WO2026006524A2 - Lipides cationiques de bis-ester et d'amide - Google Patents

Lipides cationiques de bis-ester et d'amide

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Publication number
WO2026006524A2
WO2026006524A2 PCT/US2025/035368 US2025035368W WO2026006524A2 WO 2026006524 A2 WO2026006524 A2 WO 2026006524A2 US 2025035368 W US2025035368 W US 2025035368W WO 2026006524 A2 WO2026006524 A2 WO 2026006524A2
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Prior art keywords
optionally substituted
alkyl
alkenyl
covalent bond
atom marked
Prior art date
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Pending
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PCT/US2025/035368
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English (en)
Inventor
Ramesh Dasari
Hongfeng Deng
Saswata KARMAKAR
Lingyao LI
Apiwat WANGWEERAWONG
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Sanofi Pasteur Inc
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Sanofi Pasteur Inc
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Publication of WO2026006524A2 publication Critical patent/WO2026006524A2/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C229/36—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
    • C07C275/16—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/20—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C275/24—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D275/00—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings
    • C07D275/02—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings not condensed with other rings

Definitions

  • the cationic lipid component of liposomes encapsulating nucleic acids plays an important role in facilitating effective encapsulation of the nucleic acid during the loading of liposomes.
  • cationic lipids may play an important role in the efficient release of the 15 nucleic acid cargo from the liposome into the cytoplasm of a target cell.
  • Various cationic lipids suitable for in vivo use have been discovered. However, there remains a need to identify lipids that can be synthesized efficiently and cheaply without the formation of potentially toxic by-products.
  • cationic lipids that contain a cyclic ring structure as a central core (lipidoids 20 like cKK-E12 - structure shown below): SUMMARY OF THE INVENTION 25 [0005]
  • the present invention provides, among other things, a novel class of cationic lipid compounds for in vivo delivery of therapeutic agents, such as nucleic acids. It is contemplated that these compounds are capable of highly effective in vivo delivery of the therapeutic agents and vaccines while maintaining a favorable safety profile. Lipid nanoparticles comprising the cationic lipids of the present invention also exhibit enhanced 2 thermostability, which is beneficial for the development of the corresponding therapeutic agents and vaccines.
  • the cationic lipids of the present invention comprise cleavable groups (e.g., esters) that are contemplated to improve biodegradability and thus contribute to their favorable 5 safety profile.
  • cationic lipids having a structure according to Formula (I): Formula (I), 10 or a pharmaceutically acceptable salt thereof, wherein: R 1 is selected from hydrogen, or optionally substituted (C 1 -C 6 )alkyl; R 2 is selected from -OH, -NH 2 , optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C 1 -C 3 )alkylene- 15 optionally substituted aryl, or optionally substituted (C 1 -C 3 )alkylene-optionally substituted heteroaryl; b and c are integers that are each independently selected from 0, 1, 2, 3, or 4; a and d are integers that are each
  • cationic lipids having a structure according to Formula (II): 5 Formula (II) or a pharmaceutically acceptable salt thereof, wherein: e and f are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; 10 each Y 2 is independently selected from hydrogen or -OH; R 6 is selected from hydrogen or optionally substituted (C1-C6)alkyl; R 7 is selected from optionally substituted (C1-C6)alkylene-R 8 or R 8 ; R 8 is selected from optionally substituted aryl, optionally substituted heteroaryl.
  • cationic lipids having a structure according to Formula (III): 4 Formula (III) or a pharmaceutically acceptable salt thereof, wherein: 5 h and i are integers that are each independently selected from 1, 2, 3 or 4; g and j are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; k is an integer selected from 1, 2, 3, 4, 5, or 6; m is an integer selected from 0, 1, 2, 3, 4, 5, or 6; each Y 3 is independently selected from hydrogen or -OH; 10 X 2 is selected from H, NH2 and F; R 10 is selected from an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C1-C25)alkyl or optionally substituted (C2-C25)alkenyl; R 9A , R 9B , R 9C , and R 9D are each independently selected from optionally substituted (C 1 -C 25 )alkyl, optionally substituted (C 2 -C 25 )
  • cationic lipids having a structure according to Formula (IV): 5 Formula (IV) wherein: p and q are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; 5 each Y 4 is independently selected from hydrogen or -OH; R 11 is selected from hydrogen or optionally substituted (C1-C6)alkyl; X 3 is selected from a covalent bond, -O-, or -N(R 13 )-; R 13 is selected from hydrogen or optionally substituted (C1-C6)alkyl; R 12A , R 12B , R 12C , and R 12D are each independently selected from optionally substituted 10 (C1-C25)alkyl, optionally substituted (C2-C25)alkenyl, or -W 1 -X 1 ; each W 1 is independently selected from a covalent bond, optionally substituted (C1- C10)alkylene or optionally substituted (C2-C10)alkenylene; and each X
  • cationic lipids having a structure according to Formula (VI): Formula (VI) or a pharmaceutically acceptable salt thereof, 20 wherein: t and u are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; each Y 6 is independently selected from hydrogen or -OH; R 19 is selected from optionally substituted (C 1 -C 3 )alkylene-optionally substituted thiazolyl and optionally substituted (C 1 -C 3 )alkylene-substituted phenyl, wherein the phenyl is 25 substituted with one or more halogen atoms (e.g.
  • cationic lipids that are pharmaceutically acceptable salts of Formula (II).
  • cationic lipids that are pharmaceutically 10 acceptable salts of Formula (III).
  • cationic lipids that are pharmaceutically acceptable salts of Formula (IV).
  • cationic lipids that are pharmaceutically acceptable salts of Formula (V). 15
  • cationic lipids that are pharmaceutically acceptable salts of Formula (VI).
  • compositions comprising the cationic lipid of the present invention or a pharmaceutically acceptable salt thereof, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
  • the composition is a lipid nanoparticle, optionally a liposome.
  • the compositions comprising the cationic lipids of the present invention may be used in therapy.
  • the inventors of the present invention have surprisingly found that cationic lipids made from commercially available acyclic linkers have high levels of peptide or protein 25 expression when delivering mRNA encoding said peptide or protein while having a reduced size and complexity.
  • amino acid refers to any compound and/or substance that can be incorporated into a polypeptide chain. 5
  • 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 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 10 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 15 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, 20 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.
  • Aromatic Amino Acid or Residue refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring.
  • Aromatic amino acids or residues include L-amino acids, D-amino acids or racemates.
  • Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), L-His (H) and L-Trp (W).
  • 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. 5 In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a bovine, a primate, and/or a pig).
  • a mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a bovine, 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. 10 [00026] 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.
  • 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 15 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).
  • Biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism.
  • 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 25 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”).
  • circulation system e.g., serum
  • expression refers to 30 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 10 assembled protein (e.g., enzyme).
  • expression and “production,” and grammatical equivalents thereof, are used interchangeably.
  • 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.
  • 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.
  • Helper lipid The term “helper lipid” as used herein refers to any neutral or zwitterionic lipid material including cholesterol. Without wishing to be held to a particular 10 theory, helper lipids may add stability, rigidity, and/or fluidity within lipid bilayers/nanoparticles.
  • 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 15 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 20 artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • 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 25 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 30 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 35 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 11 other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
  • liposome refers to any lamellar, multilamellar, or solid nanoparticle vesicle.
  • a liposome as used herein can be 5 formed by mixing one or more lipids or by mixing one or more lipids and polymer(s).
  • a liposome suitable for the present invention contains a cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and/or optionally PEG- modified lipid(s).
  • messenger RNA mRNA
  • mRNA messenger RNA 10
  • mRNA messenger RNA 10
  • mRNA refers to a polynucleotide that encodes at least one polypeptide.
  • mRNA as used herein encompasses both modified and unmodified RNA.
  • 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, 15 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.
  • an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside 20 analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 propynyl-cytidine, C5 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, O
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into a 30 polynucleotide chain.
  • a 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), 12 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 (lncRNA), micro-RNA (miRNA) multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), 5 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
  • lncRNA micro-RNA
  • MCNA multimeric coding nucle
  • 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.
  • DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid 10 DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., P1, 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 15 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 (lncRNA), micro-RNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA 20 (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), transfer
  • a nucleic acid is a mRNA encoding a protein such as an enzyme.
  • patient refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, 25 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.
  • compositions of the compounds of this invention include those derived from suitable inorganic and 13 organic acids and bases.
  • Examples of pharmaceutically acceptable, non-toxic 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 5 malonic acid, or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or 5 malonic 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, 10 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 15 bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 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, 20 and aryl sulfonate.
  • Systemic distribution or delivery As used herein, the terms “systemic distribution” or “systemic delivery,” or grammatical equivalents thereof, refer to a delivery or 25 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.
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a 30 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.
  • 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.
  • therapeutically effective amount As used herein, the term “therapeutically 10 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 15 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 20 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 As used herein, the term aliphatic refers to C1-C50 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 30 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 alkyls e.g., linear or 30 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
  • 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 35 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 15 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’’, -CO2H, -CO2R’’, -CN, -OH, -OR’’, - OCOR’, -OCO2R’’, -NH2, -NHR’’, -N(R’’)2, -SR’’ or-SO2R’’, wherein each instance of R’’ 5 independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • R’ 5 independently is C1-C20 aliphatic (e.g., C1-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). In embodiments, R’’ independently is unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not include any heteroatoms.
  • Alkyl As used herein, the term “alkyl” 10 means acyclic linear and branched hydrocarbon groups, e.g. “C 1 -C 30 alkyl” refers to alkyl groups having 1-30 carbons.
  • An alkyl group may be linear or branched.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, pentyl, isopentyl tert-pentylhexyl, isohexyl, etc.
  • the term “lower alkyl” means an alkyl group straight chain or branched alkyl having 1 to 6 carbon atoms. Other 15 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’’, -NH 2 , -NHR’’, -N(R’’) 2 , -SR’’ or-SO 2 R’’,20 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 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). In 25 embodiments, an alkyl group is substituted with a–OH group and may also be referred to herein as a “hydroxyalkyl” group, where the prefix denotes the –OH group and “alkyl” is as described herein.
  • alkyl also refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms (“C1-C50 alkyl”). In some 30 embodiments, an alkyl group has 1 to 40 carbon atoms (“C1-C40 alkyl”). In some embodiments, an alkyl group has 1 to 30 carbon atoms (“C1-C30 alkyl”). In some embodiments, an alkyl group has 1 to 20 carbon atoms (“C1-C20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-C10 alkyl”).
  • an alkyl group has 1 to 9 carbon atoms (“C1-C9 alkyl”). In some embodiments, 35 an alkyl group has 1 to 8 carbon atoms (“C1-C8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-C7 alkyl”). In some embodiments, an alkyl group has 1 to 6 16 carbon atoms (“C1-C6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-C5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-C4 alkyl”).
  • an alkyl group has 1 to 3 carbon atoms (“C1-C3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-C2 alkyl”). In some embodiments, 5 an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”).
  • C1-C6 alkyl groups include, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2- butanyl (C5), tertiary amyl (C5), and n-hexyl (C6).
  • alkyl groups include 10 n-heptyl (C 7 ), n-octyl (C 8 ) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted C 1 -C 50 alkyl. In certain embodiments, the alkyl group is a substituted C 1 -C 50 alkyl.
  • alkylene represents a saturated divalent straight or branched chain hydrocarbon group and is exemplified by methylene, 20 ethylene, isopropylene and the like.
  • 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 herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple 25 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 30 substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, -CO2H, -CO2R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO2R’’, -NH2, -NHR’’, - N(R’’)2, -SR’’ or -SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • R’ independently is C1-C20 aliphatic (e.g., C1-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 35 alkyl). In embodiments, R’’ independently is unsubstituted C1-C3 alkyl. In certain embodiments, an alkylene, alkenylene, or alkynylene is unsubstituted. In certain 17 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. “C2-C30 alkenyl” refers to an alkenyl group having 2-30 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 10 substituent groups as described herein.
  • 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’’, -CO 2 H, -CO 2 R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO 2 R’’, -NH 2 , -NHR’’, -N(R’’) 2 , -SR’’ or- SO 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 alkyl
  • R’’ independently is an 15 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).
  • an alkenyl group is substituted with a–OH group and may also be referred to herein as a “hydroxyalkenyl” group, where the 20 prefix denotes the –OH group and “alkenyl” is as described herein.
  • alkenyl also refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon- carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-C50 alkenyl”).
  • an alkenyl group has 2 to 40 carbon atoms (“C 2 -C 40 alkenyl”).
  • an alkenyl group has 2 to 30 carbon atoms (“C2-C30 alkenyl”). In some embodiments, an alkenyl group has 2 to 20 carbon atoms (“C2-C20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-C10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-C9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some 30 embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-C7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms (“C2-C6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-C4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-C3 alkenyl”). In some 35 embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon- carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1- 18 butenyl).
  • Examples of C2-C4 alkenyl groups include, without limitation, ethenyl (C2), 1- propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like.
  • Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl 5 include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.
  • each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents.
  • the alkenyl group is an unsubstituted C2-C50 alkenyl.
  • the alkenyl group is a substituted C2-C50 alkenyl.
  • 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 30 alkynyl”, refers to an alkynyl group having 2-30 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 15 comprises one carbon-carbon triple bond.
  • An alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein.
  • 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’’, -CO 2 H, -CO 2 R’’, -CN, -OH, -OR’’, -OCOR’’, - OCO 2 R’’, -NH 2 , -NHR’’, -N(R’’) 2 , -SR’’ or-SO 2 R’’, wherein each instance of R’’ independently 20 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, 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 25 herein).
  • alkynyl also refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon- carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-C50 alkynyl”).
  • An alkynyl group that has one or more 30 triple bonds, and one or more double bonds is also referred to as an “ene-yne”.
  • an alkynyl group has 2 to 40 carbon atoms (“C2-C40 alkynyl”).
  • an alkynyl group has 2 to 30 carbon atoms (“C2-C30 alkynyl”). In some embodiments, an alkynyl group has 2 to 20 carbon atoms (“C2-C20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 alkynyl”). In some 35 embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-C9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some 19 embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-C7 alkynyl”).
  • an alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-C4 alkynyl”). In some 5 embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-- carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • C2-C4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2- propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like.
  • C2-C6 alkenyl groups 10 include the aforementioned C 2 -C 4 alkynyl groups as well as pentynyl (C 5 ), hexynyl (C 6 ), and the like. Additional examples of alkynyl include heptynyl (C 7 ), octynyl (C 8 ), and the like.
  • each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents.
  • the alkynyl group is an unsubstituted C 2 -C 50 alkynyl. 15 In certain embodiments, the alkynyl group is a substituted C 2 -C 50 alkynyl.
  • Aryl refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein said ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic and wherein each ring in the 20 system contains 4 to 7 ring members.
  • an aryl group has 6 ring carbon atoms (“C 6 aryl,” e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C10 aryl,” e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl,” e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or 25 heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • aryls include phenyl, naphthyl, and anthracene.
  • aryl also refers to a radical of a monocyclic or polycyclic 30 shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”).
