • each of R 1 , R 2 and R 3 is the aliphatic chain of lauric, tridecyclic, myristic, or pentadecylic acid. In some embodiments, each of R 1 , R 2 and R 3 is the aliphatic chain of lauric or myristic acid. In some embodiments, each of R 1 , R 2 and R 3 is the aliphatic chain of stearic, nonadecylic, arachidic, heneicosylic, behenic, triosylic, lignoceric, oleic, linoleic, pentacosylic or cerotic acid.
  • each of R 1 , R 2 and R 3 is the aliphatic chain of lignoceric, oleic, linoleic, pentacosylic or cerotic acid. In some embodiments, each of R 1 , R 2 and R 3 is the aliphatic chain of oleic, linoleic or pentacosylic acid. In some embodiments, each of R 1 , R 2 and R 3 is the aliphatic chain of oleic or linoleic acid. In some embodiments, each of R 1 , R 2 and R 3 is the aliphatic chain of oleic acid.
  • each of R 1 , R 2 and R 3 is the aliphatic chain of linoleic acid.
  • each of R 1 , R 2 and R 3 independently is any of the following aliphatic chains.
  • each of R 1 , R 2 and R 3 is the same aliphatic chain.
  • Exemplary cationic lipids include cationic lipids (la)-(21a) (Table A).
  • Exemplary cationic lipids include cationic lipids (lb)-(21b) (Table B).
  • Exemplary cationic lipids include cationic lipids according to formula (Ilia) such as cationic lipids (22)-(34) (Table C):
  • a cationic lipid is cationic lipid (22). In embodiments, a cationic lipid is cationic lipid (23). In embodiments, a cationic lipid is cationic lipid (24). In embodiments, a cationic lipid is cationic lipid (25). In embodiments, a cationic lipid is cationic lipid (26). In embodiments, a cationic lipid is cationic lipid (27). In embodiments, a cationic lipid is cationic lipid (28). In embodiments, a cationic lipid is cationic lipid (29). In embodiments, a cationic lipid is cationic lipid (30).
  • a cationic lipid is cationic lipid (31). In embodiments, a cationic lipid is cationic lipid (32). In embodiments, a cationic lipid is cationic lipid (33). In embodiments, a cationic lipid is cationic lipid (34).
  • Exemplary cationic lipids include cationic lipids according to formula (lllb) such as cationic lipids (35)-(47) (Table D):
  • a cationic lipid is cationic lipid (35). In embodiments, a cationic lipid is cationic lipid (36). In embodiments, a cationic lipid is cationic lipid (37). In embodiments, a cationic lipid is cationic lipid (38). In embodiments, a cationic lipid is cationic lipid (39). In embodiments, a cationic lipid is cationic lipid (40). In embodiments, a cationic lipid is cationic lipid (41). In embodiments, a cationic lipid is cationic lipid (42). In embodiments, a cationic lipid is cationic lipid (43).
  • a cationic lipid is cationic lipid (44). In embodiments, a cationic lipid is cationic lipid (45). In embodiments, a cationic lipid is cationic lipid (46). In embodiments, a cationic lipid is cationic lipid (47).
  • Exemplary cationic lipids include cationic lipids according to formula (lllc) such as cationic lipids
  • a cationic lipid is cationic lipid (48). In embodiments, a cationic lipid is cationic lipid (49). In embodiments, a cationic lipid is cationic lipid (50). In embodiments, a cationic lipid is cationic lipid (51). In embodiments, a cationic lipid is cationic lipid (52). In embodiments, a cationic lipid is cationic lipid (53). In embodiments, a cationic lipid is cationic lipid (54). In embodiments, a cationic lipid is cationic lipid (55). In embodiments, a cationic lipid is cationic lipid (56).
  • a cationic lipid is cationic lipid (57). In embodiments, a cationic lipid is cationic lipid (58). In embodiments, a cationic lipid is cationic lipid (59). In embodiments, a cationic lipid is cationic lipid (60).
  • Exemplary cationic lipids include cationic lipids according to formula (llld) such as cationic lipids (61)-(71) (Table F):
  • a cationic lipid is cationic lipid (61). In embodiments, a cationic lipid is cationic lipid (62). In embodiments, a cationic lipid is cationic lipid (63). In embodiments, a cationic lipid is cationic lipid (64). In embodiments, a cationic lipid is cationic lipid (65). In embodiments, a cationic lipid is cationic lipid (66). In embodiments, a cationic lipid is cationic lipid (67). In embodiments, a cationic lipid is cationic lipid (68). In embodiments, a cationic lipid is cationic lipid (69). In embodiments, a cationic lipid is cationic lipid (70). In embodiments, a cationic lipid is cationic lipid (71).
  • Exemplary cationic lipids include cationic lipids according to formula (llle) such as cationic lipids (72)-(82) (Table F):
  • a cationic lipid is cationic lipid (72). In embodiments, a cationic lipid is cationic lipid (73). In embodiments, a cationic lipid is cationic lipid (74). In embodiments, a cationic lipid is cationic lipid (75). In embodiments, a cationic lipid is cationic lipid (76). In embodiments, a cationic lipid is cationic lipid (77). In embodiments, a cationic lipid is cationic lipid (78). In embodiments, a cationic lipid is cationic lipid (79). In embodiments, a cationic lipid is cationic lipid (80). In embodiments, a cationic lipid is cationic lipid (81). In embodiments, a cationic lipid is cationic lipid (82).
  • Exemplary cationic lipids include cationic lipids according to formula (lllf) such as cationic lipids (83)-(93) (Table H):
  • a cationic lipid is cationic lipid (83). In embodiments, a cationic lipid is cationic lipid (84). In embodiments, a cationic lipid is cationic lipid (85). In embodiments, a cationic lipid is cationic lipid (86). In embodiments, a cationic lipid is cationic lipid (87). In embodiments, a cationic lipid is cationic lipid (88). In embodiments, a cationic lipid is cationic lipid (89). In embodiments, a cationic lipid is cationic lipid (90). In embodiments, a cationic lipid is cationic lipid (91). In embodiments, a cationic lipid is cationic lipid (92). In embodiments, a cationic lipid is cationic lipid (93).
  • Exemplary cationic lipids include cationic lipids according to formula (lllg) such as cationic lipids (94)-(106) (Table I):
  • a cationic lipid is cationic lipid (94). In embodiments, a cationic lipid is cationic lipid (95). In embodiments, a cationic lipid is cationic lipid (96). In embodiments, a cationic lipid is cationic lipid (97). In embodiments, a cationic lipid is cationic lipid (98). In embodiments, a cationic lipid is cationic lipid (99). In embodiments, a cationic lipid is cationic lipid (100). In embodiments, a cationic lipid is cationic lipid (101). In embodiments, a cationic lipid is cationic lipid (102).
  • a cationic lipid is cationic lipid (103). In embodiments, a cationic lipid is cationic lipid (104). In embodiments, a cationic lipid is cationic lipid (105). In embodiments, a cationic lipid is cationic lipid (106). [0193] Exemplary cationic lipids include cationic lipids according to formula (lllh) such as cationic lipids
  • a cationic lipid is cationic lipid (107). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (108). In embodiments, a cationic lipid is cationic lipid (109). In embodiments, a cationic lipid is cationic lipid (110). In embodiments, a cationic lipid is cationic lipid (111). In embodiments, a cationic lipid is cationic lipid (112). In embodiments, a cationic lipid is cationic lipid (113). In embodiments, a cationic lipid is cationic lipid (114). In embodiments, a cationic lipid is cationic lipid (115). In embodiments, a cationic lipid is cationic lipid (116). In embodiments, a cationic lipid is cationic lipid (117). In embodiments, a cationic lipid is cationic lipid (118). In embodiments, a cationic lipid is cationic lipid (119).
  • Exemplary cationic lipids include cationic lipids according to formula (llli) such as cationic lipids
  • a cationic lipid is cationic lipid (120). In embodiments, a cationic lipid is cationic lipid (121). In embodiments, a cationic lipid is cationic lipid (122). In embodiments, a cationic lipid is cationic lipid (123). In embodiments, a cationic lipid is cationic lipid (124). In embodiments, a cationic lipid is cationic lipid (125). In embodiments, a cationic lipid is cationic lipid (126). In embodiments, a cationic lipid is cationic lipid (127). In embodiments, a cationic lipid is cationic lipid (128).
  • a cationic lipid is cationic lipid (129). In embodiments, a cationic lipid is cationic lipid (130). In embodiments, a cationic lipid is cationic lipid (131). In embodiments, a cationic lipid is cationic lipid (132).
  • Exemplary cationic lipids include cationic lipids according to formula (lllj) such as cationic lipids (133)-(143) (Table L): f lllj), where B, R 1 , R 2 , and R 3 are independently as described herein, including the exemplified groups of Table L.
  • a cationic lipid is cationic lipid (133). In embodiments, a cationic lipid is cationic lipid (134). In embodiments, a cationic lipid is cationic lipid (135). In embodiments, a cationic lipid is cationic lipid (136). In embodiments, a cationic lipid is cationic lipid (137). In embodiments, a cationic lipid is cationic lipid (138). In embodiments, a cationic lipid is cationic lipid (139). In embodiments, a cationic lipid is cationic lipid (140). In embodiments, a cationic lipid is cationic lipid (141). In embodiments, a cationic lipid is cationic lipid (142). In embodiments, a cationic lipid is cationic lipid (143).
  • Exemplary cationic lipids include cationic lipids according to formula (lllk) such as cationic lipids (144)-(154) (Table M):
  • a cationic lipid is cationic lipid (144). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (145). In embodiments, a cationic lipid is cationic lipid (146). In embodiments, a cationic lipid is cationic lipid (147). In embodiments, a cationic lipid is cationic lipid (148). In embodiments, a cationic lipid is cationic lipid (149). In embodiments, a cationic lipid is cationic lipid (150). In embodiments, a cationic lipid is cationic lipid (151). In embodiments, a cationic lipid is cationic lipid (152). In embodiments, a cationic lipid is cationic lipid (153). In embodiments, a cationic lipid is cationic lipid (154).
  • Exemplary cationic lipids include cationic lipids according to formula (Him) such as cationic lipids (155)-(165) (Table N):
  • a cationic lipid is cationic lipid (155). In embodiments, a cationic lipid is cationic lipid (156). In embodiments, a cationic lipid is cationic lipid (157). In embodiments, a cationic lipid is cationic lipid (158). In embodiments, a cationic lipid is cationic lipid (159). In embodiments, a cationic lipid is cationic lipid (160). In embodiments, a cationic lipid is cationic lipid (161). In embodiments, a cationic lipid is cationic lipid (162). In embodiments, a cationic lipid is cationic lipid (163). In embodiments, a cationic lipid is cationic lipid (164). In embodiments, a cationic lipid is cationic lipid (165).
  • Exemplary cationic lipids include cationic lipids according to formula (llln) such as cationic lipids ( 166)-( 178) (Table O):
  • a cationic lipid according to formula (llln) has the following structure, (llln-1).
  • each of R 1 , R 2 , and R 3 is any of the exemplified aliphatic groups described hereinin (e.g., Table O).
  • a cationic lipid is cationic lipid (166). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (167). In embodiments, a cationic lipid is cationic lipid (168). In embodiments, a cationic lipid is cationic lipid (169). In embodiments, a cationic lipid is cationic lipid (170). In embodiments, a cationic lipid is cationic lipid (171). In embodiments, a cationic lipid is cationic lipid (172). In embodiments, a cationic lipid is cationic lipid (173). In embodiments, a cationic lipid is cationic lipid (174). In embodiments, a cationic lipid is cationic lipid (175). In embodiments, a cationic lipid is cationic lipid (176). In embodiments, a cationic lipid is cationic lipid (177). In embodiments, a cationic lipid is cationic lipid (178).
  • Exemplary cationic lipids include cationic lipids according to formula (lllo) such as cationic lipids (179)-(191) (Table P):
  • a cationic lipid is cationic lipid (179). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (181). In embodiments, a cationic lipid is cationic lipid (182). In embodiments, a cationic lipid is cationic lipid (183). In embodiments, a cationic lipid is cationic lipid (184). In embodiments, a cationic lipid is cationic lipid (185). In embodiments, a cationic lipid is cationic lipid (186). In embodiments, a cationic lipid is cationic lipid (187). In embodiments, a cationic lipid is cationic lipid (188). In embodiments, a cationic lipid is cationic lipid (189). In embodiments, a cationic lipid is cationic lipid (190). In embodiments, a cationic lipid is cationic lipid (191).
  • Exemplary cationic lipids include cationic lipids according to formula (lllp) such as cationic lipids (192)-(204) (Table Q): (lllp), where B, B', R 2 , and R 3 are independently as described herein, including the exemplified groups of Table Q.
  • formula (lllp) such as cationic lipids (192)-(204) (Table Q): (lllp), where B, B', R 2 , and R 3 are independently as described herein, including the exemplified groups of Table Q.
  • a cationic lipid is cationic lipid (192). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (193). In embodiments, a cationic lipid is cationic lipid (194). In embodiments, a cationic lipid is cationic lipid (195). In embodiments, a cationic lipid is cationic lipid (196). In embodiments, a cationic lipid is cationic lipid (197). In embodiments, a cationic lipid is cationic lipid (198). In embodiments, a cationic lipid is cationic lipid (199). In embodiments, a cationic lipid is cationic lipid (200). In embodiments, a cationic lipid is cationic lipid (201). In embodiments, a cationic lipid is cationic lipid (202). In embodiments, a cationic lipid is cationic lipid (203). In embodiments, a cationic lipid is cationic lipid (204).
  • Exemplary cationic lipids include cationic lipids according to formula (lllq) such as cationic lipids (205)-(215) (Table R): (lllq), where B, R 1 , R 2 , and R 3 are independently as described herein, including the exemplified groups of Table R.
  • formula (lllq) such as cationic lipids (205)-(215) (Table R): (lllq), where B, R 1 , R 2 , and R 3 are independently as described herein, including the exemplified groups of Table R.
  • a cationic lipid according to formula (lllq) has the following structure,
  • each of R 1 , R 2 , and R 3 is any of the exemplified aliphatic groups described in, e.g., Table R.
  • a cationic lipid is cationic lipid (205). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (206). In embodiments, a cationic lipid is cationic lipid (207). In embodiments, a cationic lipid is cationic lipid (208). In embodiments, a cationic lipid is cationic lipid (209). In embodiments, a cationic lipid is cationic lipid (210). In embodiments, a cationic lipid is cationic lipid (211). In embodiments, a cationic lipid is cationic lipid (212). In embodiments, a cationic lipid is cationic lipid (213). In embodiments, a cationic lipid is cationic lipid (214). In embodiments, a cationic lipid is cationic lipid (215).
  • Exemplary cationic lipids include cationic lipids according to formula (lllr) such as cationic lipids (216)-(226) (Table S):
  • a cationic lipid is cationic lipid (216). In embodiments, a cationic lipid is cationic lipid (217). In embodiments, a cationic lipid is cationic lipid (218). In embodiments, a cationic lipid is cationic lipid (219). In embodiments, a cationic lipid is cationic lipid (220). In embodiments, a cationic lipid is cationic lipid (221). In embodiments, a cationic lipid is cationic lipid (222). In embodiments, a cationic lipid is cationic lipid (223). In embodiments, a cationic lipid is cationic lipid (224). In embodiments, a cationic lipid is cationic lipid (225). In embodiments, a cationic lipid is cationic lipid (226).
  • Exemplary cationic lipids include cationic lipids according to formula (Ills) such as cationic lipids
  • a cationic lipid is cationic lipid (227). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (228). In embodiments, a cationic lipid is cationic lipid (229). In embodiments, a cationic lipid is cationic lipid (230). In embodiments, a cationic lipid is cationic lipid (231). In embodiments, a cationic lipid is cationic lipid (232). In embodiments, a cationic lipid is cationic lipid (233). In embodiments, a cationic lipid is cationic lipid (234). In embodiments, a cationic lipid is cationic lipid (235). In embodiments, a cationic lipid is cationic lipid (236). In embodiments, a cationic lipid is cationic lipid (237).
  • Exemplary cationic lipids include cationic lipids according to formula (lilt) such as cationic lipids (238)-(261) (Table U): (lilt), wherein each R is independently as described herein, including the exemplified groups of Table U.
  • a cationic lipid is cationic lipid (238). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (240). In embodiments, a cationic lipid is cationic lipid (241). In embodiments, a cationic lipid is cationic lipid (242). In embodiments, a cationic lipid is cationic lipid (243). In embodiments, a cationic lipid is cationic lipid (244). In embodiments, a cationic lipid is cationic lipid (245). In embodiments, a cationic lipid is cationic lipid (246). In embodiments, a cationic lipid is cationic lipid (247). In embodiments, a cationic lipid is cationic lipid (248).
  • a cationic lipid is cationic lipid (249). In embodiments, a cationic lipid is cationic lipid (250). In embodiments, a cationic lipid is cationic lipid (251). In embodiments, a cationic lipid is cationic lipid (252). In embodiments, a cationic lipid is cationic lipid (253). In embodiments, a cationic lipid is cationic lipid (254). In embodiments, a cationic lipid is cationic lipid (255). In embodiments, a cationic lipid is cationic lipid (256). In embodiments, a cationic lipid is cationic lipid (257).
  • a cationic lipid is cationic lipid (258). In embodiments, a cationic lipid is cationic lipid (259). In embodiments, a cationic lipid is cationic lipid (260). In embodiments, a cationic lipid is cationic lipid (261).
  • Exemplary cationic lipids include cationic lipids according to formula (lllu) such as cationic lipids (262)-(285) (Table V): (lllu), wherein each R is independently as described herein, including the exemplified groups of Table V.
  • a cationic lipid is cationic lipid (262). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (264). In embodiments, a cationic lipid is cationic lipid (265). In embodiments, a cationic lipid is cationic lipid (266). In embodiments, a cationic lipid is cationic lipid (267). In embodiments, a cationic lipid is cationic lipid (268). In embodiments, a cationic lipid is cationic lipid (269). In embodiments, a cationic lipid is cationic lipid (270). In embodiments, a cationic lipid is cationic lipid (271). In embodiments, a cationic lipid is cationic lipid (272).
  • a cationic lipid is cationic lipid (273). In embodiments, a cationic lipid is cationic lipid (274). In embodiments, a cationic lipid is cationic lipid (275). In embodiments, a cationic lipid is cationic lipid (276). In embodiments, a cationic lipid is cationic lipid (277). In embodiments, a cationic lipid is cationic lipid (278). In embodiments, a cationic lipid is cationic lipid (279). In embodiments, a cationic lipid is cationic lipid (280). In embodiments, a cationic lipid is cationic lipid (281).
  • a cationic lipid is cationic lipid (282). In embodiments, a cationic lipid is cationic lipid (283). In embodiments, a cationic lipid is cationic lipid (284). In embodiments, a cationic lipid is cationic lipid (285).
  • Exemplary cationic lipids include cationic lipids according to formula (lllv) such as cationic lipids (286)-(309) (Table W): (lllv), wherein each R is independently as described herein, including the exemplified groups of Table W.
  • a cationic lipid is cationic lipid (286). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (287). In embodiments, a cationic lipid is cationic lipid (288). In embodiments, a cationic lipid is cationic lipid (289). In embodiments, a cationic lipid is cationic lipid (290). In embodiments, a cationic lipid is cationic lipid (291). In embodiments, a cationic lipid is cationic lipid (292). In embodiments, a cationic lipid is cationic lipid (293). In embodiments, a cationic lipid is cationic lipid (294). In embodiments, a cationic lipid is cationic lipid (295). In embodiments, a cationic lipid is cationic lipid (296).
  • a cationic lipid is cationic lipid (297). In embodiments, a cationic lipid is cationic lipid (298). In embodiments, a cationic lipid is cationic lipid (299). In embodiments, a cationic lipid is cationic lipid (300). In embodiments, a cationic lipid is cationic lipid (301). In embodiments, a cationic lipid is cationic lipid (302). In embodiments, a cationic lipid is cationic lipid (303). In embodiments, a cationic lipid is cationic lipid (304). In embodiments, a cationic lipid is cationic lipid (305).
  • a cationic lipid is cationic lipid (306). In embodiments, a cationic lipid is cationic lipid (307). In embodiments, a cationic lipid is cationic lipid (308). In embodiments, a cationic lipid is cationic lipid (309).
  • Exemplary cationic lipids include cationic lipids according to formula (lllw) such as cationic lipids
  • a cationic lipid is cationic lipid (310). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (311). In embodiments, a cationic lipid is cationic lipid (312). In embodiments, a cationic lipid is cationic lipid (313). In embodiments, a cationic lipid is cationic lipid (314). In embodiments, a cationic lipid is cationic lipid (315). In embodiments, a cationic lipid is cationic lipid (316). In embodiments, a cationic lipid is cationic lipid (317). In embodiments, a cationic lipid is cationic lipid (318). In embodiments, a cationic lipid is cationic lipid (319). In embodiments, a cationic lipid is cationic lipid (320).
  • a cationic lipid is cationic lipid (321). In embodiments, a cationic lipid is cationic lipid (322). In embodiments, a cationic lipid is cationic lipid (323). In embodiments, a cationic lipid is cationic lipid (324). In embodiments, a cationic lipid is cationic lipid (325). In embodiments, a cationic lipid is cationic lipid (326). In embodiments, a cationic lipid is cationic lipid (327). In embodiments, a cationic lipid is cationic lipid (328). In embodiments, a cationic lipid is cationic lipid (329).
  • a cationic lipid is cationic lipid (330). In embodiments, a cationic lipid is cationic lipid (331). In embodiments, a cationic lipid is cationic lipid (332). In embodiments, a cationic lipid is cationic lipid (333).
  • Exemplary cationic lipids include cationic lipids according to formula (Mix) such as cationic lipids (334)-(357) (Table Y):
  • a cationic lipid is cationic lipid (334). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (335). In embodiments, a cationic lipid is cationic lipid (336). In embodiments, a cationic lipid is cationic lipid (337). In embodiments, a cationic lipid is cationic lipid (338). In embodiments, a cationic lipid is cationic lipid (339). In embodiments, a cationic lipid is cationic lipid (340). In embodiments, a cationic lipid is cationic lipid (341). In embodiments, a cationic lipid is cationic lipid (342). In embodiments, a cationic lipid is cationic lipid (343). In embodiments, a cationic lipid is cationic lipid (344).
  • a cationic lipid is cationic lipid (345). In embodiments, a cationic lipid is cationic lipid (346). In embodiments, a cationic lipid is cationic lipid (347). In embodiments, a cationic lipid is cationic lipid (348). In embodiments, a cationic lipid is cationic lipid (349). In embodiments, a cationic lipid is cationic lipid (350). In embodiments, a cationic lipid is cationic lipid (351). In embodiments, a cationic lipid is cationic lipid (352). In embodiments, a cationic lipid is cationic lipid (353).
  • a cationic lipid is cationic lipid (354). In embodiments, a cationic lipid is cationic lipid (355). In embodiments, a cationic lipid is cationic lipid (356). In embodiments, a cationic lipid is cationic lipid (357).
  • Exemplary cationic lipids include cationic lipids according to formula (Illy) such as cationic lipids (358)-(381) (Table Z):
  • each R is independently as described herein, including the exemplified groups of Table Z.
  • a cationic lipid is cationic lipid (358). In embodiments, a cationic lipid is cationic lipid (359). In embodiments, a cationic lipid is cationic lipid (360). In embodiments, a cationic lipid is cationic lipid (361). In embodiments, a cationic lipid is cationic lipid (362). In embodiments, a cationic lipid is cationic lipid (363). In embodiments, a cationic lipid is cationic lipid (364). In embodiments, a cationic lipid is cationic lipid (365). In embodiments, a cationic lipid is cationic lipid (366).
  • a cationic lipid is cationic lipid (367). In embodiments, a cationic lipid is cationic lipid (368). In embodiments, a cationic lipid is cationic lipid (369). In embodiments, a cationic lipid is cationic lipid (370). In embodiments, a cationic lipid is cationic lipid (371). In embodiments, a cationic lipid is cationic lipid (372). In embodiments, a cationic lipid is cationic lipid (373). In embodiments, a cationic lipid is cationic lipid (374). In embodiments, a cationic lipid is cationic lipid (375).
  • a cationic lipid is cationic lipid (376). In embodiments, a cationic lipid is cationic lipid (377). In embodiments, a cationic lipid is cationic lipid (378). In embodiments, a cationic lipid is cationic lipid (379). In embodiments, a cationic lipid is cationic lipid (380). In embodiments, a cationic lipid is cationic lipid (381).
  • Exemplary cationic lipids include cationic lipids according to formula (lllz) such as cationic lipids (382)-(405) (Table AA):
  • a cationic lipid is cationic lipid (382). In embodiments, a cationic lipid is cationic lipid (383). In embodiments, a cationic lipid is cationic lipid (384). In embodiments, a cationic lipid is cationic lipid (385). In embodiments, a cationic lipid is cationic lipid (386). In embodiments, a cationic lipid is cationic lipid (387). In embodiments, a cationic lipid is cationic lipid (388). In embodiments, a cationic lipid is cationic lipid (389). In embodiments, a cationic lipid is cationic lipid (390).
  • a cationic lipid is cationic lipid (391). In embodiments, a cationic lipid is cationic lipid (392). In embodiments, a cationic lipid is cationic lipid (393). In embodiments, a cationic lipid is cationic lipid (394). In embodiments, a cationic lipid is cationic lipid (395). In embodiments, a cationic lipid is cationic lipid (396). In embodiments, a cationic lipid is cationic lipid (397). In embodiments, a cationic lipid is cationic lipid (398). In embodiments, a cationic lipid is cationic lipid (399).
  • a cationic lipid is cationic lipid (400). In embodiments, a cationic lipid is cationic lipid (401). In embodiments, a cationic lipid is cationic lipid (402). In embodiments, a cationic lipid is cationic lipid (403). In embodiments, a cationic lipid is cationic lipid (404). In embodiments, a cationic lipid is cationic lipid (405).
  • Exemplary cationic lipids include cationic lipids according to formula (lllaa) such as cationic lipids (406)-(429) (Table AB):
  • a cationic lipid is cationic lipid (406). In embodiments, a cationic lipid is
  • a cationic lipid (407). In embodiments, a cationic lipid is cationic lipid (408). In embodiments, a cationic lipid is cationic lipid (409). In embodiments, a cationic lipid is cationic lipid (410). In embodiments, a cationic lipid is cationic lipid (411). In embodiments, a cationic lipid is cationic lipid (412). In embodiments, a cationic lipid is cationic lipid (413). In embodiments, a cationic lipid is cationic lipid (414). In embodiments, a cationic lipid is cationic lipid (415). In embodiments, a cationic lipid is cationic lipid (416).
