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WO2016168469A1 - Fatty acid analogs and methods of use thereof - Google Patents

Fatty acid analogs and methods of use thereof Download PDF

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Publication number
WO2016168469A1
WO2016168469A1 PCT/US2016/027544 US2016027544W WO2016168469A1 WO 2016168469 A1 WO2016168469 A1 WO 2016168469A1 US 2016027544 W US2016027544 W US 2016027544W WO 2016168469 A1 WO2016168469 A1 WO 2016168469A1
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substituted
compound
acid
mir
alkyl
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PCT/US2016/027544
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French (fr)
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Fenyong Liu
Naresh Sunkara
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The Regents Of The University Of California
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Publication of WO2016168469A1 publication Critical patent/WO2016168469A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • C07C233/38—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 carbon atom of an acyclic unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • RNA interference or post-transcriptional gene silencing is a conserved biological response to a double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein coding genes. This mechanism of gene interference holds promise in developing therapeutics for cancers, viral infections and several genetic disorders.
  • the use of RNAi technologies is especially useful for targets that usually considered undruggable.
  • the present disclosure provides lipidoid compounds, and compositions comprising the lipidoid
  • the present disclosure provides methods of making a subject lipidoid.
  • the present disclosure provides a method of delivering a target nucleic acid to a cell, using a lipidoid composition of the present disclosure.
  • the present disclosure provides a method of delivering a gene product to an individual, using a lipidoid composition of the present disclosure.
  • Figures 1A-1E depict examples of lipidoids.
  • FIG. 2 is a graph showing knockdown of green fluorescent protein (GFP) expression via siRNA
  • lipidoid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
  • Figure 3 is a graph showing expression of GFP from a plasmid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
  • FIG. 4 is a graph showing knockdown of green fluorescent protein (GFP) expression via siRNA
  • lipidoid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
  • FIG. 5 is a graph showing knockdown of green fluorescent protein (GFP) expression via siRNA
  • lipidoid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
  • Figure 6 is a graph showing expression of GFP from a plasmid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
  • Figure 7 is a graph showing serum cytokine levels in mice injected intravenously with lipidoid
  • Figure 8 is a graph showing serum cytokine levels in mice injected intravenously with lipidoid
  • Figure 9 is a graph showing organ distribution and levels of an miRNA after intravenous administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
  • Figure 10 is a graph showing organ distribution and levels of an miRNA after intravenous administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
  • Figure 11 is a graph showing organ distribution and levels of an miRNA after intravenous administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
  • Figure 12 is a graph showing organ distribution and levels of an miRNA after intranasal administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
  • Figure 13 is a collection of images showing delivery and expression of a plasmid via intradermal
  • Alkylene refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more
  • This term includes, by way of example, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene
  • Substituted alkylene refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of "substituted” below.
  • alkane refers to alkyl group and alkylene group, as defined herein.
  • alkylaminoalkyl refers to the groups R NHR - where R is alkyl group as defined herein and R is alkylene, alkenylene or alkynylene group as defined herein.
  • alkaryl or "aralkyl” refers to the groups - alkylene- aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.
  • Alkoxy refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like.
  • alkoxy also refers to the groups alkenyl-O-, cycloalkyl-O, cycloalkenyl-O, and alkynyl-
  • alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
  • substituted alkoxy refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
  • alkoxyamino refers to the group -NH-alkoxy, wherein alkoxy is defined herein.
  • haloalkoxy refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.
  • haloalkyl refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group.
  • groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoro methyl, trifluoroethyl and the like.
  • alkylalkoxy refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-0 -alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
  • alkylthioalkoxy refers to the group -alkylene-S -alkyl, alkylene-S-substituted alkyl, substituted alkylene-S -alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
  • Alkenyl refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-l-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.
  • substituted alkenyl refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxy
  • Alkynyl refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C ⁇ CH), and propargyl (-CH 2 C ⁇ CH).
  • substituted alkynyl refers to an alkynyl group as defined herein having from 1 to 5
  • substituents or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
  • Alkynyloxy refers to the group -O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.
  • Acyl refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkenyl-C
  • Acylamino refers to the groups -NR 20 C(O)alkyl, -NR 20 C(O)substituted alkyl, N R 20 C(O)cycloalkyl, - NR 20 C(O)substituted cycloalkyl, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)substituted cycloalkenyl, - NR 20 C(O)alkenyl, -NR 20 C(O)substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O)substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O)substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O)substituted heteroaryl,
  • R 20 is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
  • aminocarbonyl or the term “aminoacyl” refers to the group -C(0)NR 21 R 22 , wherein R 21 and R 22
  • Aminocarbonylamino refers to the group -NR 21 C(0)NR 22 R 23 where R 21 , R 22 , and R 23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.
  • alkoxycarbonylamino refers to the group -NRC(0)OR where each R is independently
  • alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
  • acyloxy refers to the groups alkyl-C(0)0-, substituted alkyl-C(0)0-, cycloalkyl-C(0)0-, substituted cycloalkyl-C(0)0-, aryl-C(0)0-, heteroaryl-C(0)0-, and heterocyclyl-C(0)0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
  • Aminosulfonyl refers to the group -SO 2 NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,
  • Sulfonylamino refers to the group -NR 21 SO 2 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 21 and R 22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted cyclo
  • Aryl or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl.
  • such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thi
  • Aryloxy refers to the group -O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.
  • Amino refers to the group -NH 2 .
  • substituted amino refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.
  • azido refers to the group -N 3 .
  • Carboxyl refers to -C0 2 H or salts thereof.
  • Carboxyl ester or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(0)0-alkyl, -C(0)0-substituted alkyl, -C(0)0-alkenyl, -C(0)0-substituted alkenyl, -C(0)0-alkynyl, -C(0)0-substituted alkynyl, -C(0)0-aryl, -C(0)0-substituted aryl, -C(0)0-cycloalkyl,
  • alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
  • (Carboxyl ester)oxy refers to the groups -0-C(0)0-alkyl, -(D-C(O)O-substituted alkyl, - 0-C(0)0-alkenyl, -0-C(0)0-substituted alkenyl, -0-C(0)0-alkynyl, -0-C(0)0-substituted alkynyl, -O- C(0)0-aryl, -0-C(0)0-substituted aryl, -0-C(0)0-cycloalkyl, -0-C(0)0-substituted cycloalkyl, -O- C(0)0-cycloalkenyl, -0-C(0)0-substituted cycloalkenyl, -0-C(0)0-heteroaryl, -0-C(0)0-substituted heteroaryl, -(D-C(O)O-heterocyclic, and -(D-C(O)O
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, he tero aryloxy, heterocyclyl,
  • Cycloalkenyl refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.
  • substituted cycloalkenyl refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamin
  • Cycloalkynyl refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.
  • Cycloalkoxy refers to -O-cycloalkyl
  • Cycloalkenyloxy refers to -O-cycloalkenyl.
  • Halo or "halogen” refers to fluoro, chloro, bromo, and iodo.
  • Heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic and at least one ring within the ring system is aromatic , provided that the point of attachment is through an atom of an aromatic ring.
  • the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N ⁇ 0), sulfinyl, or sulfonyl moieties.
  • This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thio
  • heteroaryl refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.
  • Heteroaryloxy refers to -O-heteroaryl.
  • Heterocycle refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms.
  • These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non- aromatic ring.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -S0 2 - moieties.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
  • heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,
  • Heterocyclyloxy refers to the group -O-heterocyclyl.
  • heterocyclylthio refers to the group heterocyclic-S-.
  • heterocyclene refers to the diradical group formed from a heterocycle, as defined herein.
  • hydroxyamino refers to the group -NHOH.
  • Neitro refers to the group -N0 2 .
  • Sulfonyl refers to the group S0 2 -alkyl, S0 2 -substituted alkyl, S0 2 -alkenyl, S0 2 -substituted alkenyl, S0 2 -cycloalkyl, S0 2 -substituted cylcoalkyl, S0 2 -cycloalkenyl, S0 2 -substituted cylcoalkenyl, S0 2 -aryl, S0 2 -substituted aryl, S0 2 -heteroaryl, S0 2 -substituted heteroaryl, S0 2 -heterocyclic, and S0 2 -substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, wherein alkyl,
  • Sulfonyloxy refers to the group -OS0 2 -alkyl, OS0 2 -substituted alkyl, OS0 2 -alkenyl, OS0 2 - substituted alkenyl, OS0 2 -cycloalkyl, OS0 2 -substituted cylcoalkyl, OS0 2 -cycloalkenyl, OS0 2 - substituted cylcoalkenyl, OS0 2 -aryl, OS0 2 -substituted aryl, OS0 2 -heteroaryl, OS0 2 -substituted heteroaryl, OS0 2 -heterocyclic, and OS0 2 substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted alkyl,
  • aminocarbonyloxy refers to the group -OC(0)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • Thiol refers to the group -SH.
  • Alkylthio or the term “thioalkoxy” refers to the group -S -alkyl, wherein alkyl is as defined herein.
  • sulfur may be oxidized to -S(O)-.
  • the sulfoxide may exist as one or more stereoisomers.
  • substituted thioalkoxy refers to the group -S-substituted alkyl.
  • thioaryloxy refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.
  • heteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.
  • heterocyclooxy refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.
  • substituted when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
  • Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 ) or an alkaline earth ion, such as [Ca [Mg or [Ba ("subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the present disclosure and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the present disclosure can serve as the counter ion for such divalent alkali earth ions).
  • an alkali ion such as K + , Na + , Li +
  • an ammonium ion such as + N(R 60 )
  • an alkaline earth ion such as [Ca [Mg or [Ba
  • subscript 0.5 means that
  • -NR 80 R 80 is meant to include -NH 2 , -NH-alkyl, ⁇ -pyrrolidinyl, ⁇ -piperazinyl, 4,/V-methyl-piperazin- 1 -yl and ⁇ -morpholinyl.
  • substituted alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -0 " M + , -OR 70 , -SR 70 , -S " M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -N0 2 , -N 3 , -S0 2 R 70 , -S0 3 " M + , -SO 3 R 70 , -OS0 2 R 70 , -OS0 3 " M + , -OSO 3 R 70 , -P0 3 "2 (M + ) 2 , -P(O)(OR 70 )O " M + , -P(O)(OR 70 ) 2 , -C(0)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C0 2
  • substituent groups for hydrogens on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -0 " M + , -OR 70 , -SR 70 , -S " M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -N0 2 , -S(0) 2 R 70 , -S(0) 2 O M + , -S(0) 2 OR 70 , -OS(0) 2 R 70 , -OS(0) 2 O M + , -OS(0) 2 OR 70 , -P(0)(0 ) 2 (M + ) 2 , -P(O)(OR 70 )O " M + , -P(O)(OR 70 )(OR 70 ), -C(0)R 70 , -C(S)R 70 , -C
  • a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
  • any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
  • salt means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime).
  • Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
  • “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
  • salt thereof means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like.
  • the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient.
  • salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
  • solvent refers to a complex formed by combination of solvent molecules with molecules or ions of the solute.
  • the solvent can be an organic compound, an inorganic compound, or a mixture of both.
  • Some examples of solvents include, but are not limited to, methanol, ⁇ , ⁇ -dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
  • Stereoisomers refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
  • pyrazoles imidazoles, benzimidazoles, triazoles, and tetrazoles.
  • “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a
  • a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.
  • treating means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
  • Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 ) 3 CCH 2 - ) ⁇
  • substituted alkyl refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -0-, -N-, -S-, -S(0) n - (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
  • R and R may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
  • the present disclosure provides lipidoid compounds, and compositions comprising the lipidoid
  • the present disclosure provides methods of making a subject lipidoid.
  • the present disclosure provides a method of delivering a target nucleic acid to a cell, using a lipidoid composition of the present disclosure.
  • the present disclosure provides a method of delivering a gene product to an individual, using a lipidoid composition of the present disclosure.
  • a lipidoid compound of the present disclosure may include a fatty acid conjugated to a charged linker.
  • the linker includes one, two or more positively charged groups capable of electrostatic interaction with a nucleic acid of interest.
  • the lipidoid includes a positively charged divalent linker that is conjugated to two fatty acids.
  • the linker is a branched multivalent linker that is conjugated to three or more fatty acid groups.
  • the positively charged group(s) of the linker are amino groups, such as secondary or tertiary amino groups which may be positively charged in an aqueous environment.
  • the positively charged group(s) of the linker are ammonium groups.
  • lipidoid compound of the present disclosure is described by the formula (I):
  • n is 0 or an integer of 1 to 6;
  • Y is selected from the group consisting of hydrogen, alkyl, substituted alkyl or a fatty acid-containing group described by the formula (II):
  • p is 0 or an integer of 1-6;
  • L 1 , L 2 and L 3 are each independently derived from a fatty acid (such as an essential fatty acid); T 1 , T 2 and ⁇ 3 are each independently a linker;
  • Z is N, sulfonium (i.e., S + ) or phosphonium (i.e., PR + );
  • R 1 , R 2 and R 3 are each independently hydrogen, an alkyl or a substituted alkyl.
  • m is 0. In certain embodiments of formula (I), m is 1. In certain embodiments of formula (I), m is 2. In certain embodiments of formula (I), m is 3. In certain embodiments of formula (I), m is 4. In certain embodiments of formula (I), m is 5. In certain embodiments of formula
  • Z is N. In certain embodiments of formula (I), Z is sulfonium. In certain embodiments of formula (I), Z is phosphonium. In certain embodiments of formula (I), Y is H. In certain embodiments of formula (I), Y is an alkyl. In certain embodiments of formula (I), Y is methyl. In certain embodiments of formula (I), Y is a substituted alkyl. In certain embodiments of formula (I), R 1 and R 2 are each hydrogen. In certain embodiments of formula (I), R 1 and R 2 are each an alkyl. In certain embodiments of formula (I), R 1 and R 2 are each methyl. In certain embodiments of formula (I), R 1 and R 2 are each a substituted alkyl.