  • an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl).
  • “Aryl” also 35 includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl 20 ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is an 5 unsubstituted C6-C14 aryl. In certain embodiments, the aryl group is a substituted C6-C14 aryl.
  • Arylene The term “arylene” as used herein refers to an aryl group that is divalent (that is, having two points of attachment to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted phenylene or substituted phenylene).
  • Carbocyclyl As used herein, “carbocyclyl” or “carbocyclic” refers to a radical10 of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C 3 -C 10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3 -C 8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3 -C 7 carbocyclyl”).
  • a carbocyclyl group has 3 to 6 ring carbon atoms 15 (“C 3 -C 6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C 4 -C 6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C 5 -C 6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C 5 -C 10 carbocyclyl”).
  • Exemplary C 3 -C 6 carbocyclyl groups include, without limitation, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C4), 20 cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like.
  • Exemplary C 3 -C 8 carbocyclyl groups include, without limitation, the aforementioned C3-C6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ), and the like.
  • Exemplary C 3 -C 10 25 carbocyclyl groups include, without limitation, the aforementioned C3-C8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged 30 or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Carbocyclyl also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of 35 carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently 21 unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents.
  • the carbocyclyl group is an unsubstituted C3-C10 carbocyclyl.
  • the carbocyclyl group is a substituted C3-C10 carbocyclyl.
  • “carbocyclyl” or “carbocyclic” is referred to as a “cycloalkyl”, i.e., a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”).
  • a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-C8 cycloalkyl”).
  • a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-C6, cycloalkyl”).
  • a cycloalkyl group has 4 to 10 6 ring carbon atoms (“C 4 -C 6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C 5 -C 6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C 5 -C 10 cycloalkyl”). Examples of C 5 -C 6 cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 5 ).
  • C 3 -C 6 cycloalkyl groups include the aforementioned C 5 -C 6 cycloalkyl groups as well as cyclopropyl (C 3 ) and cyclobutyl (C 4 ).
  • C 3 -C 8 cycloalkyl groups include the aforementioned C 3 -C 6 cycloalkyl groups as well as cycloheptyl (C 7 ) and cyclooctyl (C 8 ).
  • each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
  • the cycloalkyl group is an unsubstituted C 3 -C 10 cycloalkyl. In certain 20 embodiments, the cycloalkyl group is a substituted C 3 -C 10 cycloalkyl.
  • Halogen As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine.
  • Heteroalkyl The term “heteroalkyl” is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 14 carbon atoms in addition to 1, 2, 3 or 4 25 heteroatoms independently selected from the group consisting of N, O, S, and P.
  • Heteroalkyls include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides.
  • a heteroalkyl group may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. 30 Examples of heteroalkyls include polyethers, such as methoxymethyl and ethoxyethyl.
  • Heteroalkylene The term “heteroalkylene,” as used herein, represents a divalent form of a heteroalkyl group as described herein.
  • Heteroaryl The term “heteroaryl,” as used herein, is fully unsaturated heteroatom-containing ring wherein at least one ring atom is a heteroatom such as, but not 35 limited to, nitrogen and oxygen.
  • heteroaryl also refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, 5 wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-14 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined 10 above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, 15 and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2- indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heteroaryl”).
  • a heteroaryl group is a 5-8 membered aromatic ring system having 25 ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heteroaryl”).
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic 30 ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heteroaryl”).
  • the 5-6 membered heteroaryl 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 heteroaryl has 1 or 2 ring heteroatoms selected from 35 oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, 23 silicon, and phosphorus.
  • each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.
  • the heteroaryl group is an unsubstituted 5-14 membered heteroaryl.
  • the heteroaryl group is 5 a substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 310 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5- membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl.
  • Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered 15 heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, 20 benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl 25 and phenazinyl.
  • 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 35 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 24 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 5 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.
  • the heterocyclyl group is a substituted 3-14 10 membered heterocyclyl.
  • 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”).
  • 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 20 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 25 oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
  • 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 limitation, azirdinyl, oxiranyl, thiorenyl.
  • Exemplary 4-membered heterocyclyl groups 30 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 35 dithiolanyl.
  • Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered 25 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 5 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, 10 tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyr
  • Heterocycloalkyl is a non- 20 aromatic ring wherein at least one atom is a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon.
  • the heterocycloalkyl group can be substituted or unsubstituted.
  • alkyl, alkenyl, alkynyl, acyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein are, in certain embodiments, 25 optionally substituted.
  • Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or ’unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, 30 “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group.
  • substituted or unsubstituted
  • substituted means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the 26 substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound.
  • the present invention contemplates any and all such combinations in order to arrive at a 5 stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • halo or halogen refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
  • a “counterion” is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality.
  • Exemplary counterions include halide ions (e.g., F-, Cl-, Br-, I-), NO 3 -, ClO 4 -, OH-, H 2 PO 4 -, HSO 4 -, 20 sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).
  • halide ions e.g., F-, Cl-, Br-
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms.
  • the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, 5 incorporated herein by reference.
  • Nitrogen protecting groups such as carbamate groups include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl 20 carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl)-1-methylethyl
  • Nitrogen protecting groups such as sulfonamide groups include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,- trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),20 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,
  • nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N’-p-toluenesulfonylaminoacyl derivative, N’ - phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine30 derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4- tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1- substituted 3,5
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group).
  • Oxygen protecting 20 groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, 25 (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- 30 methoxycyclohexyl, 4- methoxytetrahydropyranyl
  • the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group).
  • Sulfur protecting groups are 33 well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • Exemplary sulfur protecting groups include, but are not limited to, alkyl, 5 benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p- hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2- quinolinylmethyl, 2-picolyl N-oxido, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzosuberyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, t-butyl, 1-adamantyl, 10 methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl,
  • Liposomal-based vehicles are considered an attractive carrier for therapeutic 25 agents and remain subject to continued development efforts. While liposomal-based vehicles that comprise certain lipid components have shown promising results with regard 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 30 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.
  • lipid compounds that demonstrate improved pharmacokinetic properties, and which are capable of delivering 35 macromolecules, such as nucleic acids, to a wide variety of cell types and tissues with enhanced efficiency.
  • novel lipid 34 compounds that are characterized as having improved safety profiles and are capable of efficiently delivering encapsulated nucleic acids and polynucleotides to targeted cells, tissues and organs.
  • Described herein is a novel class of cationic lipid compounds for improved in 5 vivo delivery of therapeutic agents, such as nucleic acids.
  • a cationic lipid described herein may be used, optionally with other lipids, to formulate a lipid-based nanoparticle (e.g., liposome) for encapsulating therapeutic agents, such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic use, such as disease treatment and prevention (vaccine) purposes.
  • therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA)
  • nucleic acids e.g., DNA, siRNA, mRNA, microRNA
  • compounds of the invention as described herein can provide one or more desired characteristics or properties. That is, in certain embodiments, compounds of the invention as described herein can be characterized as having one or more properties that afford such compounds advantages relative to other similarly classified lipids.
  • compounds disclosed herein can allow for the control and tailoring of the 15 properties of liposomal compositions (e.g., lipid nanoparticles) of which they are a component.
  • compounds 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., 20 polynucleotides) intracellularly.
  • the compounds disclosed herein have advantageous pharmacokinetic properties, biodistribution, and efficiency.
  • the present application demonstrates that the cationic lipids of the present invention are synthetically tractable from readily available starting materials.
  • the cationic lipids of the present invention have cleavable groups 25 such as ester groups and amide groups. These cleavable groups (e.g. esters and amides) are contemplated to improve biodegradability and thus contribute to the lipids’ favorable safety profiles. [000100] Provided herein are compounds which are cationic lipids.
  • the cationic lipids of the present invention include compounds having a structure according to 30 Formula (I): 35 Formula (I), or a pharmaceutically acceptable salt thereof, wherein: 5 R 1 is selected from hydrogen, or optionally substituted (C 1 -C 6 )alkyl; R 2 is selected from -OH, -NH 2 , optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C 1 -C 3 )alkylene- optionally substituted aryl, or optionally substituted (C 1 -C 3 )alkylene-optionally substituted heteroaryl; 10 b and c are integers that are each independently selected from 0, 1, 2, 3, or 4; a and d are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; each Y 1 is independently selected from hydrogen or -OH; each R 4 is independently selected from hydrogen or optionally substituted (C 1 - C 6 )alkyl.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (IA1): Formula (IA1) or a pharmaceutically acceptable salt thereof. 15 [000103]
  • the cationic lipids of the present invention include compounds having a structure according to Formula (IB): 37 or a pharmaceutically acceptable salt thereof.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (IB1): Formula (IB1) or a pharmaceutically acceptable salt thereof.
  • R 1 is hydrogen.
  • R 1 is optionally substituted (C1-C6)alkyl.
  • R 1 is methyl.
  • R 2 is -OH.
  • R 2 is -NH2.
  • R 2 is optionally substituted (C 1 -C 6 )alkyl.
  • R 2 is methyl.
  • R 2 is ethyl.
  • R 2 is isopropyl.
  • R 2 is optionally substituted aryl.
  • R 2 is optionally substituted phenyl. In embodiments, R 2 is phenyl. In embodiments, R 2 is optionally substituted (C 1 -C 3 )alkylene-optionally substituted aryl. In embodiments, R 2 is optionally substituted (C 1 -C 3 )alkylene-optionally substituted phenyl. In 38 embodiments, R 2 is optionally substituted benzyl. In embodiments, R 2 is benzyl. In embodiments, R 2 is optionally substituted heteroaryl. In embodiments, R 2 is optionally substituted (C1-C3)alkylene-optionally substituted heteroaryl. [000107] In embodiments, b is 0. In embodiments, b is 1.
  • b is 2. In 5 embodiments, b is 3. In embodiments, b is 4. [000108] In embodiments, a is 3 or 4. In embodiments, a is 1. In embodiments, a is 2. In embodiments, a is 3. In embodiments, a is 4. In embodiments, a is 5. In embodiments, a is 6. [000109] In embodiments, c is 0. In embodiments, c is 1. In embodiments, c is 2. In 10 embodiments, c is 3. In embodiments, c is 4. [000110] In embodiments, d is 3 or 4. In embodiments, d is 1. In embodiments, d is 2. In embodiments, d is 3. In embodiments, d is 4. In embodiments, d is 5.
  • d is 6. [000111] In embodiments, b and c are each 1. In embodiments, a and d are each 3 or 15 4. In embodiments, b and c are each 1 and a and d are each 3. In embodiments, b and c are each 1 and a and d are each 4. [000112] In embodiments, at least one Y 1 is -OH. In embodiments, at least one Y 1 is hydrogen. In embodiments, Y 1 is -OH. In embodiments, Y 1 is hydrogen. [000113] In embodiments, each R 4 is hydrogen. In embodiments, each R 4 is optionally 20 substituted (C 1 -C 6 )alkyl. In embodiments, each R 4 is methyl.
  • R 3A is optionally substituted (C 1 -C 25 )alkyl. In embodiments, R 3A is optionally substituted (C5-C25)alkyl. In embodiments, R 3A is optionally substituted (C5-C20)alkyl. In embodiments, R 3A is optionally substituted (C5-C15)alkyl. In embodiments, R 3A is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 3A is optionally substituted 25 (C8-C10)alkyl. [000115] In embodiments, R 3A is optionally substituted (C2-C25)alkenyl.
  • R 3A is optionally substituted (C5-C25)alkenyl. In embodiments, R 3A is optionally substituted (C5-C20)alkenyl. In embodiments, R 3A is optionally substituted (C10-C20)alkenyl. In embodiments, R 3A is optionally substituted (C15-C20)alkenyl. 30 [000116] In embodiments, R 3A is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally substituted (C1-C8)alkylene.
  • W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- 35 C8)alkenylene. In embodiments, W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 3B is optionally substituted (C1-C25)alkyl. In embodiments, R 3B is optionally substituted (C5-C25)alkyl.
  • R 3B is optionally substituted 40 (C5-C20)alkyl. In embodiments, R 3B is optionally substituted (C5-C15)alkyl. In embodiments, R 3B is optionally substituted (C6-C12)alkyl. In embodiments, R 3B is optionally substituted (C8-C10)alkyl. [000118] In embodiments, R 3B is optionally substituted (C2-C25)alkenyl. In 5 embodiments, R 3B is optionally substituted (C5-C25)alkenyl. In embodiments, R 3B is optionally substituted (C5-C20)alkenyl. In embodiments, R 3B is optionally substituted (C10-C20)alkenyl.
  • R 3B is optionally substituted (C15-C20)alkenyl. [000119] In embodiments, R 3B is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally 10 substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene. In embodiments, W 1 is optionally substituted (C 2 -C 10 )alkenylene.
  • R 3C is optionally substituted (C 1 -C 25 )alkyl. In embodiments, R 3C is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 3C is optionally substituted 15 (C 5 -C 20 )alkyl. In embodiments, R 3C is optionally substituted (C 5 -C 15 )alkyl.
  • R 3C is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 3C is optionally substituted (C 8 -C 10 )alkyl. [000121] In embodiments, R 3C is optionally substituted (C 2 -C 25 )alkenyl. In embodiments, R 3C is optionally substituted (C 5 -C 25 )alkenyl. In embodiments, R 3C is optionally 20 substituted (C 5 -C 20 )alkenyl. In embodiments, R 3C is optionally substituted (C 10 -C 20 )alkenyl. In embodiments, R 3C is optionally substituted (C 15 -C 20 )alkenyl.
  • R 3C is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C 1 -C 8 )alkylene.
  • W 1 is optionally substituted (C 1 -C 6 )alkylene.
  • W 1 is optionally substituted (C1-C5)alkylene.
  • W 1 is optionally substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene.
  • W 1 is optionally substituted (C2-C5)alkenylene.
  • R 3D is optionally substituted (C1-C25)alkyl. In embodiments, R 3D is optionally substituted (C5-C25)alkyl.
  • R 3D is optionally substituted (C5-C20)alkyl. In embodiments, R 3D is optionally substituted (C5-C15)alkyl. In embodiments, 30 R 3D is optionally substituted (C6-C12)alkyl. In embodiments, R 3D is optionally substituted (C8-C10)alkyl. [000124] In embodiments, R 3D is optionally substituted (C2-C25)alkenyl. In embodiments, R 3D is optionally substituted (C5-C25)alkenyl. In embodiments, R 3D is optionally substituted (C5-C20)alkenyl. In embodiments, R 3D is optionally substituted (C10-C20)alkenyl.
  • R 3D is optionally substituted (C15-C20)alkenyl. 43 [000125] In embodiments, R 3D is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally 5 substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • each R 3A , R 3B , R 3C and R 3D is independently selected from: 10 15 45 5 (xvii) , for example wherein each R 3A , R 3B , R 3C and R 3D is selected from options (ii) or (iii). [000127] In embodiments, R 3A , R 3B , R 3C and R 3D are the same. In embodiments, R 3A and R 3C are the same and R 3B and R 3D are the same. In embodiments, R 3A and R 3C are the same and R 3B and R 3D are different.