  • a cationic lipid is cationic lipid (417). In embodiments, a cationic lipid is cationic lipid (418). In embodiments, a cationic lipid is cationic lipid (419). In embodiments, a cationic lipid is cationic lipid (420). In embodiments, a cationic lipid is cationic lipid (421). In embodiments, a cationic lipid is cationic lipid (422). In embodiments, a cationic lipid is cationic lipid (423). In embodiments, a cationic lipid is cationic lipid (424). In embodiments, a cationic lipid is cationic lipid (425).
  • a cationic lipid is cationic lipid (426). In embodiments, a cationic lipid is cationic lipid (427). In embodiments, a cationic lipid is cationic lipid (428). In embodiments, a cationic lipid is cationic lipid (429). [0233] Exemplary cationic lipids include cationic lipids according to formula (lllaa) such as cationic lipids
  • a cationic lipid is cationic lipid (430). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (431). In embodiments, a cationic lipid is cationic lipid (432). In embodiments, a cationic lipid is cationic lipid (433). In embodiments, a cationic lipid is cationic lipid (434). In embodiments, a cationic lipid is cationic lipid (435). In embodiments, a cationic lipid is cationic lipid (436). In embodiments, a cationic lipid is cationic lipid (437). In embodiments, a cationic lipid is cationic lipid (438). In embodiments, a cationic lipid is cationic lipid (439). In embodiments, a cationic lipid is cationic lipid (440).
  • a cationic lipid is cationic lipid (441). In embodiments, a cationic lipid is cationic lipid (442). In embodiments, a cationic lipid is cationic lipid (443). In embodiments, a cationic lipid is cationic lipid (444). In embodiments, a cationic lipid is cationic lipid (445). In embodiments, a cationic lipid is cationic lipid (446). In embodiments, a cationic lipid is cationic lipid (447). In embodiments, a cationic lipid is cationic lipid (448). In embodiments, a cationic lipid is cationic lipid (449).
  • a cationic lipid is cationic lipid (450). In embodiments, a cationic lipid is cationic lipid (451). In embodiments, a cationic lipid is cationic lipid (452). In embodiments, a cationic lipid is cationic lipid (453). [0235] Exemplary cationic lipids include cationic lipids (454)-(462) of Table AD.
  • a cationic lipid is cationic lipid (454). In embodiments, a cationic lipid is
  • a cationic lipid is cationic lipid (455). In embodiments, a cationic lipid is cationic lipid (456). In embodiments, a cationic lipid is cationic lipid (457). In embodiments, a cationic lipid is cationic lipid (458). In embodiments, a cationic lipid is cationic lipid (459). In embodiments, a cationic lipid is cationic lipid (460). In embodiments, a cationic lipid is cationic lipid (461). In embodiments, a cationic lipid is cationic lipid (462).
  • Cationic lipids described herein e.g., cationic lipids of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • G cationic lipids of Formula (G), (I), (II), or (llla)-(lllab)
  • cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462) can be prepared according to methods known in the art. Additional exemplary methods are provided in the Examples described herein.
  • Scheme 1 provides an exemplary synthesis for cationic lipids described herein.
  • This compound can be treated with various electrophiles to afford cationic lipids (e.g., cationic lipids of Formula (I) and (II)) as described herein.
  • cationic lipids e.g., cationic lipids of Formula (I) and (II)
  • Compound C can be treated with an aliphatic electrophile (e.g., alkyl halides or alkenyl halides) such as Compound D to afford product Cationic Lipid E.
  • Compound C can be treated with an acylating agent such
  • Scheme 2 provides an exemplary synthesis using D-ribose and acylating agents.
  • Cationic lipids described herein e.g., a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • Nucleic acids according to the present invention may be synthesized according to any known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • a linear or circular DNA template containing 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, mutated T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7, mutated T7 or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7, mutated T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
  • Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence.
  • a desired amino acid sequence e.g., an enzyme sequence
  • Optimization algorithms may then be used for selection of suitable codons.
  • the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand.
  • the optimized RNA sequence can be established and
  • a secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
  • nucleic acid in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain.
  • DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • PCR polymerase chain reaction
  • RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA,
  • RNAs according to the present invention may be synthesized according to any of a variety of known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
  • the in vitro transcribing occurs in a single batch.
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
  • Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
  • a desired amino acid sequence e.g., an enzyme sequence
  • Optimization algorithms may then be used for selection of suitable codons.
  • the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency
  • mRNA according to the present invention may be synthesized as
  • Modified mRNA comprise nucleotide modifications in the RNA.
  • a modified mRNA according to the invention can thus include nucleotide modification that are, for example, backbone modifications, sugar modifications or base modifications.
  • mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
  • purines adenine (A), guanine (G)
  • pyrimidines thymine (T), cytosine (C), uracil (U)
  • modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
  • mRNAs may contain RNA backbone modifications.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically.
  • Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates,
  • methylphosphoramidates e.g. cytidine 5'-0-(l- thiophosphate)
  • boranophosphates e.g. cytidine 5'-0-(l- thiophosphate)
  • boranophosphates e.g. cytidine 5'-0-(l- thiophosphate)
  • positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
  • mRNAs may contain sugar modifications.
  • a typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 4'-thio-ribonucleotide (see, e.g., US Patent Application Publication No.
  • mRNAs may contain modifications of the bases of the nucleotides (base modifications).
  • a modified nucleotide which contains a base modification is also called a base- modified nucleotide.
  • base-modified nucleotides include, but are not limited to, 2- amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'- triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'- triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'- triphosphate, 5-methyluridine 5'--
  • mRNA synthesis typically includes the addition of a "cap” on the N-terminal (5') end, and a “tail” on the C-terminal (3') end.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a "tail” serves to protect the mRNA from exonuclease degradation.
  • mRNAs include a 5' cap structure.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7- nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
  • mRNAs include a 3' poly(A) tail structure.
  • terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • mRNAs include a 3' poly(C) tail structure.
  • a suitable poly-C tail on the 3' terminus of 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 may be added to the poly-A tail or may substitute the poly-A tail.
  • mRNAs include a 5' and/or 3' untranslated region.
  • a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element.
  • a 5' untranslated region may be between about 50 and 500 nucleotides in length.
  • a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs.
  • a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.
  • mRNAs include a 5' cap structure.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7- nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
  • Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m 7 G(5')ppp(5')N, where N is any nucleoside.
  • the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase.
  • the addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription.
  • the terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp.
  • a common cap for mRNA produced by in vitro transcription is m 7 G(5')ppp(5')G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5'-termini.
  • the prevailing method for the in vitro synthesis of caPPEd mRNA employs a pre-formed dinucleotide of the form m 7 G(5')ppp(5')G ("m 7 GpppG”) as an initiator of transcription.
  • ARCA Anti-Reverse Cap Analog
  • modified ARCA which is generally a modified cap analog in which the 2' or 3' OH group is replaced with -OCH 3 .
  • Additional cap analogs include, but are not limited to, a 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 analog e.g., m 2 - 7 GpppG
  • trimethylated cap analog e.g., m 2 - 2 ' 7 GpppG
  • dimethylated symmetrical cap analogs e.g., m 7 Gpppm 7 G
  • anti reverse cap analogs e.g., ARCA; m 7 , 2Ome GpppG, m 72 d GpppG, m 7 ' 3 0me GpppG, m 7 - 3 d GpppG and their tetraphosphate derivatives
  • a suitable cap is a 7-methyl guanylate ("m 7 G") linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in m 7 G(5')ppp(5')N, where N is any nucleoside.
  • m 7 G 7-methyl guanylate
  • a preferred embodiment of a m 7 G cap utilized in embodiments of the invention is m 7 G(5')ppp(5')G.
  • the cap is a CapO structure.
  • CapO structures lack a 2'-0-methyl residue of the ribose attached to bases 1 and 2.
  • the cap is a Capl structure.
  • Capl structures have a 2'-0-methyl residue at base 2.
  • the cap is a Cap2 structure.
  • Cap2 structures have a 2'-0-methyl residue attached to both bases 2 and 3.
  • m 7 G cap analogs are known in the art, many of which are commercially available. These include the m 7 GpppG described above, as well as the ARCA 3'-OCH 3 and 2'-OCH 3 cap analogs (Jemielity, J.
  • cap analogs for use in embodiments of the 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 (including biotinylated cap analogs) described in U.S. Patent Nos. 8,093,367 and 8,304,529, incorporated by reference herein.
  • a "tail” serves to protect the mRNA from exonuclease degradation.
  • poly A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A tail can be added to an mRNA molecule thus rendering the RNA more stable.
  • Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products.
  • Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Flarbor
  • mRNAs include a 3' poly(A) tail structure.
  • the length of the poly A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides.
  • a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • mRNAs include a 3' poly(C) tail structure.
  • a suitable poly-C tail on the 3' terminus of 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 may be added to the poly-A tail or may substitute the poly-A tail.
  • the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein.
  • the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.
  • mRNAs include a 5' and/or 3' untranslated region.
  • a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element.
  • a 5' untranslated region may be between about 50 and 500 nucleotides in length.
  • a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs.
  • a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.
  • Exemplary 3' and/or 5' UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule.
  • a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide.
  • IE1 immediate-early 1
  • hGH human growth hormone
  • modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.
  • cationic lipids described herein described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • encapsulated materials e.g., one or more polynucleotides such as mRNA
  • cationic lipids described herein are characterized as resulting in one or more of receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and
  • a nucleic acid e.g., mRNA encoding a protein (e.g., a full length, fragment or portion of a protein) as described herein may be delivered via a delivery vehicle comprising a cationic lipid as described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)- (lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • a delivery vehicle comprising a cationic lipid as described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)- (lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • delivery vehicle As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalent, are used interchangeably.
  • the present invention provides a composition (e.g., a pharmaceutical composition) comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)- (lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) and one or more polynucleotides.
  • a composition may further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
  • a composition exhibits an enhanced (e.g., increased) ability to transfect one or more target cells.
  • methods of transfecting one or more target cells generally comprise the step of contacting the one or more target cells with the cationic lipids and/or pharmaceutical compositions disclosed herein (e.g., a liposomal formulation comprising a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) encapsulating one or more polynucleotides) such that the one or more target cells are transfected with the materials encapsulated therein (e.g., one or more polynucleotides).
  • a liposomal formulation comprising a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic
  • transfect refers to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or polynucleotides) into a cell, or preferably into a target cell.
  • the introduced polynucleotide may be stably or transiently maintained in the target cell.
  • transfection efficiency refers to the relative amount of such encapsulated material (e.g., polynucleotides) up-taken by, introduced into and/or expressed by the target cell which is subject to transfection. In practice, transfection efficiency may be estimated by the amount of a reporter polynucleotide product produced by the target cells following transfection.
  • the compounds and pharmaceutical compositions described herein demonstrate high transfection efficiencies thereby improving the likelihood that appropriate dosages of the encapsulated materials (e.g., one or more
  • polynucleotides will be delivered to the site of pathology and subsequently expressed, while at the same time minimizing potential systemic adverse effects or toxicity associated with the compound or their encapsulated contents.
  • the production of the product e.g., a polypeptide or protein
  • the production of the product may be preferably stimulated and the capability of such target cells to express the polynucleotide and produce, for example, a polypeptide or protein of interest is enhanced.
  • transfection of a target cell by one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of the protein or enzyme encoded by such mRNA.
  • delivery vehicles described herein may be prepared to preferentially distribute to other target tissues, cells or organs, such as the heart, lungs, kidneys, spleen.
  • the lipid nanoparticles of the present invention may be prepared to achieve enhanced delivery to the target cells and tissues.
  • polynucleotides e.g., mRNA
  • encapsulated in one or more of the compounds or pharmaceutical and liposomal compositions described herein can be delivered to and/or transfect targeted cells or tissues.
  • the encapsulated polynucleotides are capable of being expressed and functional polypeptide products produced (and in some instances excreted) by the target cell, thereby conferring a beneficial property to, for example the target cells or tissues.
  • encapsulated polynucleotides may encode, for example, a hormone, enzyme, receptor, polypeptide, peptide or other protein of interest.
  • a composition is a suitable delivery vehicle.
  • a suitable delivery vehicle In embodiments, a
  • composition is a liposomal delivery vehicle, e.g., a lipid nanoparticle.
  • liposomal delivery vehicle and “liposomal composition” are used interchangeably.
  • Enriching liposomal compositions with one or more of the cationic lipids disclosed herein may be used as a means of improving (e.g., reducing) the toxicity or otherwise conferring one or more desired properties to such enriched liposomal composition (e.g., improved delivery of the encapsulated polynucleotides to one or more target cells and/or reduced in vivo toxicity of a liposomal composition).
  • the compounds described herein are cationic lipids that may be used as a component of a liposomal composition to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).
  • encapsulated materials e.g., one or more therapeutic agents
  • liposomal delivery vehicles e.g., lipid nanoparticles
  • lipid nanoparticles are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).
  • Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue.
  • compositions e.g., liposomal compositions
  • encapsulate materials such as for example, one or more biologically-active
  • polynucleotides e.g., mRNA
  • a composition (e.g., a pharmaceutical composition) comprises an mRNA
  • a 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., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)).
  • a composition comprises an mRNA encoding for a protein (e.g., any protein described herein).
  • a composition comprises an mRNA encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a composition comprises an mRNA encoding for ornithine transcarbamylase (OTC) protein.
  • OTC ornithine transcarbamylase
  • an mRNA encodes for an antigen from an infectious agent.
  • a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises any cationic lipid (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)- (462)) as described herein.
  • a cationic lipid e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)- (462)
  • a nucleic acid is an mRNA encoding a peptide or polypeptide.
  • an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell (e.g., an mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein).
  • an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell (e.g., an mRNA encodes ornithine transcarbamylase (OTC) protein).
  • OTC ornithine transcarbamylase
  • an mRNA encodes for an antigen from an infectious agent. Still other exemplary mRNAs are described herein.
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a net positive charge e.g., a lipid nanoparticle
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a net negative charge e.g., a net negative charge
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a net neutral charge e.g., a lipid nanoparticle
  • a lipid nanoparticle that encapsulates a nucleic acid comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • the amount of a cationic lipid as described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) in a composition can be described as a percentage ("wt%") of the combined dry weight of all lipids of a composition (e.g., the combined dry weight of all lipids present in a liposomal composition).
  • wt% percentage of the combined dry weight of all lipids of a composition
  • a cationic lipid as
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a composition e.g., a liposomal composition
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) is present in an amount that is about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition).
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) is present in an amount that is about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
  • the amount of a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) is present in an amount that is at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the
  • the amount of a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) is present in an amount that is no more than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the
  • a composition e.g., a liposomal delivery vehicle such as a lipid nanoparticle
  • a cationic lipid described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • a delivery vehicle e.g., a liposomal delivery vehicle such as a lipid nanoparticle
  • a delivery vehicle comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • a cationic lipid described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
  • the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
  • the amount of a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) in a composition also can be described as a percentage ("mol%") of the combined molar amounts of total lipids of a composition (e.g., the combined molar amounts of all lipids present in a liposomal delivery vehicle).
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a composition such as a liposomal delivery vehicle.
  • a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) is present in an amount that is 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 amounts of all lipids present in a composition such as a liposomal delivery vehicle.
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) is present in an amount that is 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
  • a cationic lipid described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a composition e.g., a liposomal delivery vehicle
  • cationic lipids described herein 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 lipids in the lipid nanoparticle.
  • cationic lipid described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a composition e.g., a liposomal delivery vehicle
  • the amount of a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) is present in an amount that is 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 total lipids in a composition (e.g., a liposomal
  • the amount of a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) is present in an amount that is 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 total lipids in a composition (e.g., a lipo
  • the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
  • a composition further comprises one more lipids (e.g., one more lipids
  • 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).
  • such pharmaceutical (e.g., liposomal) compositions comprise one or more of a PEG-modified lipid, a non-cationic lipid and a cholesterol lipid.
  • such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids.
  • such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids and one or more cholesterol lipids.
  • a composition that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)) and one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)
  • lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a
  • a composition that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)); one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid; and further comprises a cholesterol-based lipid.
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)
  • a lipid nanoparticle that encapsulates a nucleic acid comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), as well as 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.
  • a cationic lipids described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • a lipid nanoparticle comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), a non-cationic lipid (e.g., DOPE), a PEGylated lipid (e.g., DMG-PEG2000), and a cholesterol-based lipid (e.g., cholesterol).
  • a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • DOPE DOPE
  • PEGylated lipid e.g., DMG-PEG2000
  • cholesterol-based lipid e.g., cholesterol
  • the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.
  • a composition may comprise one or more further cationic lipids.
  • liposomes may comprise one or more further cationic lipids.
  • cationic lipid refers to any of a number of lipid species that have 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.
  • compositions and methods of the invention employ a lipid nanoparticles comprising a further cationic lipid described in U.S.
  • a composition (e.g., a liposomal composition) comprises a further cationic lipid described in WO 2013/063468, filed October 26, 2012 and in U.S. provisional application 61/953,516, filed March 14, 2014, both of which are incorporated by reference herein.
  • a composition e.g., a liposomal composition
  • a composition comprises a further cationic lipid CKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).
  • CKK-E12 a further cationic lipid CKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione.
  • the structure of CKK-E12 is shown below:
  • a further cationic lipid may be N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride or "DOTMA" (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride
  • DOTMA can be formulated alone or can be combined with a neutral lipid (e.g., dioleoylphosphatidyl-ethanolamine or "DOPE") or still other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells.
  • a neutral lipid e.g., dioleoylphosphatidyl-ethanolamine or "DOPE”
  • DOPE dioleoylphosphatidyl-ethanolamine
  • Suitable further cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS"; 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" (Behr et al. Proc. Nat.'l Acad. Sci.
  • Additional exemplary further cationic lipids also include l,2-distearyloxy-N,N-dimethyl-3- aminopropane or "DSDMA”; l,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA”; 1 ,2- dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA”; l,2-dilinolenyloxy-N,N-dimethyl-3- aminopropane or "DLenDMA”; N-dioleyl-N,N-dimethylammonium chloride or "DODAC”;N,N- distearyl-N,N-dimethylarnrnonium bromide or "DDAB”; N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE”;
  • one or more of the further cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • the one or more further cationic lipids may be chosen from XTC (2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1 ,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3- oxo-3-(undecylamino)propyl)-N l,N16-diundec
  • the percentage of total cationic lipids in a 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 total lipids as measured by molar ratios (mol%) or by weight (wt%).
  • the percentage of total cationic lipids in a 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 total lipids as measured by molar ratios (mol%) or by weight (wt%).
  • total cationic lipid(s) constitute(s) about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight. In some embodiments, the cationic lipid constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of a composition (e.g., a liposomal composition) by molar ratio. In some embodiments, total cationic lipid(s) constitute(s) about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight. In some embodiments, the cationic lipid constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of a composition (e.g., a liposomal composition) by weight.
  • compositions may also comprise one or more non-cationic molecules
  • non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a number 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),
  • DPPG dipalmitoylphosphatidylglycerol
  • DOPE dioleoylphosphatidylethanolamine
  • palmitoyloleoylphosphatidylcholine POPC
  • palmitoyloleoyl-phosphatidylethanolamine POPE
  • dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate DOPE-mal
  • dipalmitoyl phosphatidyl ethanolamine DPPE
  • dimyristoylphosphoethanolamine DMPE
  • distearoyl-phosphatidyl-ethanolamine DSPE
  • 16-O-monomethyl PE 16-O-dimethyl PE
  • 18-1-trans PE l-stearoyl-2-oleoyl-phosphatidyethanolamine
  • SOPE l-stearoyl-2-oleoyl-phosphatidyethanolamine
  • a non-cationic or helper lipid is dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • the percentage of non-cationic lipid in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%.
  • the percentage total non-cationic lipids in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipid in a 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%.
  • the percentage total non-cationic lipids in a liposome may 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%.
  • a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • the percentage of non-cationic lipid in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%.
  • the percentage of non- cationic lipid in a liposome is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may 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%.
  • a composition (e.g., a liposomal composition) comprises one or more cholesterol-based lipids.
  • suitable cholesterol-based lipids include cholesterol and , for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al.
  • a cholesterol-based lipid is cholesterol.
  • a cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome.
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%.
  • the percentage of cholesterol- based lipid in the lipid nanoparticle may 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%.
  • a cholesterol-based lipid may be present in a weight ratio (wt%) of
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be 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%.
  • a composition (e.g., a liposomal composition) comprises one or more PEGylated lipids.
  • PEG polyethylene glycol
  • derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the liposome.
  • particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C M or Cig).
  • Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
  • a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K.
  • the addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
  • a PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000).
  • a PEG-modified phospholipid and derivatized lipids of the present invention may be present in a molar ratio (mol%) from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the composition (e.g., a liposomal composition).
  • a PEG-modified phospholipid and derivatized lipids of the present invention may be present in a weight ratio (wt%) from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the composition (e.g., a liposomal composition).
  • Cationic lipids described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • compositions e.g., to construct liposomal compositions
  • encapsulated materials e.g., one or more therapeutic polynucleotides
  • a liposomal composition e.g., a lipid nanoparticle
  • the phase transition in the lipid bilayer of the one or more target cells may facilitate the delivery of the encapsulated materials (e.g., one or more therapeutic polynucleotides encapsulated in a lipid nanoparticle) into the one or more target cells.
  • the encapsulated materials e.g., one or more therapeutic polynucleotides encapsulated in a lipid nanoparticle
  • cationic lipids described herein e.g., a cationic lipid of
  • Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) may be used to prepare liposomal vehicles that are characterized by their reduced toxicity in vivo.
  • the reduced toxicity is a function of the high transfection efficiencies associated with the compositions disclosed herein, such that a reduced quantity of such
  • composition may administered to the subject to achieve a desired therapeutic response or outcome.
  • compositions comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)- (462)) and nucleic acids provided by the present invention may be used for various therapeutic purposes.
  • a cationic lipid described herein e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)- (462)
  • nucleic acids provided by the present invention
  • a cationic lipid e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands or stabilizing reagents.
  • a cationic lipid e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • G cationic lipid of Formula (G), (I), (II), or (llla)-(lllab)
  • cationic lipids la)-(21a), (lb)-(21b), and (22)-(462)
  • a composition comprising a cationic lipid e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)- (21a), (lb)-(21b), and (22)-(462)
  • a cationic lipid e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)- (21a), (lb)-(21b), and (22)-(462)
  • Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal,
  • intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle.
  • the administration results in delivery of the nucleic acids to a muscle cell.
  • the administration results in delivery of the nucleic acids to a hepatocyte (i.e., liver cell).
  • administration is intramuscular.
  • administration is intravenous.
  • compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into a targeted tissue, preferably in a sustained release formulation.
  • Local delivery can be affected in various ways, depending on the tissue to be targeted.
  • Exemplary tissues in which delivered mRNA may 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.
  • the tissue to be targeted in the liver are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.
  • compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
  • administration is via pulmonary delivery.
  • pulmonary delivery refers to delivery to lung via, e.g., nasal cavity, trachea, bronchi, bronchioles, and/or other pulmonary system.
  • a composition described herein is formulated for nebulization.
  • the delivery vehicle may be in an aerosolized composition which can be inhaled.
  • pulmonary delivery involves inhalation (e.g., for nasal, tracheal, or bronchial delivery).
  • a composition is nebulized prior to inhalation.
  • the present invention provides methods for delivering a composition having full-length mRNA molecules encoding a peptide or polypeptide of interest for use in the treatment of a subject, e.g., a human subject or a cell of a human subject or a cell that is treated and delivered to a human subject.
  • the present invention provides a method for producing a therapeutic composition comprising full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell.
  • the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP-binding cassette sub-family A member 3 protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for dynein axonemal intermediate chain 1 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for dynein axonemal heavy chain 5 (DNAH5) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for alpha-l-antitrypsin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for forkhead box P3 (FOXP3) protein.
  • FOXP3 forkhead box P3
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes one or more surfactant protein, e.g., one or more of surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.
  • one or more surfactant protein e.g., one or more of surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell.
  • peptides and polypeptides can include those associated with a urea cycle disorder, associated with a lysosomal storage disorder, with a glycogen storage disorder, associated with an amino acid metabolism disorder, associated with a lipid metabolism or fibrotic disorder, associated with methylmalonic acidemia, or associated with any other metabolic disorder for which delivery to or treatment of the liver or a liver cell with enriched full-length mRNA provides therapeutic benefit.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a urea cycle disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ornithine transcarbamylase (OTC) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arginosuccinate synthetase 1 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for carbamoyl phosphate synthetase I protein.
  • OTC ornithine transcarbamylase
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arginosuccinate lyase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arginase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a lysosomal storage disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for alpha galactosidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glucocerebrosidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for iduronate-2-sulfatase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for iduronidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for N- acetyl-alpha-D-glucosaminidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for heparan N-sulfatase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for galactosamine-6 sulfatase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for beta-galactosidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for lysosomal lipase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for transcription factor EB (TFEB).
  • TFEB transcription factor EB
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a glycogen storage disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for acid alpha-glucosidase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glucose-6-phosphatase (G6PC) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for liver glycogen phosphorylase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for muscle phosphoglycerate mutase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glycogen debranching enzyme.
  • G6PC glucose-6-phosphatase
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with amino acid metabolism. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for phenylalanine hydroxylase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glutaryl-CoA dehydrogenase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for propionyl-CoA caboxylase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for oxalase alanine-glyoxylate aminotransferase enzyme.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a lipid metabolism or fibrotic disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a mTOR inhibitor. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATPase phospholipid transporting 8B1 (ATP8B1) protein.
  • ATP8B1 ATP8B1
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for one or more NF-kappa B inhibitors, such as one or more of l-kappa B alpha, interferon-related development regulator 1 (IFRD1), and Sirtuin 1 (SIRT1).
  • NF-kappa B inhibitors such as one or more of l-kappa B alpha, interferon-related development regulator 1 (IFRD1), and Sirtuin 1 (SIRT1).
  • IFRD1 interferon-related development regulator 1
  • SIRT1 Sirtuin 1
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with methylmalonic acidemia.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for methylmalonyl CoA mutase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for methylmalonyl CoA epimerase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA for which delivery to or treatment of the liver can provide therapeutic benefit.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP7B protein, also known as Wilson disease protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for porphobilinogen deaminase enzyme.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for one or clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for human hemochromatosis (FIFE) protein.
  • FIFE human hemochromatosis
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the cardiovasculature of a subject or a cardiovascular cell.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for vascular endothelial growth factor A protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for relaxin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for bone morphogenetic protein-9 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for bone morphogenetic protein-2 receptor protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the muscle of a subject or a muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for dystrophin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for frataxin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the cardiac muscle of a subject or a cardiac muscle cell.