  • Y is a fatty acid-containing group described by the formula (II).
  • p is 0. In certain embodiments of formula (II), p is 1. In certain embodiments of formula (II), p is 2. In certain embodiments of formula (II), p is 3. In certain embodiments of formula (II), p is 4. In certain embodiments of formula (II), p is 5. In certain embodiments of formula
  • R 3 is hydrogen. In certain embodiments of formula (II), R 3 is an alkyl. In certain embodiments of formula (II), R 3 is methyl. In certain embodiments of formula (II), R 3 is a substituted alkyl.
  • T 1 , T 2 and T 3 are each independently a linker (e.g., as described herein).
  • linker refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length.
  • a linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom.
  • one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom.
  • the bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group.
  • a linker may include, without limitations, poly(ethylene glycol); ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (iso- propyl), n-butyl, n-pentyl, 1 , 1 -dimethylethyl (t-butyl), and the like.
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
  • a linker may be cleavable or non-cleavable.
  • a linker includes a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • linker includes an alkyl or substituted alkyl group.
  • linker includes an alkenyl or substituted alkenyl group.
  • linker includes an alkynyl or substituted alkynyl group. In certain embodiments, linker includes an alkoxy or substituted alkoxy group. In certain embodiments, linker includes an amino or substituted amino group. In certain embodiments, linker includes a carboxyl or carboxyl ester group. In certain embodiments, linker includes an acyl amino group. In certain embodiments, linker includes an alkylamide or substituted alkylamide group. In certain embodiments, linker includes an aryl or substituted aryl group. In certain embodiments, linker includes a heteroaryl or substituted heteroaryl group. In certain embodiments, linker includes a cycloalkyl or substituted cycloalkyl group. In certain embodiments, linker includes a heterocyclyl or substituted heterocyclyl group.
  • linker includes a polymer.
  • the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like.
  • the polymer is a polyalkylene glycol.
  • the polymer is a polyethylene glycol.
  • Any convenient fatty acids may be utilized in the preparation of the subject lipidoid compounds.
  • L 1 , L 2 and L 3 may be independently derived from any convenient fatty acids.
  • Fatty acids of interest include, but are not limited to, short chain polyunsaturated fatty acids such as omega-3 fatty acids and omega-6 fatty acids, long-chain polyunsaturated fatty acids such as omega-9 fatty acids.
  • “Fatty acids” refer to a family of carboxylic acids having a hydrocarbon chain of from about 12 to about 24 carbons in length. Unsaturated fatty acids have at least one carbon-carbon double bond in the hydrocarbon chain. Unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs).
  • Unsaturated fatty acids are designated by the position of the first double bond from the methyl end of the hydrocarbon chain.
  • Omega-3 fatty acids have a first double bond at the third carbon from the methyl end of the chain; and include, e.g., a-linolenic acid (octadeca-9, 12,15-trienoic acid), stearidonic acid (octadeca-6,9,12,15-tetraenoic acid), eicosapentaenoic acid (eicosa-5, 8,11, 14,17- pentaenoic acid; "EPA"), docosapentaenoic acid (docosa-7, 10, 13, 16, 19-pentaenoic acid), eicosatetraenoic acid (eicosa-8, 11,14,17-tetraenoic acid), and docosahexaenoic acid (docosa-4,7, 10,13, 16, 19-hexaenoic acid; "D
  • Omega-6 fatty acids have a first double bond at the sixth carbon from the methyl end of the chain; and include, e.g., linoleic acid (9, 12-octadecadienoic acid), ⁇ -linolenic acid (6,9, 12-octadecatrienoic acid; GLA), eicosadienoic acid (11, 14-eicosadienoic acid), dihomo-y-linolenic acid (8,11,14- eicosatrienoic acid), arachidonic acid (5,8, 11,14-eicosatetraenoic acid), docosadienoic acid (13,16- docosadienoic acid), adrenic acid (7,10,13,16-docosatetraenoic acid), docosapentaenoic acid
  • Omega-9 fatty acids have a first double bond at the ninth carbon from the methyl end of the chain; and include, e.g., oleic acid (c «-9-octadecenoic acid); eicosenoic acid (cis-l 1-eicosenoic acid); mead acid (all- cis-5,8,11-eicosatrienoic acid); erucic acid (c «-13-docosenoic acid); and nervonic acid (cis-15- tetracosenoic acid).
  • lipoic acid refers to a-lipoic acid, which is a chiral molecule also known as thioctic acid; l,2-diethylene-3 pentanoic acid; l,2-diethylene-3 valeric acid; and 6,8-thioctic acid. Unless specified the term “lipoic acid” encompasses the racemic mixture as well as any other (non-50/50) mixture of the enantiomers including substantially pure forms of either the -(+) or the S-(-) enantiomer.
  • L 1 , L 2 and L 3 are independently derived from an essential fatty acid.
  • An essential fatty acid is a fatty acid that a human or other animal must ingest in its diet because the fatty acid is not synthesized in vivo.
  • Suitable fatty acids include, but are not limited to, eicosapentaenoic acid, octadecanoic acid, eicosatetraenoic acid, docosahexaenoic acid, arachidonic acid, calendic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosoapentaenoic acid,tetracosatetraenoic acid, tetracosapentaenoic acid, hexadecatrienoic acid, stearidonic acid, eicosatrienoic acid, tetracosahexaenoic acid, 5-Dodecenoic acid, 7-tetradecenoic acid, 9-hexadecenoic acid, 11- octadecenoic acid, 13-eicosenoic acid, 15-decosenoic acid, elaidic acid, gonodoic
  • the essential fatty acid is an omega-3 fatty acid. In certain embodiments, the essential fatty acid is an omega-6 fatty acid. In some embodiments, the essential fatty acid is selected from the group consisting of linoleic acid, linolenic acid and oleic acid. In certain embodiments, the essential fatty acid is linoleic acid. In certain embodiments, the essential fatty acid is linolenic acid. In certain embodiments, the essential fatty acid is oleic acid.
  • the lipidoid compound is described by the formula (III):
  • Z is N.
  • Y is H.
  • Y is an alkyl.
  • Y is methyl.
  • Y is a substituted alkyl.
  • R 1 and R 2 are each hydrogen.
  • R 1 and R 2 are each an alkyl.
  • R 1 and R 2 are each methyl.
  • R 1 and R 2 are each a substituted alkyl.
  • Z is sulfonium. In some embodiments of Formula (III), Z is S + and Y is alkyl or substituted alkyl. In some embodiments of Formula (III), Z is phosphonium. In some embodiments of Formula (III), Z is P + and Y is two groups, each group independently an alkyl or a substituted alkyl, e.g., -ZY- is -P + R 2 - In some embodiments of Formula (III), Y is a fatty acid-containing group described by formula (II).
  • the lipidoid compound is described by the formula (IV):
  • L 1 , V2, R 1 , R2% T 1 and 2 are as described for formula (I); and R 4 is H, an alkyl or a substituted alkyl.
  • R 4 is H.
  • R 4 is an alkyl.
  • R 4 is methyl.
  • R 4 is a substituted alkyl.
  • the lipidoid compound is described by the formula (V):
  • L 1 - L 3 , R 1 - R 3 and T 1 - T 3 are as described for formulae (I) and (II).
  • L 1 - L 3 are each independently selected from the group consisting of linoleic acid, linolenic acid and oleic acid. In certain embodiments, L 1 - L 3 are each linoleic acid. In certain embodiments, L 1 - L 3 are each linolenic acid. In certain embodiments, L 1 - L 3 are each oleic acid. In some embodiments of Formulae (IV) and (V), T 1 - T 3 are each independently a Ci_ 6 alkyl.
  • T 1 - T 3 are each independently -(CH 2 ) n -, where n is an integer from 1 to 6. In certain instances, n is 2. In certain instances, n is 3. In certain instances, n is 4. In certain instances, n is 5. In certain instances, n is 6.
  • R 1 - R 4 are each independently hydrogen or methyl. In some embodiments of Formulae (IV) and (V), R : -R 3 are each H and R 4 is methyl.
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure: NKS09
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure: NKS 16
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure: NKS20
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure:
  • the lipidoid compound has the following structure: NKS36
  • the lipidoid compound has the following structure:
  • the present disclosure includes a lipoid composition that contains a lipoid compound, as described above, wherein the lipoid compound is non-covalently bound to a target nucleic acid.
  • Non-covalent binding of the lipidoid compound and the target nucleic acid may be via any convenient non-covalent interactions, such as electrostatic interactions.
  • the target nucleic acid is a target RNA.
  • the target nucleic acid is selected from the group consisting of: a DNA, an siRNA, an shRNA, a miRNA, and an antisense nucleic acid.
  • the DNA includes a nucleotide sequence encoding a gene product.
  • the gene product is a polypeptide.
  • the polypeptide is a therapeutic polypeptide.
  • the gene product is an RNA.
  • lipidoid compound further comprising nanoparticles of the lipidoid compound.
  • Any convenient lipidoid nanoparticles may be prepared from the subject lipidoid compounds and utilized in the subject compositions. Methods and materials that may find use in nanoparticles of the subject lipidoid compounds include, but are not limited to, those nanoparticle methods and materials described by Whitehead et al. in "Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity", Nature
  • the present disclosure includes a lipoid composition that comprises a lipoid compound, as described above, wherein the lipoid compound is non-covalently bound to a target nucleic acid.
  • the nucleic acid may be any nucleic acid suitable for non-covalent binding to the present lipoid compounds.
  • the target nucleic acid contains a target RNA (e.g., RNA encoding a polypeptide, a regulatory or inhibitory RNA, a ribozyme, a Ul adaptor, riboswitches, etc.), or a target DNA (e.g., DNA encoding a gene product such as a polypeptide or a regulatory RNA, or a DNA encoding an inhibitory RNA etc.).
  • the target nucleic acid contains a nucleic acid aptamer.
  • the target nucleic acid contains a binding partner for a binding moiety.
  • a target nucleic acid can be a guide RNA that comprises: i) a nucleotide sequence that binds to an endonuclease, such as a CRISPR/Cas9 endonuclease; and ii) a nucleotide sequence that binds a target DNA sequence, e.g., a DNA sequence in the genome of a cell.
  • an endonuclease such as a CRISPR/Cas9 endonuclease
  • a target DNA sequence e.g., a DNA sequence in the genome of a cell.
  • the target DNA may be linear (e.g., a polymerase chain reaction (PCR) product, a linearized plasmid, etc.) or circular (e.g., a plasmid, a cosmid, etc.).
  • the target nucleic acid may be single stranded or double stranded (partially or completely).
  • the length of the target nucleic acid may be any length suitable for non-covalently binding to the present lipoid compounds and for performing the function of the target nucleic acid in a target cell.
  • the length of the target nucleic acid is 5 nucleotides or more, e.g., 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 25 nucleotides or more, 30 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 500 nucleotides or more, 1,000 nucleotides or more, 3,000 nucleotides or more, 5,000 nucleotides or more, 8,000 nucleotides or more, or 10,000 nucleotides or more, and in some cases is 50,000 nucleotides or less, 20,000 nucleotides or less, 8,000 nucleotides or less, 6,000 nucleotides or less, 4,000 nucleotides or less, 2,000 nucleotides or less, 1,000 nucleotides or less, 750 nucleotides or less, 500 nucleotides or less, 250 nucleotides or less, 100 nucleotides
  • the length of the target nucleic acid is in the range of 10 to 50,000 nucleotides, e.g., 10 to 20,000 nucleotides, 15 to 10,000 nucleotides, 15 to 6,000 nucleotides, 10 to 1,000 nucleotides, 15 to 500 nucleotides, 18 to 200 nucleotides, 1,000 to 30,000 nucleotides, 2,000 to 10,000 nucleotides, including 3,000 to 8,000 nucleotides.
  • the target nucleic acid contains a therapeutic target nucleic acid (e.g., DNA
  • DNA encoding a therapeutic polypeptide, DNA encoding a therapeutic regulatory or inhibitory RNA, a therapeutic regulatory or inhibitory RNA, etc.).
  • the target nucleic acid contains a target RNA (e.g., RNA encoding a polypeptide, a regulatory or inhibitory RNA, a ribozyme, a Ul adaptor, riboswitches, etc.).
  • a target RNA e.g., RNA encoding a polypeptide, a regulatory or inhibitory RNA, a ribozyme, a Ul adaptor, riboswitches, etc.
  • target RNAs suitable for non-covalently binding to the present lipoid compounds include, but are not limited to, micro RNA (miRNA; including pri-miRNA and pre-miRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), Ul adaptors, ribozymes, riboswitches.
  • the target nucleic acid contains an siRNA.
  • Small interference RNA is a double-stranded RNA containing a sense strand having a base sequence corresponding to a part of a target gene and an antisense strand thereof.
  • siRNA can induce sequence-specific post-transcriptional gene silencing (i.e., RNA interference) in cells (e.g., eukaryotic cells) (See, e.g., Fire A. et al., 1998, Nature, 391, 806-811).
  • siRNA contained in the lipoid composition may include a base sequence that is fully complementary to a region of the sense strand of a target gene. The length of the complementarity may be 17 to 32 bases, e.g., 18 to 30 bases, or 19 to 25 bases.
  • the siRNA may target any suitable gene for silencing in a target cell. Any suitable method may be used to design the siRNA based on a target gene sequence. Suitable methods are described in, e.g., Ui-Tei et al. (Nucleic Acids Res., 32: 936-948, 2004); Reynolds et al. (Nat. Biotechnol., 22: 326-330, 2004); and Amarzguioui et al. (Biochem. Biophys. Res. Commun., 316: 1050-1058, 2004), which are incorporated herein by reference.