  • the cationic lipids of the present invention also include compounds having a structure according to Formula (II): Formula (II) or a pharmaceutically acceptable salt thereof, 15 wherein: e and f are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; each Y 2 is independently selected from hydrogen or -OH; 46 R 6 is selected from hydrogen or optionally substituted (C1-C6)alkyl; R 7 is selected from optionally substituted (C1-C6)alkylene-R 8 or R 8 ; R 8 is selected from optionally substituted aryl, optionally substituted heteroaryl.
  • Formula (II) Formula (II) or a pharmaceutically acceptable salt thereof, 15 wherein: e and f are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; each Y 2 is independently selected from hydrogen or -OH; 46 R 6 is selected from hydrogen or optionally substituted (C1-C6)alkyl; R 7 is selected from optionally substituted (C1-C6)alkylene-R 8 or
  • the cationic lipids of the present invention include compounds having a structure according to Formula (IIB): Formula (IIB) 10 or a pharmaceutically acceptable salt thereof.
  • e is 3 or 4.
  • e is 1.
  • e is 2.
  • e is 3.
  • e is 4.
  • e is 5.
  • e is 6.
  • f is 3 or 4.
  • f is 1.
  • f is 2.
  • f is 3.
  • f is 4.
  • f is 5.
  • f is 6.
  • e and f are each 3 or 4.
  • each R 6 is hydrogen. In embodiments, each R 6 is optionally substituted (C1-C6)alkyl. In embodiments, each R 6 is methyl. [000137] In embodiments, R 7 is optionally substituted (C1-C6)-alkylene-R 8 . In embodiments, R 7 is R 8 . In embodiments, R 8 is optionally substituted aryl.
  • R 7 is –(CH2)-optionally substituted aryl. In embodiments, R 7 is -(CH2)2-optionally substituted aryl. In embodiments, R 7 is -(CH2)3-optionally substituted aryl. In embodiments, R 7 is –(CH2)4-optionally substituted aryl. In embodiments, R 7 is – (CH2)-optionally substituted heteroaryl. In embodiments, R 7 is -(CH2)2-optionally substituted 10 heteroaryl. [000139] In embodiments, R 7 is –(CH 2 )-optionally substituted phenyl.
  • R 7 is -(CH 2 ) 2 -optionally substituted phenyl. In embodiments, R 7 is -(CH 2 ) 3 -optionally substituted phenyl. In embodiments, R 7 is –(CH 2 ) 4 -optionally substituted phenyl. In embodiments, R 7 is –(CH 2 )-optionally substituted imidazolyl. In embodiments, R 7 is -(CH 2 ) 2 - 15 optionally substituted imidazolyl. In embodiments, . embodiments, . embodiments, R 7 is . In embodiments, R 7 is . In embodiments, R 7 is . In embodiments, R 7 is . In embodiments, R 7 is
  • R 7 is –(CH 2 )-optionally substituted phenyl. In embodiments, R 7 is -(CH 2 ) 2 -optionally substituted phenyl. In embodiments, R 7 is -(CH 2 ) 3 -optionally 5 substituted phenyl. In embodiments, R 7 is –(CH 2 ) 4 -optionally substituted phenyl. In embodiments, R 7 is –(CH 2 )-optionally substituted imidazolyl. In embodiments, R 7 is -(CH 2 ) 2 - optionally substituted imidazolyl. In embodiments, R 7 is –(CH 2 )-optionally substituted indolyl.
  • R 7 is -(CH 2 ) 2 -optionally substituted indolyl. In embodiments, R 7 is -(CH 2 ) 3 - optionally substituted indolyl. In embodiments, . embodiments, 10 , . , 50 5 . , . [000141]
  • R 5A is optionally substituted (C 1 -C 25 )alkyl. In embodiments, R 5A is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 5A is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 5A is optionally substituted (C 5 -C 15 )alkyl.
  • R 5A is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 5A is optionally substituted 10 (C 8 -C 10 )alkyl. 51 [000142] In embodiments, R 5A is optionally substituted (C2-C25)alkenyl. In embodiments, R 5A is optionally substituted (C5-C25)alkenyl. In embodiments, R 5A is optionally substituted (C5-C20)alkenyl. In embodiments, R 5A is optionally substituted (C10-C20)alkenyl. In embodiments, R 5A is optionally substituted (C15-C20)alkenyl.
  • R 5A is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C1-C8)alkylene.
  • W 1 is optionally substituted (C1-C6)alkylene.
  • W 1 is optionally substituted (C1-C5)alkylene.
  • W 1 is optionally substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- 10 C 8 )alkenylene.
  • W 1 is optionally substituted (C 2 -C 6 )alkenylene.
  • W 1 is optionally substituted (C 2 -C 5 )alkenylene.
  • R 5B is optionally substituted (C 1 -C 25 )alkyl.
  • R 5B is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 5B is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 5B is optionally substituted (C 5 -C 15 )alkyl. In embodiments, R 5B is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 5B is optionally substituted (C 8 -C 10 )alkyl. 15 [000145] In embodiments, R 5B is optionally substituted (C 2 -C 25 )alkenyl. In embodiments, R 5B is optionally substituted (C 5 -C 25 )alkenyl.
  • R 5B is optionally substituted (C 5 -C 20 )alkenyl. In embodiments, R 5B is optionally substituted (C 10 -C 20 )alkenyl. In embodiments, R 5B is optionally substituted (C 15 -C 20 )alkenyl. [000146] In embodiments, R 5B is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In 20 embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene.
  • R 5C is optionally substituted (C 1 -C 25 )alkyl. In embodiments, 25 R 5C is optionally substituted (C5-C25)alkyl. In embodiments, R 5C is optionally substituted (C5-C20)alkyl. In embodiments, R 5C is optionally substituted (C5-C15)alkyl. In embodiments, R 5C is optionally substituted (C6-C12)alkyl.
  • R 5C is optionally substituted (C8-C10)alkyl. [000148] In embodiments, R 5C is optionally substituted (C2-C25)alkenyl. In 30 embodiments, R 5C is optionally substituted (C5-C25)alkenyl. In embodiments, R 5C is optionally substituted (C5-C20)alkenyl. In embodiments, R 5C is optionally substituted (C10-C20)alkenyl. In embodiments, R 5C is optionally substituted (C15-C20)alkenyl. [000149] In embodiments, R 5C is -W 1 -X 1 . In embodiments, W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally 35 substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally 54 substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- C8)alkenylene. In embodiments, W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 5D is optionally substituted (C1-C25)alkyl. In embodiments, R 5D is optionally substituted (C5-C25)alkyl.
  • R 5D is optionally substituted 5 (C5-C20)alkyl. In embodiments, R 5D is optionally substituted (C5-C15)alkyl. In embodiments, R 5D is optionally substituted (C6-C12)alkyl. In embodiments, R 5D is optionally substituted (C8-C10)alkyl. [000151] In embodiments, R 5D is optionally substituted (C2-C25)alkenyl. In embodiments, R 5D is optionally substituted (C5-C25)alkenyl. In embodiments, R 5D is optionally 10 substituted (C 5 -C 20 )alkenyl.
  • R 5D is optionally substituted (C 10 -C 20 )alkenyl. In embodiments, R 5D is optionally substituted (C 15 -C 20 )alkenyl. [000152] In embodiments, R 5D is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In 15 embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene.
  • each R 5A , R 5B , R 5C and R 5D is independently selected from: 20 25 57 5 , for example wherein each 10 R 5A , R 5B , R 5C and R 5D is option (iii).
  • R 5A , R 5B , R 5C and R 5D are the same.
  • the cationic lipids of the present invention also include compounds having a 15 structure according to Formula (III): 58 Formula (III) or a pharmaceutically acceptable salt thereof, wherein: 5 h and i are integers that are each independently selected from 1, 2, 3 or 4; g and j are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; k is an integer selected from 1, 2, 3, 4, 5, or 6; m is an integer selected from 0, 1, 2, 3, 4, 5, or 6; each Y 3 is independently selected from hydrogen or -OH; 10 X 2 is selected from H, NH2 and F; R 10 is selected from an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C1-C25)alkyl or optionally substituted (C2-C25)alkenyl; R
  • the cationic lipids of the present invention include compounds having a structure according to Formula (IIIA): 59 Formula (IIIA) or a pharmaceutically acceptable salt thereof.
  • the cationic lipids of the 10 present invention include compounds having a structure according to Formula (IIIA1):
  • the cationic lipids of the present invention include 5 compounds having a structure according to Formula (IIIA1i): Formula (IIIA1i) or a pharmaceutically acceptable salt thereof.
  • h is 1 or 2.
  • h is 1.
  • h is 2.
  • h is 3.
  • h is 4.
  • g is 2, 3 or 4.
  • g is 3 or 4.
  • g is 3.
  • g is 4.
  • g is 1.
  • g is 2.
  • g is 5.
  • g is 6.
  • i is 1 or 2. In embodiments, i is 1. In embodiments, i is 2. In embodiments, i is 3. In embodiments, i is 4. [000162] In embodiments, j is 2, 3 or 4. In embodiments, j is 3 or 4. In embodiments, j is 3. In embodiments, j is 4. In embodiments, j is 1. In embodiments, j is 2. In embodiments, j 10 is 5. In embodiments, j is 6. [000163] In embodiments, k is 1, 2 or 3. In embodiments, k is 2. In embodiments, k is 1. In embodiments, k is 3. In embodiments, k is 4. In embodiments, k is 5. In embodiments, k is 6.
  • m is 0, 1 or 2. In embodiments, m is 1. In embodiments, m is 15 0. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. [000165] In embodiments, g and j are each 3. In embodiments, h and i are each 1. In embodiments, g and j are each 3 and h and i are each 1. In embodiments, h and i are each 1. In embodiments, g and j are each 3 and h and i are each 1, and k is 2. In embodiments, h 20 and i are each 1.
  • g and j are each 3 and h and i are each 1, k is 2 and , is 1.
  • at least one Y 3 is -OH.
  • at least one Y 3 is hydrogen.
  • Y 3 is -OH.
  • Y 3 is hydrogen.
  • X 2 is hydrogen.
  • X 2 is NH 2 .
  • 25 X 2 is F.
  • X 2 is selected from NH2 and F.
  • R 10 is optionally substituted aryl.
  • R 10 is optionally substituted heteroaryl.
  • R 10 is selected from an optionally substituted aryl or optionally substituted heteroaryl. In embodiments, R 10 is optionally substituted alkyl. In embodiments, R 10 is optionally substituted alkenyl. 30 [000169] In embodiments, R 10 is optionally substituted phenyl. In embodiments, R 10 is optionally substituted imidazolyl. In embodiments, R 10 is optionally substituted indolyl. In embodiments, . embodiments, . 62 [000170] In embodiments, R 9A is optionally substituted (C1-C25)alkyl. In embodiments, R 9A is optionally substituted (C5-C25)alkyl.
  • R 9A is optionally substituted 5 (C5-C20)alkyl. In embodiments, R 9A is optionally substituted (C5-C15)alkyl. In embodiments, R 9A is optionally substituted (C6-C12)alkyl. [000171] In embodiments, R 9A is optionally substituted (C2-C25)alkenyl. In embodiments, R 9A is optionally substituted (C5-C25)alkenyl. In embodiments, R 9A is optionally substituted (C5-C20)alkenyl. In embodiments, R 9A is optionally substituted (C10-C20)alkenyl.
  • R 9A is optionally substituted (C15-C20)alkenyl.
  • R 9A is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C1-C8)alkylene.
  • W 1 is optionally substituted (C1-C6)alkylene.
  • W 1 is optionally substituted (C1-C5)alkylene.
  • W 1 is optionally15 substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 9B is optionally substituted (C 1 -C 25 )alkyl.
  • R 9B is optionally substituted (C5-C25)alkyl. In embodiments, R 9B is optionally substituted (C5-C20)alkyl. In embodiments, R 9B is optionally substituted (C5-C15)alkyl. In embodiments, R 9B is optionally substituted (C6-C12)alkyl. [000174] In embodiments, R 9B is optionally substituted (C2-C25)alkenyl. In embodiments, R 9B is optionally substituted (C5-C25)alkenyl. In embodiments, R 9B is optionally 30 substituted (C5-C20)alkenyl.
  • R 9B is optionally substituted (C10-C20)alkenyl. In embodiments, R 9B is optionally substituted (C15-C20)alkenyl. [000175] In embodiments, R 9B is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In 35 embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally substituted (C2-C10)alkenylene.
  • R 9C is optionally substituted (C1-C25)alkyl. In embodiments, R 9C is optionally substituted (C5-C25)alkyl. In embodiments, R 9C is optionally substituted (C5-C20)alkyl. In embodiments, R 9C is optionally substituted (C5-C15)alkyl. In embodiments, R 9C is optionally substituted (C6-C12)alkyl.
  • R 9C is optionally substituted (C2-C25)alkenyl. In embodiments, R 9C is optionally substituted (C5-C25)alkenyl. In embodiments, R 9C is optionally substituted (C5-C20)alkenyl. In embodiments, R 9C is optionally substituted (C10-C20)alkenyl. In embodiments, R 9C is optionally substituted (C15-C20)alkenyl. [000178] In embodiments, R 9C is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In 10 embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene.
  • W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene. In embodiments, W 1 is optionally substituted (C 2 -C 10 )alkenylene. In embodiments, W 1 is optionally substituted (C 2 - C 8 )alkenylene. In embodiments, W 1 is optionally substituted (C 2 -C 6 )alkenylene. In 15 embodiments, W 1 is optionally substituted (C 2 -C 5 )alkenylene.
  • R 9D is optionally substituted (C 1 -C 25 )alkyl.
  • R 9D is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 9D is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 9D is optionally substituted (C 5 -C 15 )alkyl. In embodiments, R 9D is optionally substituted (C 6 -C 12 )alkyl. [000180] In embodiments, R 9D is optionally substituted (C 2 -C 25 )alkenyl. In embodiments, R 9D is optionally substituted (C 5 -C 25 )alkenyl. In embodiments, R 9D is optionally 20 substituted (C 5 -C 20 )alkenyl.
  • R 9D is optionally substituted (C 10 -C 20 )alkenyl. In embodiments, R 9D is optionally substituted (C 15 -C 20 )alkenyl. [000181] In embodiments, R 9D is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In 25 embodiments, W 1 is optionally substituted (C1-C5)alkylene.
  • each R 9A , R 9B , R 9C and R 9D is independently selected from: 30 , 68 5 10 , for example wherein each R 9A , R 9B , R 9C and R 9D is option (iii). 69 [000183] In embodiments, R 9A , R 9B , R 9C and R 9D are the same.
  • R 9A and R 9B are the same and R 9C and R 9D are the same. In embodiments, R 9A and R 9C are the same and R 9B and R 9D are the same. In embodiments, R 9A and R 9C are the same and R 9B and R 9D are different.