  • the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for a protein that modulates one or both of a potassium channel and a sodium channel in muscle tissue or in a muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein that modulates a Kv7.1 channel in muscle tissue or in a muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for a protein that modulates a Navl.5 channel in muscle tissue or in a muscle cell.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the nervous system of a subject or a nervous system cell.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for survival motor neuron 1 protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for survival motor neuron 2 protein.
  • the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for frataxin protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for CLN3 protein.
  • ABCD1 ATP binding cassette subfamily D member 1
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the blood or bone marrow of a subject or a blood or bone marrow cell.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for beta globin protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Bruton's tyrosine kinase protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for one or clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the kidney of a subject or a kidney cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for collagen type IV alpha 5 chain (COL4A5) protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the eye of a subject or an eye cell.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP-binding cassette sub-family A member 4 (ABCA4) protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for retinoschisin protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for retinal pigment epithelium-specific 65 kDa (RPE65) protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for centrosomal protein of 290 kDa (CEP290).
  • an mRNA encodes for an antigen from an infectious agent.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from an infectious agent, such as a virus.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from influenza virus.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from respiratory syncytial virus.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from rabies virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from cytomegalovirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from rotavirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus, or hepatis C virus.
  • a hepatitis virus such as hepatitis A virus, hepatitis B virus, or hepatis C virus.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from human papillomavirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a human metapneumovirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from malaria virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from zika virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from chikungunya virus.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen associated with a cancer of a subject or identified from a cancer cell of a subject. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen determined from a subject's own cancer cell, i.e., to provide a personalized cancer vaccine. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen expressed from a mutant KRAS gene.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody.
  • the antibody can be a bi-specific antibody.
  • the antibody can be part of a fusion protein.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to 0X40.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to VEGF.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to tissue necrosis factor alpha.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to CD3. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to CD19.
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an immunomodulator. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Interleukin 12. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Interleukin 23. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Interleukin 36 gamma. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a constitutively active variant of one or more stimulator of interferon genes (STING) proteins.
  • STING interferon genes
  • the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an endonuclease. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an RNA-guided DNA endonuclease protein, such as Cas 9 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a meganuclease protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a transcription activator-like effector nuclease protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a zinc finger nuclease protein.
  • compositions and methods of the invention provide for delivery of mRNA encoding a secreted protein.
  • the compositions and methods of the invention provide for delivery of mRNA encoding one or more secreted proteins listed in Table 1; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein Table 1.
  • Secreted Proteins secreted Proteins
  • compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more additional exemplary proteins listed in Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 2 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 2 (or a homolog thereof) along with other components set out herein.
  • the Uniprot IDs set forth in Table 1 and Table 2 refer to the human versions the listed proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also generally available for various animals, including various mammals and animals of veterinary or industrial interest.
  • compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more proteins chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of the secreted proteins listed in Table 1 and Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 and Table 2 along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in
  • mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs.
  • animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.
  • compositions and methods of the invention provide for the delivery of m RNA encoding a lysosomal protein chosen from Table 3.
  • the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more lysosomal and/or related proteins listed in Table 3; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 3 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 3 (or a homolog thereof) along with other components set out herein.
  • the protein listed in Table 3 and encoded by mRNA in the compositions and methods of the invention is a human protein. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest as described above.
  • compositions and methods of the invention provide for the delivery of mRNA encoding a therapeutic protein (e.g., cytosolic, transmembrane or secreted) such as those listed in Table 4.
  • a therapeutic protein e.g., cytosolic, transmembrane or secreted
  • the compositions and methods of the invention provide for the delivery of an mRNA encoding a therapeutic protein useful in treating a disease or disorder (i.e., indication) listed in Table 4; thus, compositions of the invention may comprise an mRNA encoding a therapeutic protein listed or not listed in Table 4 (or a homolog thereof, as discussed below) along with other components set out herein for treating a disease or disorder (i.e., indication) listed in Table 4, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a such a protein (or a homolog thereof, as discussed below) along with other components set out herein for treatment of a disease or disorder listed in Table 4.
  • the present invention is used to prevent, treat and/or cure a subject affected with a disease or disorder listed or associated with the proteins listed in Tables 1, 2, 3, or 4.
  • an 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).
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • ASS1 argininosuccinate synthetase
  • SNS1 survival motor neuron 1
  • PAH phenylalanine hydroxylase
  • a cationic lipid e.g., cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)
  • cationic lipids la)-(21a), (lb)-(21b), and (22)-(462)
  • Example 7 Exemplary Synthesis of Lipids Comprising Aliphatic Lipid Arms - Tetraacetylribose Method
  • reaction mixture was diluted with saturated sodium dihydrogen phosphate and extracted with ethyl acetate, and the combined organic layers were washed with brine. After concentration, 1.13 g desired product A27 was obtained as colorless oil, which was used for the next step without purification.
  • Example 8 Lipid Nanoparticle Formulation Using Ribose Cationic Lipids
  • Cationic lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art.
  • suitable methods include methods described in
  • Process A relates to a conventional method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles.
  • an ethanol lipid solution and an aqueous buffered solution of mRNA were prepared separately.
  • a solution of mixture of lipids (cationic lipid, helper lipids, zwitterionic lipids, PEG lipids etc.) was prepared by dissolving lipids in ethanol.
  • the mRNA solution was prepared by dissolving the mRNA in citrate buffer, resulting in mRNA at a
  • the mixtures were then both heated to 65 ° C prior to mixing. Then, these two solutions were mixed using a pump system. In some instances, the two solutions were mixed using a gear pump system. In certain embodiments, the two solutions were mixing using a T junction (or "Y" junction). The mixture was then purified by diafiltration with a TFF process. The resultant formulation concentrated and stored at 2-8 °C until further use.
  • a second exemplary process for lipid nanoparticle formulation is Process B of WO 2018/089801 (see, e.g., Example 2 and Figure 2 of WO 2018/089801).
  • Process B (“B”) refers to a process of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA.
  • mRNA messenger RNA
  • a range of different conditions, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations, may be employed in Process B.
  • lipids dissolved in ethanol and citrate buffer were mixed using a pump system. The instantaneous mixing of the two streams resulted in the formation of empty lipid nanoparticles, which was a self- assembly process.
  • the resultant formulation mixture was empty lipid nanoparticles in citrate buffer containing alcohol.
  • the formulation was then subjected to a TFF purification process wherein buffer exchange occurred.
  • the resulting suspension of pre-formed empty lipid nanoparticles was then mixed with mRNA using a pump system.
  • heating the solution post-mixing resulted in a higher percentage of lipid nanoparticles containing mRNA and a higher total yield of m RNA.
  • IV Intravenous
  • lipid nanoparticle formulations comprising a ribose cationic lipid and mRNA encoding hEPO (Table 5) was undertaken in order to study mRNA delivery and resultant hEPO expression.
  • Male CD1 mice at 6-8 weeks old were given a single intravenous injection of the LNP formulations at a dosage level of 1 mg/kg.
  • Blood samples were collected by tail snip at 6 and 24 hours post-dose.

Abstract

Disclosed are cationic lipids which are compounds of Formula (I'). Cationic lipids provided herein can be useful for delivery and expression of mRNA and encoded protein, e.g., as a component of liposomal delivery vehicle, and accordingly can be useful for treating various diseases, disorders and conditions, such as those associated with deficiency of one or more proteins.

Description

RIBOSE CATIONIC LIPIDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit of U.S. Patent Application No. 62/672,194, filed May 16, 2018; U.S. Patent Application No. 62/740,095, filed October 2, 2018; and U.S. Patent Application No. 62/749,442, filed October 23, 2018, each of which is incorporated by reference in its entirety.
BACKGROUND
[0002] Delivery of nucleic acids has been explored extensively as a potential therapeutic option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for treatment of various diseases, including for those associated with deficiency of one or more proteins.
SUMMARY
[0003] The present invention provides, among other things, cationic lipids useful in for delivery of mRNA. Delivery of mRNA provided by cationic lipids described herein can result in targeted delivery, reduce administration frequency, improve patient tolerability, and provide more potent and less toxic mRNA therapy for the treatment of a variety of diseases, including but not limited to cancer, cardiovascular, cystic fibrosis, infectious, and neurological diseases.
[0004] In one aspect, the present invention provides a cationic lipid of Formula (G):
Figure imgf000002_0001
wherein:
Rx is independently -H, -L^R1, or -L5A-L5B-B';
each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NRL-; each L4A and L5A is independently -C(O)-, -C(0)0-, or -C(0)NRL-;
each L4B and L5B is independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene;
each B and B' is NR4R5, a 5- to 10-membered nitrogen-containing heterocyclyl, or a 5- to 10- membered nitrogen-containing heteroaryl;
each R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
[0005] In embodiments, the present invention provides a cationic lipid of Formula
Figure imgf000003_0001
Figure imgf000003_0002
[0006] In embodiments, the present invention provides a cationic lipid of Formula (la):
Figure imgf000003_0003
wherein each of L1, L2, L3, R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0007] In embodiments, the present invention provides a cationic lipid of Formula (lb):
Figure imgf000003_0004
wherein: each of R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0008] In embodiments, the present invention provides a cationic lipid of Formula (lc):
Figure imgf000003_0005
wherein each of R1, R2, R3, B, L4A and L4B is independently as defined herein. [0009] In embodiments, the present invention provides a cationic lipid of Formula (Id):
Figure imgf000004_0001
wherein each of R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0010] In embodiments, the present invention provides a cationic lipid of Formula (le):
Figure imgf000004_0002
wherein each of R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0011] In embodiments, each L3 is independently -C(O)- or a covalent bond.
[0012] In embodiments, L4A is -C(O)-.
[0013] In embodiments, L4B is unsubstituted C1-C6 alkylene; C2-C6 alkenylene; or C2-C6 alkynylene.
In embodiments, L4B is -CH2-. ln embodiments, L4B is -CH2CH2-. In embodiments, L4B is
-CH2CH2CH2-. In embodiments, L4B is -CH2CH2CH2CH2-
[0014] In embodiments, B is NR4R5, and each R4 and R5 is independently hydrogen or unsubstituted Ci- C6 alkyl. In embodiments, B is N(CHB)2.
[0015] In embodiments, B is NR4R5, and R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl. In embodiments, B is piperdinyl or piperazinyl. In
embodiments, B is 4-methylpiperazinyl.
[0016] In another aspect, the present invention provides a cationic lipid of Formula (I I):
Figure imgf000004_0003
wherein: RA is hydrogen or -lAR1;
RB is hydrogen or -L2-R2;
Rc is hydrogen or -L3-R3;
each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NRL-;
L4 is independently Ci-Cio alkylene;
each R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and
each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
[0017] In embodiments, RA is -lAR1; RB is -L2-R2; and Rc -L3-R3.
[0018] In embodiments, the present invention provides a cationic lipid of Formula (lla):
Figure imgf000005_0001
wherein each RA, RB, Rc, L4 , R4 and R5 is as independently as defined herein.
[0019] In embodiments, the present invention provides a cationic lipid of Formula (Mb):
Figure imgf000005_0002
wherein each RA, RB, Rc, L4 , R4 and R5 independently as defined herein.
[0020] In embodiments, the present invention provides a cationic lipid of Formula (lie):
Figure imgf000005_0003
wherein each RA, RB, Rc, L4 , R4 and R5 is independently as defined herein. [0021] In embodiments, the present invention provides a cationic lipid of Formula (lid):
Figure imgf000006_0001
(lid),
wherein each RA, RB, Rc, L4 , R4 and R5 is independently as defined herein.
[0022] In embodiments, the present invention provides a cationic lipid of Formula (lie):
Figure imgf000006_0002
wherein each RA, RB, Rc, L4 , R4 and R5 is independently as defined herein.
[0023] In embodiments, L4 is unsubstituted C1-C6 alkylene. In embodiments, L4B is -CH2- ,ln embodiments, L4B is -CH2CH2-. In embodiments, L4B is -CH2CH2CH2-.
[0024] In embodiments, each R4 and R5 is independently hydrogen or unsubstituted C1-C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens. In embodiments, each R4 and R5 is CH3.
[0025] In embodiments, R4 and R5 combine to form a 5- to 10-membered heterocyclyl. In
embodiments, R4 and R5 combine to form a piperdinyl or piperazinyl. In embodiments, R4 and R5 combine to form 4-methylpiperazinyl.
[0026] In embodiments, each R1, R2, and R3 is independently C6-C22 alkyl, C6-C22 alkenyl, or C6-C22
alkynyl. In embodiments, each R1, R2, and R3 is unsubstituted linear C6-C22 alkyl, unsubstituted linear C6-C22 alkenyl, unsubstituted linear C6-C22 alkynyl, unsubstituted branched C6-C22 alkyl, unsubstituted branched C6-C22 alkenyl, or unsubstituted branched C6-C22 alkynyl. In embodiments, each R1, R2, and R3 is unsubstituted linear C6-C22 alkyl. In embodiments, each R1, R2, and R3 is unsubstituted linear C6- C22 alkenyl. In embodiments, each R1, R2, and R3 is unsubstituted linear C6-C22 alkynyl. In embodiments, each R1, R2, and R3 is unsubstituted branched C6-C22 alkyl. In embodiments, each R1, R2, and R3 is unsubstituted branched C6-C22 alkenyl. In embodiments, each R1, R2, and R3 is unsubstituted branched C6-C22 alkynyl.
[0027] In embodiments, each R1, R2, and R3 is unsubstituted C6-C22 alkyl. In embodiments, R6 is
unsubstituted linear C6-C14 alkyl. In embodiments, R6 is unsubstituted branched C6-C14 alkyl. In embodiments, each R1, R2, and R3 is (CH2)7CH3, (O-hJgCHs, (CH2)uCH3, (CH2)i3CH3, (CH2)I5CH3, or (CH2)i7CH3. In embodiments, each R1, R2, and R3 is (CH2)6CH3, (CH2)8CH3, (CH2)IOCH3, (CH2)i2CH3, (CH2)I4CH3, or (CH2)I6CH3.
[0028] In embodiments, each R1, R2, and R3 is C6-C12 alkyl substituted by -0(C0)R6 or -C(0)OR6, wherein R6 is unsubstituted C6-C14 alkyl. In embodiments, each R1, R2, and R3 is (CH2)80C(0)(CH2)6CH3, (CH2)90C(0)(CH2)6CH3, (CH2)7C(0)0(CH2)2CH(C5HII)2, or (CH2)8C(0)0(CH2)2CH(C5HII)2.
[0029] In embodiments, each R1, R2, and R3 is unsubstituted C6-C22 alkenyl (e.g., a monoalkenyl, a dienyl, or a trienyl).
[0030] In embodiments, each R1, R2, and R3 is:
Figure imgf000007_0001
[0031] In embodiments, each R1, R2, and R3 is:
Figure imgf000007_0002
[0032] In embodiments, each
Figure imgf000008_0001
[0033] In another aspect, the invention provides a cationic lipid selected from the group consisting of:
Figure imgf000008_0002
Figure imgf000009_0002
[0034] In another aspect, the invention provides a cationic lipid selected from the group consisting of:
Figure imgf000009_0001
Figure imgf000009_0003
Figure imgf000010_0004
[0035] In embodiments, a cationic lipid has a structure according to one of the following formulas,
Figure imgf000010_0001
[0036] In embodiments, a cationic lipid has a structure according to one of the following formulas,
Figure imgf000010_0002
[0037] In embodiments, a cationic lipid is selected from the group consisting of:
Figure imgf000010_0003
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0002
[0038] In embodiments, a cationic lipid is selected from the group consisting of:
Figure imgf000014_0001
(int),
Figure imgf000014_0003
Figure imgf000015_0001
[0039] In embodiments, a cationic lipid has a structure according to the following formula,
Figure imgf000016_0001
[0040] In embodiments, a cationic lipid has a structure according to the following formula,
Figure imgf000016_0002
[0041] In embodiments, a cationic lipid has a structure according to the following formula,
Figure imgf000016_0003
[0042] In embodiments, a cationic lipid has a structure according to the following formula,
Figure imgf000016_0004
[0043] In embodiments, L5B is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-. [0044] In embodiments, B' is NR4R5, and each R4 and R5 is independently hydrogen or unsubstituted Ci- C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens. In embodiments, B' is N(CHB)2.
[0045] In embodiments, L4B is -CH2-. In embodiments, L4B is -CH2CH2-. In embodiments, L4B is - CH2CH2CH2-. In embodiments, L4B is -CH2CH2CH2CH2-.
[0046] In embodiments, B' is NR4R5, and each R4 and R5 is independently hydrogen or unsubstituted Ci- C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens. In embodiments, B' is N(CHB)2.
[0047] In embodiments, each of L2 and L3 is a covalent bond.
[0048] In embodiments, each of L2 and L3 is -C(O)-.
[0049] In embodiments, each of R2 and R3 is independently C6-C22 alkyl, C6-C22 alkenyl, or C6-C22 alkynyl.
[0050] In embodiments, each R2 and R3 is unsubstituted linear C6-C22 alkyl, unsubstituted linear C6-C22 alkenyl, unsubstituted linear C6-C22 alkynyl, unsubstituted branched C6-C22 alkyl, unsubstituted branched C6-C22 alkenyl, or unsubstituted branched C6-C22 alkynyl. In embodiments, each R2 and R3 is unsubstituted linear C6-C22 alkyl. In embodiments, each R2 and R3 is unsubstituted linear C6-C22 alkenyl. In embodiments, each R2 and R3 is unsubstituted linear C6-C22 alkynyl. In embodiments, each R2 and R3 is unsubstituted branched C6-C22 alkyl. In embodiments, each R2 and R3 is unsubstituted branched C6-C22 alkenyl. In embodiments, each R2 and R3 is unsubstituted branched C6-C22 alkynyl.
[0051] In embodiments, each of R2 and R3 is unsubstituted C6-C22 alkyl. In embodiments, each of R2 and R3 is (CH2)7CH3, (CH2)gCH3, (CH2)nCH3, (CH2)IBCH3, (CH2)I5CH3, or (CH2)i7CH3. In embodiments, each of R2 and R3 is (CH2)6CH3, (CH2)8CH3, (CH2)IOCH3, (CH2)i2CH3, (CH2)I4CH3, or (CH2)I6CH3. In embodiments, each of R2 and R3 is C6-C12 alkyl substituted by -0(CO)R6 or -C(0)OR6, wherein R6 is unsubstituted C6- C14 alkyl. In embodiments, R6 is unsubstituted linear C6-C14 alkyl. In embodiments, R6 is
unsubstituted branched C6-C14 alkyl. In embodiments, each of R2 and R3 is (CH2)80C(0)(CH2)6CH3, (CH2)90C(0)(CH2)6CH3, (CH2)7C(0)0(CH2)2CH(C5HII)2, or (CH2)8C(0)0(CH2)2CH(C5HII)2.
[0052] In embodiments, each of R2 and R3 is unsubstituted C6-C22 alkenyl. In embodiments, a C6-C22 alkenyl is a monoalkenyl, a dienyl, or a trienyl.
[0053] In embodiments, each of R2 and R3 is
Figure imgf000018_0001
[0054] In embodiments, each of R2 and R3 is
Figure imgf000018_0002
[0055] In embodiments, a cationic lipid selected from the group consisting of cationic lipids (22)-(462).
[0056] In another aspect, the invention features a compound having the following structure,
Figure imgf000018_0003
salt thereof.
[0057] In another aspect, the invention features a compound having the following structure,
Figure imgf000018_0004
salt thereof.
[0058] In some aspects, the present invention provides methods of preparing cationic lipids described herein (e.g., a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
[0059] In some aspects, the present invention provides a composition such as a pharmaceutical
composition comprising a cationic lipid of the present invention and one or more polynucleotides.
[0060] In some embodiments, a 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 described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids. In embodiments, the liposome comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), one or more non- cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. In embodiments, an mRNA encodes for cystic fibrosis transmembrane conductance regulator (CFTR) protein. In embodiments, an mRNA encodes for ornithine transcarbamylase (OTC) protein. In embodiments, an mRNA encodes for an antigen from an infectious agent.
[0061] In embodiments, a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid as described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)). In embodiments, a composition further comprises one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In embodiments, a composition further comprises one more lipids selected from the group consisting of one or more non-cationic lipids and one or more PEG- modified lipids. In embodiments, a nucleic acid is an mRNA encoding a peptide or polypeptide. In embodiments, an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell (e.g., an mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein). In embodiments, an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell (e.g., an mRNA encodes ornithine transcarbamylase (OTC) protein). In embodiments, an mRNA encodes for an antigen from an infectious agent.
[0062] In some aspects, the present invention provides methods of treating a disease in a subject comprising administering to the subject a composition (e.g., a pharmaceutical composition) as described herein.
[0063] In embodiments, a composition (e.g., a pharmaceutical composition) is formulated for
intravenous (IV) administration.
[0064] In embodiments, a composition (e.g., a pharmaceutical composition) is formulated for
intramuscular (IM) administration. [0065] In embodiments, a composition (e.g., a pharmaceutical composition) is formulated for administration by inhalation. In embodiments, a composition (e.g., a pharmaceutical composition) is formulated for nebulization.
BRIEF DESCRIPTION OF DRAWINGS
[0066] FIG. 1 shows hEPO protein expression following intravenous (IV) administration of lipid
nanoparticle formulations comprising a cationic lipid described herein and mRNA encoding hEPO. Protein expression was determined using ELISA.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Definitions
[0067] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.
[0068] Amino acid: As used herein, the term "amino acid," in its broadest sense, refers to any
compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some
embodiments, an 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. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- 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 change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities ( e.g ., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
[0069] Animal : As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some
embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
[0070] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" 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 in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0071] Biologically active: As used herein, the term "biologically active" refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
[0072] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery"). [0073] Expression: As used herein, "expression" of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms "expression" and "production," and grammatical equivalent, are used inter-changeably.
[0074] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
[0075] Half-life: As used herein, the term "half-life" is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
[0076] Improve, increase, or reduce: As used herein, the terms "improve," "increase" or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
[0077] In Vitro: As used herein, the term "in vitro” refers to events that occur in an artificial
environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi cellular organism.
[0078] In Vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
[0079] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients ( e.g ., buffer, solvent, water, etc.).
[0080] messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. The term "modified mRNA" related to mRNA comprising at least one chemically modified nucleotide. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides [e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5 '-N- phosphoramidite linkages).
[0081] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any
compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acids (RNA), including but not limited to any one or more of interference RNAs (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (IncRNA), micro-RNA (miRNA) multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and
complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In embodiments, DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. In embodiments, RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), micro- RNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA, retrotransposons, a viral genome, a viroid, satellite RNA, or derivatives of these groups. In some embodiments, a nucleic acid is a mRNA encoding a protein such as an enzyme.
[0082] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a
provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals {e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.
[0083] Pharmaceutically acceptable: The term "pharmaceutically acceptable", as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0084] Pharmaceutically acceptable salt: Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic 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, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, 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 salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
[0085] Systemic distribution or delivery : As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of "local distribution or delivery."
[0086] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
[0087] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total 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 completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
[0088] Target tissues: As used herein, the term "target tissues" refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.
[0089] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
[0090] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
[0091] Aliphatic: As used herein, the term aliphatic refers to C1-C40 hydrocarbons and includes both saturated and unsaturated hydrocarbons. An aliphatic may be linear, branched, or cyclic. For example, C1-C20 aliphatics can include C1-C20 alkyls (e.g., linear or branched C1-C20 saturated alkyls), C2-C20 alkenyls (e.g., linear or branched C4-C20 dienyls, linear or branched C6-C20 trienyls, and the like), and C2-C20 alkynyls (e.g., linear or branched C2-C20 alkynyls). C1-C20 aliphatics can include C3-C20 cyclic aliphatics (e.g., C3-C20 cycloalkyls, C4-C20 cycloalkenyls, or C8-C20 cycloalkynyls). In certain embodiments, the aliphatic may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide. An aliphatic group is unsubstituted or substituted with one or more substituent groups as described herein. For example, an aliphatic may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2FI, -CO2R', -CN, -OFI, -OR', -OCOR', -OCO2R', -NFI2,
-NFIR', -N(R')2, -SR' or-S02R', wherein each instance of R' independently is C1-C20 aliphatic (e.g., Ci- C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not include any heteroatoms.
[0092] Alkyl: As used herein, the term "alkyl" means acyclic linear and branched hydrocarbon groups, e.g. "C1-C20 alkyl" refers to alkyl groups having 1-20 carbons. An alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentylhexyl, Isohexyletc. Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. An alkyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -IMH2, -NHR', -N(R')2, -SR' or-S02R', wherein each instance of R' independently is Ci- C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R'
independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is unsubstituted C1-C3 alkyl. In embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
[0093] Alkylene: The term "alkylene," as used herein, represents a saturated divalent straight or
branched chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene and the like. Likewise, the term "alkenylene" as used herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, and the term "alkynylene" herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur in any stable point along the chain. In certain embodiments, an alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide. For example, an alkylene, alkenylene, or alkynylene 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', -OC02R', -NH2, -NHR', -N(R')2, -SR' or-S02R', wherein each instance of R' independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is unsubstituted C1-C3 alkyl. In certain embodiments, an alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, an alkylene, alkenylene, or alkynylene does not include any heteroatoms. [0094] Alkenyl : As used herein, "alkenyl" means any linear or branched hydrocarbon chains having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, e.g. "C2-C20 alkenyl" refers to an alkenyl group having 2-20 carbons. For example, an alkenyl group 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 comprises 1, 2, or 3 carbon-carbon double bond. In embodiments, the alkenyl comprises a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated. An alkenyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkenyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6
independently selected substituents) of halogen, -COR', -CO2FI, -CO2R', -CN, -OH, -OR', -OCOR', - OCO2R', -IM H2, -NH R', -N(R')2, -SR' or-S02R', wherein each instance of R' independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
[0095] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain of either linear or branched configuration, having one or more carbon-carbon triple bonds occurring in any stable point along the chain, e.g. "C2-C20 alkynyl" refers to an alkynyl group having 2-20 carbons. Examples of an alkynyl group include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex- 5-ynyl, etc. In embodiments, an alkynyl comprises one carbon-carbon triple bond. An alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkynyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6
independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -N H2, -NH R', -N(R')2, -SR' or-S02R', wherein each instance of R' independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is unsubstituted C1-C3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
[0096] Cycloalkyl: As used herein, the term "cycloalkyl" means a nonaromatic, saturated, cyclic group, e.g. "C3-C10 cycloalkyl." In embodiments, a cycloalkyl is monocyclic. In embodiments, a cycloalkyl is polycyclic (e.g., bicyclic or tricyclic). In polycyclic cycloalkyl groups, individual rings can be fused, bridged, or spirocyclic. Examples of a cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornanyl, bicyclo[3.2.1]octanyl, octahydro-pentalenyl, and spiro[4.5]decanyl, and the like. The term "cycloalkyl" may be used interchangeably with the term "carbocycle". A cycloalkyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, a cycloalkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -IM H2, -NH R', -N(R')2, -SR' or-S02R', wherein each instance of R' independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R' independently is unsubstituted C1-C3 alkyl. In embodiments, the cycloalkyl is unsubstituted. In embodiments, the cycloalkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
[0097] Heterocyclyl: As used herein, "heterocyclyl" refers to a radical of a 3- to 14-membered non aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3-14 membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits.
A heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl") or tricyclic system ("tricyclic heterocyclyl")), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclylis independently unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents. In certain
embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
[0098] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system
having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heterocyclyl"). In some embodiments,
a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some
embodiments, the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6
membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
[0099] Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without
limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5- membered heterocyclyl groups containing 1 heteroatom include, without limitation
tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5- membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. Exemplary 7- membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-l,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H- benzo[e][l,4]diazepinyl, l,4,5,7-tetrahydropyrano[3,4-b] pyrrolyl, 5,6-dihydro-4H-furo[3,2- b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-lH- pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-lH-pyrrolo-[2,3- b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno [3,2-b]pyridinyl, 1, 2,3,4- tetrahydro-l,6-naphthyridinyl, and the like.
[0100] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine.
Cationic Lipids
[0101] Liposomal-based vehicles are considered an attractive carrier for therapeutic agents and remain subject to continued development efforts. While liposomal-based vehicles that comprise a cationic lipid component have shown promising results with regards to encapsulation, stability and site localization, there remains a great need for improvement of liposomal-based delivery systems. For example, a significant drawback of liposomal delivery systems relates to the construction of liposomes that have sufficient cell culture or in vivo stability to reach desired target cells and/or intracellular compartments, and the ability of such liposomal delivery systems to efficiently release their encapsulated materials to such target cells.
[0102] In particular, there remains a need for improved cationic lipids that demonstrate improved pharmacokinetic properties and which are capable of delivering macromolecules, such as nucleic acids to a wide variety cell types and tissues with enhanced efficiency. Importantly, there also remains a particular need for novel cationic lipids that are characterized as having reduced toxicity and are capable of efficiently delivering encapsulated nucleic acids and polynucleotides to targeted cells, tissues and organs.
[0103] Described herein are novel cationic lipids, compositions comprising such lipids, and related methods of their use. In embodiments, the compounds described herein are useful as liposomal compositions or as components of liposomal compositions to facilitate the delivery to, and subsequent transfection of one or more target cells.
[0104] Cationic lipids disclosed herein comprise a basic, ionizable functional group (e.g., an amine or a nitrogen-containing heteroaryl as described herein), which is present in neutral or charged form. [0105] In embodiments, cationic lipids described herein can provide one or more desired characteristics or properties. That is, in certain embodiments, cationic lipids described herein can be characterized as having one or more properties that afford such compounds advantages relative to other similarly classified lipids. For example, cationic lipids disclosed herein can allow for the control and tailoring of the properties of liposomal compositions (e.g., lipid nanoparticles) of which they are a component. In particular, cationic lipids disclosed herein can be characterized by enhanced transfection efficiencies and their ability to provoke specific biological outcomes. Such outcomes can include, for example enhanced cellular uptake, endosomal/lysosomal disruption capabilities and/or promoting the release of encapsulated materials (e.g., polynucleotides) intracellularly.
Cationic Lipids of Formula (P. (I). (II). and (III)
[0106] In one aspect, the present invention provides a cationic lipid of Formula (G):
Figure imgf000032_0001
wherein:
Rx is independently -H, -l^-R1, or -L5A-L5B-B';
each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NRL-; each L4A and L5A is independently -C(O)-, -C(0)0-, or -C(0)NRL-;
each L4B and L5B is independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene;
each B and B' is NR4R5 , a 5- to 10-membered nitrogen-containing heterocyclyl, or a 5- to 10- membered nitrogen-containing heteroaryl;
each R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and
each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
[0107] In embodiments, Rx is -H. In embodiments, Rx is -l^-R1. In embodiments, Rx is -L5A-L5B-B'. [0108] In some embodiments, the present invention provides a cationic lipid of Formula (I):
Figure imgf000033_0001
[0109] In embodiments, L1 is a covalent bond. In embodiments, L1 is -C(O)-. In embodiments, L1 is - C(0)0-. In embodiments, L s -C(0)S-. In embodiments, L s -C(0)NRL. In embodiments, L s a covalent bond or -C(O)-.
[0110] In embodiments, R1 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, R1 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, R1 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, R1 is C6-C30 alkenyl (e.g., C6-C22 alkenyl). In embodiments, R1 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, R1 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, R1 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, R1 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, R1 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl). In embodiments, a R1 group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group. In embodiments, a R1 group (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
[0111] In embodiments, L2 is a covalent bond. In some, L2 is -C(O)-. In embodiments, L2 is -C(0)0-. In embodiments, L2 is -C(0)S-. In some embodiments, L2 is -C(0)NRL. In embodiments, L2 is a covalent bond or -C(O)-.
[0112] In embodiments, R2 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, R2 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, R2 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, R2 is C6-C30 alkenyl (e.g., C6-C22 alkenyl). In embodiments, R2 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, R2 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, R2 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, R2 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, R2 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl). In embodiments, a R2 group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group. In embodiments, a R2 group (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group. [0113] In embodiments, L3 is a covalent bond. In some, L3 is -C(O)-. In embodiments, L3 is -C(0)0-. In embodiments, L3 is -C(0)S-. In some embodiments, L3 is -C(0)NRL. In embodiments, L3 is a covalent bond or -C(O)-.
[0114] In embodiments, -
Figure imgf000034_0001
integer of 0-6.
[0115] In embodiments, -
Figure imgf000034_0002
integer of 0-6.
[0116] In embodiments, -
Figure imgf000034_0003
Figure imgf000034_0004
[0117] In embodiments, R3 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, R3 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, R3 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, R3 is C6-C30 alkenyl (e.g., C6-C22 alkenyl). In embodiments, R3 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, R3 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, R3 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, R3 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, R3 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl). In embodiments, a R3 group (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group. In embodiments, a R3 group (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
[0118] In embodiments, each of L1, L2, and L3 is the same group. In embodiments, each of L1, L2, and L3 is a covalent bond. In some, each of L1, L2, and L3 is -C(O)-. In embodiments, each of L1, L2, and L3 is - C(0)0-. In embodiments, each of L1, L2, and L3 is -C(0)S-. In some embodiments, each of L1, L2, and L3 is -C(0)NRl. In embodiments, each of L1, L2, and L3 is a covalent bond or each of L1, L2, and L3 is
-C(O)-.
[0119] In embodiments, each of R1, R2, and R3 is the same group. In embodiments, each of R1, R2, and R3 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, each of R1, R2, and R3 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, each of R1, R2, and R3 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, each of R1, R2, and R3 is C6-C30 alkenyl (e.g., C6- C22 alkenyl). In embodiments, each of R1, R2, and R3 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, each of R1, R2, and R3 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, each of R1, R2, and R3 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, each of R1, R2, and R3 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, each of R1, R2, and R3 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl). In embodiments, each R1, R2, and R3 (e.g., as described herein) is a branched group; for example, a branched alkyl, branched alkenyl, or branched alkynyl group. In embodiments, each R1, R2, and R3 (e.g., as described herein) is a linear group; for example, a linear alkyl, linear alkenyl, or linear alkynyl group.
[0120] In embodiments, L4A is -C(O)-. In embodiments, L4A is -C(0)0-. In embodiments, L4A is -C(0)NRL-.
[0121] In embodiments, L4B is C1-C20 alkylene (e.g., C1-C6 alkylene). In embodiments, L4B is
unsubstituted C1-C20 alkylene (e.g., unsubstituted C1-C6 alkylene). In embodiments, L4B is substituted C1-C20 alkylene (e.g., substituted C1-C6 alkylene). In embodiments, L4B is C2-C20 alkenylene (e.g., C2-C6 alkenylene). In embodiments, L4B is unsubstituted C2-C20 alkenylene (e.g., unsubstituted C2-C6 alkenylene). In embodiments, L4B is substituted C2-C20 alkenylene (e.g., substituted C2-C6 alkenylene). In embodiments, L4B is C2-C20 alkynylene (e.g., C2-C6 alkynylene). In embodiments, L4B is unsubstituted C2-C20 alkynylene (e.g., unsubstituted C2-C6 alkynylene). In embodiments, L4B is substituted C2-C20 alkynylene (e.g., substituted C2-C6 alkynylene). In embodiments, L4B is -CH2- ln embodiments, L4B is -CH2CH2-. In embodiments, L4B is -CH2CH2CH2-.
[0122] In embodiments, B is NR4R5. In embodiments, each R4 and R5 is independently hydrogen or unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl such as CH3), or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
[0123] In embodiments, B is NR4R5, and R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl. In embodiments, R4 and R5 combine to form a 5- to 10- membered heterocyclyl. In embodiments, R4 and R5 combine to form a 5- to 10-membered heterocyclyl comprising one or two nitrogens in the ring or rings. In embodiments, R4 and R5 combine to form a piperdinyl or piperazinyl. In embodiments, R4 and R5 combine to form an unsubstituted piperdinyl. In embodiments, R4 and R5 combine to form an unsubstituted piperazinyl. In embodiments, R4 and R5 combine to form a substituted piperdinyl. In embodiments, R4 and R5 combine to form a substituted piperazinyl. In embodiments, R4 and R5 combine to form 4- methylpiperazinyl.
[0124] In embodiments, L5A is -C(O)-. In embodiments, L5A is -C(0)0-. In embodiments, L5A is -C(0)NRL-.
In embodiments, L5A and L4A are the same. In embodiments, L5A and L4A are different.
[0125] In embodiments, L5B is C1-C20 alkylene (e.g., C1-C6 alkylene). In embodiments, L5B is
unsubstituted C1-C20 alkylene (e.g., unsubstituted C1-C6 alkylene). In embodiments, L5B is substituted C1-C20 alkylene (e.g., substituted C1-C6 alkylene). In embodiments, L5B is C2-C20 alkenylene (e.g., C2-C6 alkenylene). In embodiments, L5B is unsubstituted C2-C20 alkenylene (e.g., unsubstituted C2-C6 alkenylene). In embodiments, L5B is substituted C2-C20 alkenylene (e.g., substituted C2-C6 alkenylene). In embodiments, L5B is C2-C20 alkynylene (e.g., C2-C6 alkynylene). In embodiments, L5B is unsubstituted C2-C20 alkynylene (e.g., unsubstituted C2-C6 alkynylene). In embodiments, L5B is substituted C2-C20 alkynylene (e.g., substituted C2-C6 alkynylene). In embodiments, L5B is -CH2- ln embodiments, L5B is -CH2CH2-. In embodiments, L5B is -CH2CH2CH2-. In embodiments, L5B and L4B are the same. In embodiments, L5B and L4B are different.
[0126] In embodiments, B' is NR4R5. In embodiments, each R4 and R5 is independently hydrogen or unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl such as CH3). In embodiments, B' and B are the same. In embodiments, B' and B are different.
[0127] In embodiments, R4 is hydrogen or C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R4 is
hydrogen. In embodiments, R4 is C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R4 is unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl). In embodiments, R4 is substituted C1-C10 alkyl (e.g., substitute C1-C6 alkyl). In embodiments, R4 is C2-C10 alkenyl (e.g., C2-C6 alkenyl). In embodiments, R4 is unsubstituted C2-C10 alkenyl (e.g., unsubstituted C2-C6 alkenyl). In embodiments, R4 is
unsubstituted C2-C10 alkenyl (e.g., substituted C2-C6 alkenyl). In embodiments, R4 is C2-C10 alkynyl (e.g., C2-C6 alkynyl). In embodiments, R4 is unsubstituted C2-C10 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, R4 is substituted C2-C10 alkynyl (e.g., substituted C2-C6 alkynyl). In embodiments, R4 is CH3.
[0128] In embodiments, R5 is hydrogen or C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R5 is
hydrogen. In embodiments, R5 is C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R5 is unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl). In embodiments, R5 is substituted C1-C10 alkyl (e.g., substitute C1-C6 alkyl). In embodiments, R5 is C2-C10 alkenyl (e.g., C2-C6 alkenyl). In embodiments, R5 is unsubstituted C2-C10 alkenyl (e.g., unsubstituted C2-C6 alkenyl). In embodiments, R5 is
unsubstituted C2-C10 alkenyl (e.g., substituted C2-C6 alkenyl). In embodiments, R5 is C2-C10 alkynyl (e.g., C2-C6 alkynyl). In embodiments, R5 is unsubstituted C2-C10 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, R5 is substituted C2-C10 alkynyl (e.g., substituted C2-C6 alkynyl). In embodiments, R5 is CH3.
[0129] In embodiments, B is N(CHB)2.
[0130] In embodiments, B is 5- to 10-membered nitrogen-containing heteroaryl. In embodiments, B is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl, or isoquinolyl. In embodiments, B is an unsubstituted heteroaryl that is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl, or isoquinolyl. In embodiments, B is a substituted heteroaryl that is pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolyl, quinolyl, or isoquinolyl. In embodiments B is imidazolyl (e.g., unsubstituted imidazolyl or substituted imidazolyl). In embodiments B is pyridyl (e.g., unsubstituted pyridyl or unsubstituted pyridyl).
[0131] In embodiments, B is 5- to 10-membered nitrogen-containing heterocyclyl. In embodiments, B is pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydro-l,3,5-triazinyl, azepanyl, diazepanyl, azocanyl, azonanyl, and the like. In embodiments, B is piperdinyl (e.g., unsubstituted piperdinyl or substituted piperdinyl). In embodiments, B is piperazinyl (e.g., unsubstituted piperazinyl or substituted piperazinyl).
[0132] In embodiments, RL is independently hydrogen. In embodiments, RL is independently C1-C20 alkyl (e.g., C1-C6 alkyl). In embodiments, RL is independently unsubstituted C1-C20 alkyl (e.g., unsubstituted C1-C6 alkyl). In embodiments, RL is independently substituted C1-C20 alkyl (e.g., substituted C1-C6 alkyl). In embodiments, RL is independently C2-C20 alkenyl (e.g., C2-C6 alkenyl). In embodiments, RL is independently unsubstituted C2-C20 alkenyl (e.g., unsubstituted C2-C6 alkenyl). In embodiments, RL is independently substituted C2-C20 alkenyl (e.g., substituted C2-C6 alkenyl). In embodiments, RL is independently C2-C20 alkynyl (e.g., C2-C6 alkynyl). In embodiments, RL is independently unsubstituted C2-C20 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, RL is independently substituted C2-C20 alkynyl (e.g., substituted C2-C6 alkynyl). [0133] In embodiments, a cationic lipid of Formula (I) has a structure according to Formula (la):
Figure imgf000038_0001
wherein each of L1, L2, L3, R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0134] In embodiments, a cationic lipid of Formula (I) has a structure according to Formula (lb):
Figure imgf000038_0002
wherein each of R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0135] In embodiments, a cationic lipid of Formula (I) has a structure according to Formula (lc):
Figure imgf000038_0003
wherein:
each of R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0136] In embodiments, a cationic lipid of Formula (I) has a structure according to Formula (Id):
Figure imgf000038_0004
wherein each of R1, R2, R3, B, L4A and L4B is independently as defined herein.
[0137] In embodiments, the present invention provides a cationic lipid of Formula (le):
Figure imgf000038_0005
wherein each of R1, R2, R3, B, L4A and L4B is independently as defined herein. [0138] In embodiments, a cationic lipid of Formula (I) has a structure according to Formula (II):
Figure imgf000039_0001
wherein:
RA is hydrogen or -AR1;
RB is hydrogen or -L2-R2;
Rc is hydrogen or -L3-R3;
each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or -C(0)NRL-;
L4 is independently C1-C10 alkylene;
each R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
[0139] In embodiments, RA is -AR1; RB is -L2-R2; and Rc -L3-R3.
[0140] In embodiments, RA is hydrogen. In embodiments, RA is -AR1.
[0141] In embodiments, L1 is a covalent bond or -C(O)-. In embodiments, L1 is a covalent bond. In embodiments, L1 is -C(O)-. In embodiments, L1 is -C(0)0-. In embodiments, L1 is -C(0)S-. In embodiments, L1 is -C(0)NRL-.
[0142] In embodiments, R1 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, R1 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, R1 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, R1 is C6-C30 alkenyl (e.g., C6-C22 alkenyl). In embodiments, R1 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, R1 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, R1 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, R1 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, R1 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl).
[0143] In embodiments, RB is hydrogen. In embodiments, RB is -AR2. [0144] . In embodiments, L2 is a covalent bond or -C(O)-. In embodiments, L2 is a covalent bond. In some, L2 is -C(O)-. In embodiments, L2 is -C(0)0-. In embodiments, L2 is -C(0)S-. In some embodiments, L2 is -C(0)NRL.
[0145] In embodiments, R2 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, R2 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, R2 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, R2 is C6-C30 alkenyl (e.g., C6-C22 alkenyl). In embodiments, R2 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, R2 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, R2 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, R2 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, R2 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl).
[0146] In embodiments, Rc is hydrogen. In embodiments, Rc is -L3-R3.
[0147] In embodiments, L3 is a covalent bond or -C(O)-. In embodiments, L3 is a covalent bond. In
some, L3 is -C(O)-. In embodiments, L3 is -C(0)0-. In embodiments, L3 is -C(0)S-. In some embodiments, L3 is -C(0)NRL.
[0148] In embodiments, R3 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, R3 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, R3 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, R3 is C6-C30 alkenyl (e.g., C6-C22 alkenyl). In embodiments, R3 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, R3 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, R3 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, R3 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, R3 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl).
[0149] In embodiments, each of L1, L2, and L3 is the same group. In embodiments, each of L1, L2, and L3 is a covalent bond. In some, each of L1, L2, and L3 is -C(O)-. In embodiments, each of L1, L2, and L3 is - C(0)0-. In embodiments, each of L1, L2, and L3 is -C(0)S-. In some embodiments, each of L1, L2, and L3 is -C(0)NRl. In embodiments, each of L1, L2, and L3 is a covalent bond or each of L1, L2, and L3 is -C(O)-.
[0150] In embodiments, each of R1, R2, and R3 is the same group. In embodiments, each of R1, R2, and R3 is C6-C30 alkyl (e.g., C6-C22 alkyl). In embodiments, each of R1, R2, and R3 is unsubstituted C6-C30 alkyl (e.g., unsubstituted C6-C22 alkyl). In embodiments, each of R1, R2, and R3 is substituted C6-C30 alkyl (e.g., substituted C6-C22 alkyl). In embodiments, each of R1, R2, and R3 is C6-C30 alkenyl (e.g., C6- C22 alkenyl). In embodiments, each of R1, R2, and R3 is unsubstituted C6-C30 alkenyl (e.g., unsubstituted C6-C22 alkenyl). In embodiments, each of R1, R2, and R3 is substituted C6-C30 alkenyl (e.g., substituted C6-C22 alkenyl). In embodiments, each of R1, R2, and R3 is C6-C30 alkynyl (e.g., C6-C22 alkynyl). In embodiments, each of R1, R2, and R3 is unsubstituted C6-C30 alkynyl (e.g., unsubstituted C6-C22 alkynyl). In embodiments, each of R1, R2, and R3 is substituted C6-C30 alkynyl (e.g., substituted C6-C22 alkynyl).
[0151] In embodiments, L4 is independently C1-C10 alkylene (e.g., C1-C6 alkylene). In embodiments, L4 is independently unsubstituted C1-C10 alkylene (e.g., unsubstituted C1-C6 alkylene). In embodiments, L4 is independently substituted C1-C10 alkylene (e.g., substituted C1-C6 alkylene). In embodiments, L4 is -CH2CH2-.
[0152] In embodiments, B is NR4R5. In embodiments, each R4 and R5 is independently hydrogen or unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl such as CH3), or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
[0153] In embodiments, R4 is hydrogen or C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R4 is
hydrogen. In embodiments, R4 is C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R4 is unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl). In embodiments, R4 is substituted C1-C10 alkyl (e.g., substitute C1-C6 alkyl). In embodiments, R4 is C2-C10 alkenyl (e.g., C2-C6 alkenyl). In embodiments, R4 is unsubstituted C2-C10 alkenyl (e.g., unsubstituted C2-C6 alkenyl). In embodiments, R4 is
unsubstituted C2-C10 alkenyl (e.g., substituted C2-C6 alkenyl). In embodiments, R4 is C2-C10 alkynyl (e.g., C2-C6 alkynyl). In embodiments, R4 is unsubstituted C2-C10 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, R4 is substituted C2-C10 alkynyl (e.g., substituted C2-C6 alkynyl). In embodiments, R4 is CH3.
[0154] In embodiments, R5 is hydrogen or C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R5 is
hydrogen. In embodiments, R5 is C1-C10 alkyl (e.g., C1-C6 alkyl). In embodiments, R5 is unsubstituted C1-C10 alkyl (e.g., unsubstituted C1-C6 alkyl). In embodiments, R5 is substituted C1-C10 alkyl (e.g., substitute C1-C6 alkyl). In embodiments, R5 is C2-C10 alkenyl (e.g., C2-C6 alkenyl). In embodiments, R5 is unsubstituted C2-C10 alkenyl (e.g., unsubstituted C2-C6 alkenyl). In embodiments, R5 is
unsubstituted C2-C10 alkenyl (e.g., substituted C2-C6 alkenyl). In embodiments, R5 is C2-C10 alkynyl (e.g., C2-C6 alkynyl). In embodiments, R5 is unsubstituted C2-C10 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, R5 is substituted C2-C10 alkynyl (e.g., substituted C2-C6 alkynyl). In embodiments, R5 is CH3.
[0155] In embodiments, R4 and R5 combine to form a 5- to 10-membered heterocyclyl. In
embodiments, R4 and R5 combine to form a 5- to 10-membered heterocyclyl comprising one or two nitrogens in the ring or rings. In embodiments, R4 and R5 combine to form a piperdinyl or piperazinyl. In embodiments, R4 and R5 combine to form an unsubstituted piperdinyl. In
embodiments, R4 and R5 combine to form an unsubstituted piperazinyl. In embodiments, R4 and R5 combine to form a substituted piperdinyl. In embodiments, R4 and R5 combine to form a substituted piperazinyl. In embodiments, R4 and R5 combine to form 4-methylpiperazinyl.
[0156] In embodiments, RL is independently hydrogen. In embodiments, RL is independently C1-C20 alkyl (e.g., C1-C6 alkyl). In embodiments, RL is independently unsubstituted C1-C20 alkyl (e.g., unsubstituted C1-C6 alkyl). In embodiments, RL is independently substituted C1-C20 alkyl (e.g., substituted C1-C6 alkyl). In embodiments, RL is independently C2-C20 alkenyl (e.g., C2-C6 alkenyl). In embodiments, RL is independently unsubstituted C2-C20 alkenyl (e.g., unsubstituted C2-C6 alkenyl). In embodiments, RL is independently substituted C2-C20 alkenyl (e.g., substituted C2-C6 alkenyl). In embodiments, RL is independently C2-C20 alkynyl (e.g., C2-C6 alkynyl). In embodiments, RL is independently unsubstituted C2-C20 alkynyl (e.g., unsubstituted C2-C6 alkynyl). In embodiments, RL is independently substituted C2-C20 alkynyl (e.g., substituted C2-C6 alkynyl).
[0157] In embodiments, a cationic lipid of Formula (II) has a structure according to Formula (lla):
Figure imgf000042_0001
(lla),
wherein each RA, RB, Rc, L4 , R4 and R5 is as independently as defined herein.
[0158] In embodiments, a cationic lipid of Formula (II) has a structure according to Formula (lib):
Figure imgf000042_0002
wherein each RA, RB, Rc, L4 , R4 and R5 independently as defined herein. [0159] In embodiments, a cationic lipid of Formula (II) has a structure according to Formula (lie):
Figure imgf000043_0001
(lie),
wherein each RA, RB, Rc, L4 , R4 and R5 is independently as defined herein.
[0160] In embodiments, a cationic lipid of Formula (II) has a structure according to Formula (lid):
Figure imgf000043_0002
(lid),
wherein each RA, RB, Rc, L4 , R4 and R5 is independently as defined herein.
[0161] In embodiments, a cationic lipid of Formula (II) has a structure according to Formula (lie):
Figure imgf000043_0003
wherein each RA, RB, Rc, L4 , R4 and R5 is independently as defined herein.
[0162] In embodiments, a C6-C30 alkyl (e.g., R1, R2, and/or R3) is a Cg-26 alkyl. In embodiments, a C6-C30 alkyl (e.g., R1, R2, and/or R3) is a straight-chain Cg-26 alkyl.
[0163] In embodiments, a C6-C30 alkyl (e.g., R1, R2, and/or R3) is CFIsiCFhJeCFh-, CFIsiCFhJyCFh-,
CH3(CH2)gCH2-, CH3(CH2)9CH2-, CH3(CH2)IOCH2-, CH3(CH2)IICH2-, CH3(CH2)i2CH2-, CH3(CH2)i3CH2-, CH3(CH2)i4CH2-, CH3(CH2)i5CH2-, CH3(CH2)i6CH2-, CH3(CH2)i7CH2-, CH3(CH2)i8CH2-, CH3(CH2)i9CH2-, CH3(CH2)2OCH2-, CH3(CH2)2iCH2-, CH3(CH2)22CH2-, CH3(CH2)23CH2- or CH3(CH2)24CH2-.
[0164] In embodiments, a C6-C30 alkyl (e.g., R1, R2, and/or R3) is CH3(CH2)i3CH2-, CH3(CH2)i4CH2-, CH3(CH2)i5CH2-, CH3(CH2)i6CH2-, CH3(CH2)i7CH2- or CH3(CH2)igCH2-. [0165] In embodiments, a C6-C30 alkyl (e.g., R1, R2, and/or R3) is CH3(CH2)i4CH2-, CH3(CH2)i5CH2- or CH3(CH2)i6CH2-.
[0166] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is a Cg-26 alkenyl having one or two
carbon-carbon double bonds.
[0167] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is c/s-CH3(CH2)3CH=CH(CH2)7CH2-, cis- CH3(CH2)5CH=CH(CH2)7CH2-, C/S-CH3(CH2)8CH=CH(CH2)4CH2-, C/S-CH3(CH2)7CH=CH(CH2)7CH2-, cis- CH3(CH2)9CH=CH(CH2)7CH2-, C/S-CH3(CH2)7CH=CH(CH2)9CH2-, tror)S-CH3(CH2)7CH=CH(CH2)7CH2-, tror)S-CH3(CH2)5CH=CH(CH2)9CH2-, c/s-CH3(CH2)9CH=CH(CH2)7CH2-, C/S-CH3(CH2)7CH=CH(CH2)IICH2-, cis- CH3(CH2)7CH=CH(CH2)I3CH2-, C/ 'S-CH3(CH2)4CH=CHCH2CH=CH(CH2)7CH2-,
c/ 's-CH3(CH2)4CH=CHCH2CH=CH(CH2)9CH2- or C/ 'S-CH3(CH2)4CH=CHCH2CH=CH(CH2)IICH2-.