  • web sites on which siRNA can be designed have been made available to public by a variety of research institutes or companies, and effective siRNA can be designed on the web. Representative examples of siRNA designing web sites include siDirect
  • the siRNA may further include other functional nucleic acids, such as RNA
  • aptamers or single-stranded miRNA precursors are aptamers or single-stranded miRNA precursors.
  • the siRNA included in the target nucleic acid of the present composition may target any suitable gene.
  • Target genes include any gene encoding a target gene product (RNA or protein) that is deleterious (e.g., pathological); a target gene product that is malfunctioning; a target gene product.
  • Target gene products include, but are not limited to, huntingtin; hepatitis C virus; human immunodeficiency virus; amyloid precursor protein; tau; a protein that includes a polyglutamine repeat; a herpes virus (e.g., varicella zoster); any pathological virus; and the like.
  • siRNA is useful for treating a variety of disorders and conditions, including, but not limited to,
  • neurodegenerative diseases e.g., a trinucleotide-repeat disease, such as a disease associated with polyglutamine repeats, e.g., Huntington's disease , spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), etc.; an acquired pathology (e.g., a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural, or molecular biological state) such as a viral infection, e.g., hepatitis that occurs or may occur as a result of an HCV infection, acquired immunodeficiency syndrome, which occurs as a result of an HIV infection; and the like.
  • a trinucleotide-repeat disease such as a disease associated with polyglutamine repeats, e.g., Huntington's disease , spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA
  • an siRNA is directed against one or more members of the following classes of proteins: developmental proteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., adhesion
  • polygalacturonases polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases).
  • an siRNA is directed against a member of a signal transduction pathway, e.g., the insulin pathway, including AKT1-3, CBL, CBLB, EIF4EBP1, FOXOIA, FOX03A, FRAP1, GSK3A, GSK3B, IGF1, IGF1R, INPP5D, INSR, IRS 1, MLLT7, PDPK1, PIK3CA, PIK3CB, PIK3R1, PIK3R2, PPP2R2B, PTEN, RPS6, RPS6KA1, RPX6KA3, SGK, TSC1 , TSC2, and XPOl); an apoptotic pathway (CASP3,6,7,8,9, DSH1/2, PI 10, P85, PDK1/2, CATENIN, HSP90, CDC37, P23, BAD, BCLXL, BCL2, SMAC, and others); and pathways involved in DNA damage, cell cycle, and the like (p53, M
  • genes involved in immune system function including TNFR1 , IL-IR, IRAKI/2, TRAF2, TRAF6, TRADD, FADD, ⁇ , ⁇ , ⁇ , ⁇ , IkBa, IkB , p50, p65, Rac, RhoA, Cdc42, ROCK, Pakl/2/3/4/5/6, cIAP, HDACl/2, CBP, ⁇ -TrCP, Rip2/4, and others are also important targets for siRNAs, where such siRNAs can be useful in treating immune system disorders.
  • siRNAs specific for gene products involved in apoptosis such as Dshl 2, PTEN, PI 10 (pan), P85, PDK1/2, Aktl , Akt2, Akt (pan), p70 s6 , GSK3 , PP2A (cat), ⁇ -catenin, HSP90, Cdc37/p50, P23, Bad, BclxL, Bcl2, Smac/Diablo, and Askl are useful in the treatment of diseases that involve defects in programmed cell death (e.g. in the treatment of cancer).
  • siRNA directed against p53, MDM2, Chkl 2, BRCAl/2, ATM, ATR, p ⁇ 5 m ⁇ P27, P21 , Skp2, Cdc25C/A, 14-3-3sigma/8, PLK, Rb, Cdk4, Glut4, iNOS, mTOR, FKBP, PPARy, RXRa, ERa, and related genes can be used to treat diseases associated with disruptions in DNA repair, and cell cycle abnormalities, where such diseases include cancer.
  • Examples of such siRNAs and targets are known in the art; see, e.g., US Patent Publication No. 2005/0246794 and 2011/0003704.
  • a target DNA that includes a nucleic acid encoding an siRNA is useful for treating disorders resulting from or associated with dysregulated cell cycle, e.g., cancer.
  • an siRNA is directed against transcription elongation factors, such as CDK9, cyclin Tl, Spt4, Spt5, Spt6.
  • the target nucleic acid contains an miRNA.
  • miRNA is a single- stranded non-coding RNA that is 21 to 23 bases in length, is present in vivo, and regulates the expression of a given gene.
  • Such RNA is known to form a complex by binding to mRNA of a target gene and a protein factor and to inhibit the translation of the target gene.
  • Endogenous miRNA may be transcribed from the genome as a single-stranded precursor referred to as pri-miRNA, further processed into a single- stranded precursor referred to as pre-miRNA with the use of an endonuclease referred to as Drosha in the nucleus, and converted into mature double-stranded miRNA by the action of an endonuclease referred to as Dicer outside the nucleus.
  • One strand thereof is incorporated into an RISC (RNA-induced silencing complex) and regulates the expression of the target gene as a mature single-stranded miRNA.
  • RISC RNA-induced silencing complex
  • a target nucleic acid may contain a mature double-stranded miRNA that has a base sequence identical to that of wild-type mature double-stranded miRNA. In such a case, such sequence may be designed based on the base sequence of miRNA encoded in the genome.
  • a target nucleic acid may contain a sequence of a single-stranded miRNA precursor that has the same base sequence as that of wild-type miRNA encoded in the genome.
  • a miRNA included in the target nucleic acid of the present composition may be any suitable miRNA.
  • miRNAs include, but are not limited to, miR-34, miR-124, miR-155, miR-181, miR-221, miR- 122a, miR-32, miR-20a, miR-34a, miR-27b, miR-17-5p, miR-29a, miR-29b, miR-29c, miR-149, miR- 324-5p, miR-378, let-7a, let-7b, let-7c, let-7e, let-7f, let-7i, miR-101, miR-103, miR-106a, miR-lOa, miR- 10b, miR-124a, miR-125a, miR-126, miR-132, miR-133a, miR-141, miR-146a, miR-146b, miR-148a, miR-148b, miR-151, miR-152, miR-15a, miR-15b, miR-181b, miR-182, miR-183, miR-18a
  • the target nucleic acid contains an shRNA.
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA
  • a sense region is base-paired with an antisense region to form a stem structure in a molecule, and the spacer sequence forms a loop structure therein.
  • an shRNA molecule has a hairpin-shaped stem-loop structure as a whole.
  • a spacer sequence may be 3 to 24 bases long, such as 4 to 15 bases long.
  • a spacer sequence may be any suitable sequence, provided that the siRNA or mature double-stranded miRNA is capable of base pairing.
  • the shRNA may conveniently include any siRNA targeting a gene, as described above, or include any suitable miRNA, as described above.
  • the target nucleic acid is a target DNA (e.g., DNA encoding a gene product such as a polypeptide or a regulatory RNA, or a DNA encoding an inhibitory RNA etc.).
  • a target DNA e.g., DNA encoding a gene product such as a polypeptide or a regulatory RNA, or a DNA encoding an inhibitory RNA etc.
  • the target nucleic acid contains a ribozyme or deoxyribozyme.
  • ribozyme refers to RNA having catalytic functions, e.g., specifically cleaving a target RNA sequence
  • deoxyribozyme refers to DNA having catalytic functions, e.g., specifically cleaving a target RNA sequence.
  • a ribozyme may be constituted in the form of single-stranded RNA
  • a deoxyribozyme may be constituted in the form of single-stranded DNA.
  • the target nucleic acid may contain any suitable ribozyme or deoxyribozyme for use in the present lipoid composition.
  • a ribozyme may be a hammerhead ribozyme, a VS ribozyme, a Leadzyme, a hairpin ribozyme, etc.
  • Exemplary ribozymes include, but are not limited to, hammerhead ribozymes configured to target HIV-1 tat/vpr RNA, hammerhead ribozymes configured to target surviving mRNA, and hairpin ribozymes targeting 5'- and 3'-untranslated regions (UTRs) of hepatitis C virus (HCV), etc.
  • the target nucleic acid contains an Ul adaptor.
  • a "Ul adaptor” is a bifunctional single-stranded nucleic acid of about 25 bases, and it includes a 5 '-"target domain" complementary to the 3 '-terminal exon in the mRNA precursor of the target gene and a 3'-"Ul domain” having a sequence complementary to the 5' region of Ul snRNA (Goraczniak R. et al., 2009, Nat. Biotechnol., Vol. 27, pp. 257-263).
  • a Ul snRNP containing Ul snRNA binds to a region in the vicinity of a poly A signal of the mRNA precursor of the target gene, and polyadenylation of such mRNA is specifically inhibited.
  • the mRNA precursor of the target gene is destabilized and degraded in the nucleus.
  • the Ul adaptor may target any convenient gene, such as those targeted by siRNA described above.
  • the target nucleic acid contains a nucleic acid aptamer.
  • nucleic acid aptamer refers to a nucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of the target molecule.
  • the nucleic acid aptamer may be an RNA aptamers or a DNA aptamer.
  • the nucleic acid aptamer non-covalently bound to the lipoid compounds may contain DNA, RNA, or a combination thereof.
  • a nucleic acid aptamer may have a higher specificity and affinity to a target molecule than an antibody. In some cases, the nucleic acid aptamer can specifically bind to a target molecule with high affinity. Thus, the nucleic aptamer may enable selective suppression of functions of a given protein among highly homologous proteins.
  • any suitable nucleic acid aptamer may be used in the present lipoid composition for binding non- covalently to the lipoid compounds.
  • Various nucleic acids as well as methods of generating target-specific aptamers are known. See, e..g, Jayasena, 1999, Clin. Chem. 45: 1628-1650, which is incorporated herein by reference.
  • an RNA aptamer may be prepared via in vitro selection making use of the systematic evolution of ligands by exponential enrichment (SELEX) method.
  • the SELEX method comprises selecting an RNA molecule bound to a target molecule from an RNA pool composed of RNA molecules each having random sequence regions and primer-binding regions at both ends thereof, amplifying the recovered RNA molecule via RT-PCR, performing transcription using the obtained cDNA molecule as a template, and using the resultant as an RNA pool for the subsequent procedure. Such procedure is repeated several times to several tens of times to select RNA with a stronger ability to bind to a target molecule.
  • the base sequence lengths of the random sequence region and the primer binding region are not particularly limited. In general, the random sequence region comprises 20 to 80 bases and the primer binding region comprises 15 to 40 bases.
  • Specificity to a target molecule may be enhanced by prospectively mixing molecules similar to the target molecule with RNA pools and using a pool comprising RNA molecules that did not bind to the molecule of interest.
  • An RNA molecule that was obtained as a final product by such technique is used as an RNA aptamer.
  • the SELEX method is a known technique, and a specific method may be implemented in accordance with, for example, Pan et al. (Proc. Natl. Acad. Sci. U.S.A., 1995, 92: 11509-11513), incorporated herein by reference.
  • the target nucleic acid is a binding partner for a binding moiety.
  • the target nucleic acid may contain a nucleotide sequence that is specifically bound by a binding moiety, such as a nucleic acid-binding protein, other nucleic acids, or small molecules.
  • a binding moiety may include a transcription factor, a restriction enzyme, etc.
  • the target nucleic acid may contain any convenient nucleotide sequence that is specifically bound by a binding moiety.
  • Exemplary nucleotide sequences that bind a binding moiety include, but are not limited to, binding sites for endogenous transcription factors, such as E2F, Stat3, cAMP response element, Ets-1, Ap-1, NF- ⁇ , GATA-3, STAT-1, STAT-6, TCF; and binding sites for transcriptional regulators used by infectious organisms, such as WhiB7 (Actinomycetes), FadR (E. coli), YycG/YycF (S. aureus, B. subtilis, S. pneumoniae, S. pyogenes, Listeria monocytogenes), Sigma 54 or Sig B (P. aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae), Fur (S.
  • endogenous transcription factors such as E2F, Stat3, cAMP response element, Ets-1, Ap-1, NF- ⁇ , GATA-3, STAT-1, STAT-6, TCF
  • binding sites for transcriptional regulators used by infectious organisms such
  • aureus E. coli, Helicobacter pylori, B. subtilis
  • TcdR Clostridium difficile
  • C. botulinium where the homologue is BotR
  • C. tetani TetR
  • C. perfringens Vfr (P. aeruginosa and E. coll)
  • NtrC Klebsiella pneumonia
  • ArsR Helicobacter pylori, H. acinonychis and H. felis).
  • the target nucleic acid is a target DNA that contains a nucleotide sequence
  • the target DNA contains a nucleotide sequence encoding for a polypeptide gene product, such as a therapeutic polypeptide.
  • exemplary polypeptides that may be encoded in the target DNA include, but are not limited to, the peptidyl hormones activin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, calcitonin gene -related peptide, calcitonin N-terminal flanking peptide, ciliary neurotrophic factor (CNTF), corticotropin (adrenocorticotropin hormone, ACTH), corticotropin-releasing factor (CRF or CRH), epidermal growth factor (EGF), follicle-stimulating hormone (FSH), gastrin, gastrin inhibitory peptide (GIP), gastrin-releasing peptide, gonadotropin-releasing factor (GnRF
  • polypeptides suitable to be encoded in the target DNA include the cytokines, e.g., colony stimulating factor 4, heparin binding neurotrophic factor (HBNF), interferon-a, interferon a-2a, interferon a-2b, interferon a-n3, interferon- ⁇ , etc., interleukin- 1 , interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, etc., tumor necrosis factor, tumor necrosis factor-a, granuloycte colony-stimulating factor (G-CSF), granulocyte-macrophage colony- stimulating factor (GM- CSF), macrophage colony-stimulating factor, midkine (MD), and thymopoietin.