  • the cationic lipids of the present invention also include compounds having a structure according to Formula (IV): Formula (IV) wherein: 10 p and q are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; each Y 4 is independently selected from hydrogen or -OH; R 11 is selected from hydrogen or optionally substituted (C1-C6)alkyl; X 3 is selected from a covalent bond, -O-, or -N(R 13 )-; R 13 is selected from hydrogen or optionally substituted (C1-C6)alkyl; 15 R 12A , R 12B , R 12C , and R 12D are each independently selected from optionally substituted (C 1 -C 25 )alkyl, optionally substituted (C 2 -C 25 )alkenyl, or -W 1 -X 1 ; each W 1 is independently selected from a covalent bond, optionally substituted (C 1 - C 10 )alkylene or optionally substituted (C 2 -C
  • 25 the cationic lipids of the present invention include compounds having a structure according to Formula (IVA): 70 Formula (IVA) or a pharmaceutically acceptable salt thereof.
  • p is 3. In embodiments, p is 1. In embodiments, p is 2. In 5 embodiments, p is 4. In embodiments, p is 5. In embodiments, p is 6. [000187] In embodiments, q is 3. In embodiments, q is 1. In embodiments, q is 2. In embodiments, q is 4. In embodiments, q is 5. In embodiments, q is 6.
  • p and q are each 3. [000189] In embodiments, at least one Y 4 is -OH. In embodiments, at least one Y 4 is 10 hydrogen. In embodiments, Y 4 is -OH. In embodiments, Y 4 is hydrogen. [000190] In embodiments, R 11 is hydrogen. In embodiments, In embodiments, R 11 is optionally substituted (C1-C6)alkyl. In embodiments, R 11 is methyl. [000191] In embodiments, X 3 is a covalent bond. In embodiments, X 3 is -O-. In embodiments, X 3 is -N(R 13 )-. In embodiments, X 3 is -N(H)-.
  • R 13 is hydrogen. [000193] In embodiments, R 12A is optionally substituted (C 1 -C 25 )alkyl. In embodiments, R 12A is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 12A is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 12A is optionally substituted (C 5 -C 15 )alkyl. In embodiments, R 12A is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 12A is optionally substituted 20 (C 8 -C 10 )alkyl.
  • R 12A is optionally substituted (C 2 -C 25 )alkenyl. In embodiments, R 12A is optionally substituted (C 5 -C 25 )alkenyl. In embodiments, R 12A is optionally substituted (C 5 -C 20 )alkenyl. In embodiments, R 12A is optionally substituted (C 10 - C 20 )alkenyl. In embodiments, R 12A is optionally substituted (C 15 -C 20 )alkenyl. 25 [000195] In embodiments, R 12A is -W 1 -X 1 . In embodiments, W 1 is a covalent bond.
  • W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene. In embodiments, W 1 is optionally 71 substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- C8)alkenylene. In embodiments, W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 12B is optionally substituted (C1-C25)alkyl. In embodiments, R 12B is optionally substituted (C5-C25)alkyl.
  • R 12B is optionally substituted 5 (C5-C20)alkyl. In embodiments, R 12B is optionally substituted (C5-C15)alkyl. In embodiments, R 12B is optionally substituted (C6-C12)alkyl. In embodiments, R 12B is optionally substituted (C8-C10)alkyl. [000197] In embodiments, R 12B is optionally substituted (C2-C25)alkenyl. In embodiments, R 12B is optionally substituted (C5-C25)alkenyl. In embodiments, R 12B is 10 optionally substituted (C 5 -C 20 )alkenyl.
  • R 12B is optionally substituted (C 10 - C 20 )alkenyl. In embodiments, R 12B is optionally substituted (C 15 -C 20 )alkenyl. [000198] In embodiments, R 12B is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In 15 embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene.
  • R 12C is optionally substituted (C 1 -C 25 )alkyl. In embodiments, R 12C is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 12C is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 12C is optionally substituted (C 5 -C 15 )alkyl.
  • R 12C is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 12C is optionally substituted (C 8 -C 10 )alkyl. [000200] In embodiments, R 12C is optionally substituted (C2-C25)alkenyl. In embodiments, R 12C is optionally substituted (C5-C25)alkenyl. In embodiments, R 12C is optionally substituted (C 5 -C 20 )alkenyl. In embodiments, R 12C is optionally substituted (C 10 - 25 C20)alkenyl. In embodiments, R 12C is optionally substituted (C15-C20)alkenyl.
  • R 12C is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C1-C8)alkylene.
  • W 1 is optionally substituted (C1-C6)alkylene.
  • W 1 is optionally substituted (C1-C5)alkylene.
  • W 1 is optionally30 substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene.
  • W 1 is optionally substituted (C2-C5)alkenylene.
  • R 12D is optionally substituted (C1-C25)alkyl. In embodiments, R 12D is optionally substituted (C5-C25)alkyl.
  • R 12D is optionally substituted (C5-C20)alkyl. In embodiments, R 12D is optionally substituted (C5-C15)alkyl. In embodiments, R 12D is optionally substituted (C6-C12)alkyl. In embodiments, R 12D is optionally substituted 35 (C8-C10)alkyl. 75 [000203] In embodiments, R 12D is optionally substituted (C2-C25)alkenyl. In embodiments, R 12D is optionally substituted (C5-C25)alkenyl. In embodiments, R 12D is optionally substituted (C5-C20)alkenyl. In embodiments, R 12D is optionally substituted (C10- C20)alkenyl.
  • R 12D is optionally substituted (C15-C20)alkenyl. 5 [000204] In embodiments, R 12D is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- 10 C 8 )alkenylene.
  • each R 12A , R 12B , R 12C and R 12D is independently selected from: 15 , 20 (x) , 77 5 (xvii) , for example wherein each R 12A , R 12B , R 12C and R 12D is option (iii).
  • R 12A , R 12B , R 12C and R 12D are the same.
  • R 12A and R 12B are the same and R 12C and R 12D are the same.
  • R 12A and R 12C are the same and R 12B and R 12D are the same.
  • R 12A and R 12C are the same and R 12B and R 12D are different.
  • the cationic lipids of the present invention include 5 compounds having a structure according to Formula (VA) wherein R 15 is optionally substituted (C1-C3)alkylene-optionally substituted indolyl.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (VA) wherein R 15 is unsubstituted (C1)alkylene-unsubstituted indolyl.
  • the cationic lipids of the present invention include 10 compounds having a structure according to Formula (VA1): or a pharmaceutically acceptable salt thereof.
  • the cationic lipids of the present invention include 15 compounds having a structure according to Formula (VB): 80 Formula (VB) or a pharmaceutically acceptable salt thereof.
  • r is 3 or 4.
  • r is 3.
  • r is 4.
  • r is 1.
  • r is 2.
  • r is 5.
  • r is 6.
  • s is 3 or 4.
  • s is 3.
  • s is 4.
  • s is 1.
  • s is 2.
  • s is 5.
  • s is 6.
  • s is 6.
  • r and s are each independently 3 or 4. In embodiments, r 10 and s are each 3. In embodiments, r and s are each 4. [000219] In embodiments, Y 5 is hydrogen. [000220] In embodiments, R 15 is an optionally substituted (C1-C3)alkylene-optionally substituted indolyl. In embodiments, R 15 is an unsubstituted (C 1 )alkylene-unsubstituted indolyl. 15 [000221] In embodiments, R 14A is optionally substituted (C5-C25)alkyl. In embodiments, R 14A is optionally substituted (C 5 -C 20 )alkyl.
  • R 14A is optionally substituted (C 5 -C 15 )alkyl. In embodiments, R 14A is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 14A is optionally substituted (C 8 -C 10 )alkyl. [000222] In embodiments, R 14A is optionally substituted (C 5 -C 25 )alkenyl. In 20 embodiments, R 14A is optionally substituted (C 5 -C 20 )alkenyl. In embodiments, R 14A is optionally substituted (C 10 -C 20 )alkenyl. In embodiments, R 14A is optionally substituted (C 15 - C 20 )alkenyl.
  • R 14A is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C 1 -C 10 )alkylene.
  • W 1 is optionally 25 substituted (C 1 -C 8 )alkylene.
  • W 1 is optionally substituted (C 1 -C 6 )alkylene.
  • W 1 is optionally substituted (C 1 -C 5 )alkylene.
  • W 1 is optionally 81 substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 14B is optionally substituted (C5-C25)alkyl.
  • R 14B is optionally substituted (C5-C20)alkyl. In embodiments, R 14B is optionally substituted 5 (C5-C15)alkyl. In embodiments, R 14B is optionally substituted (C6-C12)alkyl. In embodiments, R 14B is optionally substituted (C8-C10)alkyl. [000225] In embodiments, R 14B is optionally substituted (C5-C25)alkenyl. In embodiments, R 14B is optionally substituted (C5-C20)alkenyl. In embodiments, R 14B is optionally substituted (C10-C20)alkenyl.
  • R 14B is optionally substituted (C15- 10 C 20 )alkenyl. [000226] In embodiments, R 14B is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 6 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene. In embodiments, W 1 is optionally15 substituted (C 2 -C 10 )alkenylene.
  • R 14C is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 14C is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 14C is optionally substituted (C 5 -C 15 )alkyl. In embodiments, R 14C is optionally substituted (C 6 -C 12 )alkyl.
  • R 14C is optionally substituted (C 8 -C 10 )alkyl. 20 [000228] In embodiments, R 14C is optionally substituted (C 5 -C 25 )alkenyl. In embodiments, R 14C is optionally substituted (C 5 -C 20 )alkenyl. In embodiments, R 14C is optionally substituted (C10-C20)alkenyl. In embodiments, R 14C is optionally substituted (C15- C20)alkenyl. [000229] In embodiments, R 14C is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In 25 embodiments, W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- C8)alkenylene. In embodiments, W 1 is optionally substituted (C2-C6)alkenylene. In 30 embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 14D is optionally substituted (C5-C25)alkyl.
  • 30 R 14D is optionally substituted (C5-C20)alkyl.
  • R 14D is optionally substituted (C5-C15)alkyl. In embodiments, R 14D is optionally substituted (C6-C12)alkyl. In embodiments, R 14D is optionally substituted (C8-C10)alkyl. [000231] In embodiments, R 14D is optionally substituted (C5-C25)alkenyl. In embodiments, R 14D is optionally substituted (C5-C20)alkenyl. In embodiments, R 14D is35 optionally substituted (C10-C20)alkenyl. In embodiments, R 14D is optionally substituted (C15- C20)alkenyl.
  • R 14D is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C1-C8)alkylene.
  • W 1 is optionally substituted (C1-C6)alkylene.
  • W 1 is optionally substituted (C1-C5)alkylene.
  • W 1 is optionally 5 substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene.
  • W 1 is optionally substituted (C2-C5)alkenylene.
  • each R 14A , R 14B , R 14C and R 14D is independently selected from: 10 15 87 5 (xvii) , for example wherein each R 14A , R 14B , R 14C and R 14D is option (x), or wherein R 14A and R 14C are option (xv) and R 14B and R 14D are option (viii).
  • R 14A , R 14B , R 14C and R 14D are the same.
  • R 14A and R 14B are the same and R 14C and R 14D are the same.
  • R 14A and R 14C 10 are the same and R 14B and R 14D are the same.
  • R 14A and R 14C are the same and R 14B and R 14D are different.
  • the cationic lipids of the present invention also include compounds having a structure according to Formula (VI): 15 Formula (VI) or a pharmaceutically acceptable salt thereof, wherein: t and u are integers that are each independently selected from 1, 2, 3, 4, 5 or 6; each Y 6 is independently selected from hydrogen or -OH; 88 R 19 is selected from optionally substituted (C1-C3)alkylene-optionally substituted thiazolyl and optionally substituted (C1-C3)alkylene-substituted phenyl, wherein the phenyl is substituted with one or more halogen atoms (e.g.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (VI) wherein R 19 is unsubstituted (C 1 )alkylene-substituted phenyl, wherein the phenyl is substituted with one fluoro 15 substituent.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (VI) wherein Y 6 is H and R 19 is unsubstituted (C 1 )alkylene-substituted phenyl, wherein the phenyl is substituted with one fluoro substituent.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (VIA): Formula (VIA) or a pharmaceutically acceptable salt thereof. 25 [000239] In embodiments, the cationic lipids of the present invention include compounds having a structure according to Formula (VIA1): 89 Formula (VIA1) or a pharmaceutically acceptable salt thereof. [000240] In embodiments, the cationic lipids of the present invention include 5 compounds having a structure according to Formula (VI) wherein R 19 is unsubstituted (C1)alkylene-unsubstituted thiazolyl.
  • the cationic lipids of the present invention include compounds having a structure according to Formula (VI) wherein Y 6 is H and R 19 is unsubstituted (C1)alkylene-unsubstituted thiazolyl. 10 [000242] In embodiments, the cationic lipids of the present invention include compounds having a structure according to Formula (VIB): Formula (VIB) or a pharmaceutically acceptable salt thereof. 15 [000243] In embodiments, the cationic lipids of the present invention include compounds having a structure according to Formula (VIB1): 90 Formula (VIB1) or a pharmaceutically acceptable salt thereof. [000244] In embodiments, t is 4. In embodiments, t is 1.
  • t is 2. In 5 embodiments, t is 3. In embodiments, t is 5. In embodiments, t is 6. [000245] In embodiments, u is 3. In embodiments, u is 1. In embodiments, u is 2. In embodiments, u is 4. In embodiments, u is 5. In embodiments, u is 6. [000246] In embodiments, t is 4 and u is 3. [000247] In embodiments, Y 6 is hydrogen. 10 [000248] In embodiments, R 19 is optionally substituted (C 1 -C 3 )alkylene-optionally substituted thiazolyl.
  • R 19 is unsubstituted (C 1 )alkylene-unsubstituted thiazolyl. [000249] In embodiments, R 19 is optionally substituted (C 1 -C 3 )alkylene-substituted phenyl, wherein the phenyl is substituted with one or more halogen atoms (e.g. -F). In 15 embodiments, R 19 is unsubstituted (C 1 )alkylene-substituted phenyl, wherein the phenyl is substituted with one fluoro substituent. In embodiments, the alkylene and the fluoro are para (1,4-arrangement) on the substituted phenyl.
  • R 18A is optionally substituted (C 5 -C 25 )alkyl. In embodiments, R 18A is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 18A is optionally substituted 20 (C 5 -C 15 )alkyl. In embodiments, R 18A is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 18A is optionally substituted (C 8 -C 10 )alkyl. [000251] In embodiments, R 18A is optionally substituted (C 5 -C 25 )alkenyl. In embodiments, R 18A is optionally substituted (C 5 -C 20 )alkenyl.
  • R 18A is optionally substituted (C 10 -C 20 )alkenyl. In embodiments, R 18A is optionally substituted (C 15 - 25 C 20 )alkenyl. [000252] In embodiments, R 18A is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene.
  • R 18B is optionally substituted (C5-C25)alkyl. In embodiments, R 18B is optionally substituted (C5-C20)alkyl. In embodiments, R 18B is optionally substituted 5 (C5-C15)alkyl. In embodiments, R 18B is optionally substituted (C6-C12)alkyl. In embodiments, R 18B is optionally substituted (C8-C10)alkyl.