[0168] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is c/s-CH3(CH2)3CH=CH(CH2)7CH2-, cis- CH3(CH2)5CH=CH(CH2)7CH2-, C/S-CH3(CH2)8CH=CH(CH2)4CH2-, C/S-CH3(CH2)7CH=CH(CH2)7CH2-, cis- CH3(CH2)9CH=CH(CH2)7CH2-, tror)S-CH3(CH2)7CH=CH(CH2)7CH2-,
c/s c/s-CH3(CH2)4CH=CHCH2CH=CH(CH2)7CH2- or c/s c/s-CH3(CH2)4CH=CHCH2CH=CH(CH2)9CH2-.
[0169] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is c/s-CH3(CH2)7CH=CH(CH2)7CH2-, cis- CH3(CH2)9CH=CH(CH2)7CH2-, c/ 's-CH3(CH2)4CH=CHCH2CH=CH(CH2)7CH2- or
cis,cis-CH3(CH2UCH=CHCH2CH=CH(CH2hCH2-.
[0170] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is c/s-CH3(CH2)7CH=CH(CH2)7CH2- or o·5,o·5-aH3(aH2)4eH=aH-aH2aH=aH(aH2)7eH2-.
[0171] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is Cg-26 aliphatic having three, four, five or six carbon-carbon double bonds.
[0172] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is
C/S C/S C/S-CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2-,
C/S C/S C/S-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-,
C/S C/S C/S-CH3(CH2)4CH=CHCH2CH=CHCH2CH2CH=CH(CH2)3CH2-,
irons, tror)s,tror)s-CH3(CH2)7CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-,
C/S,C/S,C/S-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CH(CH2)6CH2-,
C/S,C/S,C/S-CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)9CH2-,
C/S,C/S,C/S,C/S-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-,
C/S,C/S,C/S,C/S-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-,
C/S,C/S,C/S,C/S-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)6CH2-, c/s/c/s/trons/trons/c/s-CH3(CH2)4CH=CHCH=CHCH=CHCH=CHCH2CH=CH(CH2)3CH2-,
s c/s c/s s s-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3CH2-,
ds/ds/ds/ds/ds-CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2-,
ds ds ds ds ds-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)5CH2-,
ds ds ds ds ds-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2-,
ds/ds/ds/ds/ds/ds-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2-, or ds/ds/ds/ds/ds/ds- CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-.
[0173] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is
ds/ds/ds-CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2-,
ds ds ds ds-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-,
ds,ds, irons, iror)s ds-CH3(CH2)4CH=CHCH=CHCH=CHCH=CHCH2CH=CH(CH2)3CH2-, cis,cis,cis,cis,cis,cis- CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CH2-.
[0174] In embodiments, a C6-C30 alkenyl (e.g., R1, R2, and/or R3) is
ds/ds/ds-CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7CH2- or
ds/ds/ds/ds-CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH2-.
[0175] In some embodiments, each of R1, R2 and R3 independently is an aliphatic chain of a saturated or unsaturated fatty acid, i.e., R'-(CH2)- for a fatty acid R'-C(O)-. In some embodiments, each of R1, R2 and R3 independently is the aliphatic chain of caprylic, pelargonic, capric, undecylic, lauric, tridecyclic, myristic, pentadecylic, margaric, stearic, nonadecylic, arachidic, heneicosylic, behenic, triosylic, lignoceric, oleic, linoleic, pentacosylic or cerotic acid. In some embodiments, each of R and R1 is the aliphatic chain of caprylic, pelargonic, capric, undecylic, lauric, tridecyclic, myristic, pentadecylic, or margaric acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of lauric, tridecyclic, myristic, or pentadecylic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of lauric or myristic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of stearic, nonadecylic, arachidic, heneicosylic, behenic, triosylic, lignoceric, oleic, linoleic, pentacosylic or cerotic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of lignoceric, oleic, linoleic, pentacosylic or cerotic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of oleic, linoleic or pentacosylic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of oleic or linoleic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of oleic acid. In some embodiments, each of R1, R2 and R3 is the aliphatic chain of linoleic acid. [0176] In some embodiments (e.g., any of Formulas (G), (I), and (II) and any of formulas (llla)-(lllaa)), each of R1, R2 and R3 independently is any of the following aliphatic chains. In some embodiments, each of R1, R2 and R3 is the same aliphatic chain.
Figure imgf000046_0001
Figure imgf000047_0001
Exemplary Cationic Lipids
[0177] Exemplary cationic lipids include cationic lipids (la)-(21a) (Table A).
Figure imgf000047_0002
Table A. Cationic Lipids (la)-(21a)
Figure imgf000047_0003
Figure imgf000048_0002
[0178] Exemplary cationic lipids include cationic lipids (lb)-(21b) (Table B).
Figure imgf000048_0001
Table B. Cationic Lipids (lb)-(21b)
Figure imgf000048_0003
Figure imgf000049_0002
[0179] Exemplary cationic lipids include cationic lipids according to formula (Ilia) such as cationic lipids (22)-(34) (Table C):
Figure imgf000049_0001
(Ilia), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table C. Table C. Formula (Ilia) Cationic Lipids
Figure imgf000050_0001
Figure imgf000051_0001
[0180] In embodiments, a cationic lipid is cationic lipid (22). In embodiments, a cationic lipid is cationic lipid (23). In embodiments, a cationic lipid is cationic lipid (24). In embodiments, a cationic lipid is cationic lipid (25). In embodiments, a cationic lipid is cationic lipid (26). In embodiments, a cationic lipid is cationic lipid (27). In embodiments, a cationic lipid is cationic lipid (28). In embodiments, a cationic lipid is cationic lipid (29). In embodiments, a cationic lipid is cationic lipid (30). In embodiments, a cationic lipid is cationic lipid (31). In embodiments, a cationic lipid is cationic lipid (32). In embodiments, a cationic lipid is cationic lipid (33). In embodiments, a cationic lipid is cationic lipid (34).
[0181] Exemplary cationic lipids include cationic lipids according to formula (lllb) such as cationic lipids (35)-(47) (Table D):
Figure imgf000052_0001
(lllb), where B, R2, and R3 are independently as described herein, including the exemplified groups of Table D.
Table D. Formula (lllb) Cationic Lipids
Figure imgf000052_0002
Figure imgf000053_0001
Figure imgf000054_0001
[0182] In embodiments, a cationic lipid is cationic lipid (35). In embodiments, a cationic lipid is cationic lipid (36). In embodiments, a cationic lipid is cationic lipid (37). In embodiments, a cationic lipid is cationic lipid (38). In embodiments, a cationic lipid is cationic lipid (39). In embodiments, a cationic lipid is cationic lipid (40). In embodiments, a cationic lipid is cationic lipid (41). In embodiments, a cationic lipid is cationic lipid (42). In embodiments, a cationic lipid is cationic lipid (43). In embodiments, a cationic lipid is cationic lipid (44). In embodiments, a cationic lipid is cationic lipid (45). In embodiments, a cationic lipid is cationic lipid (46). In embodiments, a cationic lipid is cationic lipid (47).
[0183] Exemplary cationic lipids include cationic lipids according to formula (lllc) such as cationic lipids
(48)-(60) (Table E):
Figure imgf000055_0001
(lllc), where B, B', R2, and R3 are independently as described herein, including the exemplified groups of Table E.
Table E. Formula (lllc) Cationic Lipids
Figure imgf000055_0002
Figure imgf000056_0001
55
Figure imgf000057_0002
[0184] In embodiments, a cationic lipid is cationic lipid (48). In embodiments, a cationic lipid is cationic lipid (49). In embodiments, a cationic lipid is cationic lipid (50). In embodiments, a cationic lipid is cationic lipid (51). In embodiments, a cationic lipid is cationic lipid (52). In embodiments, a cationic lipid is cationic lipid (53). In embodiments, a cationic lipid is cationic lipid (54). In embodiments, a cationic lipid is cationic lipid (55). In embodiments, a cationic lipid is cationic lipid (56). In embodiments, a cationic lipid is cationic lipid (57). In embodiments, a cationic lipid is cationic lipid (58). In embodiments, a cationic lipid is cationic lipid (59). In embodiments, a cationic lipid is cationic lipid (60).
[0185] Exemplary cationic lipids include cationic lipids according to formula (llld) such as cationic lipids (61)-(71) (Table F):
Figure imgf000057_0001
(llld), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table F. Table F. Formula (Mid) Cationic Lipids
Figure imgf000058_0001
Figure imgf000059_0001
[0186] In embodiments, a cationic lipid is cationic lipid (61). In embodiments, a cationic lipid is cationic lipid (62). In embodiments, a cationic lipid is cationic lipid (63). In embodiments, a cationic lipid is cationic lipid (64). In embodiments, a cationic lipid is cationic lipid (65). In embodiments, a cationic lipid is cationic lipid (66). In embodiments, a cationic lipid is cationic lipid (67). In embodiments, a cationic lipid is cationic lipid (68). In embodiments, a cationic lipid is cationic lipid (69). In embodiments, a cationic lipid is cationic lipid (70). In embodiments, a cationic lipid is cationic lipid (71).
[0187] Exemplary cationic lipids include cationic lipids according to formula (llle) such as cationic lipids (72)-(82) (Table F):
Figure imgf000060_0001
(llle), where B, R2, and R3 are independently as described herein, including the exemplified groups of Table F.
Table G. Formula (llle) Cationic Lipids
Figure imgf000060_0002
Figure imgf000061_0001
Figure imgf000062_0002
[0188] In embodiments, a cationic lipid is cationic lipid (72). In embodiments, a cationic lipid is cationic lipid (73). In embodiments, a cationic lipid is cationic lipid (74). In embodiments, a cationic lipid is cationic lipid (75). In embodiments, a cationic lipid is cationic lipid (76). In embodiments, a cationic lipid is cationic lipid (77). In embodiments, a cationic lipid is cationic lipid (78). In embodiments, a cationic lipid is cationic lipid (79). In embodiments, a cationic lipid is cationic lipid (80). In embodiments, a cationic lipid is cationic lipid (81). In embodiments, a cationic lipid is cationic lipid (82).
[0189] Exemplary cationic lipids include cationic lipids according to formula (lllf) such as cationic lipids (83)-(93) (Table H):
Figure imgf000062_0001
(lllf), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table H. Table H. Formula (lllf) Cationic Lipids
Figure imgf000063_0001
Figure imgf000064_0001
63 [0190] In embodiments, a cationic lipid is cationic lipid (83). In embodiments, a cationic lipid is cationic lipid (84). In embodiments, a cationic lipid is cationic lipid (85). In embodiments, a cationic lipid is cationic lipid (86). In embodiments, a cationic lipid is cationic lipid (87). In embodiments, a cationic lipid is cationic lipid (88). In embodiments, a cationic lipid is cationic lipid (89). In embodiments, a cationic lipid is cationic lipid (90). In embodiments, a cationic lipid is cationic lipid (91). In embodiments, a cationic lipid is cationic lipid (92). In embodiments, a cationic lipid is cationic lipid (93).
[0191] Exemplary cationic lipids include cationic lipids according to formula (lllg) such as cationic lipids (94)-(106) (Table I):
Figure imgf000065_0001
(lllg), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table I.
Table I. Formula (lllg) Cationic Lipids
Figure imgf000065_0002
Figure imgf000066_0001
65
Figure imgf000067_0001
[0192] In embodiments, a cationic lipid is cationic lipid (94). In embodiments, a cationic lipid is cationic lipid (95). In embodiments, a cationic lipid is cationic lipid (96). In embodiments, a cationic lipid is cationic lipid (97). In embodiments, a cationic lipid is cationic lipid (98). In embodiments, a cationic lipid is cationic lipid (99). In embodiments, a cationic lipid is cationic lipid (100). In embodiments, a cationic lipid is cationic lipid (101). In embodiments, a cationic lipid is cationic lipid (102). In embodiments, a cationic lipid is cationic lipid (103). In embodiments, a cationic lipid is cationic lipid (104). In embodiments, a cationic lipid is cationic lipid (105). In embodiments, a cationic lipid is cationic lipid (106). [0193] Exemplary cationic lipids include cationic lipids according to formula (lllh) such as cationic lipids
( 107)-( 119) (Table J):
Figure imgf000068_0001
(lllh), where B, R2, and R3 are independently as described herein, including the exemplified groups of Table J.
Table J. Formula (lllh) Cationic Lipids
Figure imgf000068_0002
Figure imgf000069_0001

Figure imgf000070_0001
[0194] In embodiments, a cationic lipid is cationic lipid (107). In embodiments, a cationic lipid is
cationic lipid (108). In embodiments, a cationic lipid is cationic lipid (109). In embodiments, a cationic lipid is cationic lipid (110). In embodiments, a cationic lipid is cationic lipid (111). In embodiments, a cationic lipid is cationic lipid (112). In embodiments, a cationic lipid is cationic lipid (113). In embodiments, a cationic lipid is cationic lipid (114). In embodiments, a cationic lipid is cationic lipid (115). In embodiments, a cationic lipid is cationic lipid (116). In embodiments, a cationic lipid is cationic lipid (117). In embodiments, a cationic lipid is cationic lipid (118). In embodiments, a cationic lipid is cationic lipid (119).
[0195] Exemplary cationic lipids include cationic lipids according to formula (llli) such as cationic lipids
(120)-(132) (Table K):
Figure imgf000071_0001
(llli), where B, B', R2, and R3 are independently as described herein, including the exemplified groups of Table K.
Table K. Formula (llli) Cationic Lipids
Figure imgf000071_0002
Figure imgf000072_0001
71
Figure imgf000073_0001
[0196] In embodiments, a cationic lipid is cationic lipid (120). In embodiments, a cationic lipid is cationic lipid (121). In embodiments, a cationic lipid is cationic lipid (122). In embodiments, a cationic lipid is cationic lipid (123). In embodiments, a cationic lipid is cationic lipid (124). In embodiments, a cationic lipid is cationic lipid (125). In embodiments, a cationic lipid is cationic lipid (126). In embodiments, a cationic lipid is cationic lipid (127). In embodiments, a cationic lipid is cationic lipid (128). In embodiments, a cationic lipid is cationic lipid (129). In embodiments, a cationic lipid is cationic lipid (130). In embodiments, a cationic lipid is cationic lipid (131). In embodiments, a cationic lipid is cationic lipid (132).
[0197] Exemplary cationic lipids include cationic lipids according to formula (lllj) such as cationic lipids (133)-(143) (Table L):
Figure imgf000074_0001
f lllj), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table L.
Table L Formula (lllj) Cationic Lipids
Figure imgf000074_0002
Figure imgf000075_0001
[0198] In embodiments, a cationic lipid is cationic lipid (133). In embodiments, a cationic lipid is cationic lipid (134). In embodiments, a cationic lipid is cationic lipid (135). In embodiments, a cationic lipid is cationic lipid (136). In embodiments, a cationic lipid is cationic lipid (137). In embodiments, a cationic lipid is cationic lipid (138). In embodiments, a cationic lipid is cationic lipid (139). In embodiments, a cationic lipid is cationic lipid (140). In embodiments, a cationic lipid is cationic lipid (141). In embodiments, a cationic lipid is cationic lipid (142). In embodiments, a cationic lipid is cationic lipid (143).
[0199] Exemplary cationic lipids include cationic lipids according to formula (lllk) such as cationic lipids (144)-(154) (Table M):
Figure imgf000076_0001
including the exemplified groups of Table M.
Table M. Formula (lllk) Cationic Lipids
Figure imgf000076_0002
Figure imgf000077_0001
Figure imgf000078_0002
[0200] In embodiments, a cationic lipid is cationic lipid (144). In embodiments, a cationic lipid is
cationic lipid (145). In embodiments, a cationic lipid is cationic lipid (146). In embodiments, a cationic lipid is cationic lipid (147). In embodiments, a cationic lipid is cationic lipid (148). In embodiments, a cationic lipid is cationic lipid (149). In embodiments, a cationic lipid is cationic lipid (150). In embodiments, a cationic lipid is cationic lipid (151). In embodiments, a cationic lipid is cationic lipid (152). In embodiments, a cationic lipid is cationic lipid (153). In embodiments, a cationic lipid is cationic lipid (154).
[0201] Exemplary cationic lipids include cationic lipids according to formula (Him) such as cationic lipids (155)-(165) (Table N):
Figure imgf000078_0001
are independently as described herein, including the exemplified groups of Table H. Table N. Formula (Him) Cationic Lipids
Figure imgf000079_0001
Figure imgf000080_0001
 [0202] In embodiments, a cationic lipid is cationic lipid (155). In embodiments, a cationic lipid is cationic lipid (156). In embodiments, a cationic lipid is cationic lipid (157). In embodiments, a cationic lipid is cationic lipid (158). In embodiments, a cationic lipid is cationic lipid (159). In embodiments, a cationic lipid is cationic lipid (160). In embodiments, a cationic lipid is cationic lipid (161). In embodiments, a cationic lipid is cationic lipid (162). In embodiments, a cationic lipid is cationic lipid (163). In embodiments, a cationic lipid is cationic lipid (164). In embodiments, a cationic lipid is cationic lipid (165).
[0203] Exemplary cationic lipids include cationic lipids according to formula (llln) such as cationic lipids ( 166)-( 178) (Table O):
Figure imgf000081_0001
(llln), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table O.
[0204] In embodiments, a cationic lipid according to formula (llln) has the following structure,
Figure imgf000081_0002
(llln-1). In embodiments, each of R1, R2, and R3 is any of the exemplified aliphatic groups described hereinin (e.g., Table O).
Table O. Formula (llln) and (llln-1) Cationic Lipids
Figure imgf000081_0003
Figure imgf000082_0001
81
Figure imgf000083_0001
[0205] In embodiments, a cationic lipid is cationic lipid (166). In embodiments, a cationic lipid is
cationic lipid (167). In embodiments, a cationic lipid is cationic lipid (168). In embodiments, a cationic lipid is cationic lipid (169). In embodiments, a cationic lipid is cationic lipid (170). In embodiments, a cationic lipid is cationic lipid (171). In embodiments, a cationic lipid is cationic lipid (172). In embodiments, a cationic lipid is cationic lipid (173). In embodiments, a cationic lipid is cationic lipid (174). In embodiments, a cationic lipid is cationic lipid (175). In embodiments, a cationic lipid is cationic lipid (176). In embodiments, a cationic lipid is cationic lipid (177). In embodiments, a cationic lipid is cationic lipid (178).
[0206] Exemplary cationic lipids include cationic lipids according to formula (lllo) such as cationic lipids (179)-(191) (Table P):
Figure imgf000084_0001
(lllo), where B, R2, and R3 are independently as described herein, including the exemplified groups of Table P.
Table P. Formula (lllo) Cationic Lipids
Figure imgf000084_0002
Figure imgf000085_0001
Figure imgf000086_0001
[0207] In embodiments, a cationic lipid is cationic lipid (179). In embodiments, a cationic lipid is
cationic lipid (180). In embodiments, a cationic lipid is cationic lipid (181). In embodiments, a cationic lipid is cationic lipid (182). In embodiments, a cationic lipid is cationic lipid (183). In embodiments, a cationic lipid is cationic lipid (184). In embodiments, a cationic lipid is cationic lipid (185). In embodiments, a cationic lipid is cationic lipid (186). In embodiments, a cationic lipid is cationic lipid (187). In embodiments, a cationic lipid is cationic lipid (188). In embodiments, a cationic lipid is cationic lipid (189). In embodiments, a cationic lipid is cationic lipid (190). In embodiments, a cationic lipid is cationic lipid (191).
[0208] Exemplary cationic lipids include cationic lipids according to formula (lllp) such as cationic lipids (192)-(204) (Table Q):
Figure imgf000087_0001
(lllp), where B, B', R2, and R3 are independently as described herein, including the exemplified groups of Table Q.
Table Q. Formula (lllp) Cationic Lipids
Figure imgf000087_0002
Figure imgf000088_0001
Figure imgf000089_0001
[0209] In embodiments, a cationic lipid is cationic lipid (192). In embodiments, a cationic lipid is
cationic lipid (193). In embodiments, a cationic lipid is cationic lipid (194). In embodiments, a cationic lipid is cationic lipid (195). In embodiments, a cationic lipid is cationic lipid (196). In embodiments, a cationic lipid is cationic lipid (197). In embodiments, a cationic lipid is cationic lipid (198). In embodiments, a cationic lipid is cationic lipid (199). In embodiments, a cationic lipid is cationic lipid (200). In embodiments, a cationic lipid is cationic lipid (201). In embodiments, a cationic lipid is cationic lipid (202). In embodiments, a cationic lipid is cationic lipid (203). In embodiments, a cationic lipid is cationic lipid (204).
[0210] Exemplary cationic lipids include cationic lipids according to formula (lllq) such as cationic lipids (205)-(215) (Table R):
Figure imgf000090_0001
(lllq), where B, R1, R2, and R3 are independently as described herein, including the exemplified groups of Table R.
[0211] In embodiments, a cationic lipid according to formula (lllq) has the following structure,
Figure imgf000090_0002
(lllq-1). In embodiments, each of R1, R2, and R3 is any of the exemplified aliphatic groups described in, e.g., Table R.
Table R. Formula (lllq) Cationic Lipids
Figure imgf000090_0003
Figure imgf000091_0001
90
Figure imgf000092_0002
[0212] In embodiments, a cationic lipid is cationic lipid (205). In embodiments, a cationic lipid is
cationic lipid (206). In embodiments, a cationic lipid is cationic lipid (207). In embodiments, a cationic lipid is cationic lipid (208). In embodiments, a cationic lipid is cationic lipid (209). In embodiments, a cationic lipid is cationic lipid (210). In embodiments, a cationic lipid is cationic lipid (211). In embodiments, a cationic lipid is cationic lipid (212). In embodiments, a cationic lipid is cationic lipid (213). In embodiments, a cationic lipid is cationic lipid (214). In embodiments, a cationic lipid is cationic lipid (215).
[0213] Exemplary cationic lipids include cationic lipids according to formula (lllr) such as cationic lipids (216)-(226) (Table S):
Figure imgf000092_0001
(lllr), where B, R2, and R3 are independently as described herein, including the exemplified groups of Table S. Table S. Formula (lllr) Cationic Lipids
Figure imgf000093_0001
Figure imgf000094_0001
[0214] In embodiments, a cationic lipid is cationic lipid (216). In embodiments, a cationic lipid is cationic lipid (217). In embodiments, a cationic lipid is cationic lipid (218). In embodiments, a cationic lipid is cationic lipid (219). In embodiments, a cationic lipid is cationic lipid (220). In embodiments, a cationic lipid is cationic lipid (221). In embodiments, a cationic lipid is cationic lipid (222). In embodiments, a cationic lipid is cationic lipid (223). In embodiments, a cationic lipid is cationic lipid (224). In embodiments, a cationic lipid is cationic lipid (225). In embodiments, a cationic lipid is cationic lipid (226).
[0215] Exemplary cationic lipids include cationic lipids according to formula (Ills) such as cationic lipids
(227)-(237) (Table T):
Figure imgf000095_0001
are independently as described herein, including the exemplified groups of Table T.
Table T. Formula (Ills) Cationic Lipids
Figure imgf000095_0002
Figure imgf000096_0001
Figure imgf000097_0001
[0216] In embodiments, a cationic lipid is cationic lipid (227). In embodiments, a cationic lipid is
cationic lipid (228). In embodiments, a cationic lipid is cationic lipid (229). In embodiments, a cationic lipid is cationic lipid (230). In embodiments, a cationic lipid is cationic lipid (231). In embodiments, a cationic lipid is cationic lipid (232). In embodiments, a cationic lipid is cationic lipid (233). In embodiments, a cationic lipid is cationic lipid (234). In embodiments, a cationic lipid is cationic lipid (235). In embodiments, a cationic lipid is cationic lipid (236). In embodiments, a cationic lipid is cationic lipid (237). [0217] Exemplary cationic lipids include cationic lipids according to formula (lilt) such as cationic lipids (238)-(261) (Table U):
Figure imgf000098_0001
(lilt), wherein each R is independently as described herein, including the exemplified groups of Table U.
Table U. Formula (lilt) Cationic Lipids
Figure imgf000098_0002
Figure imgf000099_0001
[0218] In embodiments, a cationic lipid is cationic lipid (238). In embodiments, a cationic lipid is
cationic lipid (239). In embodiments, a cationic lipid is cationic lipid (240). In embodiments, a cationic lipid is cationic lipid (241). In embodiments, a cationic lipid is cationic lipid (242). In embodiments, a cationic lipid is cationic lipid (243). In embodiments, a cationic lipid is cationic lipid (244). In embodiments, a cationic lipid is cationic lipid (245). In embodiments, a cationic lipid is cationic lipid (246). In embodiments, a cationic lipid is cationic lipid (247). In embodiments, a cationic lipid is cationic lipid (248). In embodiments, a cationic lipid is cationic lipid (249). In embodiments, a cationic lipid is cationic lipid (250). In embodiments, a cationic lipid is cationic lipid (251). In embodiments, a cationic lipid is cationic lipid (252). In embodiments, a cationic lipid is cationic lipid (253). In embodiments, a cationic lipid is cationic lipid (254). In embodiments, a cationic lipid is cationic lipid (255). In embodiments, a cationic lipid is cationic lipid (256). In embodiments, a cationic lipid is cationic lipid (257). In embodiments, a cationic lipid is cationic lipid (258). In embodiments, a cationic lipid is cationic lipid (259). In embodiments, a cationic lipid is cationic lipid (260). In embodiments, a cationic lipid is cationic lipid (261).
[0219] Exemplary cationic lipids include cationic lipids according to formula (lllu) such as cationic lipids (262)-(285) (Table V):
Figure imgf000100_0001
(lllu), wherein each R is independently as described herein, including the exemplified groups of Table V.
Table V. Formula (lllu) Cationic Lipids
Figure imgf000100_0002
Figure imgf000101_0001
[0220] In embodiments, a cationic lipid is cationic lipid (262). In embodiments, a cationic lipid is
cationic lipid (263). In embodiments, a cationic lipid is cationic lipid (264). In embodiments, a cationic lipid is cationic lipid (265). In embodiments, a cationic lipid is cationic lipid (266). In embodiments, a cationic lipid is cationic lipid (267). In embodiments, a cationic lipid is cationic lipid (268). In embodiments, a cationic lipid is cationic lipid (269). In embodiments, a cationic lipid is cationic lipid (270). In embodiments, a cationic lipid is cationic lipid (271). In embodiments, a cationic lipid is cationic lipid (272). In embodiments, a cationic lipid is cationic lipid (273). In embodiments, a cationic lipid is cationic lipid (274). In embodiments, a cationic lipid is cationic lipid (275). In embodiments, a cationic lipid is cationic lipid (276). In embodiments, a cationic lipid is cationic lipid (277). In embodiments, a cationic lipid is cationic lipid (278). In embodiments, a cationic lipid is cationic lipid (279). In embodiments, a cationic lipid is cationic lipid (280). In embodiments, a cationic lipid is cationic lipid (281). In embodiments, a cationic lipid is cationic lipid (282). In embodiments, a cationic lipid is cationic lipid (283). In embodiments, a cationic lipid is cationic lipid (284). In embodiments, a cationic lipid is cationic lipid (285).