  • cytokines e.g., colony stimulating factor 4, heparin binding neurotrophic factor (HBNF), interferon-a, interferon a-2a,
  • polypeptides suitable to be encoded in the target DNA include endorphins (e.g., dermorphin, dynorphin, a-endorphin, ⁇ - endorphin, ⁇ -endorphin, sigma-endorphin, [Leu 5 ]enkephalin,
  • a target DNA contains a nucleotide sequence encoding a gene product that is used in laboratory research, such as fluorescent proteins (e.g., green fluorescent protein (GFP)); indicators (e.g., calcium indicators, voltage indicators); and other genetically engineered proteins (e.g., light-sensitive ion channels, voltage-gated ion channels, transcription factors, signaling proteins, cytoskeletal proteins, enzymes, etc.).
  • fluorescent proteins e.g., green fluorescent protein (GFP)
  • indicators e.g., calcium indicators, voltage indicators
  • other genetically engineered proteins e.g., light-sensitive ion channels, voltage-gated ion channels, transcription factors, signaling proteins, cytoskeletal proteins, enzymes, etc.
  • a target DNA containing a nucleotide sequence encoding a gene product is configured such that the gene product is expressed in a target cell.
  • the nucleotide sequence encoding a gene product may be operably linked to one or more control elements, e.g. a promoter sequence, enhancer sequence, introns, etc., that promote expression of the gene product from the target DNA in a target cell.
  • control elements can include control sequences normally associated with the selected gene product (e.g., endogenous cellular control elements).
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
  • Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter that is heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • synthetic promoters hybrid promoters, and the like.
  • sequences derived from nonviral genes such as the murine metallothionein gene, can also be used.
  • the target DNA includes an expression vector that contains a nucleotide sequence encoding a gene product and one or more control elements that promote expression of the gene product in a target cell.
  • a cell type-specific or a tissue-specific promoter will be operably linked to the nucleotide sequence encoding a gene product, such that the gene product is produced selectively or preferentially in a particular cell type(s) or tissue(s).
  • an inducible promoter will be operably linked to the nucleotide sequence encoding a gene product.
  • a target nucleic acid (e.g., a target DNA, a target RNA, an antisense nucleic acid) comprises one or more modifications, e.g., a base modification, a backbone modification, etc.
  • a nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable.
  • linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. Modified backbones and modified internucleoside linkages
  • nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages.
  • Nucleic acids (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,
  • aminoalkylphosphotriesters methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
  • Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
  • MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,67
  • target nucleic acids e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid
  • a subject nucleic acid e.g., a subject target RNA; a subject antisense nucleic acid
  • a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.
  • Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have
  • backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • a subject target nucleic acid can be a nucleic acid mimetic.
  • the term "mimetic" as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA peptide nucleic acid
  • the backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone.
  • the heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262.
  • morpholino nucleic acid having heterocyclic bases attached to the morpholino ring.
  • a number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid.
  • One class of linking groups has been selected to give a non-ionic oligomeric compound.
  • the non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins.
  • Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey,
  • Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.
  • a further class of polynucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA).
  • CeNA cyclohexenyl nucleic acids
  • CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.
  • a further modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • the linkage can be a methylene (-CH 2 -), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456).
  • Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).
  • LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • a subject target nucleic acid can also include one or more substituted sugar moieties.
  • Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O- alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub. l to do alkyl or C 2 to do alkenyl and alkynyl.
  • Particularly suitable are 0((CH 2 ) n O) m CH 3 , 0(CH 2 ) n OCH 3 , 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 , 0(CH 2 ) n ONH 2 , and 0(CH 2 ) n ON((CH 2 ) n CH 3 ) 2 , where n and m are from 1 to about 10.
  • Suitable polynucleotides comprise a sugar substituent group selected from: d to do lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a suitable modification includes 2'-methoxyethoxy (2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • a further suitable modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'- DMAEOE), i.e., 2'-0-CH 2 -0-CH 2 -N(CH 3 ) 2 .
  • Suitable sugar substituent groups include methoxy (-0-CH 3 ), aminopropoxy (—0 CH 2 CH 2
  • 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • a suitable 2'-arabino modification is 2'-F.
  • Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • a subject target nucleic acid may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.
  • nucleobases are useful for increasing the binding affinity of an oligomeric compound (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid).
  • an oligomeric compound e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid.
  • these include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C.
  • a subject target nucleic acid e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid
  • a subject target nucleic acid involves chemically linking to the polynucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject antisense nucleic acid or target protector nucleic acid.
  • Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.
  • the present disclosure provides a method of delivering a gene product to an individual, the method
  • the present disclosure provides a method of delivering a target nucleic acid to a cell, the method comprising contacting a cell with the lipidoid composition of the present disclosure to intracellularly deliver the target nucleic acid to the cell.
  • a lipidoid composition of the present disclosure can be provided together with a pharmaceutically
  • an "active agent” will refer to an agent discussed herein, e.g., a lipidoid composition of the present disclosure, unless otherwise specified.
  • a lipidoid composition of the present disclosure can be formulated in a variety of ways.
  • the form (e.g., liquid, solid, pill, capsule) and composition of the formulation will vary according to the method of administration used.
  • the nucleic acid can be formulated as a tablet, pill, capsule, solution (e.g., gel, syrup, slurry, or suspension), or other suitable form.
  • a lipidoid composition of the present disclosure is formulated to facilitate delivery to the surface of the intestinal cells.
  • the formulation can contain components in addition to a lipidoid composition of the present disclosure, where the additional components aid in the delivery of the lipidoid composition, e.g., delivery to an intestinal cell.
  • the lipidoid composition of the present disclosure acid can be present in a pharmaceutical composition with additional components such as, but not limited to, stabilizing compounds and/or biocompatible pharmaceutical carriers, e.g., saline, buffered saline, dextrose, or water.
  • the lipidoid composition of the present disclosure can also be administered alone or in combination with other agents, including other therapeutic agents.
  • the formulation can also contain organic and inorganic compounds to, for example, facilitate nucleic acid delivery to and uptake by the target cell (e.g., detergents, salts, chelating agents, etc.).
  • a formulation comprising a lipidoid composition of the present disclosure can include: a) a lipidoid
  • composition of the present disclosure and b) one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative.
  • a buffer a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative.
  • Suitable buffers include, but are not limited to, (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2- hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3- propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulf
  • a formulation comprising a lipidoid composition of the present disclosure can include a lipidoid composition of the present disclosure in an amount of from about 0.001 % to about 90% (w/w).
  • a lipidoid composition of the present disclosure can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • a lipidoid composition of the present disclosure or a formulation comprising a lipidoid
  • composition of the present disclosure can be administered to an individual in need thereof by any of a variety of routes of administration. Suitable routes of administration include enteral and parenteral routes. Administration can be via a local or a systemic route of administration.
  • routes of administration include enteral and parenteral routes.
  • Administration can be via a local or a systemic route of administration.
  • a lipidoid composition of the present disclosure, or a formulation comprising a lipidoid composition of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; and intracranial, e.g., intrathecal or intraventricular, administration.
  • Intratumoral and peritumoral administration is also contemplated.
  • compositions and their subsequent administration are within the skill of those in the art. Dosing is dependent on several criteria, including severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual lipidoid compositions, and can generally be estimated based on EC50s or IC50s found to be effective in vitro and in vivo animal models.
  • a suitable dose of a lipidoid composition of the present disclosure is from 0.01 ⁇ g to
  • 100 g per kg of body weight from 0.1 ⁇ g to 10 g per kg of body weight, from 1 ⁇ g to 1 g per kg of body weight, from 10 ⁇ g to 100 mg per kg of body weight, from 100 ⁇ g to 10 mg per kg of body weight, or from 100 ⁇ g to 1 mg per kg of body weight.
  • Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • a lipidoid composition of the present disclosure is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, from 0.1 ⁇ g to 10 g per kg of body weight, from 1 ⁇ g to 1 g per kg of body weight, from 10 ⁇ g to 100 mg per kg of body weight, from 100 ⁇ g to 10 mg per kg of body weight, or from 100 ⁇ g to 1 mg per kg of body weight.
  • a lipidoid composition of the present disclosure is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).
  • the duration of administration of lipidoid composition of the present disclosure can vary, depending on any of a variety of factors, e.g., patient response, etc.
  • a lipidoid composition of the present disclosure can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.
  • the present disclosure includes methods of making a lipidoid compound, e.g., as described herein.
  • the method includes: contacting a fatty acid ester with an amino linker to produce a fatty acid contacted mixture; and heating the mixture under an inert atmosphere for a time sufficient to produce the lipidoid compound, e.g., as described herein.
  • the subject methods of making may provide for preparation of the lipidoid compounds from fatty acid ester and amino linker starting materials in a single step without the need for extensive purification.
  • the fatty acid ester is an alkyl ester or a substituted alkyl ester. In some instances, the fatty acid ester is a lower alkyl ester. In certain instances, the fatty acid ester is a methyl ester or an ethyl ester of a fatty acid of interest. In some instances, the fatty acid ester is an aryl, substituted aryl, heteroaryl or substituted heteroaryl ester. In some instances, the fatty acid ester is an activated ester that comprises a convenient leaving group.
  • amino linker is described by formula (VI):
  • Z is N.
  • Y is H.
  • Y is an alkyl.
  • Y is methyl.
  • Y is a substituted alkyl.
  • R 1 and R 2 are each hydrogen.
  • R 1 and R 2 are each an alkyl.
  • R 1 and R 2 are each methyl.
  • R 1 and R 2 are each a substituted alkyl.
  • any convenient methods may be utilized in contacting the fatty acid with the amino linker to produce a fatty acid contacted mixture.
  • the fatty acid contacted mixture further includes a solvent. Any convenient solvents in which one or more of the components of the fatty acid contacted mixture is soluble may be utilized. In certain instances, one of the starting material components of the mixture can act as a solvent.
  • any convenient methods may be utilized in heating the mixture under an inert atmosphere for a time sufficient to produce the lipidoid compound, e.g., as described herein. Heating may be performed at any convenient temperature between room temperature and the boiling point of one of the components of the mixture and/or the solvent. In some embodiments, the heating achieves a temperature of 50°C or more, such as 80°C or more, 90°C or more, 100°C or more, such as 110 °C or more, 120 °C or more, 130 °C or more, or even 150 °C or more.
  • the heating is maintained at a temperature in the range of 100 to 200 °C, such as in the range of 100 to 160 °C, such as in the range of 130-160 °C.
  • an inert atmosphere is meant that the mixture is heated in the absence of oxygen.
  • the mixture may be purged and/or heated under an inert gas, such as nitrogen or helium.
  • the heating may be maintained for any convenient length of time depending on a variety of factors, such as the particular components, the solvent, the temperature, etc. In certain embodiments, the heating is maintained for 1 hour or more, such as 3 hours or more, 6 hours or more, overnight, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or even more.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • the amines chosen contain short carbon chains with two to three amine moieties and the fatty acid ester compounds include a tail of varying chain lengths and optionally feature various functional groups and varying degrees of saturation.
  • the resulting novel essential fatty acid lipidoid compounds were particularly useful in delivering negatively charged agents given the secondary and tertiary amines available for protonation thus forming a cationic moiety.
  • the essential fatty acid lipidoid compounds may be used to delivery DNA, RNA, or other polynucleotides to a subject or to a cell.
  • two different fatty acid esters may be used in the reaction mixture to prepare an essential fatty acid lipidoid compound with two different tails.
  • NKS lipidoids were synthesized by addition of fatty acid esters to respective amines drop wise in a ratio of either, 1 : 1 or 1 :2 or 1 :3 according to the number of amine sites open for substitution in 3-inl Teflon-lined glass screw- ⁇ vial. No solvent or catalyst was used for these reactions.
  • the reaction vial was heated at 140-160°C for 72h while stirring under N 2 .
  • the product was purified by recrystallization using hexanes and acetone. For higher purity, the reaction mixture was purified using flash
  • NKS lipidoids were analyzed and verified using ⁇ , l3 C nuclear magnetic resonance (NMR) and high-resolution mass spectrometry.
  • Hela or Hela-GFP cells were analyzed 48 hours after transfection. Cells were detached from the plate by trypsin treatment (100 uIJwell of 0.25% IX (Invitrogen/Gibco®)) at 37°C for 10 minutes, followed by addition of media. Samples were transferred into centrifuge tubes and centrifuged at 300g, 4°C for 10 minutes (Eppendorf 5417R). The supernatant (media) was removed and cells were washed with PBS (Dulbecco's Phosphate-Buffered Saline (Invitrogen/Gibco®)).
  • PBS Dulbecco's Phosphate-Buffered Saline
  • the cells were then fixed with 4% formaldehyde (37% formaldehyde (Fischer Scientific, F79-500) diluted into PBS) and refrigerated at 4°C for 1 hour. The 4% formaldehyde was removed, and the cells were re-suspended in PBS. The resulting samples were assayed by flow cytometer (Beckman-Coulter, EPICS® XL-MCLTM) and Beckman-Coulter System II software. Data from each sample was collected for up to 10 4 cells over 5 minutes of time.
  • 4% formaldehyde 37% formaldehyde (Fischer Scientific, F79-500) diluted into PBS
  • the resulting samples were assayed by flow cytometer (Beckman-Coulter, EPICS® XL-MCLTM) and Beckman-Coulter System II software. Data from each sample was collected for up to 10 4 cells over 5 minutes of time.
  • mice Studies in animals. Four- to six-week-old BALB/c mice (Jackson Laboratory) were used to determine the toxicity and efficiency of delivering RNAi facilitated by omega-fatty acid lipidoids. In these experiments, mice were first anesthetized with isoflurane, and then the formulations developed using lipidoids NKS 11, NKS21 and NKS22 were then slowly injected in separate experiments into the tail veins of the animals by using a syringe with hypodermic needle of 27 -gauge size.