  • R 18B is optionally substituted (C5-C25)alkenyl. In embodiments, R 18B is optionally substituted (C5-C20)alkenyl. In embodiments, R 18B is optionally substituted (C10-C20)alkenyl. In embodiments, R 18B is optionally substituted (C15- 10 C 20 )alkenyl. [000255] In embodiments, R 18B is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C 1 -C 10 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 8 )alkylene.
  • W 1 is optionally substituted (C 1 -C 6 )alkylene. In embodiments, W 1 is optionally substituted (C 1 -C 5 )alkylene. In embodiments, W 1 is optionally15 substituted (C 2 -C 10 )alkenylene. In embodiments, W 1 is optionally substituted (C 2 - C 8 )alkenylene. In embodiments, W 1 is optionally substituted (C 2 -C 6 )alkenylene. In embodiments, W 1 is optionally substituted (C 2 -C 5 )alkenylene.
  • R 18C is optionally substituted (C 5 -C 25 )alkyl.
  • R 18C is optionally substituted (C 5 -C 20 )alkyl. In embodiments, R 18C is optionally substituted (C 5 -C 15 )alkyl. In embodiments, R 18C is optionally substituted (C 6 -C 12 )alkyl. In embodiments, R 18C is optionally substituted (C 8 -C 10 )alkyl. 20 [000257] In embodiments, R 18C is optionally substituted (C 5 -C 25 )alkenyl. In embodiments, R 18C is optionally substituted (C 5 -C 20 )alkenyl. In embodiments, R 18C is optionally substituted (C10-C20)alkenyl.
  • R 18C is optionally substituted (C15- C20)alkenyl.
  • R 18C is -W 1 -X 1 .
  • W 1 is a covalent bond.
  • W 1 is optionally substituted (C1-C10)alkylene.
  • W 1 is optionally substituted (C1-C8)alkylene.
  • W 1 is optionally substituted (C1-C6)alkylene.
  • W 1 is optionally substituted (C1-C5)alkylene.
  • W 1 is optionally substituted (C2-C10)alkenylene.
  • W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene. In 30 embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • R 18D is optionally substituted (C5-C25)alkyl.
  • R 18D is optionally substituted (C5-C20)alkyl. In embodiments, R 18D is optionally substituted (C5-C15)alkyl. In embodiments, R 18D is optionally substituted (C6-C12)alkyl. In embodiments, R 18D is optionally substituted (C8-C10)alkyl. [000260] In embodiments, R 18D is optionally substituted (C5-C25)alkenyl. In embodiments, R 18D is optionally substituted (C5-C20)alkenyl. In embodiments, R 18D is35 optionally substituted (C10-C20)alkenyl.
  • R 18D is optionally substituted (C15- C20)alkenyl. 95 [000261] In embodiments, R 18D is -W 1 -X 1 . In embodiments, W 1 is a covalent bond. In embodiments, W 1 is optionally substituted (C1-C10)alkylene. In embodiments, W 1 is optionally substituted (C1-C8)alkylene. In embodiments, W 1 is optionally substituted (C1-C6)alkylene. In embodiments, W 1 is optionally substituted (C1-C5)alkylene. In embodiments, W 1 is optionally 5 substituted (C2-C10)alkenylene. In embodiments, W 1 is optionally substituted (C2- C8)alkenylene.
  • W 1 is optionally substituted (C2-C6)alkenylene. In embodiments, W 1 is optionally substituted (C2-C5)alkenylene.
  • each R 18A , R 18B , R 18C and R 18D is independently selected from: 10 15 97 5 (xvii) , for example wherein R 18A and R 18C are option (xi) and R 18B and R 18D are option (viii).
  • R 18A , R 18B , R 18C and R 18D are the same.
  • R 18A and R 18B are the same and R 18C and R 18D are the same.
  • R 18A and R 18C are the same and R 18B and R 18D are the same.
  • R 18A and R 18C are the same 10 and R 18B and R 18D are different.
  • the substituents are not optionally substituted.
  • the cationic lipids of the present invention have any one of the structures in Table A or Table B, or a pharmaceutically acceptable salt thereof.
  • the cationic lipids of the present invention have any one of the structures in Table A, Table B or Table C, or a pharmaceutically acceptable salt thereof.
  • a composition comprising a cationic lipid of the present invention, and further comprising: (i) one or more non-cationic lipids, 98 (ii) one or more cholesterol-based lipids and (iii) one or more PEG-modified lipids.
  • this composition is a lipid nanoparticle, optionally a liposome.
  • the one or more cationic lipid(s) constitute(s) about 30 mol %-60 5 mol % of the lipid nanoparticle.
  • the one or more non-cationic lipid(s) constitute(s) 10 mol%-50 mol% of the lipid nanoparticle.
  • the one or more PEG-modified lipid(s) constitute(s) 1 mol%-10 mol% of the lipid nanoparticle.
  • the cholesterol-based lipid constitutes 10 mol%-50 mol% of the lipid nanoparticle.
  • the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein.
  • the peptide is an antigen.
  • the lipid nanoparticle encapsulates an mRNA encoding a peptide or protein.
  • the phrase “encapsulation percentage” refers to the fraction of therapeutic agent (e.g. mRNA) that is effectively encapsulated within a liposomal-based vehicle (e.g. a 15 lipid nanoparticle) relative to the initial fraction of therapeutic agent present in the lipid phase.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at least 50%.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at least 55%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 60%. In embodiments, the lipid nanoparticles have an 20 encapsulation percentage for mRNA of at least 65%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 70%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 75%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 80%.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at 25 least 85%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 90%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 95%. In embodiments, the encapsulation percentage is calculated by performing the Ribogreen assay (Invitrogen) with and without the presence of 0.1% Triton-X 100. 30 [000271] In embodiments, the composition of the present invention is for use in a vaccine. [000272] In embodiments, the composition of the present invention is for use in therapy.
  • the composition of the present invention is for use in a 35 method of treating or preventing a disease amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the disease is (a) a protein 99 deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
  • a method for treating or preventing a disease comprises administering to a subject in need thereof a composition of 5 the present invention and wherein the disease is amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
  • the composition is administered intranasally, intravenously, 10 intrathecally or intramuscularly, or by pulmonary delivery, optionally through nebulization.
  • the composition is administered intramuscularly.
  • the cationic lipids of the present invention include compounds selected from those depicted in Table A or Table B, or a pharmaceutically 15 acceptable salt thereof.
  • the cationic lipids of the present invention include compounds selected from those depicted in Table A, Table B or Table C, or a pharmaceutically acceptable salt thereof.
  • Exemplary compounds include those described in Table A, or a 20 pharmaceutically acceptable salt thereof.
  • Table A 100 101 102 103 104 105 106 107 108 [000279] Any of the compounds identified in Table A above may be provided in the form of a pharmaceutically acceptable salt and such salts are intended to be encompassed by the present invention. 5 Table B 109 110
  • 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 15 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 25 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.
  • 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 30 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 35 may be synthesized as unmodified or modified mRNA.
  • Modified mRNA comprises nucleotide 121 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 5 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-d
  • compositions comprising such lipids
  • 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 macropinocytosis, fusogenicity, endosomal or lysosomal disruption and/or releasable properties that afford such compounds advantages relative 35 other similarly classified lipids.
  • 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 compound of the invention as described herein.
  • delivery vehicle comprising a compound of the invention as described herein.
  • delivery vehicle comprising a compound of the invention as described herein.
  • delivery vehicle comprising a compound of the invention as described herein.
  • the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle,” 5 or grammatical equivalents thereof, are used interchangeably.
  • the present invention provides a composition (e.g., a pharmaceutical composition) comprising a compound described herein 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 10 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. 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 15 compositions disclosed herein (e.g., a liposomal formulation comprising a compound described herein 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).
  • the terms “transfect” or “transfection” refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or 20 polynucleotides) into a cell (e.g., 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 25 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 30 systemic adverse effects or toxicity associated with the compound or their encapsulated contents.
  • the encapsulated materials e.g., one or more polynucleotides
  • the production of the product 35 (e.g., a polypeptide or protein) encoded by such polynucleotide may be stimulated and the capability of such target cells to express the polynucleotide and produce, for example, a 123 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 10 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.
  • Such encapsulated polynucleotides may encode, for example, a hormone, enzyme, receptor, polypeptide, peptide or other 15 protein of interest.
  • Liposomal Delivery Vehicles [000296]
  • a composition is a suitable delivery vehicle.
  • 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 the safety profile or otherwise conferring one or more desired properties to such enriched liposomal composition (e.g., 25 improved delivery of the encapsulated polynucleotides to one or more target cells and/or reduced in vivo toxicity of a liposomal composition).
  • the compounds of the invention as described 30 herein 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
  • target cells e.g., by permeating or fusing with the lipid membranes of such target cells.
  • liposomal delivery vehicles e.g., lipid nanoparticles
  • are 35 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 124 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 5 the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue.
  • compositions are loaded with or otherwise encapsulate materials, such as for example, one or more biologically-active polynucleotides (e.g., mRNA).
  • a composition e.g., a pharmaceutical composition
  • 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 wherein at least one cationic lipid is a compound of the invention as described herein.
  • a 15 composition comprises an mRNA encoding for a peptide or protein (e.g., any peptide or protein described herein).
  • a composition comprises an mRNA encoding for a peptide (e.g., any peptide described herein).
  • a composition comprises an mRNA encoding for a protein (e.g., any protein described herein).
  • a composition e.g., a pharmaceutical composition
  • 20 comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises a compound described herein.
  • a nucleic acid is an mRNA encoding a peptide or protein.
  • an mRNA encodes a peptide or protein for use in the delivery to or treatment of the lung of a subject or a lung cell. In embodiments, an mRNA encodes a peptide or 25 protein for use in the delivery to or treatment of the liver of a subject or a liver cell. Still other exemplary mRNAs are described herein.
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • can 30 have a net negative charge.
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a lipid nanoparticle that encapsulates a nucleic acid comprises one or more compounds of the invention as 35 described herein.
  • the amount of a compound of the invention as described herein 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).
  • a compound of the invention as described herein 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).
  • a compound of the invention as described herein is present 10 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 compound of the invention as described herein 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 15 20 wt%, or about 10 wt% to about 20 wt% of the combined dry weight of all lipids present in a composition such as a liposomal delivery vehicle.
  • the amount of a compound of the invention as described herein 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%, 20 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).
  • a composition e.g., a liposomal composition
  • the amount of a compound of the invention as described 25 herein 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 30 composition (e.g., a liposomal composition).
  • a 30 composition e.g., a liposomal composition
  • a composition e.g., a liposomal delivery vehicle such as a lipid nanoparticle
  • a delivery vehicle comprises about 0.5 wt%, about 1 35 wt%, about 3 wt%, about 5 wt%, or about 10 wt% of a compound described herein.
  • a delivery vehicle e.g., a liposomal delivery vehicle such as a lipid 126 nanoparticle
  • a delivery vehicle 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 compound described herein.
  • 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 compound of the invention as described herein 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).
  • mol% a percentage of the combined molar amounts of total lipids of a composition
  • a 10 compound of the invention as described herein is present in an amount that is about 0.5 mol% to about 50 mol% (e.g., about 0.5 mol% to about 20 mol%) of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
  • a compound of the invention as described herein is present in an amount that is about 0.5 mol% to about 5 mol%, about 1 mol% to about 10 mol%, 15 about 5 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol %, or about 45 mol% to about 60 mol% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
  • a compound of the 20 invention as described herein is present in an amount that is about 1 mol% to about 60 mol%, 1 mol% to about 50 mol%, 1 mol% to about 40 mol%, 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%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol% or about 5 mol% to about 25 mol% of the combined molar amounts of all 25 lipids present in a composition such as a liposomal delivery vehicle [000318]
  • a compound of the invention as described herein 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 50 mol%, or from about 5 mol% to about 50 mol%
  • a compound of the invention as described herein can comprise greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than 35 about 1 mol%, greater than about 5 mol%, greater than about 10 mol%, greater than about 127 20 mol%, greater than about 30 mol%, or greater than about 40 mol% of the total amount of lipids in the lipid nanoparticle.
  • a compound as described can comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 5 45 mol%, or less than about 40 mol%, or less than about 35 mol %, less than about 30 mol%, or 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).
  • a composition e.g., a liposomal delivery vehicle
  • the amount of a compound of the invention as described 10 herein 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 molar amounts 15 of total lipids in a composition (e.g., a liposomal composition).
  • a composition e.g., a liposomal composition
  • the amount of a compound of the invention as described herein 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 20 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 molar amounts of total lipids in a composition (e.g., a liposomal composition).
  • a composition e.g., a liposomal composition
  • a composition of the invention comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the invention as described herein.
  • a composition suitable for practicing the invention has four lipid components comprising a compound of the 30 invention as described herein as the cationic lipid component, a non-cationic lipid, a cholesterol-based lipid, and a PEG-modified lipid.
  • the non-cationic lipid may be DOPE or DEPE.
  • the cholesterol-based lipid may be cholesterol.
  • the PEG-modified lipid may be DMG-PEG2K.
  • pharmaceutical (e.g., liposomal) compositions 35 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 128 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 e.g., lipid nanoparticle
  • a 5 nucleic acid e.g., mRNA encoding a peptide or protein
  • lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.
  • a composition e.g., lipid nanoparticle
  • a nucleic acid e.g., mRNA encoding a peptide or protein
  • lipid nanoparticle that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or protein)
  • a nucleic acid e.g., mRNA encoding a peptide or protein
  • lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid
  • further comprises a cholesterol-based lipid e.g., lipid nanoparticle
  • such a composition has four lipid components comprising a compound of the invention as described herein as the cationic lipid component, a non- cationic lipid (e.g., DOPE), a cholesterol-based lipid (e.g., cholesterol) and a PEG-modified 15 lipid (e.g., DMG-PEG2K).
  • a non- cationic lipid e.g., DOPE
  • a cholesterol-based lipid e.g., cholesterol
  • PEG-modified 15 lipid e.g., DMG-PEG2K
  • a lipid nanoparticle that encapsulates a nucleic acid comprises one or more compounds of the invention as described herein, 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.
  • 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 25 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 30 composition may comprise one or more additional cationic lipids.
  • liposomes may comprise one or more additional 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 35 available.
  • Suitable additional cationic lipids for use in the compositions include the cationic lipids as described in the literature.
  • compositions may also comprise one or more helper lipids.
  • helper lipids include non-cationic lipids.
  • 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, 10 distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2- Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl- 15 phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimy
  • a non-cationic or helper lipid suitable for practicing the invention is 20 dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • DEPE 1,2-Dierucoyl-sn-glycero-3- phosphoethanolamine
  • 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 30 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 35 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 130 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- 5 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 10 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 15 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%.
  • 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 20 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%.
  • 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 25 about 30 wt%, or no more than about 40 wt%.
  • Cholesterol-based Lipids [000336]
  • a composition e.g., a liposomal composition
  • a suitable cholesterol-based 30 lipid for practicing the invention is cholesterol.
  • cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N- oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No.