[0221] Exemplary cationic lipids include cationic lipids according to formula (lllv) such as cationic lipids (286)-(309) (Table W):
Figure imgf000102_0001
(lllv), wherein each R is independently as described herein, including the exemplified groups of Table W.
Table W. Formula (lllv) Cationic Lipids
Figure imgf000102_0002
Figure imgf000103_0001
[0222] In embodiments, a cationic lipid is cationic lipid (286). In embodiments, a cationic lipid is
cationic lipid (287). In embodiments, a cationic lipid is cationic lipid (288). In embodiments, a cationic lipid is cationic lipid (289). In embodiments, a cationic lipid is cationic lipid (290). In embodiments, a cationic lipid is cationic lipid (291). In embodiments, a cationic lipid is cationic lipid (292). In embodiments, a cationic lipid is cationic lipid (293). In embodiments, a cationic lipid is cationic lipid (294). In embodiments, a cationic lipid is cationic lipid (295). In embodiments, a cationic lipid is cationic lipid (296). In embodiments, a cationic lipid is cationic lipid (297). In embodiments, a cationic lipid is cationic lipid (298). In embodiments, a cationic lipid is cationic lipid (299). In embodiments, a cationic lipid is cationic lipid (300). In embodiments, a cationic lipid is cationic lipid (301). In embodiments, a cationic lipid is cationic lipid (302). In embodiments, a cationic lipid is cationic lipid (303). In embodiments, a cationic lipid is cationic lipid (304). In embodiments, a cationic lipid is cationic lipid (305). In embodiments, a cationic lipid is cationic lipid (306). In embodiments, a cationic lipid is cationic lipid (307). In embodiments, a cationic lipid is cationic lipid (308). In embodiments, a cationic lipid is cationic lipid (309).
[0223] Exemplary cationic lipids include cationic lipids according to formula (lllw) such as cationic lipids
(310)-(333) (Table X):
Figure imgf000104_0001
(lllw), wherein each R is independently as described herein, including the exemplified groups of Table X.
Table X. Formula (lllw) Cationic Lipids
Figure imgf000104_0002
Figure imgf000105_0002
[0224] In embodiments, a cationic lipid is cationic lipid (310). In embodiments, a cationic lipid is
cationic lipid (311). In embodiments, a cationic lipid is cationic lipid (312). In embodiments, a cationic lipid is cationic lipid (313). In embodiments, a cationic lipid is cationic lipid (314). In embodiments, a cationic lipid is cationic lipid (315). In embodiments, a cationic lipid is cationic lipid (316). In embodiments, a cationic lipid is cationic lipid (317). In embodiments, a cationic lipid is cationic lipid (318). In embodiments, a cationic lipid is cationic lipid (319). In embodiments, a cationic lipid is cationic lipid (320). In embodiments, a cationic lipid is cationic lipid (321). In embodiments, a cationic lipid is cationic lipid (322). In embodiments, a cationic lipid is cationic lipid (323). In embodiments, a cationic lipid is cationic lipid (324). In embodiments, a cationic lipid is cationic lipid (325). In embodiments, a cationic lipid is cationic lipid (326). In embodiments, a cationic lipid is cationic lipid (327). In embodiments, a cationic lipid is cationic lipid (328). In embodiments, a cationic lipid is cationic lipid (329). In embodiments, a cationic lipid is cationic lipid (330). In embodiments, a cationic lipid is cationic lipid (331). In embodiments, a cationic lipid is cationic lipid (332). In embodiments, a cationic lipid is cationic lipid (333).
[0225] Exemplary cationic lipids include cationic lipids according to formula (Mix) such as cationic lipids (334)-(357) (Table Y):
Figure imgf000105_0001
wherein each R is independently as described herein, including the exemplified groups of Table Y. Table Y. Formula (Mix) Cationic Lipids
Figure imgf000106_0001
Figure imgf000107_0002
[0226] In embodiments, a cationic lipid is cationic lipid (334). In embodiments, a cationic lipid is
cationic lipid (335). In embodiments, a cationic lipid is cationic lipid (336). In embodiments, a cationic lipid is cationic lipid (337). In embodiments, a cationic lipid is cationic lipid (338). In embodiments, a cationic lipid is cationic lipid (339). In embodiments, a cationic lipid is cationic lipid (340). In embodiments, a cationic lipid is cationic lipid (341). In embodiments, a cationic lipid is cationic lipid (342). In embodiments, a cationic lipid is cationic lipid (343). In embodiments, a cationic lipid is cationic lipid (344). In embodiments, a cationic lipid is cationic lipid (345). In embodiments, a cationic lipid is cationic lipid (346). In embodiments, a cationic lipid is cationic lipid (347). In embodiments, a cationic lipid is cationic lipid (348). In embodiments, a cationic lipid is cationic lipid (349). In embodiments, a cationic lipid is cationic lipid (350). In embodiments, a cationic lipid is cationic lipid (351). In embodiments, a cationic lipid is cationic lipid (352). In embodiments, a cationic lipid is cationic lipid (353). In embodiments, a cationic lipid is cationic lipid (354). In embodiments, a cationic lipid is cationic lipid (355). In embodiments, a cationic lipid is cationic lipid (356). In embodiments, a cationic lipid is cationic lipid (357).
[0227] Exemplary cationic lipids include cationic lipids according to formula (Illy) such as cationic lipids (358)-(381) (Table Z):
Figure imgf000107_0001
(Illy), wherein each R is independently as described herein, including the exemplified groups of Table Z.
Table Z. Formula (Illy) Cationic Lipids
Figure imgf000107_0003
Figure imgf000108_0001
[0228] In embodiments, a cationic lipid is cationic lipid (358). In embodiments, a cationic lipid is cationic lipid (359). In embodiments, a cationic lipid is cationic lipid (360). In embodiments, a cationic lipid is cationic lipid (361). In embodiments, a cationic lipid is cationic lipid (362). In embodiments, a cationic lipid is cationic lipid (363). In embodiments, a cationic lipid is cationic lipid (364). In embodiments, a cationic lipid is cationic lipid (365). In embodiments, a cationic lipid is cationic lipid (366). In embodiments, a cationic lipid is cationic lipid (367). In embodiments, a cationic lipid is cationic lipid (368). In embodiments, a cationic lipid is cationic lipid (369). In embodiments, a cationic lipid is cationic lipid (370). In embodiments, a cationic lipid is cationic lipid (371). In embodiments, a cationic lipid is cationic lipid (372). In embodiments, a cationic lipid is cationic lipid (373). In embodiments, a cationic lipid is cationic lipid (374). In embodiments, a cationic lipid is cationic lipid (375). In embodiments, a cationic lipid is cationic lipid (376). In embodiments, a cationic lipid is cationic lipid (377). In embodiments, a cationic lipid is cationic lipid (378). In embodiments, a cationic lipid is cationic lipid (379). In embodiments, a cationic lipid is cationic lipid (380). In embodiments, a cationic lipid is cationic lipid (381).
[0229] Exemplary cationic lipids include cationic lipids according to formula (lllz) such as cationic lipids (382)-(405) (Table AA):
Figure imgf000109_0001
(lllz), wherein each R is independently as described herein, including the exemplified groups of Table AA.
Table AA. Formula (lllz) Cationic Lipids
Figure imgf000109_0002
Figure imgf000110_0001
[0230] In embodiments, a cationic lipid is cationic lipid (382). In embodiments, a cationic lipid is cationic lipid (383). In embodiments, a cationic lipid is cationic lipid (384). In embodiments, a cationic lipid is cationic lipid (385). In embodiments, a cationic lipid is cationic lipid (386). In embodiments, a cationic lipid is cationic lipid (387). In embodiments, a cationic lipid is cationic lipid (388). In embodiments, a cationic lipid is cationic lipid (389). In embodiments, a cationic lipid is cationic lipid (390). In embodiments, a cationic lipid is cationic lipid (391). In embodiments, a cationic lipid is cationic lipid (392). In embodiments, a cationic lipid is cationic lipid (393). In embodiments, a cationic lipid is cationic lipid (394). In embodiments, a cationic lipid is cationic lipid (395). In embodiments, a cationic lipid is cationic lipid (396). In embodiments, a cationic lipid is cationic lipid (397). In embodiments, a cationic lipid is cationic lipid (398). In embodiments, a cationic lipid is cationic lipid (399). In embodiments, a cationic lipid is cationic lipid (400). In embodiments, a cationic lipid is cationic lipid (401). In embodiments, a cationic lipid is cationic lipid (402). In embodiments, a cationic lipid is cationic lipid (403). In embodiments, a cationic lipid is cationic lipid (404). In embodiments, a cationic lipid is cationic lipid (405).
[0231] Exemplary cationic lipids include cationic lipids according to formula (lllaa) such as cationic lipids (406)-(429) (Table AB):
Figure imgf000111_0001
(lllaa), wherein each R is independently as described herein, including the exemplified groups of Table AB.
Table AB. Formula (lllaa) Cationic Lipids
Figure imgf000111_0002
Figure imgf000112_0001
[0232] In embodiments, a cationic lipid is cationic lipid (406). In embodiments, a cationic lipid is
cationic lipid (407). In embodiments, a cationic lipid is cationic lipid (408). In embodiments, a cationic lipid is cationic lipid (409). In embodiments, a cationic lipid is cationic lipid (410). In embodiments, a cationic lipid is cationic lipid (411). In embodiments, a cationic lipid is cationic lipid (412). In embodiments, a cationic lipid is cationic lipid (413). In embodiments, a cationic lipid is cationic lipid (414). In embodiments, a cationic lipid is cationic lipid (415). In embodiments, a cationic lipid is cationic lipid (416). In embodiments, a cationic lipid is cationic lipid (417). In embodiments, a cationic lipid is cationic lipid (418). In embodiments, a cationic lipid is cationic lipid (419). In embodiments, a cationic lipid is cationic lipid (420). In embodiments, a cationic lipid is cationic lipid (421). In embodiments, a cationic lipid is cationic lipid (422). In embodiments, a cationic lipid is cationic lipid (423). In embodiments, a cationic lipid is cationic lipid (424). In embodiments, a cationic lipid is cationic lipid (425). In embodiments, a cationic lipid is cationic lipid (426). In embodiments, a cationic lipid is cationic lipid (427). In embodiments, a cationic lipid is cationic lipid (428). In embodiments, a cationic lipid is cationic lipid (429). [0233] Exemplary cationic lipids include cationic lipids according to formula (lllaa) such as cationic lipids
(430)-(453) (Table AC):
Figure imgf000113_0001
(lllab), wherein each R is independently as described herein, including the exemplified groups of Table AC.
Table AC. Formula (lllab) Cationic Lipids
Figure imgf000113_0002
Figure imgf000114_0001
[0234] In embodiments, a cationic lipid is cationic lipid (430). In embodiments, a cationic lipid is
cationic lipid (431). In embodiments, a cationic lipid is cationic lipid (432). In embodiments, a cationic lipid is cationic lipid (433). In embodiments, a cationic lipid is cationic lipid (434). In embodiments, a cationic lipid is cationic lipid (435). In embodiments, a cationic lipid is cationic lipid (436). In embodiments, a cationic lipid is cationic lipid (437). In embodiments, a cationic lipid is cationic lipid (438). In embodiments, a cationic lipid is cationic lipid (439). In embodiments, a cationic lipid is cationic lipid (440). In embodiments, a cationic lipid is cationic lipid (441). In embodiments, a cationic lipid is cationic lipid (442). In embodiments, a cationic lipid is cationic lipid (443). In embodiments, a cationic lipid is cationic lipid (444). In embodiments, a cationic lipid is cationic lipid (445). In embodiments, a cationic lipid is cationic lipid (446). In embodiments, a cationic lipid is cationic lipid (447). In embodiments, a cationic lipid is cationic lipid (448). In embodiments, a cationic lipid is cationic lipid (449). In embodiments, a cationic lipid is cationic lipid (450). In embodiments, a cationic lipid is cationic lipid (451). In embodiments, a cationic lipid is cationic lipid (452). In embodiments, a cationic lipid is cationic lipid (453). [0235] Exemplary cationic lipids include cationic lipids (454)-(462) of Table AD.
Table AD. Exemplary Cationic Lipids
Figure imgf000115_0001
Figure imgf000116_0001
[0236] In embodiments, a cationic lipid is cationic lipid (454). In embodiments, a cationic lipid is
cationic lipid (455). In embodiments, a cationic lipid is cationic lipid (456). In embodiments, a cationic lipid is cationic lipid (457). In embodiments, a cationic lipid is cationic lipid (458). In embodiments, a cationic lipid is cationic lipid (459). In embodiments, a cationic lipid is cationic lipid (460). In embodiments, a cationic lipid is cationic lipid (461). In embodiments, a cationic lipid is cationic lipid (462).
Synthesis of Cationic Lipids
[0237] Cationic lipids described herein (e.g., cationic lipids of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) can be prepared according to methods known in the art. Additional exemplary methods are provided in the Examples described herein.
[0238] Scheme 1 provides an exemplary synthesis for cationic lipids described herein.
Scheme 1. Exemplary Synthesis of Cationic Lipids
Figure imgf000117_0001
RY - C1-C2g aliphatic group
LG : = leaving group
[0239] In Scheme 1, combination of a carbohydrate such as Compound A with a cationic electrophile (e.g., Compound B, 3-(dimethylamino)propionic acid, or a salt thereof) can afford Compound C.
This compound can be treated with various electrophiles to afford cationic lipids (e.g., cationic lipids of Formula (I) and (II)) as described herein. For example, Compound C can be treated with an aliphatic electrophile (e.g., alkyl halides or alkenyl halides) such as Compound D to afford product Cationic Lipid E. Alternatively, Compound C can be treated with an acylating agent such
Compound F (e.g., acyl halides) to afford product Cationic Lipid G.
[0240] Scheme 2 provides an exemplary synthesis using D-ribose and acylating agents.
Scheme 2. Exemplary Synthesis of Ribose Cationic Lipids
Figure imgf000117_0002
CH3
Compound B Rx = C1-C29 aliphatic group [0241] In Scheme 2, ribose (Compound Al) can be combined with 3-(dimethylamino)propionic acid (Compound B) using iodine and tri(p-tolyl)phosphine in dichloromethane to afford Compound Cl. Treatment of Compound Cl with excess acyl halide (Compound FI) under basic conditions can afford peracylated Cationic Lipid Gl.
Nucleic Acids
[0242] Cationic lipids described herein (e.g., a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) can be used to prepare compositions useful for the delivery of nucleic acids.
Synthesis of Nucleic Acids
[0243] Nucleic acids according to the present invention may be synthesized according to any known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing 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, mutated T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
[0001] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7, mutated T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
[0244] Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA. [0245] As described above, the term "nucleic acid," in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA, retrotransposons, a viral genome, a viroid, satellite RNA, or derivatives of these groups. In some embodiments, a nucleic acid is a mRNA encoding a protein.
Synthesis of mRNA
[0246] m RNAs according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application. The exact conditions will vary according to the specific application. The presence of these reagents is undesirable in the final product according to several embodiments and may thus be referred to as impurities and a preparation containing one or more of these impurities may be referred to as an impure preparation. In some embodiments, the in vitro transcribing occurs in a single batch.
[0247] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
[0248] Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
Modified mRNA
[0249] In some embodiments, mRNA according to the present invention may be synthesized as
unmodified or modified mRNA. Modified mRNA comprise nucleotide modifications in the RNA. A modified mRNA according to the invention can thus include nucleotide modification that are, for example, backbone modifications, sugar modifications or base modifications. In some
embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2- methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2- methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5- methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl- pseudouracil, queosine, .beta.-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g., from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.
[0250] In some embodiments, mRNAs may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates,
methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-0-(l- thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
[0251] In some embodiments, mRNAs may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 4'-thio-ribonucleotide (see, e.g., US Patent Application Publication No. US 2016/0031928, incorporated by reference herein), 2'-deoxy-2'- fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'- triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-0-alkyloligoribonucleotide, 2'-deoxy-2'-C- alkyloligoribonucleotide (2'-0-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'- C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'- triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'- deoxyuridine 5'-triphosphate).
[0252] In some embodiments, mRNAs may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base- modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2- amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'- triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'- triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'- triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'- triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7- deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, Nl-methyladenosine 5'-triphosphate, Nl-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, 06-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.
[0253] Typically, mRNA synthesis includes the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from exonuclease degradation. [0254] Thus, in some embodiments, mRNAs include a 5' cap structure. A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7- nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
[0255] In some embodiments, mRNAs include a 3' poly(A) tail structure. A poly-A tail on the 3'
terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3' poly(C) tail structure. A suitable poly-C tail on the 3' terminus of 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 may be added to the poly-A tail or may substitute the poly-A tail.
[0256] In some embodiments, mRNAs include a 5' and/or 3' untranslated region. In some
embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.
[0257] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.
Cap structure
[0258] In some embodiments, mRNAs include a 5' cap structure. A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7- nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. [0259] Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp.
[0260] A common cap for mRNA produced by in vitro transcription is m7G(5')ppp(5')G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5'-termini. The prevailing method for the in vitro synthesis of caPPEd mRNA employs a pre-formed dinucleotide of the form m7G(5')ppp(5')G ("m7GpppG") as an initiator of transcription.
[0261] To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog ("ARCA") or modified ARCA, which is generally a modified cap analog in which the 2' or 3' OH group is replaced with -OCH3.
[0262] Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG);
dimethylated cap analog (e.g., m2-7GpppG), trimethylated cap analog (e.g., m2-2'7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2OmeGpppG, m72 dGpppG, m7'3 0meGpppG, m7-3 dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties”,
RNA, 9: 1108-1122 (2003)).
[0263] In some embodiments, a suitable cap is a 7-methyl guanylate ("m7G") linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleoside. A preferred embodiment of a m7G cap utilized in embodiments of the invention is m7G(5')ppp(5')G.
[0264] In some embodiments, the cap is a CapO structure. CapO structures lack a 2'-0-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, the cap is a Capl structure. Capl structures have a 2'-0-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. Cap2 structures have a 2'-0-methyl residue attached to both bases 2 and 3. [0265] A variety of m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG described above, as well as the 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 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 (including biotinylated cap analogs) described in U.S. Patent Nos. 8,093,367 and 8,304,529, incorporated by reference herein.
Tail structure
[0266] Typically, the presence of a "tail" serves to protect the mRNA from exonuclease degradation.
The poly A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A tail can be added to an mRNA molecule thus rendering the RNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Flarbor
Laboratory Press: 1991 edition)).
[0267] In some embodiments, mRNAs include a 3' poly(A) tail structure. Typically, the length of the poly A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3' poly(C) tail structure. A suitable poly-C tail on the 3' terminus of 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 may be added to the poly-A tail or may substitute the poly-A tail.
[0268] In some embodiments, the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein. For example, since the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.
5' and 3' Untranslated Region
[0269] In some embodiments, mRNAs include a 5' and/or 3' untranslated region. In some
embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.
[0270] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.
[0271] Exemplary 3' and/or 5' UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.
Pharmaceutical Formulations of Cationic Lipids and Nucleic Acids
[0272] In certain embodiments cationic lipids described herein described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), as well as pharmaceutical and liposomal compositions comprising such lipids, can be used in formulations to facilitate the delivery of encapsulated materials (e.g., one or more polynucleotides such as mRNA) to, and subsequent transfection of one or more target cells. For example, in certain embodiments cationic lipids described herein (and compositions such as liposomal compositions comprising such lipids) are characterized as resulting in 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 afford such compounds advantages relative other similarly classified lipids. [0273] According to the present invention, a nucleic acid, e.g., mRNA encoding a protein (e.g., a full length, fragment or portion of a protein) as described herein may be delivered via a delivery vehicle comprising a cationic lipid as described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)- (lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
[0274] As used herein, the terms "delivery vehicle," "transfer vehicle," "nanoparticle" or grammatical equivalent, are used interchangeably.
[0275] For example, the present invention provides a composition (e.g., a pharmaceutical composition) comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)- (lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) and one or more polynucleotides. A composition (e.g., a pharmaceutical composition) may further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
[0276] In certain embodiments a composition exhibits an enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally comprise the step of contacting the one or more target cells with the cationic lipids and/or pharmaceutical compositions disclosed herein (e.g., a liposomal formulation comprising a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) encapsulating one or more polynucleotides) such that the one or more target cells are transfected with the materials encapsulated therein (e.g., one or more polynucleotides). As used herein, the terms "transfect" or "transfection" refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or polynucleotides) into a cell, or preferably into a target cell. The introduced polynucleotide may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulated material (e.g., polynucleotides) up-taken by, introduced into and/or expressed by the target cell which is subject to transfection. In practice, transfection efficiency may be estimated by the amount of a reporter polynucleotide product produced by the target cells following transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein demonstrate high transfection efficiencies thereby improving the likelihood that appropriate dosages of the encapsulated materials (e.g., one or more
polynucleotides) will be delivered to the site of pathology and subsequently expressed, while at the same time minimizing potential systemic adverse effects or toxicity associated with the compound or their encapsulated contents. [0277] Following transfection of one or more target cells by, for example, the polynucleotides encapsulated in the one or more lipid nanoparticles comprising the pharmaceutical or liposomal compositions disclosed herein, the production of the product (e.g., a polypeptide or protein) encoded by such polynucleotide may be preferably stimulated and the capability of such target cells to express the polynucleotide and produce, for example, a polypeptide or protein of interest is enhanced. For example, transfection of a target cell by one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of the protein or enzyme encoded by such mRNA.
[0278] Further, delivery vehicles described herein (e.g., liposomal delivery vehicles) may be prepared to preferentially distribute to other target tissues, cells or organs, such as the heart, lungs, kidneys, spleen. In embodiments, the lipid nanoparticles of the present invention may be prepared to achieve enhanced delivery to the target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical and liposomal compositions described herein can be delivered to and/or transfect targeted cells or tissues. In some
embodiments, the encapsulated polynucleotides (e.g., mRNA) are capable of being expressed and functional polypeptide products produced (and in some instances excreted) by the target cell, thereby conferring a beneficial property to, for example the target cells or tissues. Such
encapsulated polynucleotides (e.g., mRNA) may encode, for example, a hormone, enzyme, receptor, polypeptide, peptide or other protein of interest.
Liposomal Delivery Vehicles
[0279] In some embodiments, a composition is a suitable delivery vehicle. In embodiments, a
composition is a liposomal delivery vehicle, e.g., a lipid nanoparticle.
[0280] The terms "liposomal delivery vehicle" and "liposomal composition" are used interchangeably.
[0281] Enriching liposomal compositions with one or more of the cationic lipids disclosed herein may be used as a means of improving (e.g., reducing) the toxicity or otherwise conferring one or more desired properties to such enriched liposomal composition (e.g., improved delivery of the encapsulated polynucleotides to one or more target cells and/or reduced in vivo toxicity of a liposomal composition). Accordingly, also contemplated are pharmaceutical compositions, and in particular liposomal compositions, that comprise one or more of the cationic lipids disclosed herein.
[0282] Thus, in certain embodiments, the compounds described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) are cationic lipids that may be used as a component of a liposomal composition to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).
[0283] As used herein, liposomal delivery vehicles, e.g., lipid nanoparticles, are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue.
[0284] In certain embodiments, such compositions (e.g., liposomal compositions) are loaded with or otherwise encapsulate materials, such as for example, one or more biologically-active
polynucleotides (e.g., mRNA).
[0285] In embodiments, a composition (e.g., a pharmaceutical composition) comprises an mRNA
encoding a protein, encapsulated within a liposome. In embodiments, a 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., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)). In embodiments, a composition comprises an mRNA encoding for a protein (e.g., any protein described herein). In embodiments, a composition comprises an mRNA encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein. In embodiments, a composition comprises an mRNA encoding for ornithine transcarbamylase (OTC) protein. In embodiments, an mRNA encodes for an antigen from an infectious agent.
[0286] In embodiments, a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises any cationic lipid (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)- (462)) as described herein.
[0287] In embodiments, a nucleic acid is an mRNA encoding a peptide or polypeptide. In
embodiments, an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell (e.g., an mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein). In embodiments, an mRNA encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell (e.g., an mRNA encodes ornithine transcarbamylase (OTC) protein). In embodiments, an mRNA encodes for an antigen from an infectious agent. Still other exemplary mRNAs are described herein.
[0288] In embodiments, a liposomal delivery vehicle (e.g., a lipid nanoparticle) can have a net positive charge.
[0289] In embodiments, a liposomal delivery vehicle (e.g., a lipid nanoparticle) can have a net negative charge.
[0290] In embodiments, a liposomal delivery vehicle (e.g., a lipid nanoparticle) can have a net neutral charge.
[0291] In embodiments, a lipid nanoparticle that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)).
[0292] For example, the amount of a cationic lipid as described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) in a composition can be described as a percentage ("wt%") of the combined dry weight of all lipids of a composition (e.g., the combined dry weight of all lipids present in a liposomal composition).
[0293] In embodiments of the pharmaceutical compositions described herein, a cationic lipid as
described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is 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 a composition (e.g., a liposomal composition).
[0294] In embodiments, a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition). In embodiments, a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle. [0295] In embodiments, the amount of a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in a composition (e.g., a liposomal composition).
[0296] In embodiments, the amount of a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is no more than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in a composition (e.g., a liposomal composition).
[0297] In embodiments, a composition (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.1 wt% to about 20 wt% (e.g., about 0.1 wt% to about 15 wt%) of a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)). In embodiments, a delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)). In embodiments, a delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)). In embodiments, the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
[0298] The amount of a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) in a composition also can be described as a percentage ("mol%") of the combined molar amounts of total lipids of a composition (e.g., the combined molar amounts of all lipids present in a liposomal delivery vehicle).
[0299] In embodiments of pharmaceutical compositions described herein, a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is about 0.5 mol% to about30 mol% (e.g., about 0.5 mol% to about20 mol%) of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
[0300] In embodiments, a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is 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 amounts of all lipids present in a composition such as a liposomal delivery vehicle. In embodiments, a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is 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
[0301] In certain embodiments, a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) 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 lipids in a composition (e.g., a liposomal delivery vehicle).
[0302] In certain embodiments, cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) 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 lipids in the lipid nanoparticle.