  • RNA from liver, spleen, kidney, lung and salivary glands was isolated using Qiagen RNeasy® kit following the manufacturer's protocol. miR98 was used as an internal control for reverse transcription. Taqman® miRNA Reverse transcription kit (Applied Biosystems) was used for reverse transcription. Quantification of miRNA was performed using Taqman® miRNA assay kit (Applied Biosystems, per protocol).
  • mice Five mice were used for experimental conditions and 3 mice were used as controls that received the miR124 via PBS. 24h after the administration, the mice were euthanized and the lungs were collected. To make sure that the miR124 was indeed taken up by the cells, the lungs were perfused using 1 % saline solution before collecting the RNA. Other organs like kidneys, liver and spleen were tested for any traces of miR124.
  • a library of lipidoid molecules (Figure 1) was generated using nucleophilic acyl substitution reactions.
  • lipidoids were tested for their ability to complex with small interfering RNA (siRNA). (35) This led to the identification of three molecules that were efficiently complexing with nucleic acids such as siRNA, miRNA and plasmids.
  • the results from these in vitro studies were either comparable or better than the current accepted industry standard, Lipofectamine® 2000. While Lipofectamine® 2000 is contraindicated for in vivo delivery, NKS 11, NKS21 and NKS22 proved to be safe and efficient for delivery of polynucleotides.
  • HeLa-GFP HeLa cell line
  • GFP green fluorescent protein
  • the efficiency of delivery was verified by treating HeLa-GFP cells with siRNA-lipidoid complexes containing GFP-targeting siRNA (siGFP), and then measuring the reduction in GFP expression by HeLa- GFP cells.
  • the complexes were prepared by simple mixing and sonicating at room temperature for 5 minutes.
  • Flow cytometry was used to analyze the transfection efficiency (TE) of the lipidoids, 48h after
  • Figure 2 depicts GFP knockdown via siRNA delivered by NKS11.
  • FIG. 1 Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS 11 mediated siRNA delivery is overlaid with
  • Lipofectamine® 2000 mediated transfection and untreated HeLa cells expressing GFP, 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
  • NKS 11 As a further proof of TE of NKS 11 , the delivery of a 5.5kBp plasmid pRLK-GFP into HeLa cells was tested. As shown in Figure 3 below, NKS 11 successfully delivered pRLK-GFP at the levels of lipofectamine® 2000 (Invitrogen, San Diego, CA), 48h after transfection. As shown in figure 3, the lipidoids NKS 11 is efficiently transfected and expressing GFP protein in HeLa cells.
  • Figure 3 In vitro delivery of pRK-9-flag®-EGFP plasmid in HeLa cells, a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS 11 mediated siRNA delivery (black) is overlaid with Lipofectamine® 2000 mediated transfection (blue) and untreated HeLa cells expressing GFP (red), 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
  • the pRK-9-flag®-EGFP plasmid is labeled as "GFP plasmid”; the pRK-9-flag®-EGFP plasmid and "GFP plasmid” are the same plasmids. NKS21
  • Figure 4 depicts in vitro delivery of siRNA using NKS21.
  • FIG. 4 In vitro delivery of siRNA. a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS21 mediated siRNA delivery is overlaid with
  • Lipofectamine® 2000 mediated transfection and untreated HeLa cells expressing GFP, 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
  • Figure 5 depicts GFP knockdown via siRNA delivered by NKS22.
  • Figure 5 In vitro delivery of siRNA. a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS22 mediated siRNA delivery is overlaid with
  • Lipofectamine® 2000 mediated transfection and untreated HeLa cells expressing GFP (red), 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
  • Figure 6 depicts GFP plasmid update using NKS22.
  • FIG. 6 In vitro delivery of pRK-9-flag®-EGFP plasmid in HeLa cells, a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS22 mediated siRNA delivery (black) is overlaid with Lipofectamine® 2000 mediated transfection (blue) and untreated HeLa cells expressing GFP (red), 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
  • the pRK-9-flag®-EGFP plasmid is labeled as "GFP plasmid”; the pRK-9-flag®-EGFP plasmid and "GFP plasmid” are the same plasmids.
  • mice received 15mg/Kg, 30mg/Kg, 60mg/Kg, or lOOmg/Kg. The mice were observed for any loss of weight or any drastic observable health changes. No death or other observable changes in weight was observed. It was concluded that these delivery agents were safe for further experiments.
  • mice Female BALB/c mice were injected intravenously (lateral tail vein) with 100 ⁇ g of NKS 11, NKS21,
  • NKS22 formulation prepared by adding 1 % PEG in acetate buffer, in separate experiments.
  • cytokine induction a group of mice were injected with 20 ⁇ g of lipopolysaccharide (LPS) from Escherichia coli 0111 :B4.
  • LPS lipopolysaccharide
  • mice were injected with PBS to get a base level of these cytokines. Since there is usually a dramatic increase in cytokine levels within a few hours of
  • cytokine levels were evaluated 2h and 6h after injecting the delivery agents NKS 11, NKS21, NKS22 and the controls. Mice were then anaesthetized with isoflurane and blood was collected via cardiac stick at set time points after injection (2h and 6h). Two mice were used for each time point. Serum was separated by centrifugation for cytokine analysis. Serum cytokine levels (TNF-a and IFN- ⁇ ) were determined using eBioscience mouse ELISA kits and Spectramax® M2 microplate reader. It was found that the immunostimulation due to NKS 11, NKS21 and NKS 22 were at the level of PBS or lower ( Figures 7 and 8).
  • FIG. 7 Cytokine levels in the serum in Balb/c mice at 2h and 6 h post intravenous injection of a single dose (4mg/Kg body weight of mice) of formulated NKS 11, NKS21, NKS 22 and PBS. 0.8 mg/Kg of Lipopolysaccharide (LPS) was used as a positive control for TNF-a induction. Averages and standard deviations of 2 mice per group are shown.
  • LPS Lipopolysaccharide
  • FIG. 8 Cytokine levels in the serum in BALB/c mice at 2h and 6 h post intravenous injection of a single dose (4mg/Kg body weight of mice) of formulated NKS 11, NKS21, NKS 22 and PBS. 0.8 mg/Kg of lipopolysaccharide (LPS) was used as a positive control for IFN- ⁇ induction. Averages and standard deviations of 2 mice per group are shown.
  • LPS lipopolysaccharide
  • Micro RN As are non-coding RNAs of around 19-25 nucleotide long transcripts that are known to negatively regulate gene expression, that affects processes like cell proliferation, differentiation, survival and motility (45).
  • Aberrantly expressed miRNAs lead to human diseases and correcting these miRNA deficiencies by antagonizing or restoring miRNA function by delivering endogenous miRNAs may result in therapeutics for various diseases (46).
  • systemic delivery of miR-34a mimics has resulted in robust inhibition of non-small cell lung cancer (NSCLC) xenografts in mice using neutral lipid emulsion (47).
  • NSCLC non-small cell lung cancer
  • microRNA 124 (miR-124) was selected. miR-124 was chosen for study because it is expressed only by the cells of the central nervous system and hence facilitates differentiation between mimics of miR-124 delivered using these lipidoids from those that are endogenously expressed (48). Mice were injected with 15 ⁇ g of miR-124 using formulation developed using 75 ⁇ g NKS lipidoids and 0.1 % PEG, which is equivalent to about 5 mg/Kg of mouse body weight, assuming a mouse weighs around 25g.
  • mice were euthanized after 24h; and five organs- salivary gland, lungs, spleen, kidneys and liver, were collected, subjected to RNA isolation and quantitative reverse transcriptase PCR (qRT- PCR).
  • qRT- PCR quantitative reverse transcriptase PCR
  • Each mouse received a dose of lmg/kg of miR124.
  • 24h after the administration the mice were euthanized and total RNA was extracted from the organs. Organs were perfused with 1 % saline solution before subjected to RNA isolation.
  • Mice that received naked miR124 acted as negative controls for endogenous miR-124 expression levels in these tissues under these conditions.
  • miR-124 copy numbers were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph.
  • FIG. 10 Biodistribution of systemically delivered microRNAs (miRNA) mimics using NKS21.
  • miR124 Delivery of miR124 to various organs- lungs, liver, spleen, kidneys and salivary glands.
  • Each mouse received a dose of lmg/kg of miR124. 24h after the administration, the mice were euthanized and total RNA was extracted from the organs. Organs were perfused with 1 % saline solution before subjected to RNA isolation. Mice that received naked miR124 acted as negative controls for endogenous miR-124 expression levels in these tissues under these conditions.
  • miR-124 copy numbers were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph.
  • FIG. 11 Biodistribution of systemically delivered microRNAs (miRNA) mimics using NKS22.
  • miR124 Delivery of miR124 to various organs- lungs, liver, spleen, kidneys and salivary glands.
  • Each mouse received a dose of lmg/kg of miR124. 24h after the administration, the mice were euthanized and total RNA was extracted from the organs. Organs were perfused with 1 % saline solution before subjected to RNA isolation. Mice that received naked miR124 acted as negative controls for endogenous miR-124 expression levels in these tissues under these conditions.
  • miR-124 copy numbers were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph.
  • RNAi agents Intranasal delivery of RNAi agents is a promising non-invasive strategy for delivering directly to the lungs by avoiding the challenges of systemic delivery.
  • the dose of RNAi required for efficacy is substantially less as this route provides direct access to lung epithelial cells, making it a very attractive delivery route for several respiratory tract disorders like chronic obstructive pulmonary disease, cystic fibrosis, asthma, as well as lung cancers and viral infections in the lung (49).
  • Figure 12 Intranasal delivery of miR-124 into lungs 24h after administration in BALB/C mice using
  • Topical application of medications for various ailments ranging from skin diseases to rheumatoid arthritis has been known for centuries (51-54), making skin a very attractive route for drug delivery.
  • drugs For topically administered drugs to be effective, they should be able to penetrate stratum corneum, the outermost layer of skin that acts as a barrier (52).
  • stratum corneum the outermost layer of skin that acts as a barrier (52).
  • Several chemical compounds that act as penetration-enhancers and their mechanisms of action have been reported (44).
  • Fatty acids of carboxylic acids, especially the unsaturated ones like oleic acid, linoleic acid, and lauric acid have been found to be enhancing penetration of stratum corneum, because of their potential to disturb the lipid packing order within the bilayer (51, 52).
  • Figure 13 Transdermal Delivery of eGFP expressing plasmid pRLK.
  • RNAi RNA interference

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Abstract

The present disclosure provides lipidoid compounds, and compositions comprising the lipidoid compounds and a nucleic acid. The present disclosure provides methods of making a subject lipidoid. The present disclosure provides a method of delivering a target nucleic acid to a cell, using a lipidoid composition of the present disclosure. The present disclosure provides a method of delivering a gene product to an individual, using a lipidoid composition of the present disclosure.

Description

FATTY ACID ANALOGS AND METHODS OF USE THEREOF CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/149,320, filed
April 17, 2015, which application is incorporated herein by reference in its entirety.
INTRODUCTION
[0002] RNA interference (RNAi) or post-transcriptional gene silencing is a conserved biological response to a double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein coding genes. This mechanism of gene interference holds promise in developing therapeutics for cancers, viral infections and several genetic disorders. The use of RNAi technologies is especially useful for targets that usually considered undruggable.
[0003] There is a need in the art for delivery systems for nucleic acid-based therapeutics.
SUMMARY
[0004] The present disclosure provides lipidoid compounds, and compositions comprising the lipidoid
compounds and a nucleic acid. The present disclosure provides methods of making a subject lipidoid. The present disclosure provides a method of delivering a target nucleic acid to a cell, using a lipidoid composition of the present disclosure. The present disclosure provides a method of delivering a gene product to an individual, using a lipidoid composition of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] Figures 1A-1E depict examples of lipidoids.
[0006] Figure 2 is a graph showing knockdown of green fluorescent protein (GFP) expression via siRNA
delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
[0007] Figure 3 is a graph showing expression of GFP from a plasmid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
[0008] Figure 4 is a graph showing knockdown of green fluorescent protein (GFP) expression via siRNA
delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
[0009] Figure 5 is a graph showing knockdown of green fluorescent protein (GFP) expression via siRNA
delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
[0010] Figure 6 is a graph showing expression of GFP from a plasmid delivered by a lipidoid in cultured cells, according to an embodiment of the present disclosure.
[0011] Figure 7 is a graph showing serum cytokine levels in mice injected intravenously with lipidoid
formulations, according to an embodiment of the present disclosure.
[0012] Figure 8 is a graph showing serum cytokine levels in mice injected intravenously with lipidoid
formulations, according to an embodiment of the present disclosure. [0013] Figure 9 is a graph showing organ distribution and levels of an miRNA after intravenous administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
[0014] Figure 10 is a graph showing organ distribution and levels of an miRNA after intravenous administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
[0015] Figure 11 is a graph showing organ distribution and levels of an miRNA after intravenous administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
[0016] Figure 12 is a graph showing organ distribution and levels of an miRNA after intranasal administration of a formulation of the miRNA with a lipidoid, according to an embodiment of the present disclosure.
[0017] Figure 13 is a collection of images showing delivery and expression of a plasmid via intradermal
administration of a formulation of the plasmid with a lipidoid, according to an embodiment of the present disclosure.
DEFINITIONS
[0018] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0021] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a lipidoid" includes a plurality of lipidoids and reference to "the gene product" includes reference to one or more gene products and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. [0022] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-0-C(0)-.
[0023] "Alkylene" refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more
preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -0-, -NR10-, -NR10C(O)-, -C(0)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene
(-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(0)-),
(-C(CH3)2CH2C(0)NH-), (-CH(CH3)CH2-), and the like.
[0024] "Substituted alkylene" refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of "substituted" below.
[0025] The term "alkane" refers to alkyl group and alkylene group, as defined herein.
[0026] The term "alkylaminoalkyl", "alkylaminoalkenyl" and "alkylaminoalkynyl" refers to the groups R NHR - where R is alkyl group as defined herein and R is alkylene, alkenylene or alkynylene group as defined herein.