  • 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 5 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%. 10 [000338] 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.
  • 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 15 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
  • a suitable PEG-modified or PEGylated lipid for practicing the invention is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).
  • DMG-PEG2K 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
  • PEG-CER derivatized ceramides
  • C8 PEG-2000 ceramide N-octanoyl- sphingosine-1-[succinyl(methoxy polyethylene glycol)-2000]
  • C8 PEG-2000 ceramide is also contemplated by the present invention in combination with one or more of compounds of the invention as described herein and, in some embodiments, other lipids together which comprise the liposome.
  • particularly useful exchangeable lipids are 30 PEG-ceramides having shorter acyl chains (e.g., C 14 or C 18 ).
  • Contemplated further 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 5 lipid is PEGylated cholesterol or PEG-2K.
  • 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).
  • PEG-modified phospholipid and derivatized lipids of the present invention may be present in a molar ratio (mol%) from about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, or about 3% to about 5% of the total lipid present in the composition (e.g., a liposomal composition).
  • compositions e.g., to construct liposomal compositions
  • encapsulated materials e.g., one or more therapeutic polynucleotides
  • target cells e.g., by permeating or fusing with the lipid 20 membranes of such target cells.
  • 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 25 a lipid nanoparticle) into the one or more target cells.
  • the encapsulated materials e.g., one or more therapeutic polynucleotides encapsulated in 25 a lipid nanoparticle
  • compounds of the invention as described herein 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 30 reduced quantity of such composition may be administered to the subject to achieve a desired therapeutic response or outcome.
  • pharmaceutical formulations comprising a compound described and nucleic acids provided by the present invention may be used for various therapeutic disease and/or disease prevention purposes.
  • a 35 compound described herein and nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands or stabilizing reagents.
  • a compound described herein can be formulated via pre-mixed lipid solution.
  • a composition comprising a compound described herein 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 5 Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.
  • 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, 10 intravenous, intraperitoneal, or intranasal.
  • the 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).
  • a common route for administering a liposomal composition of the invention may be intravenous delivery, in particular when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamylase (OTC) deficiency).
  • OTC ornithine transcarbamylase
  • the liposomal composition may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis).
  • a liposomal composition of the invention is typically administered intramuscularly.
  • a liposomal composition of the invention may be administered intranasally for vaccination.
  • Diseases or disorders affecting the eye may be treated by administering a liposomal composition of the invention intravitreally.
  • pharmaceutical formulations of the invention may 25 be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into a targeted tissue (e.g., in a sustained release formulation).
  • tissue to be targeted can be affected in various ways, depending on the tissue to be targeted.
  • exemplary tissues in which mRNA may be delivered and/or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, 30 skin, and/or cerebrospinal fluid.
  • the tissue to be targeted in the liver include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, 30 skin, and/or cerebrospinal fluid.
  • the tissue to be targeted in the liver include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, 30 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, 35 tablet or capsule form for administration to the stomach or intestines, can be supplied in 134 suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
  • Compositions described herein can comprise mRNA encoding peptides including those described herein (e.g., a polypeptide such as a protein).
  • a mRNA encodes a polypeptide.
  • a mRNA encodes a peptide.
  • the peptide is an antigen.
  • a mRNA encodes a protein.
  • the present invention provides methods for delivering a composition having 10 full-length mRNA molecules encoding a peptide or protein 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 route of delivery used in the methods of the invention allows for non- invasive, self-administration of the compounds of the invention.
  • the methods involve intranasal, intratracheal or pulmonary administration by aerosolization, nebulization, or instillation of a compositions comprising mRNA encoding a therapeutic peptide or protein in a suitable transfection or lipid carrier vehicles as described above.
  • the peptide or protein is encapsulated with a liposome.
  • the liposome comprises a lipid, which is a compound of the invention.
  • administration of a compound of the invention includes administration of a composition comprising a compound of the invention.
  • the local cells and tissues of the lung represent a potential target 25 capable of functioning as a biological depot or reservoir for production and secretion of the protein encoded by the mRNA
  • administration of the compounds of the invention to the lung via aerosolization, nebulization, or instillation results in the distribution of even non-secreted proteins outside the lung cells.
  • nanoparticle compositions of the 30 invention pass, through the lung airway-blood barrier, resulting in translation of the intact nanoparticle to non-lung cells and tissues, such as, e.g., the heart, the liver, the spleen, where it results in the production of the encoded peptide or protein in these non-lung tissues.
  • the utility of the compounds of the invention and methods of the invention extend beyond production of therapeutic protein in lung cells and tissues of the lung and can be 35 used to delivery to non-lung target cells and/or tissues. They are useful in the management and treatment of a large number of diseases.
  • the compounds of the 135 invention, used in the methods of the invention result in the distribution of the mRNA encapsulated nanoparticles and production of the encoded peptide or protein in the liver, spleen, heart, and/or other non-lung cells.
  • the compounds of the invention may be employed in 10 the methods of the invention to specifically target peripheral cells or tissues. Following the pulmonary delivery, it is contemplated the compounds of the invention cross the lung airway- blood barrier and distribute into cells other than the local lung cells.
  • the compounds disclosed herein may be administered to a subject by way of the pulmonary route of administration, using a variety of approach known by those skilled in the art (e.g., by 15 inhalation), and distribute to both the local target cells and tissues of the lung, as well as in peripheral non-lung cells and tissues (e.g., cells of the liver, spleen, kidneys, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma).
  • peripheral non-lung cells and tissues e.g., cells of the liver, spleen, kidneys, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma.
  • both the local cells of the lung and the peripheral non-lung cells can serve as biological reservoirs or depots capable of producing and/or secreting a translation product encoded by one or more 20 polynucleotides.
  • the present invention is not limited to the treatment of lung diseases or conditions, but rather can be used as a non-invasive means of facilitating the delivery of polynucleotides, or the production of peptides or proteins encoded thereby, in peripheral organs, tissues and cells (e.g., hepatocytes) which would otherwise be achieved only by systemic administration.
  • Exemplary peripheral non-lung cells include, but are not 25 limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
  • the peptide or protein product encoded by the mRNA (e.g., a functional protein or enzyme) is detectable in the peripheral target tissues for at least about one to seven days or longer following administration of the compound to the subject.
  • the amount of peptide or protein product necessary to achieve a therapeutic effect will vary depending on the condition being treated, 35 the peptide or protein encoded, and the condition of the patient.
  • the peptide or protein product may be detectable in the peripheral target tissues at a concentration (e.g., a 136 therapeutic concentration) of at least 0.025-1.5 ⁇ g/ml (e.g., at least 0.050 ⁇ g/ml, at least 0.075 ⁇ g/ml, at least 0.1 ⁇ g/ml, at least 0.2 ⁇ g/ml, at least 0.3 ⁇ g/ml, at least 0.4 ⁇ g/ml, at least 0.5 ⁇ g/ml, at least 0.6 ⁇ g/ml, at least 0.7 ⁇ g/ml, at least 0.8 ⁇ g/ml, at least 0.9 ⁇ g/ml, at least 1.0 ⁇ g/ml, at least 1.1 ⁇ g/ml, at least 1.2 ⁇ g/ml, at least 1.3 ⁇ g/ml, at least 1.4 ⁇ g/ml, or 5 at least 1.5 ⁇ g/ml), for at least about 1, 2, 3, 4, 5,
  • nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and inhalation of an 10 aerosol mist produced by a liquid nebulizer or the use of a dry powder apparatus such as that described in U.S. patent 5,780,014, incorporated herein by reference.
  • the compounds of the invention may be formulated such that they may be aerosolized or otherwise delivered as a particulate liquid or solid prior to or upon administration to the subject.
  • Such compounds may be administered with the 15 assistance of one or more suitable devices for administering such solid or liquid particulate compositions (such as, e.g., an aerosolized aqueous solution or suspension) to generate particles that are easily respirable or inhalable by the subject.
  • suitable devices e.g., a metered dose inhaler, jet-nebulizer, ultrasonic nebulizer, dry-powder- inhalers, propellant-based inhaler or an insufflator
  • a 20 predetermined mass, volume or dose of the compositions e.g., about 0.5 mg/kg of mRNA per dose
  • the compounds of the invention are administered to a subject using a metered dose inhaler containing a suspension or solution comprising the compound and a suitable propellant.
  • the compounds of the invention may be formulated as a particulate powder 25 (e.g., respirable dry particles) intended for inhalation.
  • compositions of the invention formulated as respirable particles are appropriately sized such that they may be respirable by the subject or delivered using a suitable device (e.g., a mean D50 or D90 30 embodiments, the compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).
  • the compounds of the invention are administered to a subject such that a concentration of at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/kg, at least 5.0 mg/kg, at least 6.0 mg/kg, at least 7.0 mg/kg, at least 8.0 35 mg/kg, at least 9.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 137 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, or at least 100 mg/kg body weight
  • the compounds of the invention are administered to a subject such that a total amount of at least 5 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 10 mg mRNA is administered in one or more doses.
  • reaction mixture was stirred at room temperature for 16 hours. The progress of reaction was monitored by ELSD and TLC.
  • the reaction mixture was diluted with ice cold 20 water and extracted diethyl ether. The organic layer was basify using triethyl amine and washed with brine solution. The ether layer was concentrated under vacuum to yield N-(3- ⁇ 4- [bis(2-hydroxydecyl)amino]butyrylamino ⁇ -2-hydroxy-2-methylpropyl)4-[bis(2- hydroxydecyl)amino]butyramide (180 mg, 77.56 % Yield) as an oily liquid.
  • reaction mixture was stirred at room temperature for 16 hours. The progress of reaction was monitored using ELSD and TLC.
  • the reaction mixture was diluted with ice cold water and extracted with diethyl ether. The organic layer was basify using triethyl amine to pH 8-9 and was organic layer was washed with brine solution. The organic layer was concentrated25 under vacuum to yield N-(3- ⁇ 5-[bis(2-hydroxydodecyl)amino]valerylamino ⁇ -2-hydroxy-2- methylpropyl)5-[bis(2-hydroxydodecyl)amino]valeramide (430 mg, 98.72 % Yield) as pale yellow liquid.
  • reaction mixture was stirred at room temperature for 16 hours. The progress of reaction was monitored by ELSD/TLC. After completion of the reaction, the 25 reaction mixture was diluted with ice cold water and extracted with ethyl acetate (3x100 mL). The organic layer was dried with Na2SO4, filtered and concentrated.
  • reaction mass was allowed to stir at RT for 16 h.
  • the progress of reaction was monitored by TLC.
  • reaction mixture was quench by saturated sodium bicarbonate 15 solution up to pH 8 and extracted with ethyl acetate (3x20 mL). The organic layer was dried over anhydride sodium sulphate, filtered and concentrated under reduced pressure to get crude compound.
  • Example 4 Synthesis of Compound 4 ⁇ 52 Intermediate [2]: 153 [000395]
  • a mixture of 2-(chloromethyl)-2-methyloxirane [1] 1.0 g, 9.39 mmol
  • ammonium hydroxide 10 mL
  • the reaction mixture was 5 concentrated to remove the excess ammonia to obtain 1,3-diamino-2-methyl-2-propanol [2] (0.8 g crude), which was forward to the next step without further purification.
  • reaction mixture was stirred for 15 min at RT and added 1,3-diamino-2-methyl-2- propanol (170 mg, 1.63 mmol).
  • the reaction mixture was stirred at RT for 16 hr.
  • the 155 progress of reaction was monitored by TLC and ELSD.
  • the reaction mass was quenched with water and extracted with DCM (3x35 mL). The organic layer was dried over sodium sulphate, concentrated under reduced pressure to get crude.
  • reaction mixture was stirred for 16 hr at RT. The progress of reaction was monitored by TLC and ELSD. The reaction mass quenched by saturated 20 sodium bicarbonate and extracted with ethyl acetate (3x25 mL). The organic layer was dried over sodium sulphate, concentrated under reduced pressure to get crude. The crude was soluble in pentane and wash by ACN, pentane layer was concentrated under reduced pressure to give desired product 1,5-bis( ⁇ 5-[bis(2-hydroxydodecyl)amino]pentyl ⁇ ) 3-hydroxy- 3-methylpentanedioate (0.290 g, 64.23 % Yield) as a pale yellow liquid.
  • reaction mass was diluted with diethyl ether (50 mL) and washed with ice cold water (25 mL), saturated sodium bicarbonate (15 mL), Milli Q water (2x10 mL). The organic layer was collected, dried over10 sodium sulphate and evaporate under reduced pressure to obtain 3-(bis(2- hydroxydodecyl)amino)propyl ((3-(bis(2-hydroxydodecyl)amino)propyl)carbamoyl)-L- phenylalaninate (0.3 g, 61.34 % Yield) as pale yellow liquid.
  • reaction mixture was diluted with diethyl ether (50 mL) and washed with cold water (2x10 mL). The diethyl ether layer was quenched with cold saturated sodium bicarbonate solution up to pH 8. Separate the ether layer and wash with cold water (2x10 mL). The organic layer was dried over anhy. sodium sulphate, concentrate under 10 reduced pressure to get crude.
  • the reaction mixture was stirred at room temperature for 16 h. The progress of reaction was monitored by TLC/ELSD.
  • the reaction mixture was diluted with diethyl ether (50 mL) and washed with cold water (2x5 mL).
  • To the organic layer was added cold saturated sodium bicarbonate solution (pH ⁇ 8).
  • the organic layer was separated and washed with cold water (2x5 mL).
  • the organic layer was dried over anhy. sodium sulphate, 20 concentrate under reduced pressure to get crude.
  • the crude was purified by flash column chromatography (SiO2: 0-20 %methanol in DCM) to obtain 3-(bis(2-hydroxydodecyl) amino)propyl ((3-(bis(2-hydroxydodecyl)amino)propyl)carbamoyl)-L-histidinate (0.4 g, 63.80% Yield) as a pale yellow liquid.
  • reaction mixture was quench with ice cold water (300.0 mL), and the organic 171 layer was separated. The organic layer was wash with saturated brine solution, and dried over sodium sulphate, filtered and concentrated under reduced pressure to give crude compound.
  • the crude was purified by flash column chromatography (0-30% Ethyl acetate in heptane) to afford 5,9-bis(decyl)-2,2,3,3,11,11,12,12-octamethyl-7-[3- 5 (triphenylmethoxy)propyl]-4,10-dioxa-7-aza-3,11-disilatridecane [5] (45.0 g, 76.72 % Yield) as a colourless liquid.
  • reaction was stirred at RT for 4 h. The progress of reaction was monitor by TLC. After completion, reaction mixture was quenched with saturated aq. NaHCO3 solution up pH 8. The compound was extracted with DCM (2x500 mL). The combined organic layer was dried over 20 anhydrous Na2SO4, filtered and evaporated to get crude compound.
  • the crude compound was purified by flash column chromatography (SiO2: 10-30% Ethyl acetate in Heptane) to afford 3-(bis ⁇ 2-[(tert-butyl)bis(methyl)siloxy]dodecyl ⁇ amino)-1-propanol [6] (15.0 g, 81.63 % Yield) as a colourless liquid.