[0303] In certain embodiments, cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) can comprise less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total amount of lipids in a composition (e.g., a liposomal delivery vehicle).
[0304] In embodiments, the amount of a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is 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 total lipids in a composition (e.g., a liposomal composition). [0305] In embodiments, the amount of a cationic lipid of Formula (I'), (I), (II), or (llla)-(lllab) (e.g., any of cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) is present in an amount that is 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 total lipids in a composition (e.g., a liposomal composition).
[0306] In embodiments, the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
[0307] In embodiments, a composition further comprises one more lipids (e.g., one 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).
[0308] In certain embodiments, such pharmaceutical (e.g., liposomal) compositions comprise one or more of a PEG-modified lipid, a non-cationic lipid and a cholesterol lipid. In embodiments, such pharmaceutical (e.g., liposomal) 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., liposomal) compositions comprise: one or more PEG-modified lipids and one or more cholesterol lipids.
[0309] In embodiments, a composition (e.g., lipid nanoparticle) that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)) and one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.
[0310] In embodiments, a composition (e.g., lipid nanoparticle) that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)- (21b), and (22)-(462)); one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid; and further comprises a cholesterol-based lipid.
[0311] In embodiments, a lipid nanoparticle that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or polypeptide) comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), as well as 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.
[0312] In embodiments of lipid nanoparticles described herein, a lipid nanoparticle comprises one or more cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), a non-cationic lipid (e.g., DOPE), a PEGylated lipid (e.g., DMG-PEG2000), and a cholesterol-based lipid (e.g., cholesterol).
[0313] According to various embodiments, the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.
Further Cationic Lipids
[0314] In addition to any of the cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)), a composition may comprise one or more further cationic lipids.
[0315] In some embodiments, liposomes may comprise one or more further cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that have 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.
[0316] Particularly suitable further cationic lipids for use in the compositions and methods of the
invention include those described in international patent publications WO 2010/053572 (and particularly, C12-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the invention employ a lipid nanoparticles comprising a further cationic lipid described in U.S.
provisional patent application 61/617,468, filed March 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-l-yl)tetracosa-15,18-dien-l- amine (HGT5000), (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl)tetracosa-4, 15,18- trien-l-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l- yl)tetracosa-5, 15, 18-trien-l-amine (HGT5002). [0317] In some embodiments, a composition (e.g., a liposomal composition) comprises a further cationic lipid described in WO 2013/063468, filed October 26, 2012 and in U.S. provisional application 61/953,516, filed March 14, 2014, both of which are incorporated by reference herein.
[0318] In particular embodiments, a composition (e.g., a liposomal composition) comprises a further cationic lipid CKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione). The structure of CKK-E12 is shown below:
Figure imgf000134_0001
[0319] In some embodiments, a further cationic lipid may be N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride or "DOTMA" (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with a neutral lipid (e.g., dioleoylphosphatidyl-ethanolamine or "DOPE") or still other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable further cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS"; 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" (Behr et al. Proc. Nat.'l Acad. Sci.
86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); l,2-dioleoyl-3- Dimethylammonium-Propane or "DODAP"; or l,2-dioleoyl-3-trimethylammonium-propane or "DOTAP".
[0320] Additional exemplary further cationic lipids also include l,2-distearyloxy-N,N-dimethyl-3- aminopropane or "DSDMA"; l,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA"; 1 ,2- dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA"; l,2-dilinolenyloxy-N,N-dimethyl-3- aminopropane or "DLenDMA"; N-dioleyl-N,N-dimethylammonium chloride or "DODAC";N,N- distearyl-N,N-dimethylarnrnonium bromide or "DDAB"; N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE"; 3-dimethylamino-2-(cholest-5-en-3-beta- oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane or "CLinDMA"; 2-[5'-(cholest-5-en-3-beta- oxy)-3'-oxapentoxy)-3-dimethy l-l-(cis,cis-9', l-2'-octadecadienoxy)propane or "CpLinDMA"; N,N- dimethyl-3,4-dioleyloxybenzylamine or "DMOBA"; 1 ,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane or "DOcarbDAP"; 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or
"DLinDAP"; l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane or "DLincarbDAP"; 1 ,2- dilinoleoylcarbamyl-3-dimethylaminopropane or "DLinCDAP"; 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane or "DLin- -DMA"; 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane or "DLin-K-XTC2-DMA"; and 2-(2,2-di((9Z,12Z)-octadeca-9,l 2-dien- l-yl)-l,3-dioxolan-4- yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (see WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication
WO2005/121348A1). In some embodiments, one or more of the further cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
[0321] In some embodiments, the one or more further cationic lipids may be chosen from XTC (2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1 ,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3- oxo-3-(undecylamino)propyl)-N l,N16-diundecyl-4, 7, 10, 13-tetraazahexadecane-l, 16-diamide), DODAP (l,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in W02010/053572, DOTAP (l,2-dioleyl-3-trimethylammonium propane), DOTMA (l,2-di-0-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L; Bremner, K.; MacLachlan, I. "Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids" J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S.C. et al. "Rational Design of Cationic Lipids for siRNA Delivery" Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K.T. et al. "Lipid-like materials for low-dose in vivo gene silencing" PNAS 2010, 107, 1864- 1869).
[0322] In some embodiments, the percentage of total cationic lipids in a composition (e.g., a liposomal 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 total lipids as measured by molar ratios (mol%) or by weight (wt%).
[0323] In some embodiments, the percentage of total cationic lipids in a composition (e.g., a liposomal 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 total lipids as measured by molar ratios (mol%) or by weight (wt%).
[0324] In some embodiments, total cationic lipid(s) constitute(s) about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight. In some embodiments, the cationic lipid constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of a composition (e.g., a liposomal composition) by molar ratio. In some embodiments, total cationic lipid(s) constitute(s) about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight. In some embodiments, the cationic lipid constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of a composition (e.g., a liposomal composition) by weight.
Non-cationic/Helper Lipids
[0325] Compositions (e.g., liposomal compositions) may also comprise 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 number 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 (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.
[0326] In embodiments, a non-cationic or helper lipid is dioleoylphosphatidylethanolamine (DOPE).
[0327] In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered. [0328] In some embodiments, a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipid in a 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 total non-cationic lipids in a liposome may 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%.
[0329] In some embodiments, a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of non- cationic lipid in a liposome is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may 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%. Cholesterol-based Lipids
[0330] In some embodiments, a composition (e.g., a liposomal 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-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al.
BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or imidazole cholesterol ester (ICE), which has the following structure,
Figure imgf000138_0001
[0331] In embodiments, a cholesterol-based lipid is cholesterol.
[0332] In some embodiments, a cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol- based lipid in the lipid nanoparticle may be 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%.
[0333] In some embodiments, a cholesterol-based lipid may be present in a weight ratio (wt%) of
about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be 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%.
PEGylated Lipids
[0334] In some embodiments, a composition (e.g., a liposomal composition) comprises one or more PEGylated lipids. [0335] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the liposome. In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., CM or Cig).
[0336] Contemplated PEG-modified lipids (also referred to herein as a PEGylated lipid, which term is interchangeable with PEG-modified lipid) include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
[0337] In embodiments, a PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000).
[0338] A PEG-modified phospholipid and derivatized lipids of the present invention may be present in a molar ratio (mol%) from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the composition (e.g., a liposomal composition).
[0339] A PEG-modified phospholipid and derivatized lipids of the present invention may be present in a weight ratio (wt%) from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the composition (e.g., a liposomal composition).
Pharmaceutical Formulations and Therapeutic Uses
[0340] Cationic lipids described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) may be used in the preparation of compositions (e.g., to construct liposomal compositions) that facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells). [0341] For example, when a liposomal composition (e.g., a lipid nanoparticle) comprises or is otherwise enriched with one or more of the compounds disclosed herein, the phase transition in the lipid bilayer of the one or more target cells may facilitate the delivery of the encapsulated materials (e.g., one or more therapeutic polynucleotides encapsulated in a lipid nanoparticle) into the one or more target cells.
[0342] Similarly, in certain embodiments cationic lipids described herein (e.g., a cationic lipid of
Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) may be used to prepare liposomal vehicles that are characterized by their reduced toxicity in vivo. In certain embodiments, the reduced toxicity is a function of the high transfection efficiencies associated with the compositions disclosed herein, such that a reduced quantity of such
composition may administered to the subject to achieve a desired therapeutic response or outcome.
[0343] Thus, pharmaceutical formulations comprising a cationic lipid described herein (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)- (462)) and nucleic acids provided by the present invention may be used for various therapeutic purposes. To facilitate delivery of nucleic acids in vivo, a cationic lipid (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) and nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands or stabilizing reagents. In some embodiments, a cationic lipid (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) can be formulated via pre-mixed lipid solution. In other embodiments, a composition comprising a cationic lipid (e.g., a cationic lipid of Formula (G), (I), (II), or (llla)-(lllab) such as cationic lipids (la)- (21a), (lb)-(21b), and (22)-(462)) can be formulated using post-insertion techniques into the lipid membrane of the nanoparticles. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.
[0344] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal,
pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In particular embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments the administration results in delivery of the nucleic acids to a muscle cell. In some embodiments the administration results in delivery of the nucleic acids to a hepatocyte (i.e., liver cell). In embodiments, administration is intramuscular. In embodiments, administration is intravenous.
[0345] Alternatively or additionally, pharmaceutical formulations of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. Exemplary tissues in which delivered mRNA may 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 embodiments, the tissue to be targeted in the liver. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
[0346] In embodiments, administration is via pulmonary delivery. As used herein, pulmonary delivery refers to delivery to lung via, e.g., nasal cavity, trachea, bronchi, bronchioles, and/or other pulmonary system. In embodiments, a composition described herein is formulated for nebulization. In embodiments, the delivery vehicle may be in an aerosolized composition which can be inhaled. In embodiments, pulmonary delivery involves inhalation (e.g., for nasal, tracheal, or bronchial delivery). In embodiments, a composition is nebulized prior to inhalation.
[0347] The present invention provides methods for delivering a composition having full-length mRNA molecules encoding a peptide or polypeptide of interest for use in the treatment of a subject, e.g., a human subject or a cell of a human subject or a cell that is treated and delivered to a human subject.
[0348] Accordingly, in certain embodiments the present invention provides a method for producing a therapeutic composition comprising full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for cystic fibrosis transmembrane conductance regulator (CFTR) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP-binding cassette sub-family A member 3 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for dynein axonemal intermediate chain 1 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for dynein axonemal heavy chain 5 (DNAH5) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for alpha-l-antitrypsin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for forkhead box P3 (FOXP3) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes one or more surfactant protein, e.g., one or more of surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.
[0349] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell. Such peptides and polypeptides can include those associated with a urea cycle disorder, associated with a lysosomal storage disorder, with a glycogen storage disorder, associated with an amino acid metabolism disorder, associated with a lipid metabolism or fibrotic disorder, associated with methylmalonic acidemia, or associated with any other metabolic disorder for which delivery to or treatment of the liver or a liver cell with enriched full-length mRNA provides therapeutic benefit.
[0350] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a urea cycle disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ornithine transcarbamylase (OTC) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arginosuccinate synthetase 1 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for carbamoyl phosphate synthetase I protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arginosuccinate lyase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arginase protein.
[0351] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a lysosomal storage disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for alpha galactosidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glucocerebrosidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for iduronate-2-sulfatase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for iduronidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for N- acetyl-alpha-D-glucosaminidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for heparan N-sulfatase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for galactosamine-6 sulfatase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for beta-galactosidase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for lysosomal lipase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for transcription factor EB (TFEB).
[0352] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a glycogen storage disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for acid alpha-glucosidase protein.
In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glucose-6-phosphatase (G6PC) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for liver glycogen phosphorylase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for muscle phosphoglycerate mutase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glycogen debranching enzyme. [0353] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with amino acid metabolism. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for phenylalanine hydroxylase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for glutaryl-CoA dehydrogenase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for propionyl-CoA caboxylase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for oxalase alanine-glyoxylate aminotransferase enzyme.
[0354] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with a lipid metabolism or fibrotic disorder. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a mTOR inhibitor. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATPase phospholipid transporting 8B1 (ATP8B1) protein.
In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for one or more NF-kappa B inhibitors, such as one or more of l-kappa B alpha, interferon-related development regulator 1 (IFRD1), and Sirtuin 1 (SIRT1). In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for PPAR-gamma protein or an active variant.
[0355] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein associated with methylmalonic acidemia. For example, in certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for methylmalonyl CoA mutase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for methylmalonyl CoA epimerase protein.
[0356] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA for which delivery to or treatment of the liver can provide therapeutic benefit. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP7B protein, also known as Wilson disease protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for porphobilinogen deaminase enzyme. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for one or clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for human hemochromatosis (FIFE) protein.
[0357] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the cardiovasculature of a subject or a cardiovascular cell. In certain
embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for vascular endothelial growth factor A protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for relaxin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for bone morphogenetic protein-9 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for bone morphogenetic protein-2 receptor protein.
[0358] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the muscle of a subject or a muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for dystrophin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for frataxin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the cardiac muscle of a subject or a cardiac muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for a protein that modulates one or both of a potassium channel and a sodium channel in muscle tissue or in a muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a protein that modulates a Kv7.1 channel in muscle tissue or in a muscle cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for a protein that modulates a Navl.5 channel in muscle tissue or in a muscle cell.
[0359] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the nervous system of a subject or a nervous system cell. For example, in certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for survival motor neuron 1 protein. For example, in certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for survival motor neuron 2 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length m RNA that encodes for frataxin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for CLN3 protein.
[0360] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the blood or bone marrow of a subject or a blood or bone marrow cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for beta globin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Bruton's tyrosine kinase protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for one or clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.
[0361] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the kidney of a subject or a kidney cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for collagen type IV alpha 5 chain (COL4A5) protein.
[0362] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the eye of a subject or an eye cell. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for ATP-binding cassette sub-family A member 4 (ABCA4) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for retinoschisin protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for retinal pigment epithelium-specific 65 kDa (RPE65) protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for centrosomal protein of 290 kDa (CEP290).
[0363] In embodiments, an mRNA encodes for an antigen from an infectious agent.
[0364] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or polypeptide for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject. For example, in certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from an infectious agent, such as a virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from influenza virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from respiratory syncytial virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from rabies virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from cytomegalovirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from rotavirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus, or hepatis C virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from human papillomavirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a human metapneumovirus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from malaria virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from zika virus. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from chikungunya virus.
[0365] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen associated with a cancer of a subject or identified from a cancer cell of a subject. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen determined from a subject's own cancer cell, i.e., to provide a personalized cancer vaccine. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen expressed from a mutant KRAS gene.
[0366] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody. In certain embodiments, the antibody can be a bi-specific antibody. In certain embodiments, the antibody can be part of a fusion protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to 0X40. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to VEGF. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to tissue necrosis factor alpha. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to CD3. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antibody to CD19.
[0367] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an immunomodulator. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Interleukin 12. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Interleukin 23. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for Interleukin 36 gamma. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a constitutively active variant of one or more stimulator of interferon genes (STING) proteins.
[0368] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an endonuclease. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an RNA-guided DNA endonuclease protein, such as Cas 9 protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a meganuclease protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a transcription activator-like effector nuclease protein. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for a zinc finger nuclease protein.
[0369] In embodiments, exemplary therapeutic uses result from the delivery of mRNA encoding a secreted protein. Accordingly, in embodiments, the compositions and methods of the invention provide for delivery of mRNA encoding a secreted protein. In some embodiments, the compositions and methods of the invention provide for delivery of mRNA encoding one or more secreted proteins listed in Table 1; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein Table 1. Secreted Proteins
Figure imgf000150_0001
Figure imgf000151_0001
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Figure imgf000156_0001
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Figure imgf000165_0001
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Figure imgf000189_0001
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Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
[0370] In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more additional exemplary proteins listed in Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 2 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 2 (or a homolog thereof) along with other components set out herein.
Table 2. Additional Exemplary Proteins
Figure imgf000198_0002
Figure imgf000199_0001
[0371] The Uniprot IDs set forth in Table 1 and Table 2 refer to the human versions the listed proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also generally available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more proteins chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of the secreted proteins listed in Table 1 and Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 and Table 2 along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in
Table 1 and Table 2 along with other components set out herein. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.
[0372] In embodiments, the compositions and methods of the invention provide for the delivery of m RNA encoding a lysosomal protein chosen from Table 3. In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more lysosomal and/or related proteins listed in Table 3; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 3 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 3 (or a homolog thereof) along with other components set out herein.
Table 3. Lysosomal and Related Proteins
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0002
[0373] Information regarding lysosomal proteins is available from Lubke et al., "Proteomics of the Lysosome," Biochim Biophys Acta. (2009) 1793: 625-635. In some embodiments, the protein listed in Table 3 and encoded by mRNA in the compositions and methods of the invention is a human protein. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest as described above.
[0374] In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a therapeutic protein (e.g., cytosolic, transmembrane or secreted) such as those listed in Table 4. In some embodiments, the compositions and methods of the invention provide for the delivery of an mRNA encoding a therapeutic protein useful in treating a disease or disorder (i.e., indication) listed in Table 4; thus, compositions of the invention may comprise an mRNA encoding a therapeutic protein listed or not listed in Table 4 (or a homolog thereof, as discussed below) along with other components set out herein for treating a disease or disorder (i.e., indication) listed in Table 4, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a such a protein (or a homolog thereof, as discussed below) along with other components set out herein for treatment of a disease or disorder listed in Table 4.
Table 4. Exemplary Indications and Related Proteins
Figure imgf000202_0001
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Figure imgf000210_0001
Figure imgf000211_0001
[0375] In some embodiments, the present invention is used to prevent, treat and/or cure a subject affected with a disease or disorder listed or associated with the proteins listed in Tables 1, 2, 3, or 4. In some embodiments, an 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).
[0376] While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.
EXAMPLES
Example 1: General Synthesis of Cationic Lipids
[0377] A cationic lipid (e.g., cationic lipids (la)-(21a), (lb)-(21b), and (22)-(462)) can be prepared according to Scheme 1 or Scheme 2 as described herein.
Example 2: Exemplary Synthesis of a Common Synthetic Intermediate [0378] Scheme 3 provides an exemplary synthesis of a common synthetic intermediate.
Scheme 3. Synthesis of ((2R,3R,4R,5S)-3,4,5-Tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2- yl)methyl 3-(dimethylamino)propanoate
Figure imgf000212_0001
A5 A7
Synthesis of (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol (A3):
Figure imgf000212_0002
[0379] To a solution of D-ribose A1 (100 g, 0.66 mole) in 300 mL pyridine was added trityl chloride A2 (180 g, 0.65 mole), and the resulting mixture was stirred overnight at room temperature. After evaporating the solvent, the residue was dissolved in dichloromethane (1.5 L), washed with Brine (500 mL x 3), and then dried over anhydrous NasSC . After filtration and concentration, the crude was dissolved in dichloromethane (1.5 L), and then the solution was slowly added into the mixture of hexanes (4.0 L) and dichloromethane (400 mL). The mixture was stirred for 1 hour while precipitate was formed. After filtration, the solid was washed with hexanes and dried under vacuum to give 104 g desired product A3 as white solid (Yield: 40%).
Synthesis of (((2S,3R,4R,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triyl)tris(oxy))tris(tert- butyldimethylsilane) (A4):
Figure imgf000213_0001
[0380] To a solution of (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol A3 (40 g,
0.102 mole) and imidazole (34.8 g, 0.51 mole) in 400 mL DMF, tert-butyldimethylsilyl chloride (76.8 g, 0.51 mol) was added at 0 °C, and the resulting mixture was stirred overnight at room
temperature. The solvent was evaporated under vacuum. The residue was dissolved in ethyl acetate (500 mL), washed with brine (300 mL x 3) and dried over anhydrous Na2S04. After the filtration, the solvent was evaporated under vacuum. The crude product was purified by column chromatography (ethyl acetate/hexanes 0-15%) to get 65 g desired product A4 as white solid (Yield: 86%).
Synthesis of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methanol (A5):
Figure imgf000213_0002
[0381] To a solution of (((2S,3R,4R,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))tris(tert-butyldimethylsilane) A4 (65 g, 88 mmol) in 600 mL dichloromethane, triethylsilane (60 mL, 0.352 mole) was added, and followed by the addition of trifluoroacetic acid (13.8 mL, 0.176 mole) at 0 °C. The resulting mixture was stirred for 4 hours at this temperature. The reaction mixture was quenched with saturated NaHCC>3 solution to pH 7. The organic phase was separated and washed with saturated NaHCOs solution (200 mL x 3) and brine (200 mL), and then dried over anhydrous MgSC . After the filtration, the solvent was evaporated under vacuum to afford 71 g of crude product A5, which was used for the next step without further purification.
Synthesis of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methyl 3- (dimethylamino)propanoate (A7):
Figure imgf000214_0001
[0382] To a solution of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2- yl)methanol A5 (71 g, 88 mmol) and 3-(dimethylamino)propanoic acid B1 (13.6 g, 88 mmol) in 500 mL dichloromethane, DMAP (10.7 g, 88 mmol) and EDCI (16.8 g, 88 mmol) were added, and the resulting mixture was stirred overnight at room temperature. The reaction mixture was washed with Brine (200 mL x 3) and dried over anhydrous Na2S04. After the filtration, the solvent was evaporated under vacuum. The crude product was purified by column chromatography (330 g silica gel, 0-100% ethyl acetate in hexanes gradient) to get 19 g desired product A7 as colorless oil (Yield: 36%).
Example 3: Exemplary Synthesis of Lipids Comprising Biodegradable Lipid Arms
Synthesis of (2S,3R,4R,5R)-5-(((3-(Dimethylamino)propanoyl)oxy)methyl) tetrahydrofuran-2,3,4-triyl tris(decanoate) (2b):
Figure imgf000214_0002
[0383] To a solution of ((2R,3R,4R,5S)-3,4,5-tris((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2- yl)methyl 3-(dimethylamino)propanoate A7 (0.8 g, 1.35 mmol) in 1 mL THF, HF-pyridine (0.72 mL, 40.5 mmol) was added, and the mixture was stirred at room temperature. After 3 hours, 2 mL dichloromethane, 4 mL pyridine and decanoyl chloride (2.3 g, 12.15 mmol) were added
subsequently, and the resulting mixture was stirred for four days at room temperature. The volatiles were evaporated under vacuum, and then the residue was dissolved in dichloromethane (50 mL), washed with Brine (50 mL x 3), and dried over anhydrous Na2S04. After filtration and concentration, the crude product was purified with column chromatography (80 g silica gel gold column, 0-10% methanol in dichloromethane gradient) to obtain 375 mg of the desired product (2b) as clear oil (Yield: 39%).
[0384] This procedure is suitable for the preparation of other lipids described herein, which can be obtained by this representative procedure in similar yields.
Example 4: Exemplary Synthesis of Lipids Comprising Biodegradable Lipid Arms
Scheme 4. Syntheses of Lipids with Biodegradable Lipid Groups
Figure imgf000215_0001
Cationic Lipid H
Figure imgf000215_0002
Synthesis of (2S,3R,4R,5R)-5-((Trityloxy)methyl)tetrahydrofuran-2,3,4-triyl trioleate (A9)
Figure imgf000216_0001
[0385] To a solution of (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol A3 (5.0 g, 1.67 mmol) in dichloromethane/pyridine (1/1 v/v, 100 mL) was added oleoyl chloride A8 (23 g, 9.4 mmol) at 0 °C, and the mixture was warmed to room temperature and stirred at room temperature for 5 days. After concentrated, the residue was dissolved in dichloromethane and washed with water and brine. After dried over sodium sulfate and concentration, the crude was purified by flash chromatography (SiC : 0-40% dichloromethane/hexanes) to get 4 g desired product A9.
Synthesis of (2S,3R,4R,5R)-5-(Hydroxymethyl)tetrahydrofuran-2,3,4-triyl trioleate (A10)
Figure imgf000216_0002
[0386] To a solution of (2S,3R,4R,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triyl trioleate A9 (0.8 g, 0.67 mmol) and triethylsilane (0.62 mL, 3.9 mmol) in 50 mL dichloromethane was added trifluoroacetic acid (0.12 mL, 1.06 mmol) at 0 °C. After stirred at this temperature for 90 min, the reaction mixture was quenched with saturated sodium bicarbonate, and then washed with saturated sodium bicarbonate and brine, dried over MgSC . After concentration, 0.81 g crude A10 was obtained, which was used for next step without purification. Synthesis of (2S,3R,4R,5R)-5-(((4-(Dimethylamino)butanoyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl trioleate (211)
Figure imgf000217_0001
[0387] A mixture of (2S,3R,4R,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4-triyl trioleate A9 (0.8 g, 0.67 mmol), EDCI (130 g, 0.67 mmol), 3-(dimethylamino)propanoic acid hydrochloride (108 mg, 0.7 mmol) and DMAP (82 mg, 0.67 mmol) in 50 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and then washed with water and brine. After concentration, the crude was purified by flash chromatography (SiC : 0-10%
MeOH/dichloromethane) to get 250 mg desired product (211) (Yield: 36%, two steps).
[0388] As shown in Scheme 4, different acylating agents can be used in place of 3-
(dimethylamino)propanoic acid hydrochloride to afford still other cationic lipids as described herein. Other lipids were prepared according the representative procedure and obtained in similar yields.
Example 5: Exemplary Synthesis of Lipids Comprising Aliphatic Lipid Arms - NaH Method
Scheme 5. Sodium Hydride Route to Lipids with Aliphatic Lipid Groups
Figure imgf000217_0002
A13 (11b) Synthesis of (2S,3R,4R,5R)-2,3,4-Tris(octyloxy)-5-((trityloxy)methyl)tetrahydrofuran (A12)
Figure imgf000218_0001
[0389] To a solution (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol A3 (3.0 g, 7.6 mmol) in 90 mL DMF was added sodium hydride (60% dispersion in oil, 2.7 g, 67.5 mmol) at 0 °C. After 30 min, 1-bromooctane All (8.1 mL, 47 mmol) and tetrabutylammonium iodide (600 mg, 1.6 mmol) were added, and the resulting solution was stirred at room temperature overnight. The reaction mixture was quenched by ice/water, and then extracted by ethyl acetate. The combined organic layers were washed with water and brine. After concentration, the crude was purified by flash column chromatography (SiC : 0-40% ethyl acetate /hexanes) to get 290 mg desired product A12 as colorless oil (Yield: 5%).