[0027] The term "alkaryl" or "aralkyl" refers to the groups - alkylene- aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.
[0028] "Alkoxy" refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O, cycloalkenyl-O, and alkynyl-
0-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
[0029] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
[0030] The term "alkoxyamino" refers to the group -NH-alkoxy, wherein alkoxy is defined herein.
[0031] The term "haloalkoxy" refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.
[0032] The term "haloalkyl" refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group. Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoro methyl, trifluoroethyl and the like.
[0033] The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-0 -alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
[0034] The term "alkylthioalkoxy" refers to the group -alkylene-S -alkyl, alkylene-S-substituted alkyl, substituted alkylene-S -alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. [0035] "Alkenyl" refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-l-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.
[0036] The term "substituted alkenyl" refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl and -S02- heteroaryl.
[0037] "Alkynyl" refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH2C≡CH).
[0038] The term "substituted alkynyl" refers to an alkynyl group as defined herein having from 1 to 5
substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02- substituted alkyl, -S02-aryl, and -S02-heteroaryl.
[0039] "Alkynyloxy" refers to the group -O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.
[0040] "Acyl" refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CH3C(0)-.
[0041] "Acylamino" refers to the groups -NR20C(O)alkyl, -NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, - NR20C(O)substituted cycloalkyl, -NR20C(O)cycloalkenyl, -NR20C(O)substituted cycloalkenyl, - NR20C(O)alkenyl, -NR20C(O)substituted alkenyl, -NR20C(O)alkynyl, -NR20C(O)substituted alkynyl, -NR20C(O)aryl, -NR20C(O)substituted aryl, -NR20C(O)heteroaryl, -NR20C(O)substituted heteroaryl,
-NR 20 C(0)heterocyclic, and -NR 20 C(0)substituted heterocyclic, wherein R 20 is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[0042] "Aminocarbonyl" or the term "aminoacyl" refers to the group -C(0)NR 21 R 22 , wherein R 21 and R 22
independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[0043] "Aminocarbonylamino" refers to the group -NR21C(0)NR22R23 where R21, R22, and R23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.
[0044] The term "alkoxycarbonylamino" refers to the group -NRC(0)OR where each R is independently
hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
[0045] The term "acyloxy" refers to the groups alkyl-C(0)0-, substituted alkyl-C(0)0-, cycloalkyl-C(0)0-, substituted cycloalkyl-C(0)0-, aryl-C(0)0-, heteroaryl-C(0)0-, and heterocyclyl-C(0)0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
[0046] "Aminosulfonyl" refers to the group -SO2NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.
[0047] "Sulfonylamino" refers to the group -NR 21 SO2R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [0048] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, - S02-substituted alkyl, -S02-aryl, -S02-heteroaryl and trihalomethyl.
[0049] "Aryloxy" refers to the group -O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.
[0050] "Amino" refers to the group -NH2.
[0051] The term "substituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.
[0052] The term "azido" refers to the group -N3.
[0053] "Carboxyl," "carboxy" or "carboxylate" refers to -C02H or salts thereof.
[0054] "Carboxyl ester" or "carboxy ester" or the terms "carboxyalkyl" or "carboxylalkyl" refers to the groups -C(0)0-alkyl, -C(0)0-substituted alkyl, -C(0)0-alkenyl, -C(0)0-substituted alkenyl, -C(0)0-alkynyl, -C(0)0-substituted alkynyl, -C(0)0-aryl, -C(0)0-substituted aryl, -C(0)0-cycloalkyl,
-C(0)0-substituted cycloalkyl, -C(0)0-cycloalkenyl, -C(0)0-substituted cycloalkenyl,
-C(0)0-heteroaryl, -C(0)0-substituted heteroaryl, -C(0)0-heterocyclic, and -C(0)0-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[0055] "(Carboxyl ester)oxy" or "carbonate" refers to the groups -0-C(0)0-alkyl, -(D-C(O)O-substituted alkyl, - 0-C(0)0-alkenyl, -0-C(0)0-substituted alkenyl, -0-C(0)0-alkynyl, -0-C(0)0-substituted alkynyl, -O- C(0)0-aryl, -0-C(0)0-substituted aryl, -0-C(0)0-cycloalkyl, -0-C(0)0-substituted cycloalkyl, -O- C(0)0-cycloalkenyl, -0-C(0)0-substituted cycloalkenyl, -0-C(0)0-heteroaryl, -0-C(0)0-substituted heteroaryl, -(D-C(O)O-heterocyclic, and -(D-C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [0056] "Cyano" or "nitrile" refers to the group -CN.
[0057] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
[0058] The term "substituted cycloalkyl" refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, he tero aryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02- substituted alkyl, -S02-aryl and -S02-heteroaryl.
[0059] "Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.
[0060] The term "substituted cycloalkenyl" refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl and -S02- heteroaryl.
[0061] "Cycloalkynyl" refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.
[0062] "Cycloalkoxy" refers to -O-cycloalkyl.
[0063] "Cycloalkenyloxy" refers to -O-cycloalkenyl.
[0064] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.
[0065] "Hydroxy" or "hydroxyl" refers to the group -OH.
[0066] "Heteroaryl" refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic and at least one ring within the ring system is aromatic , provided that the point of attachment is through an atom of an aromatic ring. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→0), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl and -S02-heteroaryl, and trihalo methyl.
[0067] The term "heteroaralkyl" refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.
[0068] "Heteroaryloxy" refers to -O-heteroaryl.
[0069] "Heterocycle," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non- aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -S02- moieties.
[0070] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6, 7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1- dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.
[0071] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-heteroaryl, and fused heterocycle.
[0072] "Heterocyclyloxy" refers to the group -O-heterocyclyl.
[0073] The term "heterocyclylthio" refers to the group heterocyclic-S-. [0074] The term "heterocyclene" refers to the diradical group formed from a heterocycle, as defined herein.
[0075] The term "hydroxyamino" refers to the group -NHOH.
[0076] "Nitro" refers to the group -N02.
[0077] "Oxo" refers to the atom (=0).
[0078] "Sulfonyl" refers to the group S02-alkyl, S02-substituted alkyl, S02-alkenyl, S02-substituted alkenyl, S02-cycloalkyl, S02-substituted cylcoalkyl, S02-cycloalkenyl, S02-substituted cylcoalkenyl, S02-aryl, S02-substituted aryl, S02-heteroaryl, S02-substituted heteroaryl, S02-heterocyclic, and S02-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-S02-, phenyl-S02-, and 4-methylphenyl-S02-.
[0079] "Sulfonyloxy" refers to the group -OS02-alkyl, OS02-substituted alkyl, OS02-alkenyl, OS02- substituted alkenyl, OS02-cycloalkyl, OS02-substituted cylcoalkyl, OS02-cycloalkenyl, OS02- substituted cylcoalkenyl, OS02-aryl, OS02-substituted aryl, OS02-heteroaryl, OS02-substituted heteroaryl, OS02-heterocyclic, and OS02 substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[0080] The term "aminocarbonyloxy" refers to the group -OC(0)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
[0081] "Thiol" refers to the group -SH.
[0082] "Thioxo" or the term "thioketo" refers to the atom (=S).
[0083] "Alkylthio" or the term "thioalkoxy" refers to the group -S -alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.
[0084] The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.
[0085] The term "thioaryloxy" refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.
[0086] The term "thioheteroaryloxy" refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.
[0087] The term "thioheterocyclooxy" refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.
[0088] In addition to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
[0089] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =0, =NR , =N-OR , =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =0, -OR™, -SR™, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -N02, =N2, -N3, -S02R™, -S020"M+, -S02OR™, -OS02R™, -OS020"M+, -OS02OR™, -P(0)(0")2(M+)2, -P(O)(OR70)O"M+, -P(0)(OR70) 2, -C(0)R™, -C(S)R™, -C(NR70)R™, -C(0)0"M+, -C(0)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(0)R70, -OC(S)R70, -OC(0)0"M+, -OC(0)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2 "M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R is independently hydrogen or R ; each R is independently R or alternatively, two R80 s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C C3 alkyl substitution; and each M+ is a counter ion with a net single positive charge. Each M+ may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as +N(R60) or an alkaline earth ion, such as [Ca [Mg or [Ba ("subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the present disclosure and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the present disclosure can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80 is meant to include -NH2, -NH-alkyl, Λ^-pyrrolidinyl, Λ^-piperazinyl, 4,/V-methyl-piperazin- 1 -yl and Λ^-morpholinyl.
[0090] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in
"substituted" alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -0"M+, -OR70, -SR70, -S"M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -N02, -N3, -S02R70, -S03 " M+, -SO3R70, -OS02R70, -OS03 "M+, -OSO3R70, -P03 "2(M+)2, -P(O)(OR70)O"M+, -P(O)(OR70)2, -C(0)R70, -C(S)R70, -C(NR70)R70, -C02 "M+, -C02R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(0)R70, -OC(S)R70, -OC02 "M+, -OC02R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2 "M+,
-NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -0"M+, -OR70, -SR70, or -S"M+.
[0091] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -0"M+, -OR70, -SR70, -S"M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -N02, -S(0)2R70, -S(0)2O M+, -S(0)2OR70, -OS(0)2R70, -OS(0)2O M+, -OS(0)2OR70, -P(0)(0 )2(M+)2, -P(O)(OR70)O"M+, -P(O)(OR70)(OR70), -C(0)R70, -C(S)R70, -C(NR70)R70, -C(0)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(0)R70, -OC(S)R70, -OC(0)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and
-NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined. [0092] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
[0093] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
[0094] The term "pharmaceutically acceptable salt" means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
[0095] The term "salt thereof means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
[0096] "Solvate" refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, Λ^,Λ^-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
[0097] "Stereoisomer" and "stereoisomers" refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
[0098] "Tautomer" refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.
[0099] It will be appreciated that the term "or a salt or solvate or stereoisomer thereof is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound.
[00100] "Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a
compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.
[00101] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to human and non-human subjects, especially mammalian subjects.
[00102] The term "treating" or "treatment" as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
[00103] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2- )■
[00104] The term "substituted alkyl" refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -0-, -N-, -S-, -S(0)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, - S02-alkyl, -S02-aryl, -S02-heteroaryl, and -NRaRb, wherein R and R may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
[00105] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub -combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
[00106] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
[00107] The present disclosure provides lipidoid compounds, and compositions comprising the lipidoid
compounds and a nucleic acid. The present disclosure provides methods of making a subject lipidoid. The present disclosure provides a method of delivering a target nucleic acid to a cell, using a lipidoid composition of the present disclosure. The present disclosure provides a method of delivering a gene product to an individual, using a lipidoid composition of the present disclosure.
LlPIDOIDS
[00108] The present disclosure provides lipidoid compounds. A lipidoid compound of the present disclosure may include a fatty acid conjugated to a charged linker. In some instances, the linker includes one, two or more positively charged groups capable of electrostatic interaction with a nucleic acid of interest. In some cases, the lipidoid includes a positively charged divalent linker that is conjugated to two fatty acids. In certain instances, the linker is a branched multivalent linker that is conjugated to three or more fatty acid groups. In certain embodiments, the positively charged group(s) of the linker are amino groups, such as secondary or tertiary amino groups which may be positively charged in an aqueous environment. In some cases, the positively charged group(s) of the linker are ammonium groups.
[00109] In some embodiments, a lipidoid compound of the present disclosure is described by the formula (I):
Figure imgf000014_0001
(I)
where:
m is 0 or an integer of 1 to 6;
Y is selected from the group consisting of hydrogen, alkyl, substituted alkyl or a fatty acid-containing group described by the formula (II):
Figure imgf000014_0002
(Π)
wherein:
p is 0 or an integer of 1-6;
L1, L2 and L3 are each independently derived from a fatty acid (such as an essential fatty acid); T1, T2 and Τ3 are each independently a linker;
Z is N, sulfonium (i.e., S+) or phosphonium (i.e., PR+); and
R1, R2 and R3 are each independently hydrogen, an alkyl or a substituted alkyl.
[00110] In certain embodiments of formula (I), m is 0. In certain embodiments of formula (I), m is 1. In certain embodiments of formula (I), m is 2. In certain embodiments of formula (I), m is 3. In certain embodiments of formula (I), m is 4. In certain embodiments of formula (I), m is 5. In certain embodiments of formula
(I) , m is 6.
[00111] In certain embodiments of formula (I), Z is N. In certain embodiments of formula (I), Z is sulfonium. In certain embodiments of formula (I), Z is phosphonium. In certain embodiments of formula (I), Y is H. In certain embodiments of formula (I), Y is an alkyl. In certain embodiments of formula (I), Y is methyl. In certain embodiments of formula (I), Y is a substituted alkyl. In certain embodiments of formula (I), R1 and R2 are each hydrogen. In certain embodiments of formula (I), R1 and R2 are each an alkyl. In certain embodiments of formula (I), R1 and R2 are each methyl. In certain embodiments of formula (I), R1 and R2 are each a substituted alkyl.
[00112] In certain embodiments of formula (I), Y is a fatty acid-containing group described by the formula (II). In certain embodiments of formula (II), p is 0. In certain embodiments of formula (II), p is 1. In certain embodiments of formula (II), p is 2. In certain embodiments of formula (II), p is 3. In certain embodiments of formula (II), p is 4. In certain embodiments of formula (II), p is 5. In certain embodiments of formula
(II) , p is 6. In certain embodiments of formula (II), R3 is hydrogen. In certain embodiments of formula (II), R3 is an alkyl. In certain embodiments of formula (II), R3 is methyl. In certain embodiments of formula (II), R3 is a substituted alkyl.
[00113] In some embodiments, T1, T2 and T3 are each independently a linker (e.g., as described herein). As used herein, the term "linker" or "linkage" refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length. A linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, poly(ethylene glycol); ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (iso- propyl), n-butyl, n-pentyl, 1 , 1 -dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable.