  • the organic layer was dried over sodium sulphate and evaporate under reduced 15 pressure to get crude.
  • the crude was purified by flash column chromatography (SiO2: 0-30% ethyl acetate in hexane) to get tert-butyl ⁇ [2-(benzyloxy)ethyl](tert- butoxycarbonylmethyl)amino ⁇ acetate [13] (2.3 g, 45.82% Yield) as a pale yellow liquid.
  • reaction mixture was stirred at RT for 16 h.
  • the progress of reaction was monitored by TLC/ELSD.
  • reaction mass quenched with water (20 mL) and extracted with DCM (2x50 mL).
  • the combined organic layer was dried over anhy. sodium sulphate and evaporate under reduced pressure to get crude.
  • reaction mixture was filtered through celite, washed the celite bed two times with methanol. The filtrate was concentrated under vacuum to give bis(3-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)propyl) 2,2'- 10 ((2-hydroxyethyl)azanediyl)diacetate [16] (3.3 g, 92.11% Yield) as a pale yellow liquid.
  • reaction mixture was stirred at RT for 30 min and 20 added bis(3-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)propyl) 2,2'-((2-hydroxyethyl) azanediyl)diacetate [16] (1.03 g, 678 ⁇ mol).
  • the reaction mixture was stirred at RT for 16 h.
  • the progress of reaction was monitored by TLC/ELSD. After completion of reaction, reaction mass quenched with 10 mL water and extracted with DCM (2x20 mL). The combined organic layer was dried over anhy. sodium sulphate and evaporated under reduced pressure 25 to get crude.
  • reaction mixture was stirred at RT for 16 hr. The progress of reaction was monitored by TLC/ELSD. After completion of reaction, reaction mass was diluted with diethyl ether (30 mL) and washed with ice cold water (10 mL), saturated sodium bicarbonate (10 mL), Milli Q water (2x15 mL). The organic layer was dried over anhy. 15 sodium sulphate and evaporated under reduced pressure to get bis(3-(bis(2- hydroxydodecyl)amino)propyl) 2,2'-((2-((L-phenylalanyl)oxy)ethyl)azanediyl) diacetate (350 mg, 73.69% Yield) as a pale yellow liquid.
  • reaction mixture was stirred at RT for 30 min and added bis(3-(bis(2-((tert- butyldimethylsilyl)oxy)dodecyl)amino)propyl) 2,2'-((2-hydroxyethyl)azanediyl)diacetate [16] (1.62 g, 1.09 mmol).
  • the reaction mixture was stirred at RT for 16 h.
  • the progress of reaction was monitored by TLC/ELSD.
  • reaction mass was quenched with water (15 mL) and extracted with DCM (2x20 mL).
  • the combined organic 15 layer was dried over sodium sulphate and concentrated under reduced pressure to get crude.
  • reaction mixture was degassed and allowed to stir at room temperature overnight under hydrogen atmosphere (balloon pressure). The progress of reaction was monitored by TLC. After completion of reaction, reaction mixture was filtered through celite and washed two times with ethyl acetate. The 10 filtrate was concentrated under vacuum get crude.
  • reaction mixture was stirred for 16 h at room temperature. The progress of reaction was monitored by TLC/ELSD. After completion of reaction, reaction mixture was diluted with diethyl ether (50 mL) and washed with cold water (2x5 mL). The cold saturated sodium bicarbonate solution (pH ⁇ 8) was added to diethyl ether layer. The organic layer was separated and wash with cold water (2x5 mL). The organic layer was dried over anhy. 10 sodium sulphate, concentrate under reduced pressure to get crude.
  • the crude was purified by flash column chromatography (SiO 2 : 0-20 %methanol in DCM) to obtain bis(3-(bis(2- hydroxydodecyl)amino)propyl) 2,2'-((2-((L-tyrosyl)oxy)ethyl)azanediyl)diacetate (0.3 g, 34.59 % Yield) as a pale yellow liquid.
  • reaction mixture was stirred at RT for 30 min and added bis(3-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)propyl)2,2'-((2- hydroxyethyl)azanediyl) diacetate [16] (967 mg, 651 ⁇ mol ) at RT.
  • the reaction mixture was 10 stirred at RT for 16 h. The progress of reaction was monitored by TLC/ELSD. After completion of reaction, reaction mass was quenched with water (20 mL) and extracted with DCM (3x30 mL). The combined organic layer was dried over anhy. sodium sulphate and evaporated under reduced pressure to get crude.
  • reaction mixture was stirred at RT for 16 h. The progress of reaction was monitored by TLC/ELSD. After completion of reaction, reaction mass quenched by saturated sodium bicarbonate solution (pH ⁇ 8) and extracted with ethyl acetate (2x15 mL). The combined organic layer was dried over anhy. sodium sulphate and evaporated under 10 reduced pressure to get crude.
  • the crude was purified by reverse phase column chromatography (water/0.1% TFA in ACN) to get bis(3-(bis(2-hydroxydodecyl) amino)propyl) 2,2'-((2-((L-tryptophyl)oxy)ethyl)azanediyl)diacetate (0.3 g, 38.05% Yield) as a pale yellow liquid.
  • reaction 5 mass was cooled to RT, added lithium(1+) hydroxide (0.232 g, 97 mmol) and water (2.5 mL).
  • the reaction mixture was allowed to stir for 4 h at RT.
  • the progress of the reaction mixture was monitored by TLC/ELSD.
  • the excess of methanol was removed under reduced pressure and residue was acidified with 1N HCl up to pH-3 and extract with ethyl acetate (2x 10 mL).
  • the organic layers were combined, dried over anhydrous sodium sulphate and 10 concentrated under a vacuum.
  • reaction mixture was quench with ice cold water (150.0 mL), and the organic layer was separated. The organic layer was wash with saturated brine solution, and dried over sodium sulphate, filtered and concentrated under reduced pressure to give crude 10 compound.
  • the crude was purified by flash column chromatography (0-30% Ethyl acetate in heptane) to afford 5,9-bis(decyl)-2,2,3,3,11,11,12,12-octamethyl-7-[3- (triphenylmethoxy)propyl]-4,10-dioxa-7-aza-3,11-disilatridecane [8] (21.5 g, 80.48 % Yield) as a colourless liquid.
  • reaction was stirred at RT for 4 h. The progress of reaction was monitor by TLC. After completion, reaction mixture was quenched with saturated aq. NaHCO3 solution up pH 8. The 25 compound was extracted with DCM (2x300 mL). The combined organic layer was dried over anhy. Na2SO4, filtered and evaporated to get crude compound.
  • the crude compound was purified by flash column chromatography (SiO2: 10-30% Ethyl acetate in Heptane) to afford 3-(bis ⁇ 2-[(tert-butyl)bis(methyl)siloxy]dodecyl ⁇ amino)-1-propanol [9] (8.0 g, 54.42 % Yield) as a colourless liquid.
  • reaction mixture was stirred at RT for 20 min and added 3-(bis ⁇ 2-[(tert- 10 butyl)bis(methyl)siloxy]dodecyl ⁇ amino)-1-propanol [9] (1.52 g, 2.26 mmol) at room temperature.
  • the reaction mixture was stirred at RT for 16 h.
  • the progress of the reaction was monitored by TLC and ELSD.
  • the reaction mixture was quenched with water and extracted with Dichloromethane. The organic layers were collected, combined, dried over Na2SO4 and concentrated under reduced pressure to get crude.
  • reaction mixture was quenched with aqueous NaHCO3 upto pH 8, and extracted with dichloromethane (2x50 mL). The organic layers was dried over Na2SO4 and concentrated under reduced pressure to get 189 the 3-(bis ⁇ 2-[(tert-butyl)bis(methyl)siloxy]dodecyl ⁇ amino)propyl (2S,3R)-2-amino-3- (benzyloxy)butyrate [12] (950 mg, 58.91%) as pale yellow liquid.
  • reaction mixture was stirred at RT for 20 min and added 3-(bis ⁇ 2-[(tert-butyl)bis(methyl)siloxy] dodecyl ⁇ amino)propyl (2S,3R)-2- amino-3-(benzyloxy)butyrate (475 mg, 1.1 mmol) at room temperature.
  • the reaction mixture 15 was stirred at RT for 16 h. The progress of the reaction was monitored by TLC and ELSD. After completion, the reaction mixture was quenched with water and extracted with Dichloromethane. The organic layers was dried over Na2SO4 and concentrated under reduced pressure to get crude.
  • reaction mixture was degassed and allowed to stir at 70 °C overnight under 70 psi hydrogen pressure in steel bomb. The progress of reaction was monitored by TLC/ELSD. After completion, reaction mixture was filtered through celite and washed with ethyl acetate. The filtrate was concentrated under reduced pressure to get 25 crude material. The crude was purified by flash column chromatography (SiO2: 0-10 % MeOH/DCM) to obtain 3-[bis(2-hydroxydodecyl)amino]propyl (2S,3R)-2- ⁇ 4-[bis(2- hydroxydodecyl)amino] butyrylamino ⁇ -3-hydroxybutyrate (180 mg, 32.72 %) as gummy liquid.
  • Example 12 Synthesis of Compound 29 5 [ 000485] To a stirred solution of 2-hexyldecanoic acid [1] (50 g, 195 mmol) in 10 dichloromethane (0.5 L, 7.81 mol), were added 1-[(cyclohexylimino)methyleneamino]cyclohexane 192 (42.2 g, 1.1 eq., 205 mmol) and N,N-dimethyl-4-pyridylamine (8.34 g, 0.35 eq., 68.2 mmol) at room temperature under nitrogen atmosphere.
  • 6-bromo-1-hexanol [2] (35.3 g, 195 mmol) was added to the resulting reaction mixture, and allowed to stir for 16 h at room temperature. Progress of reaction was monitored by ELSD/TLC (SM was consumed). 5 Reaction mixture was washed with water (2x 250 ml), dried over Na2SO4 and concentrated under reduced pressure. Resulting crude was purified by silica gel flash column chromatography using 5% ethyl acetate in n-hexane to obtain 6-bromohexyl 2-hexyldecanoate [3] (50 g, 61 % Yield) as yellow coloured liquid.
  • reaction mixture was stirred at 60°C for 48 h. The progress of the reaction was monitored by ELSD. Reaction mixture was diluted with diethyl ether (600 mL) and washed with cold water 4x 100ml). The 20 organic layer was dried over sodium sulphate and evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography using 0-2 % MeOH in DCM to obtain 6- ⁇ [6-(1-hexylnonylcarbonyloxy)hexyl](4-hydroxybutyl)amino ⁇ hexyl 2-hexyldecanoate [5] (7 g, 40.71% Yield) as yellowish gummy compound.
  • reaction mass was quenched with water (50 mL) and extracted with DCM (2x 50 10 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude. The crude was then purified by flash chromatography (SiO2: 0-20% EtOAc/Heptane) to get 7-bromoheptyl 2-octyldecanoate [8] (4.4 g, 79.7 % Yield) as pale yellow liquid.
  • reaction mixture was diluted with diethyl ether (100 mL) and washed with water (4x 50 mL). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure.
  • the crude was purified by flash column chromatography by using 0-40% ethyl acetate in heptane to obtain 25 13,13,14,14-tetramethyl-12-oxa-8-aza-13-silapentadecyl 2-octyldecanoate (4.5 g, 33 % Yield) as yellow gummy liquid.
  • reaction mixture was allowed to stir at 80°C for 16 h. Reaction progress was monitored by TLC and ELSD. After completion of reaction, reaction mixture was diluted with diethyl ether (100 mL) and washed with water (4x 50 mL). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure.
  • reaction mixture was diluted with diethyl ether (20 mL), washed with cold water (2x 20 mL), followed by saturated solution of sodium bicarbonate (2x 25 mL) 5 and water (2x 25 mL). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure.
  • the crude was purified by column chromatography using 0-2 % MeOH in DCM to obtain 6- ⁇ [6-(1-hexylnonylcarbonyloxy)hexyl](3-hydroxypropyl)amino ⁇ hexyl 2- hexyldecanoate [11] (2.4 g, 78.9 % Yield) as yellow coloured liquid.
  • reaction mixture was stirred at 0 °C for 2h. The progress of the reaction was monitored by 20 TLC. After completion of reaction, reaction mixture was quenched with cool sodium bicarbonate solution and extracted with DCM (2x 50 mL). The organic layer was washed with cold water (2x 50 mL)). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure.
  • reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by 15 TLC. After completion of reaction, reaction mixture was quenched with water (25 mL) and extracted with ethyl acetate (2x 20 mL). The organic layer was washed with cold water (2x 20 mL), dried over sodium sulfate and was evaporated under reduced pressure.
  • the compound was prep-purified in Mobile phase: A: - 10mM AA IN 20%ACN WATER, B:IPA to obtain di(3- ⁇ [7-(1-octylnonylcarbonyloxy)heptyl][5- (undecyloxycarbonyl)pentyl]amino ⁇ propyl) [(3-indolyl)methyl]malonate (145 mg, 12.15 % Yield) as colourless liquid.
  • 6-bromo-1-hexanol [4] 10 (35.3 g, 195 mmol) was added to the resulting reaction mixture, and allowed to stir for 16 h at room temperature. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with water (2x 250 ml), dried over Na 2 SO 4 and concentrated under reduced pressure. Resulting crude was purified by silica gel flash column chromatography using 5% ethyl acetate in n-hexane to obtain 6-bromohexyl 2-hexyldecanoate 15 [5] (50 g, 61 % Yield) as an yellow liquid.
  • reaction mixture was allowed to stir at 90°C for 48 h. Reaction progress was monitored by TLC and ELSD. After completion of reaction, reaction mixture was diluted with diethyl ether (2x 50 mL) and washed with water (4x 50 mL). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure.
  • reaction mixture was diluted with diethyl ether (2x 40 mL), washed with cold water (2x 30 mL), saturated solution of sodium bicarbonate (2x 30 mL) and brine (20 mL). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure.
  • the crude was purified by silica gel flash column chromatography using (0-2 % MeOH in DCM) to 20 obtain 6- ⁇ [6-(1-hexylnonylcarbonyloxy)hexyl](3-hydroxypropyl)amino ⁇ hexyl 2-hexyldecanoate [7] (3.5 g, 89.6 % Yield) as yellow liquid.
  • reaction mixture was stirred at 0 °C for 2h. The progress of the reaction was monitored by TLC. After completion of reaction, reaction mixture was quenched with cool sodium bicarbonate solution (50 mL)) and extracted with DCM (2x 40 mL). The organic layer was dried over sodium sulfate and was evaporated under reduced pressure. The crude was purified by using silica gel combi flash chromatography (0-20% MeOH in DCM) to obtain 10 di(3- ⁇ bis[6-(1-hexylnonylcarbonyloxy)hexyl]amino ⁇ propyl) malonate [9] (1.3 g, 45. 6 % Yield) as yellow liquid.
  • the compound was prep-purified in Mobile phase: A: - 0.1% TFA in water ACN, B: ACN to obtain di(3- ⁇ bis[6-(1-hexylnonylcarbonyloxy)hexyl]amino ⁇ propyl) [(3- 15 indolyl)methyl]malonate (75 mg, 6.3 % Yield) as colourless liquid.