Synthesis of ((2R,3R,4R,5S)-3,4,5-Tris(octyloxy)tetrahydrofuran-2-yl)methanol (A13)
Figure imgf000218_0002
[0390] To a solution of (2S,3R,4R,5R)-2,3,4-tris(octyloxy)-5-((trityloxy)methyl)tetrahydrofuran A12 (290 mg, 0.4 mmol) and triethylsilane (0.25 mL, 1.6 mmol) in 2 mL dichloromethane was added trifluoroacetic acid (0.06 mL, 0.8 mmol) at 0 °C. After stirred at this temperature for 1 h, the reaction mixture was quenched with saturated NaHCOs, and then washed with saturated NaHCOs and brine, dried over MgSC . After concentration, 325 mg crude A13 was obtained, which was used for next step without purification. Synthesis of ((2R,3R,4R,5S)-3,4,5-Tris(octyloxy)tetrahydrofuran-2-yl)methyl 3- (dimethylamino)propanoate (lib)
Figure imgf000219_0001
[0391] A mixture of ((2R,3R,4R,5S)-3,4,5-tris(octyloxy)tetrahydrofuran-2-yl)methanol A13 (325 mg, 0.4 mmol), EDCI (76 mg, 0.4 mmol), 3-(dimethylamino)propanoic acid hydrochloride B1 (61 mg, 0.4 mmol) and DMAP (48 mg, 0.4 mmol) in 6 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane, and then washed with water and brine. After concentration, the crude was purified by flash column chromatography (SiC : 0-10% MeOH/ dichloromethane) to get 102 mg of the desired product as colorless oil (Yield: 43%, two steps).
Example 6: Exemplary Synthesis of Lipids Comprising Aliphatic Lipid Arms - Method
Figure imgf000219_0002
Scheme 6. Ti-mediated Synthesis of Lipids with Aliphatic Arms
Figure imgf000219_0003
Synthesis of (2R,3R,4S,5R)-2-(Decyloxy)-5-((trityloxy)methyl)tetrahydrofuran-3,4-diol (A15)
Figure imgf000220_0001
[0392] To a suspension of (2R,3R,4S,5R)-5-((trityloxy)methyl)tetrahydrofuran-2,3,4-triol A3 (3.0 g, 7.65 mmol), lithium bromide (0.66 g, 7.65 mmol) and mandelic acid (0.58 g, 3.82 mmol) in 15 mL 1- decanol A14 was added titanium(IV) tert-butoxide (0.26 g, 0.76 mmol). The mixture was stirred for 2 days at room temperature. After THF was removed, the residue was purified by column
chromatography (SiC , 0-10% MeOH/ dichloromethane) to get 1.9 g desired product A15 as colorless oil (yield: 44%).
Synthesis of (2R,3R,4R,5R)-2,3,4-Tris(decyloxy)-5-((trityloxy)methyl)tetrahydrofuran (A16)
Figure imgf000220_0002
[0393] To a solution (2R,3R,4S,5R)-2-(decyloxy)-5-((trityloxy)methyl)tetrahydrofuran-3,4-diol A15 (1.7 g, 3 mmol) in DMF/THF (100 mL/100 mL) was added sodium hydride (60% dispersion in oil, 0.72 g, 18.2 mmol) at 0 °C. After 5 min, 1-bromodecane (3.78 g, 15.2 mmol) was added, and the resulting solution was stirred at room temperature overnight. The reaction mixture was quenched by ice/water, and then extracted by ethyl acetate. The combined organic layers were washed with water and brine. After concentration, the crude was purified by flash column chromatography (SiC : 0-100% dichloromethane /hexanes) to get 1.5 g desired product A16 (Yield: 55%).
Synthesis of ((2R,3R,4R,5R)-3,4,5-Tris(decyloxy)tetrahydrofuran-2-yl)methanol (A17)
Figure imgf000220_0003
[0394] To a solution of (2R,3R,4R,5R)-2,3,4-tris(decyloxy)-5-((trityloxy)methyl)tetrahydrofuran A16 (1.5 g, 1.67 mmol) and triethylsilane (1.5 mL, 9.4 mmol) in 35 mL dichloromethane was added trifluoroacetic acid (0.3 mL, 3.89 mmol) at 0 °C. After stirred at this temperature for 90 min, the reaction mixture was quenched with saturated NaHCOs, and then washed with saturated NaHCOs and brine, dried over MgS04. After concentration, 1.7 g crude A17 was obtained, which was used for next step without purification.
Synthesis of ((2R,3R,4R,5R)-3,4,5-Tris(decyloxy)tetrahydrofuran-2-yl)methyl 3- (dimethylamino)propanoate (12b)
Figure imgf000221_0001
[0395] A mixture of ((2R,3R,4R,5R)-3,4,5-tris(decyloxy)tetrahydrofuran-2-yl)methanol 17 (1.7 g, 1.67 mmol), EDCI (320 mg, 1.67 mmol), 3-(dimethylamino)propanoic acid hydrochloride 6 (260 mg, 1.9 mmol) and DMAP (200 mg, 1.67 mmol) in 100 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane, and then washed with water and brine. After concentration, the crude was purified by flash column chromatography (SiC : 0-10% MeOH/ dichloromethane) to get 460 mg desired product 12b (Yield: 36%, two steps).
[0396] Other lipids were prepared according the representative procedures in similar yields.
Example 7: Exemplary Synthesis of Lipids Comprising Aliphatic Lipid Arms - Tetraacetylribose Method
Figure imgf000222_0001
Synthesis of (2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(dec-9-en-l-yloxy)tetrahydrofuran-3,4-diyl diacetate (A21)
Figure imgf000222_0002
[0397] To a solution of 1,2,3,5-tetra-o-acetyl-beta-l-ribofuranose A19 (6.36 g, 20 mmol) and dec-9-en- l-ol A20 (4.3 mL, 24 mmol) in 100 mL dichloromethane at 0 °C was added BF -OEt (3.2 mL, 26 mmol), and the reaction mixture was kept at this temperature for 3 h. The reaction mixture was poured into ice cold saturated NaHCOs and extracted by dichloromethane. The combined organic layers were dried over sodium sulfate. After concentration, the crude was purified by flash column chromatography (SiC : ethyl acetate/hexane 0-60%) to get a mixture of desired product A21 and dec-9-en-l-ol A20 (3.12 g) as yellowish oil, which was used for the next step without further purification.
Synthesis of (2R,3R,4S,5R)-2-(Dec-9-en-l-yloxy)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (A22)
Figure imgf000223_0001
[0398] The mixture of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(dec-9-en-l-yloxy)tetrahydrofuran-3,4-diyl diacetate A21 and dec-9-en-l-ol A20 (3.12 g) was dissolved in THF/MeOH (40 mL/40 mL), and a solution of lithium hydroxide hydrate (3.16 g, 75 mmol) in 5 mL water was added. The resulting mixture was stirred at room temperature overnight. After concentrated to dryness, the residue was dissolved in dichloromethane and washed with brine. The organic layer was dried over sodium sulfate. After concentrated, the crude was purified by flash chromatography (SiC :
methanol/dichloromethane 0-20%) to get 2.08 g pure product A22 as white solid (Yield: 36% in two steps).
Synthesis of (2R,3R,4S,5R)-2-(Dec-9-en-l-yloxy)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran- 3,4-diol (A23)
Figure imgf000223_0002
[0399] To a solution of (2R,3R,4S,5R)-2-(dec-9-en-l-yloxy)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol A22 (1.63 g, 5.55 mmol) and imidazole (755 mg, 11.1 mmol) in DMF (60 mL) was added
triisopropylsilyl chloride (1.3 mL, 6.1 mmol) and stirred at room temperature overnight. The reaction mixture was concentrated to dryness. The residue was partitioned with water and ether. After separation, the organic layer was dried and concentrated. The crude was purified by flash chromatography (SiC : methanol/dichloromethane 0-10%) to get 1.76 g desired product A23 as colorless oil (Yield: 71%). Synthesis of Triisopropyl(((2R,3R,4R,5R)-3,4,5-tris(dec-9-en-l-yloxy)tetrahydrofuran-2- yl)methoxy)silane (A25)
Figure imgf000224_0001
[0400] To a solution of (2R,3R,4S,5R)-2-(dec-9-en-l-yloxy)-5-
(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3,4-diol A23 (1.76 g, 3.96 mmol) in DMF/THF (40 mL/40 mL) was added sodium hydride (0.95 g, 23.7 mmol) at 0 °C. After 10 min, 10-bromodec-l-ene A24 (4.33 g, 19.8 mmol) was added, the resulting solution was stirred at room temperature overnight. The reaction mixture was quenched by ice/water, and then extracted by ether. The combined organic layers were washed with water and brine. After concentration, the crude was purified by flash chromatography (SiC : ethyl acetate/hexane 0-5%) to get 2.45 g product A25 as colorless oil (Yield: 85%).
Synthesis of 9,9,,9"-(((2R,3R,4R,5R)-5-(((Triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))trinonanal (A26)
Figure imgf000224_0002
[0401] To a solution of triisopropyl(((2R,3R,4R,5R)-3,4,5-tris((9-(benzyloxy)nonyl)oxy)tetrahydrofuran- 2-yl)methoxy)silane A25 (2.20 g, 3.05 mmol) and 2,6-Lutidine (4.3 mL, 36.6 mmol) in 80 mL dioxane, a solution of Osmium oxide in water (4 wt%, 0.84 mL, 0.137 mmol) was added, and followed by a solution of sodium periodate in water (7.83 g in 10 mL). After the reaction mixture was stirred at room temperature for 4 h, saturated sodium sulfite was added. The resulting mixture was stirred for 10 min and extracted with dichloromethane. The organic layer was separated and washed with 2 N HCI. After dried over sodium sulfate, the solvent was removed under vacuum to get 2.30 g desired product A26 as yellow semi-solid, which was used for the next step without purification. Synthesis of 9,9,,9"-(((2R,3R,4R,5R)-5-(((Triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))trinonanoic acid (A27)
Figure imgf000225_0001
[0402] To a solution of 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))trinonanal A26 (1.0 g, 1.3 mmol) in tBuOH/water (15 mL/1 mL), sodium chlorite (0.54 g, 5.9 mmol) and sodium dihydrogen phosphate (0.95 g, 7.9 mmol) were added, and the resulting mixture was stirred at room temperature for 30 min. TLC showed clean reaction. The reaction mixture was diluted with saturated sodium dihydrogen phosphate and extracted with ethyl acetate, and the combined organic layers were washed with brine. After concentration, 1.13 g desired product A27 was obtained as colorless oil, which was used for the next step without purification.
Synthesis of Tris(3-pentyloctyl) 9,9,,9"-(((2R,3R,4R,5R)-5-
(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tris(oxy))trinonanoate (A29)
Figure imgf000225_0002
[0403] A mixture of 9,9',9"-(((2R,3R,4R,5R)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))trinonanoic acid A27 (1.13 g, 1.46 mmol), 3-pentyloctan-l-ol A28 (3.5 g, 17.5 mmol), EDCI (3.35 g, 17.5 mmol) and DMAP (215 mg, 1.75 mmol) in 100 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with 1 N HCI and extracted with dichloromethane, and the combined organic layers were washed with brine. After concentration, the crude was purified by flash chromatography (SiC : ethyl acetate/hexane 0-10%) to get 0.64 g product A29 as colorless oil (Yield: 33%). Synthesis of Tris(3-pentyloctyl) 9,9,,9"-(((2R,3R,4R,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))trinonanoate (A30)
Figure imgf000226_0001
[0404] To a solution of tris(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-
(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tris(oxy))trinonanoate A29 (0.64 g, 0.48 mmol) in 8 mL THF, was added HF-Pyridine (70%, 1 mL), and the resulting solution was stirred for 3 h. The reaction mixture was diluted with dichloromethane, saturated sodium bicarbonate was added to adjust to pH7. The organic layer was separated and dried over sodium sulfate. After concentration, the crude was purified by flash chromatography (SiC : ethyl acetate/hexane 0-15%) to get 0.51 g pure product A30 as colorless oil (Yield: 91%).
Synthesis of Tris(3-pentyloctyl) 9,9,,9"-(((2R,3R,4R,5R)-5-(((3-
(dimethylamino)propanoyl)oxy)methyl)tetrahydrofuran-2,3,4-triyl)tris(oxy))trinonanoate (21b)
Figure imgf000226_0002
[0405] A mixture of tris(3-pentyloctyl) 9,9',9"-(((2R,3R,4R,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4- triyl)tris(oxy))trinonanoate A30 (0.51 g, 0.437 mmol), 3-(dimethylamino)propanoic acid
hydrochloride B1 (67 mg g, 0.437 mmol), EDCI (84 mg, 0.437 mmol) and DMAP (53 mg, 0.437 mmol) in 30 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with dichloromethane, and the combined organic layers were washed with brine. After concentration, the crude was purified by flash chromatography (SiC : methanol/dichloromethane 0-5%) to get 322 mg desired product 21b as clear oil (Yield: 58%).
[0406] Compound 19b and 20b were prepared according the same procedures with similar yields.
Figure imgf000227_0001
Example 8: Lipid Nanoparticle Formulation Using Ribose Cationic Lipids
[0407] Cationic lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include methods described in
International Publication No. WO 2018/089801, which is hereby incorporated by reference in its entirety.
[0408] One exemplary process for lipid nanoparticle formulation is Process A of WO 2018/089801 (see, e.g., Example 1 and Figure 1 of WO 2018/089801). Process A ("A") relates to a conventional method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles. In an exemplary process, an ethanol lipid solution and an aqueous buffered solution of mRNA were prepared separately. A solution of mixture of lipids (cationic lipid, helper lipids, zwitterionic lipids, PEG lipids etc.) was prepared by dissolving lipids in ethanol. The mRNA solution was prepared by dissolving the mRNA in citrate buffer, resulting in mRNA at a
concentration of 0.0833mg/ml in citrate buffer with a pH of 4.5. The mixtures were then both heated to 65 °C prior to mixing. Then, these two solutions were mixed using a pump system. In some instances, the two solutions were mixed using a gear pump system. In certain embodiments, the two solutions were mixing using a T junction (or "Y" junction). The mixture was then purified by diafiltration with a TFF process. The resultant formulation concentrated and stored at 2-8 °C until further use.
[0409] A second exemplary process for lipid nanoparticle formulation is Process B of WO 2018/089801 (see, e.g., Example 2 and Figure 2 of WO 2018/089801). Process B ("B") refers to a process of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA. A range of different conditions, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations, may be employed in Process B. In an exemplary process, lipids dissolved in ethanol and citrate buffer were mixed using a pump system. The instantaneous mixing of the two streams resulted in the formation of empty lipid nanoparticles, which was a self- assembly process. The resultant formulation mixture was empty lipid nanoparticles in citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process wherein buffer exchange occurred. The resulting suspension of pre-formed empty lipid nanoparticles was then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution post-mixing resulted in a higher percentage of lipid nanoparticles containing mRNA and a higher total yield of m RNA.
[0410] Lipid nanoparticle formulations of Table 5 were prepared by using either Process A or Process B as described in WO 2018/089801. All of the lipid nanoparticle formulations comprised hEPO mRNA and the different lipids in following mol % ratios: Cationic Lipid: DMG-PEG2000; Cholesterol: DOPE = 40:5:25:30.
Table 5. Exemplary lipid nanoparticle formulations
Figure imgf000228_0001
Figure imgf000229_0001
Example 9: In Vivo Expression of hEPO in CD1 Mice using Ribose Lipids
[0411] Intravenous (IV) administration of lipid nanoparticle formulations comprising a ribose cationic lipid and mRNA encoding hEPO (Table 5) was undertaken in order to study mRNA delivery and resultant hEPO expression. Male CD1 mice at 6-8 weeks old were given a single intravenous injection of the LNP formulations at a dosage level of 1 mg/kg. Blood samples were collected by tail snip at 6 and 24 hours post-dose. hEPO protein expression levels measured in the sera samples by ELISA (FIG. 1). These studies show that ribose cationic lipids described herein are highly effective at delivery mRNA in vivo, resulting in high expression of the protein or polypeptide encoded by the delivered mRNA.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A cationic lipid having a structure according to Formula (G),
Figure imgf000230_0001
wherein
Rx is independently -H, -L^R1, or -L5A-L5B-B';
each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or - C(0)NRl-;
each L4A and L5A is independently -C(O)-, -C(0)0-, or -C(0)NRL-;
each L4B and L5B is independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene; each B and B' is NR4R5, a 5- to 10-membered nitrogen-containing heterocyclyl, or a 5- to 10-membered nitrogen-containing heteroaryl;
each R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl; and
each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
2. The cationic lipid of claim 1, having a structure according to Formula (I),
Figure imgf000230_0002
3. The cationic lipid of claim 1, having a structure according to Formula (la),
Figure imgf000230_0003
4. The cationic lipid of any one of claims 1-3, wherein each L3 is independently -C(O)- or a covalent bond.
5. The cationic lipid of claim 1, having a structure according to Formula (lb) or (Ic),
Figure imgf000231_0001
6. The cationic lipid of claim 5, having a structure according to Formula (Id) or (le),
Figure imgf000231_0002
7. The cationic lipid of any one of claims 1-6, wherein L4A is -C(O)-.
8. The cationic lipid of any one of claims 1-7, wherein L4B is unsubstituted C1-C6 alkylene;
C2-C6 alkenylene; or C2-C6 alkynylene.
9. The cationic lipid of claim 8, wherein L4B is -CH2-, -CH2CH2-, or -CH2CH2CH2-.
10. The cationic lipid of any one of claims 1-9, wherein
B is NR4R5, and
each R4 and R5 is independently hydrogen or unsubstituted C1-C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
11. The cationic lipid of claim 10, wherein B is N(CHB)2.
12. A cationic lipid having a structure according to Formula (II),
Figure imgf000231_0003
wherein
RA is hydrogen or -lAR1;
RB is hydrogen or -L2-R2;
Rc is hydrogen or -L3-R3;
each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(0)0-, -C(0)S-, or - C(0)NRl-; L4 is independently C1-C10 alkylene;
each R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl;
each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; or R4 and R5 combine to form a 5- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl;
each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.
13. The cationic lipid of claim 12, wherein RA is -l^-R1; RB is -L2-R2; and Rc -L3-R3.
14. The cationic lipid of claim 12 or 13, having a structure according to Formula (lla),
Figure imgf000232_0001
15. The cationic lipid of claim 12 or 13, having a structure according to Formula (lib) or (lie),
Figure imgf000232_0002
(lib).
16. The cationic lipid of claim 15, having a structure according to Formula (lid) or (lie),
Figure imgf000232_0003
(lib).
17. The cationic lipid of any one of claims 12-16, wherein L4 is unsubstituted C1-C6 alkylene.
18. The cationic lipid of claim 17, wherein L4 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.
19. The cationic lipid of any one of claims 12-18, wherein each R4 and R5 is independently hydrogen or unsubstituted C1-C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
20. The cationic lipid of claim 19, wherein each R4 and R5 is CH3.
21. The cationic lipid of any one of claims 1-20, wherein each R1, R2, and R3 is independently C6-C22 alkyl, C6-C22 alkenyl, or C6-C22 alkynyl.
22. The cationic lipid of claim 21, wherein each R1, R2, and R3 is unsubstituted linear C6-C22 alkyl, unsubstituted linear C6-C22 alkenyl, unsubstituted linear C6-C22 alkynyl, unsubstituted branched C6-C22 alkyl, unsubstituted branched C6-C22 alkenyl, or unsubstituted branched C6-C22 alkynyl.
23. The cationic lipid of claim 21, wherein each R1, R2, and R3 is unsubstituted C6-C22 alkyl.
24. The cationic lipid of claim 21, wherein each R1, R2, and R3 is (CH2)7CH3, (CI-hJgCHs, (CH2)nCH3,
(CH2)I3CH3, (CH2)I5CH3, or (CH2)i7CH3.
25. The cationic lipid of claim 21, wherein each R1, R2, and R3 is (CH2)6CH3, (CH2)8CH3, (CH2)IOCH3, (CH2)I2CH3, (CH2)I4CH3, or (CH2)I6CH3.
26. The cationic lipid of any one of claims 1-20, wherein each R1, R2, and R3 is C6-C12 alkyl substituted by -0(C0)R6 or -C(0)0R6, wherein R6 is unsubstituted C6-C14 alkyl.
27. The cationic lipid of claim 26, wherein R6 is unsubstituted linear C6-C14 alkyl.
28. The cationic lipid of claim 26, wherein R6 is unsubstituted branched C6-C14 alkyl.
29. The cationic lipid of any one of claims 26-28, wherein each R1, R2, and R3 is
(CH2)80C(0)(CH2)6CH3, (CH2)90C(0)(CH2)6CH3, (CH2)7C(0)0(CH2)2CH(C5HII)2, or
(CH2)8C(0)0(CH2)2CH(C5HII)2.
30. The cationic lipid of any one of claims 1-20, wherein each R1, R2, and R3 is unsubstituted C6-C22 alkenyl.
31. The cationic lipid of claim 30, wherein said C6-C22 alkenyl is a monoalkenyl, a dienyl, or a trienyl.
32. The cationic lipid of claim 30 or 31, wherein each R1, R2, and R3 is
Figure imgf000233_0001
33. The cationic lipid of claim 30 or 31, wherein each R1, R2, and R3 is
Figure imgf000234_0001
34. A cationic lipid selected from the group consisting of
Figure imgf000234_0002
Figure imgf000234_0003
Figure imgf000235_0002
35. A cationic lipid selected from the group consisting of
Figure imgf000235_0001
Figure imgf000235_0003
Figure imgf000236_0003
36. The cationic lipid of claim 1, having a structure according to one of the following formulas,
Figure imgf000236_0001
37. The cationic lipid of claim 36, having a structure according to one of the following formulas,
Figure imgf000236_0002
The cationic lipid of claim 36 or 37, selected from the group consisting of:
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0002
39. A cationic lipid selected from the group consisting of:
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0005
40. The cationic lipid of claim 1, having a structure according to the following formula,
Figure imgf000242_0001
41. The cationic lipid of claim 40, having a structure according to the following formula,
Figure imgf000242_0002
42. The cationic lipid of claim 1, having a structure according to the following formula,
Figure imgf000242_0003
43. The cationic lipid of claim 42, having a structure according to the following formula,
Figure imgf000242_0004
44. The cationic lipid of claim 42 or claim 43, wherein L5B is -CH2-, -CH2CH2-, or -CH2CH2CH2
45. The cationic lipid of any one of claims 42-44, wherein
B' is NR4R5, and
each R4 and R5 is independently hydrogen or unsubstituted C1-C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
46. The cationic lipid of claim 45, wherein B' is N(CHB)2.
47. The cationic lipid of any one of claims 42-46, wherein L4B is -CH2-, -CH2CH2-, or -CH2CH2CH2-.
48. The cationic lipid of any one of claims 42-47, wherein
B' is NR4R5, and
each R4 and R5 is independently hydrogen or unsubstituted C1-C6 alkyl, or R4 and R5 combine to form a 5- to 6-membered heterocyclyl comprising one or two ring nitrogens.
49. The cationic lipid of claim 48, wherein B' is N(CHB)2.
50. The cationic lipid of any one of claims 42-49, wherein each of L2 and L3 is a covalent bond.
51. The cationic lipid of any one of claims 42-49, wherein each of L2 and L3 is -C(O)-.
52. The cationic lipid of any one of claims 42-51, wherein each of R2 and R3 is independently C6-C22 alkyl, C6-C22 alkenyl, or C6-C22 alkynyl.
53. The cationic lipid of claim 48, wherein each of R2 and R3 is unsubstituted linear C6-C22 alkyl, unsubstituted linear C6-C22 alkenyl, unsubstituted linear C6-C22 alkynyl, unsubstituted branched C6-C22 alkyl, unsubstituted branched C6-C22 alkenyl, or unsubstituted branched C6-C22 alkynyl.
54. The cationic lipid of claim 52, wherein each of R2 and R3 is unsubstituted C6-C22 alkyl.
55. The cationic lipid of claim 52, wherein each of R2 and R3 is (CH2)7CH3, (CI-hJgCHa, (CH2)nCH3,
(CH2)IBCH3, (CH2)I5CH3, or (CH2)i7CH3.
56. The cationic lipid of claim 52, wherein each of R2 and R3 is (CH2)6CH3, (CH2)8CH3, (CH2)IOCH3, (CH2)I2CH3, (CH2)I4CH3, or (CH2)I6CH3.
57. The cationic lipid of any claim 52, wherein each of R2 and R3 is C6-C12 alkyl substituted by
-0(C0)R6 or -C(0)0R6, wherein R6 is unsubstituted C6-C14 alkyl.
58. The cationic lipid of claim 57, wherein R6 is unsubstituted linear C6-C14 alkyl.
59. The cationic lipid of claim 57, wherein R6 is unsubstituted branched C6-C14 alkyl.
60. The cationic lipid of any one of claims 57-59, wherein each of R2 and R3 is (CH2)80C(0)(CH2)6CH3,
(CH2)90C(0)(CH2)6CH3, (CH2)7C(0)0(CH2)2CH(C5HII)2, or (CH2)8C(0)0(CH2)2CH(C5HII)2.
61. The cationic lipid of claim 52, wherein each of R2 and R3 is unsubstituted C6-C22 alkenyl.
62. The cationic lipid of claim 61, wherein said C6-C22 alkenyl is a monoalkenyl, a dienyl, or a trienyl.
63. The cationic lipid of claim 61 or 62, wherein each of R2 and R3 is
Figure imgf000244_0001
64. The cationic lipid of claim 61 or 62, wherein each of R2 and R3 is
Figure imgf000244_0002
65. A cationic lipid selected from the group consisting of cationic lipids (22)-(462).
66. A compound having the following structure,
Figure imgf000244_0003
salt thereof.
67. A compound having the following structure,
Figure imgf000244_0004
salt thereof.
68. A composition comprising an mRNA encoding a protein, 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 of any one of claims 1-65.
69. The composition of claim 68, comprising an mRNA encoding for an antigen from an infectious agent.
70. The composition of claim 68, comprising an mRNA encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
71. The composition of claim 68, comprising an mRNA encoding for ornithine transcarbamylase (OTC) protein.
72. A composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid according to of any one of claims 1-65.
73. The composition of claim 72, further comprising one 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.
74. The composition of claim 72 or 73, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide.
75. The composition of any one of claims 72-74, comprising an mRNA encoding for an antigen from an infectious agent.
76. The composition of any one of claims 72-74, wherein the mRNA encodes a peptide or
polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell.
77. The composition of claim 76, wherein the mRNA encodes cystic fibrosis transmembrane
conductance regulator (CFTR) protein.
78. The composition of any one of claims 72-74, wherein the mRNA encodes a peptide or
polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell.
79. The composition of claim 78, wherein the mRNA encodes ornithine transcarbamylase (OTC) protein.
80. The composition of any one of claims 68-79, formulated for intravenous (IV) administration.
81. The composition of any one of claims 68-79, formulated for intramuscular (IM) administration.
82. The composition of any one of claims 68-79, formulated for administration by inhalation.
83. The composition of claim 82, wherein the composition is formulated for nebulization.
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