[00114] For instance, in certain embodiments, a linker includes a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, linker includes an alkyl or substituted alkyl group. In certain embodiments, linker includes an alkenyl or substituted alkenyl group. In certain embodiments, linker includes an alkynyl or substituted alkynyl group. In certain embodiments, linker includes an alkoxy or substituted alkoxy group. In certain embodiments, linker includes an amino or substituted amino group. In certain embodiments, linker includes a carboxyl or carboxyl ester group. In certain embodiments, linker includes an acyl amino group. In certain embodiments, linker includes an alkylamide or substituted alkylamide group. In certain embodiments, linker includes an aryl or substituted aryl group. In certain embodiments, linker includes a heteroaryl or substituted heteroaryl group. In certain embodiments, linker includes a cycloalkyl or substituted cycloalkyl group. In certain embodiments, linker includes a heterocyclyl or substituted heterocyclyl group.
[00115] In certain embodiments, linker includes a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is a polyethylene glycol.
[00116] Any convenient fatty acids may be utilized in the preparation of the subject lipidoid compounds.
As such, L1, L2 and L3 (if present) may be independently derived from any convenient fatty acids. Fatty acids of interest include, but are not limited to, short chain polyunsaturated fatty acids such as omega-3 fatty acids and omega-6 fatty acids, long-chain polyunsaturated fatty acids such as omega-9 fatty acids. "Fatty acids" refer to a family of carboxylic acids having a hydrocarbon chain of from about 12 to about 24 carbons in length. Unsaturated fatty acids have at least one carbon-carbon double bond in the hydrocarbon chain. Unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). Unsaturated fatty acids are designated by the position of the first double bond from the methyl end of the hydrocarbon chain. Omega-3 fatty acids have a first double bond at the third carbon from the methyl end of the chain; and include, e.g., a-linolenic acid (octadeca-9, 12,15-trienoic acid), stearidonic acid (octadeca-6,9,12,15-tetraenoic acid), eicosapentaenoic acid (eicosa-5, 8,11, 14,17- pentaenoic acid; "EPA"), docosapentaenoic acid (docosa-7, 10, 13, 16, 19-pentaenoic acid), eicosatetraenoic acid (eicosa-8, 11,14,17-tetraenoic acid), and docosahexaenoic acid (docosa-4,7, 10,13, 16, 19-hexaenoic acid; "DHA"). Omega-6 fatty acids have a first double bond at the sixth carbon from the methyl end of the chain; and include, e.g., linoleic acid (9, 12-octadecadienoic acid), γ-linolenic acid (6,9, 12-octadecatrienoic acid; GLA), eicosadienoic acid (11, 14-eicosadienoic acid), dihomo-y-linolenic acid (8,11,14- eicosatrienoic acid), arachidonic acid (5,8, 11,14-eicosatetraenoic acid), docosadienoic acid (13,16- docosadienoic acid), adrenic acid (7,10,13,16-docosatetraenoic acid), docosapentaenoic acid
(4,7, 10, 13, 16-docosapentaenoic acid), and calendic acid (8E, 10E, 12Z-octadecatrienoic acid), and the like. Omega-9 fatty acids have a first double bond at the ninth carbon from the methyl end of the chain; and include, e.g., oleic acid (c«-9-octadecenoic acid); eicosenoic acid (cis-l 1-eicosenoic acid); mead acid (all- cis-5,8,11-eicosatrienoic acid); erucic acid (c«-13-docosenoic acid); and nervonic acid (cis-15- tetracosenoic acid). As used herein, the term "lipoic acid" refers to a-lipoic acid, which is a chiral molecule also known as thioctic acid; l,2-diethylene-3 pentanoic acid; l,2-diethylene-3 valeric acid; and 6,8-thioctic acid. Unless specified the term "lipoic acid" encompasses the racemic mixture as well as any other (non-50/50) mixture of the enantiomers including substantially pure forms of either the -(+) or the S-(-) enantiomer.
[00117] In certain instances, L1, L2 and L3 (if present) are independently derived from an essential fatty acid. An essential fatty acid is a fatty acid that a human or other animal must ingest in its diet because the fatty acid is not synthesized in vivo. Suitable fatty acids include, but are not limited to, eicosapentaenoic acid, octadecanoic acid, eicosatetraenoic acid, docosahexaenoic acid, arachidonic acid, calendic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosoapentaenoic acid,tetracosatetraenoic acid, tetracosapentaenoic acid, hexadecatrienoic acid, stearidonic acid, eicosatrienoic acid, tetracosahexaenoic acid, 5-Dodecenoic acid, 7-tetradecenoic acid, 9-hexadecenoic acid, 11- octadecenoic acid, 13-eicosenoic acid, 15-decosenoic acid, elaidic acid, gonodoic acid, mead acid, erucic acid, and nervonic acid. In certain embodiments, the essential fatty acid is an omega-3 fatty acid. In certain embodiments, the essential fatty acid is an omega-6 fatty acid. In some embodiments, the essential fatty acid is selected from the group consisting of linoleic acid, linolenic acid and oleic acid. In certain embodiments, the essential fatty acid is linoleic acid. In certain embodiments, the essential fatty acid is linolenic acid. In certain embodiments, the essential fatty acid is oleic acid.
[00118] In some embodiments, the lipidoid compound is described by the formula (III):
Figure imgf000017_0001
(III)
wherein L1, L2, R1, R2, T1, T2, Z and Y are as described for formula (I), and in formula (II) p is 1. It is understood that any of the embodiments described herein for Formulae (I) and (II) may also apply to embodiments of Formula (III).
[00119] In some embodiments of Formula (III), Z is N. In certain embodiments of formula (III), Y is H. In certain embodiments of formula (III), Y is an alkyl. In certain embodiments of formula (III), Y is methyl. In certain embodiments of formula (III), Y is a substituted alkyl. In certain embodiments of formula (III), R1 and R2 are each hydrogen. In certain embodiments of formula (III), R1 and R2 are each an alkyl. In certain embodiments of formula (III), R1 and R2 are each methyl. In certain embodiments of formula (III), R1 and R2 are each a substituted alkyl.
[00120] In some embodiments of Formula (III), Z is sulfonium. In some embodiments of Formula (III), Z is S+ and Y is alkyl or substituted alkyl. In some embodiments of Formula (III), Z is phosphonium. In some embodiments of Formula (III), Z is P+ and Y is two groups, each group independently an alkyl or a substituted alkyl, e.g., -ZY- is -P+R2- In some embodiments of Formula (III), Y is a fatty acid-containing group described by formula (II).
[00121] In some embodiments, the lipidoid compound is described by the formula (IV):
Figure imgf000018_0001
(IV)
where: L 1 , V2, R 1 , R2% T 1 and 2 are as described for formula (I); and R 4 is H, an alkyl or a substituted alkyl. In certain embodiments of formula (IV), R4 is H. In certain embodiments of formula (IV), R4 is an alkyl. In certain embodiments of formula (IV), R4 is methyl. In certain embodiments of formula (IV), R4 is a substituted alkyl.
[00122] In some embodiments, the lipidoid compound is described by the formula (V):
L3
N-R3
Figure imgf000018_0002
(V)
where L1- L3, R1- R3 and T1 - T3 are as described for formulae (I) and (II).
[00123] In some embodiments of Formulae (IV) and (V), L1- L3 are each independently selected from the group consisting of linoleic acid, linolenic acid and oleic acid. In certain embodiments, L1- L3 are each linoleic acid. In certain embodiments, L1- L3 are each linolenic acid. In certain embodiments, L1- L3 are each oleic acid. In some embodiments of Formulae (IV) and (V), T1- T3 are each independently a Ci_6alkyl. In some embodiments of Formulae (IV) and (V), T1- T3 are each independently -(CH2)n-, where n is an integer from 1 to 6. In certain instances, n is 2. In certain instances, n is 3. In certain instances, n is 4. In certain instances, n is 5. In certain instances, n is 6.
[00124] In some embodiments of Formulae (IV) and (V), R1- R4 are each independently hydrogen or methyl. In some embodiments of Formulae (IV) and (V), R:-R3 are each H and R4 is methyl.
[00125] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000018_0003
[00126] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000018_0004
[00127] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0001
[00128] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0002
[00129] In certain embodiments, the lipidoid compound has the following structure: NKS09
O CH3 O
[00130] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0003
[00131] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0004
[00132] In certain embodiments, the lipidoid compound has the following structure: NKS 16
Figure imgf000019_0005
[00133] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0006
[00134] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0007
[00135] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000019_0008
[00136] In certain embodiments, the lipidoid compound has the following structure: NKS20
Figure imgf000020_0001
[00137] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000020_0002
[00138] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000020_0003
[00139] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000020_0004
[00140] In certain embodiments, the lipidoid compound has the following structure:
Figure imgf000020_0005
[00141] In certain embodiments, the lipidoid compound has the following structure: NKS36
Figure imgf000021_0001
[00143] In certain embodiments, the lipidoid compound has the following structure:
NKS50
Figure imgf000021_0002
LIPIDOID COMPOSITIONS
[00144] The present disclosure includes a lipoid composition that contains a lipoid compound, as described above, wherein the lipoid compound is non-covalently bound to a target nucleic acid. Non-covalent binding of the lipidoid compound and the target nucleic acid may be via any convenient non-covalent interactions, such as electrostatic interactions. In certain embodiments, the target nucleic acid is a target RNA. In certain embodiments, the target nucleic acid is selected from the group consisting of: a DNA, an siRNA, an shRNA, a miRNA, and an antisense nucleic acid. In certain embodiments, the DNA includes a nucleotide sequence encoding a gene product. In certain embodiments, the gene product is a polypeptide. In certain embodiments, the polypeptide is a therapeutic polypeptide. In certain embodiments, the gene product is an RNA.
[00145] In certain embodiments, further comprising nanoparticles of the lipidoid compound. Any convenient lipidoid nanoparticles may be prepared from the subject lipidoid compounds and utilized in the subject compositions. Methods and materials that may find use in nanoparticles of the subject lipidoid compounds include, but are not limited to, those nanoparticle methods and materials described by Whitehead et al. in "Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity", Nature
Communications 5, Article number:4277, June 2014.
Nucleic acids
[00146] The present disclosure includes a lipoid composition that comprises a lipoid compound, as described above, wherein the lipoid compound is non-covalently bound to a target nucleic acid. The nucleic acid may be any nucleic acid suitable for non-covalent binding to the present lipoid compounds. In some embodiments, the target nucleic acid contains a target RNA (e.g., RNA encoding a polypeptide, a regulatory or inhibitory RNA, a ribozyme, a Ul adaptor, riboswitches, etc.), or a target DNA (e.g., DNA encoding a gene product such as a polypeptide or a regulatory RNA, or a DNA encoding an inhibitory RNA etc.). In some embodiments, the target nucleic acid contains a nucleic acid aptamer. In some embodiments, the target nucleic acid contains a binding partner for a binding moiety. A target nucleic acid can be a guide RNA that comprises: i) a nucleotide sequence that binds to an endonuclease, such as a CRISPR/Cas9 endonuclease; and ii) a nucleotide sequence that binds a target DNA sequence, e.g., a DNA sequence in the genome of a cell.
[00147] The target DNA may be linear (e.g., a polymerase chain reaction (PCR) product, a linearized plasmid, etc.) or circular (e.g., a plasmid, a cosmid, etc.). The target nucleic acid may be single stranded or double stranded (partially or completely).
[00148] The length of the target nucleic acid (single-stranded or double stranded) may be any length suitable for non-covalently binding to the present lipoid compounds and for performing the function of the target nucleic acid in a target cell. In some instances, the length of the target nucleic acid is 5 nucleotides or more, e.g., 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 25 nucleotides or more, 30 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 500 nucleotides or more, 1,000 nucleotides or more, 3,000 nucleotides or more, 5,000 nucleotides or more, 8,000 nucleotides or more, or 10,000 nucleotides or more, and in some cases is 50,000 nucleotides or less, 20,000 nucleotides or less, 8,000 nucleotides or less, 6,000 nucleotides or less, 4,000 nucleotides or less, 2,000 nucleotides or less, 1,000 nucleotides or less, 750 nucleotides or less, 500 nucleotides or less, 250 nucleotides or less, 100 nucleotides or less, 50 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less. In some instances, the length of the target nucleic acid is in the range of 10 to 50,000 nucleotides, e.g., 10 to 20,000 nucleotides, 15 to 10,000 nucleotides, 15 to 6,000 nucleotides, 10 to 1,000 nucleotides, 15 to 500 nucleotides, 18 to 200 nucleotides, 1,000 to 30,000 nucleotides, 2,000 to 10,000 nucleotides, including 3,000 to 8,000 nucleotides.
[00149] In some embodiments, the target nucleic acid contains a therapeutic target nucleic acid (e.g., DNA
encoding a therapeutic polypeptide, DNA encoding a therapeutic regulatory or inhibitory RNA, a therapeutic regulatory or inhibitory RNA, etc.).
Target RNA
[00150] In some embodiments, the target nucleic acid contains a target RNA (e.g., RNA encoding a polypeptide, a regulatory or inhibitory RNA, a ribozyme, a Ul adaptor, riboswitches, etc.). Examples of target RNAs suitable for non-covalently binding to the present lipoid compounds include, but are not limited to, micro RNA (miRNA; including pri-miRNA and pre-miRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), Ul adaptors, ribozymes, riboswitches.
[00151] In some embodiments, the target nucleic acid contains an siRNA. Small interference RNA (siRNA) is a double-stranded RNA containing a sense strand having a base sequence corresponding to a part of a target gene and an antisense strand thereof. siRNA can induce sequence-specific post-transcriptional gene silencing (i.e., RNA interference) in cells (e.g., eukaryotic cells) (See, e.g., Fire A. et al., 1998, Nature, 391, 806-811). siRNA contained in the lipoid composition may include a base sequence that is fully complementary to a region of the sense strand of a target gene. The length of the complementarity may be 17 to 32 bases, e.g., 18 to 30 bases, or 19 to 25 bases.