  • reaction was monitored by ELSD/TLC. After completion, reaction was quenched with ice cold water and 10 washed with brine solution. The organic layer was dried over sodium sulphate, evaporated under reduced pressure. The crude was purified by flash column chromatography (SiO 2 :10- 20% EtOAc in Hexane) to afford tert-butyl (3-(bis(2-((tert- butyldimethylsilyl)oxy)dodecyl)amino)propyl)carbamate [6] (5.0 g; 87.97 % Yield) as colourless liquid.
  • reaction mass was quenched with saturated solution of sodium bicarbonate (make pH 8 to 9) and extracted with DCM (2x100 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to obtain N 1 ,N 1 -bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)propane-1,3-diamine [7] (4.79 g; 30 98.83 % Yield) as pale yellow liquid.
  • reaction mixture was quench with ice cold water (300.0 mL), and extracted DCM 212 layer. The organic layer was wash with saturated brine solution, dried over sodium sulphate, filtered and concentrated under reduced pressure.
  • the crude was purified by flash column chromatography (0-30% Ethyl acetate in heptane) to afford 2-((tert-butyldimethylsilyl)oxy)-N- (2-((tert-butyldimethylsilyl)oxy)dodecyl)-N-(3-(trityloxy)propyl)dodecan-1-amine [13] (21.5 g; 5 80.48 % Yield) as a colourless liquid.
  • reaction mixture was quenched with saturated aq. NaHCO3 20 solution up pH 8.
  • the compound was extracted with DCM (3x250 mL). The combined organic layer was dried over anhy. Na 2 SO 4 , filtered and evaporated to get crude compound.
  • reaction mass was allowed to stir at room temperature for 16 h. The progress of reaction was monitored by TLC/ELSD/CAD. After completion, reaction mass was diluted with diethyl ether (30 mL). The ether layer was washed with cold water (2x10 mL) and then wash with brine solution (2x10 10 mL). Separated ether layer was washed with saturated solution of bicarbonate (2x 25 mL) and cold water (2x 25 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure.
  • Boc-anhydride (704 mg, 3.24 mmol) was added to the resulting reaction mixture at 0 oC then allowed to stir at room temperature for 16 h. The progress of reaction was monitored by LCMS/TLC. After completion, reaction mass was diluted with water (30 mL) and washed with diethyl ether (2x 30 mL) to remove impurity. The aqueous layer was collected maintained pH upto 6-7 by using aq. Citric acid and extracted with DCM (2x 50 mL).
  • reaction mass was allowed to stir at room temperature for 16 h. The progress of reaction was monitored by TLC/ELSD/CAD. After completion, reaction mass was diluted with diethyl ether (30 mL). The ether layer was washed with cold water (2x10 mL) and then wash with brine solution (2x10 mL). Separated ether layer was washed with saturated solution of bicarbonate (2x 25 mL) 10 and cold water (2x 25 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure.
  • reaction mixture 10 was allowed to stir at 60 °C for 16 hr. The progress of reaction was monitored by ELSD and TLC. After completion, reaction mass was quenched by water (15 mL) and extracted with ethyl acetate (2x 25 mL). The organic layers were collected, dried over sodium sulphate and concentrated under reduced pressure. The crude was purified by column chromatography (SiO2: 0-30 % ethyl acetate/heptane) to afford tert-butyl ⁇ [2-(benzyloxy)ethyl](tert- 15 butoxycarbonylmethyl)amino ⁇ acetate [12] (8.1 g; 64.55 % Yield) as pale yellow liquid.
  • reaction mass was degassed and purged with Hydrogen at room temperature, then allowed to stir under hydrogen atmosphere for 16 h. After completion, reaction mass was filtered through celite by sintered funnel using THF (150 mL). The organic layers was concentrated under reduced 5 pressure to get bis(3-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)propyl) 2,2'-((2- hydroxyethyl)azanediyl)diacetate [15] (1.88 g; 79.76 % Yield) as colourless liquid.
  • reaction mixture was diluted with diethyl ether (50 mL) and washed with cold water (2x10 mL). The diethyl ether layer was quenched with cold saturated 10 sodium bicarbonate solution up to pH 8. Separate the ether layer and wash with cold water (2x10 mL). The organic layer was dried over anhy. sodium sulphate and concentrate under reduced pressure.
  • the crude was purified by flash column chromatography (SiO 2 : 0-20 % methanol in DCM) to obtain 2-[bis( ⁇ 3-[bis(2- hydroxydodecyl)amino]propoxycarbonyl ⁇ methyl)amino]ethyl 2-fluoro-3-phenylpropionate 15 (0.33 g, 57.24 % Yield) as pale yellow liquid.
  • Cationic lipids described herein can be used in the preparation of lipid 30 nanoparticles according to methods known in the art.
  • suitable methods 227 include methods described in International Publication No.
  • WO 2018/089801 which is hereby incorporated by reference in its entirety.
  • the lipid nanoparticles in the examples of the present invention were formulated using Process A of WO 2018/089801 (see, e.g., Example 1 and Figure 1 of WO 5 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.
  • an ethanol lipid solution and an aqueous buffered solution of mRNA were prepared separately.
  • a solution of mixture of lipids was prepared by dissolving lipids in ethanol. 10
  • the mRNA solution was prepared by dissolving the mRNA in citrate buffer. 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. 15 [000585]
  • Lipid nanoparticle formulations of the Study Design Table below were prepared by Process A.
  • an ethanolic solution of a mixture of lipids including DMG-PEG-2000, cationic lipid, cholesterol, and phosphatidylethanolamine (DOPE) in a ratio of 1.5:40:28.5:30 were combined with an aqueous buffered solution (1 mM Citrate and 150 mM NaCl) of target mRNA at an acidic pH (4.5) under controlled conditions to yield 20 a dispersion of uniform lipid nanoparticles (LNPs).
  • LNPs uniform lipid nanoparticles
  • the resulting nanoparticle dispersions were diluted to required concentration using the final buffer (10% Trehalose), sterile filtered and stored frozen at -80 °C until use.
  • WO2022/099003 A1 describes an in vivo assay for intramuscular administration (e.g. on page 46, paragraph [00206]). Further details of the intramuscular experiments performed in this application are provided below.
  • Test Materials remained RNase free during loading into the syringe (as 5 applicable).
  • Test Article Class of Compound Oligonucleotides ABSL-1
  • Treatment Regimen On Day 1, animals from Groups 1 – 11 were dosed via intramuscular injection (30 ⁇ L/animal) while under light isoflurane anesthesia according to 10 the study design table above. Animals in Groups 2 - 11 were injected with EPO in the right gastrocnemius muscle. Group 1 animals received saline control. Study Animals Animals: 15 [000590] Acclimation: Animals were acclimatised to the Test Facility for at least 24 hours.
  • Housing All animals were socially housed in polycarbonate cages with contact bedding in an animal housing room. 20 [000592] Food and Water: Food (Envigo irradiated 2918 diet) and filtered tap water was provided to animals ad libitum. In-Life Observations and Measures 229 [000593] Animal Health Checks: At least once daily animals received a cage side health check observation. [000594] Clinical Observations: Clinical observations were performed for all animals on Day 1 prior to dose administration and prior to euthanasia. Clinical observations were 5 performed more often if abnormal clinical signs were exhibited by animals on study. [000595] Body Weights: Body weights were recorded prior to test material administration. Body weights were rounded to the nearest 0.1g.
  • Interim Sample Collections Interim whole blood ( ⁇ 50 ⁇ L) was collected by tail snip or saphenous vein at 6 hours post dose administration ( ⁇ 5%). Blood samples were 10 collected into serum separator tubes, allowed to clot at room temperature for at least 10 minutes, centrifuged at ambient temperature at minimum 1000g for 10 minutes and the serum was extracted. All serum samples were stored at nominally -70oC until analysis hEPO by the Testing Facility. The results of the EPO analysis were included in the Data submission. 15 In-Life Sample Collection Table No.
  • ELISA Assay Human erythropoietin (hEPO) levels in sera samples were determined by ELISA kit (R&D systems, Cat# DEP-00) according to the manufactory instruction and the results were included in the Data submission. The “shaker” protocol was used. The serum samples were diluted between 1:40 and 1:100. 25 Reporting and Data Retention [000598] Data submission: A tabulated data summary of animal assignment, individual and group means (as applicable) for times of dose administration and euthanasia, body 230 weights, clinical observations in-vitro analysis and mortality (as applicable) were delivered for this study.
  • Example 19 Cellular HA expression studies [000599] Lipid nanoparticles of Table E below including influenza A hemagglutinin mRNA and new cationic lipids described herein were produced by the methods described in 10 Example 18 and tested for induction of protein expression in human skeletal muscle cells.
  • Human skeletal muscle cells (primary cell line from Lonza Bioscience – CC 2561) were seeded the day prior to transfection (Day 0) post which they were incubated overnight at 37°C/5% CO2 in a humidified incubator. The following day (Day 1), formulations were thawed and diluted in relevant diluent to 10x final concentration before diluting to 1x in 20 cell growth medium. Post preparation of the final dilution mixture of LNP and growth media, 231 old media was aspirated from the plated cells and replaced by the treatment media comprising LNP at 0.1 mg/ ml mRNA concentration, following which they were allowed to stand for fifteen minutes to avoid edge effects.
  • the cationic lipid of numbered embodiment 1, or pharmaceutically acceptable salt thereof, wherein Y 1 is -OH. 15 6.
  • the cationic lipid of any one of numbered embodiments 1, 5 or 6, or pharmaceutically 20 acceptable salt thereof, wherein R 2 is -OH. 235 8.
  • the cationic lipid of numbered embodiment 19 wherein the cationic lipid has a structure according to Formula (IIA): Formula (IIA) 5 or a pharmaceutically acceptable salt thereof.
  • 21. The cationic lipid of numbered embodiment 19 or 20, wherein the cationic lipid has a structure according to Formula (IIA1): 10 Formula (IIA1) or a pharmaceutically acceptable salt thereof.
  • 22. The cationic lipid of any one of numbered embodiments 19 to 21, or pharmaceutically acceptable salt thereof, wherein e is from 2-4, for example wherein e is 3. 15 23.
  • R 7 is 5 selected from –(CH2)-optionally substituted aryl, -(CH2)2-optionally substituted aryl, -(CH2)3- optionally substituted aryl, –(CH2)4-optionally substituted aryl, –(CH2)-optionally substituted heteroaryl or -(CH2)2-optionally substituted heteroaryl.
  • R 7 is15 selected from –(CH 2 )-optionally substituted phenyl, -(CH 2 ) 2 -optionally substituted phenyl, - (CH 2 ) 3 -optionally substituted phenyl, –(CH 2 ) 4 -optionally substituted phenyl, –(CH 2 )-optionally substituted imidazolyl, -(CH 2 ) 2 -optionally substituted imidazolyl, –(CH 2 )-optionally substituted indolyl, -(CH 2 ) 2 -optionally substituted indolyl, or -(CH 2 ) 3 -optionally substituted indolyl.
  • the cationic lipid of numbered embodiment 57 wherein the cationic lipid has a structure according to Formula (IVA): 10 Formula (IVA) or a pharmaceutically acceptable salt thereof.
  • 15 71 The cationic lipid of any one of numbered embodiments 57-69, or pharmaceutically acceptable salt thereof, wherein R 12A and R 12C are the same and R 12B and R 12D are the same. 252 72.
  • 76. The cationic lipid of any one of numbered embodiments 73-75, wherein the cationic lipid has a structure according to Formula (VA1): 10 or a pharmaceutically acceptable salt thereof. 77.
  • the cationic lipid of any one of numbered embodiments 73-83, or pharmaceutically acceptable salt thereof, wherein R 14A , R 14B , R 14C , and R 14D are each independently selected from -W 1 -X 1 . 255 85.
  • each R 14A , R 14B , R 14C , and R 14D is independently selected from: 5 10 256 , 5 (xv ) , for example wherein each R 14A , R 14B , R 14C and R 14D is option (x), or wherein R 14A and R 14C are option (xv) and R 14B and R 14D are option (viii).
  • the cationic lipid of any one of numbered embodiments 88-94, or pharmaceutically acceptable salt thereof, wherein R 18A , R 18B , R 18C , and R 18D are each independently selected from -W 1 -X 1 . 20 260 96.
  • 15 99. A compound selected from those listed in (a) Table A and Table B, or a pharmaceutically acceptable salt thereof; or (b) Table A, Table B and Table C, or a pharmaceutically acceptable salt thereof.
  • 20 100. A composition comprising the cationic lipid of any one of the preceding numbered embodiments and further comprising (i) one or more non-cationic lipids, (ii) one or more cholesterol-based lipids, and 262 (iii) one or more PEG-modified lipids. 101.
  • composition of numbered embodiment 100 wherein the composition is a lipid nanoparticle, optionally a liposome. 5 102.
  • the composition of numbered embodiment 101 wherein the one or more cationic lipid(s) constitute(s) about 30 mol %-60 mol % of the lipid nanoparticle.
  • 103 The composition of numbered embodiment 101 or 102, wherein the one or more non- 10 cationic lipid(s) constitute(s) 10 mol %-50 mol % of the lipid nanoparticle.
  • 104 The composition of any one of numbered embodiments 101-103, wherein the one or more PEG-modified lipid(s) constitute(s) 1 mol %-10 mol % of the lipid nanoparticle. 15 105.
  • composition of any one of numbered embodiments 101-104, wherein the cholesterol-based lipid constitutes 10 mol %-50 mol% of the lipid nanoparticle.
  • 106. The composition of any one of numbered embodiments 101-105, wherein the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein. 20 107.
  • composition of numbered embodiment 107 wherein the lipid nanoparticles have 25 an encapsulation percentage for mRNA of (a) at least 70%; (b) at least 75%; (c) at least 80%; (d) at least 85%; 30 (e) at least 90%; or (f) at least 95%.
  • a method for treating or preventing a disease wherein said method comprises 15 administering to a subject in need thereof the composition of any one of numbered embodiments 107-108 and wherein the disease is amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
  • the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
  • composition is administered intranasally, intravenously, intrathecally or intramuscularly, or by pulmonary delivery, optionally through nebulization. 25 116. The method of numbered embodiment 114, wherein the composition is administered intramuscularly. 30

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Abstract

La présente invention concerne, en partie, des composés lipidiques cationiques de formules (I), (II), (III), (IV), (V) ou (VI) ou un sel pharmaceutiquement acceptable de ceux-ci. Les composés selon l'invention peuvent être utiles pour l'administration et l'expression d'ARNm et de protéine codée, par exemple , en tant que composant d'un véhicule d'administration liposomal, et peuvent par conséquent être utiles pour traiter diverses maladies, troubles et états, tels que ceux associés à une carence en une ou plusieurs protéines.
PCT/US2025/035368 2024-06-26 2025-06-26 Lipides cationiques de bis-ester et d'amide Pending WO2026006524A2 (fr)

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