[00152] The siRNA may target any suitable gene for silencing in a target cell. Any suitable method may be used to design the siRNA based on a target gene sequence. Suitable methods are described in, e.g., Ui-Tei et al. (Nucleic Acids Res., 32: 936-948, 2004); Reynolds et al. (Nat. Biotechnol., 22: 326-330, 2004); and Amarzguioui et al. (Biochem. Biophys. Res. Commun., 316: 1050-1058, 2004), which are incorporated herein by reference. In addition, web sites on which siRNA can be designed have been made available to public by a variety of research institutes or companies, and effective siRNA can be designed on the web. Representative examples of siRNA designing web sites include siDirect
(http(colon)//design(dot)RNAi(dot)jp/), siSearch
(http(colon)//www(dot)epigeneticstation(dot)com/epigenetic-links/detail/link-203(dot)html), the siDESIGN Center (http(colon)//www(dot)dharmacon(dot)com/designcenter/designcenterpage(dot)aspx), the siRNA Selection Server (http(colon)//jura(dot)wi(dot)mit(dot)edu/bioc/siRNAext/), and the Gene Specific siRNA Selector (http(colon)//bioinfo(dot)wistar(dot)upenn(dot)edu/siRNA/siRNA(dot)htm).
[00153] In some embodiments, the siRNA may further include other functional nucleic acids, such as RNA
aptamers or single-stranded miRNA precursors.
[00154] The siRNA included in the target nucleic acid of the present composition may target any suitable gene.
Target genes include any gene encoding a target gene product (RNA or protein) that is deleterious (e.g., pathological); a target gene product that is malfunctioning; a target gene product. Target gene products include, but are not limited to, huntingtin; hepatitis C virus; human immunodeficiency virus; amyloid precursor protein; tau; a protein that includes a polyglutamine repeat; a herpes virus (e.g., varicella zoster); any pathological virus; and the like.
[00155] siRNA is useful for treating a variety of disorders and conditions, including, but not limited to,
neurodegenerative diseases, e.g., a trinucleotide-repeat disease, such as a disease associated with polyglutamine repeats, e.g., Huntington's disease , spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), etc.; an acquired pathology (e.g., a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural, or molecular biological state) such as a viral infection, e.g., hepatitis that occurs or may occur as a result of an HCV infection, acquired immunodeficiency syndrome, which occurs as a result of an HIV infection; and the like.
[00156] In certain embodiments, an siRNA is directed against one or more members of the following classes of proteins: developmental proteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53, and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextriinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hernicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases,
polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases).
[00157] In some embodiments, an siRNA is directed against a member of a signal transduction pathway, e.g., the insulin pathway, including AKT1-3, CBL, CBLB, EIF4EBP1, FOXOIA, FOX03A, FRAP1, GSK3A, GSK3B, IGF1, IGF1R, INPP5D, INSR, IRS 1, MLLT7, PDPK1, PIK3CA, PIK3CB, PIK3R1, PIK3R2, PPP2R2B, PTEN, RPS6, RPS6KA1, RPX6KA3, SGK, TSC1 , TSC2, and XPOl); an apoptotic pathway (CASP3,6,7,8,9, DSH1/2, PI 10, P85, PDK1/2, CATENIN, HSP90, CDC37, P23, BAD, BCLXL, BCL2, SMAC, and others); and pathways involved in DNA damage, cell cycle, and the like (p53, MDM2, CHKl/2, BRCAl/2, ATM, ATR, P15INK4, P27, P21 , SKP2, CDC25C/A, 14-3-3, PLK, RB, CDK4, GLUT4, Inos, Mtor, FKBP, PPAR, RXR, ER). Similarly, genes involved in immune system function including TNFR1 , IL-IR, IRAKI/2, TRAF2, TRAF6, TRADD, FADD, ΙΚΚε, ΓΚΚγ, ΙΚΚβ, ΙΚΚα, IkBa, IkB , p50, p65, Rac, RhoA, Cdc42, ROCK, Pakl/2/3/4/5/6, cIAP, HDACl/2, CBP, β-TrCP, Rip2/4, and others are also important targets for siRNAs, where such siRNAs can be useful in treating immune system disorders. siRNAs specific for gene products involved in apoptosis, such as Dshl 2, PTEN, PI 10 (pan), P85, PDK1/2, Aktl , Akt2, Akt (pan), p70s6 , GSK3 , PP2A (cat), β-catenin, HSP90, Cdc37/p50, P23, Bad, BclxL, Bcl2, Smac/Diablo, and Askl are useful in the treatment of diseases that involve defects in programmed cell death (e.g. in the treatment of cancer). siRNA directed against p53, MDM2, Chkl 2, BRCAl/2, ATM, ATR, p\5m \ P27, P21 , Skp2, Cdc25C/A, 14-3-3sigma/8, PLK, Rb, Cdk4, Glut4, iNOS, mTOR, FKBP, PPARy, RXRa, ERa, and related genes can be used to treat diseases associated with disruptions in DNA repair, and cell cycle abnormalities, where such diseases include cancer. Examples of such siRNAs and targets are known in the art; see, e.g., US Patent Publication No. 2005/0246794 and 2011/0003704. For example, a target DNA that includes a nucleic acid encoding an siRNA is useful for treating disorders resulting from or associated with dysregulated cell cycle, e.g., cancer. In some embodiments, an siRNA is directed against transcription elongation factors, such as CDK9, cyclin Tl, Spt4, Spt5, Spt6.
[00158] In some embodiments, the target nucleic acid contains an miRNA. miRNA (micro RNA) is a single- stranded non-coding RNA that is 21 to 23 bases in length, is present in vivo, and regulates the expression of a given gene. Such RNA is known to form a complex by binding to mRNA of a target gene and a protein factor and to inhibit the translation of the target gene. Endogenous miRNA may be transcribed from the genome as a single-stranded precursor referred to as pri-miRNA, further processed into a single- stranded precursor referred to as pre-miRNA with the use of an endonuclease referred to as Drosha in the nucleus, and converted into mature double-stranded miRNA by the action of an endonuclease referred to as Dicer outside the nucleus. One strand thereof is incorporated into an RISC (RNA-induced silencing complex) and regulates the expression of the target gene as a mature single-stranded miRNA.
[00159] A target nucleic acid may contain a mature double-stranded miRNA that has a base sequence identical to that of wild-type mature double-stranded miRNA. In such a case, such sequence may be designed based on the base sequence of miRNA encoded in the genome. A target nucleic acid may contain a sequence of a single-stranded miRNA precursor that has the same base sequence as that of wild-type miRNA encoded in the genome.
[00160] A miRNA included in the target nucleic acid of the present composition may be any suitable miRNA.
Exemplary miRNAs include, but are not limited to, miR-34, miR-124, miR-155, miR-181, miR-221, miR- 122a, miR-32, miR-20a, miR-34a, miR-27b, miR-17-5p, miR-29a, miR-29b, miR-29c, miR-149, miR- 324-5p, miR-378, let-7a, let-7b, let-7c, let-7e, let-7f, let-7i, miR-101, miR-103, miR-106a, miR-lOa, miR- 10b, miR-124a, miR-125a, miR-126, miR-132, miR-133a, miR-141, miR-146a, miR-146b, miR-148a, miR-148b, miR-151, miR-152, miR-15a, miR-15b, miR-181b, miR-182, miR-183, miR-18a, miR-191, miR-193b, miR-195, miR-196a, miR-196b, miR-199a, miR-200a, miR-200c, miR-203, miR-205, miR-21, miR-214, miR-218, miR-22, miR-222, miR-24, miR-25, miR-26a, miR-27a, miR-29b, miR-29c, miR-30a- 3p, miR-30a-5p, miR-30d, miR-320, miR-324-5p, miR-328, miR-331, miR-335, miR-340, miR-342, miR- 345, miR-362, miR-365, miR-375, miR-378, miR-422a, miR-422b, miR-425, miR-429, miR-500, miR-7, miR-9*, miR-92, miR-93, miR-98, miR-153-2, miR-33a, let-7d, miR-24-1, miR-27b, miR-23b, miR- 181a, miR-199b, miR-31, let-7g, miR-32a-l, miR-33b, miR-100, miR-125b-l, miR-135-1, miR-142as, miR-142s, miR-144, miR-301, miR-297-3, miR-17, miR-18, miR-19a, miR-19bl, miR-20, miR-92-1, miR-128a, miR-7-3, miR-123, miR-161, miR-177, miR-212, miR-99a, miR-125b-2, miR-210, miR-135-2, miR-208, miR-211, miR-180, miR-145, miR-143, miR-127, miR-136, miR-138-1, miR-154, miR-134, miR-299, miR-92-2, miR-19b-2, miR-108-1, miR-193, miR-29a, miR-29b, miR- 129-1, miR-96, miR-32, miR- 159-1, miR- 192, miR- 186, miR- 194, miR-215, miR-106b, miR-196-2, miR- 190, miR-105-1, miR- 175, miR-148, miR-196-1, miR-202, miR-139.
[00161] In some embodiments, the target nucleic acid contains an shRNA. shRNA (short hairpin RNA) is a single- stranded RNA comprising siRNA or mature double-stranded miRNA ligated by an adequate short spacer sequence. Accordingly, a sense region is base-paired with an antisense region to form a stem structure in a molecule, and the spacer sequence forms a loop structure therein. Thereby, an shRNA molecule has a hairpin-shaped stem-loop structure as a whole. A spacer sequence may be 3 to 24 bases long, such as 4 to 15 bases long. A spacer sequence may be any suitable sequence, provided that the siRNA or mature double-stranded miRNA is capable of base pairing.
[00162] The shRNA may conveniently include any siRNA targeting a gene, as described above, or include any suitable miRNA, as described above.
[00163] In some embodiments, the target nucleic acid is a target DNA (e.g., DNA encoding a gene product such as a polypeptide or a regulatory RNA, or a DNA encoding an inhibitory RNA etc.).
[00164] In some embodiments, the target nucleic acid contains a ribozyme or deoxyribozyme. The term
"ribozyme" refers to RNA having catalytic functions, e.g., specifically cleaving a target RNA sequence, and "deoxyribozyme" refers to DNA having catalytic functions, e.g., specifically cleaving a target RNA sequence. Thus, a ribozyme may be constituted in the form of single-stranded RNA, and a deoxyribozyme may be constituted in the form of single-stranded DNA. The target nucleic acid may contain any suitable ribozyme or deoxyribozyme for use in the present lipoid composition. A ribozyme may be a hammerhead ribozyme, a VS ribozyme, a Leadzyme, a hairpin ribozyme, etc. Exemplary ribozymes include, but are not limited to, hammerhead ribozymes configured to target HIV-1 tat/vpr RNA, hammerhead ribozymes configured to target surviving mRNA, and hairpin ribozymes targeting 5'- and 3'-untranslated regions (UTRs) of hepatitis C virus (HCV), etc.
[00165] In some embodiments, the target nucleic acid contains an Ul adaptor. A "Ul adaptor" is a bifunctional single-stranded nucleic acid of about 25 bases, and it includes a 5 '-"target domain" complementary to the 3 '-terminal exon in the mRNA precursor of the target gene and a 3'-"Ul domain" having a sequence complementary to the 5' region of Ul snRNA (Goraczniak R. et al., 2009, Nat. Biotechnol., Vol. 27, pp. 257-263). Upon introduction of the Ul adaptor into an organism, a Ul snRNP containing Ul snRNA binds to a region in the vicinity of a poly A signal of the mRNA precursor of the target gene, and polyadenylation of such mRNA is specifically inhibited. As a result, the mRNA precursor of the target gene is destabilized and degraded in the nucleus. The Ul adaptor may target any convenient gene, such as those targeted by siRNA described above.
[00166] In some embodiments, the target nucleic acid contains a nucleic acid aptamer. The term "nucleic acid aptamer" refers to a nucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of the target molecule. The nucleic acid aptamer may be an RNA aptamers or a DNA aptamer. The nucleic acid aptamer non-covalently bound to the lipoid compounds may contain DNA, RNA, or a combination thereof.
[00167] A nucleic acid aptamer may have a higher specificity and affinity to a target molecule than an antibody. In some cases, the nucleic acid aptamer can specifically bind to a target molecule with high affinity. Thus, the nucleic aptamer may enable selective suppression of functions of a given protein among highly homologous proteins.
[00168] Any suitable nucleic acid aptamer may be used in the present lipoid composition for binding non- covalently to the lipoid compounds. Various nucleic acids as well as methods of generating target-specific aptamers are known. See, e..g, Jayasena, 1999, Clin. Chem. 45: 1628-1650, which is incorporated herein by reference. For example, an RNA aptamer may be prepared via in vitro selection making use of the systematic evolution of ligands by exponential enrichment (SELEX) method. The SELEX method comprises selecting an RNA molecule bound to a target molecule from an RNA pool composed of RNA molecules each having random sequence regions and primer-binding regions at both ends thereof, amplifying the recovered RNA molecule via RT-PCR, performing transcription using the obtained cDNA molecule as a template, and using the resultant as an RNA pool for the subsequent procedure. Such procedure is repeated several times to several tens of times to select RNA with a stronger ability to bind to a target molecule. The base sequence lengths of the random sequence region and the primer binding region are not particularly limited. In general, the random sequence region comprises 20 to 80 bases and the primer binding region comprises 15 to 40 bases. Specificity to a target molecule may be enhanced by prospectively mixing molecules similar to the target molecule with RNA pools and using a pool comprising RNA molecules that did not bind to the molecule of interest. An RNA molecule that was obtained as a final product by such technique is used as an RNA aptamer. The SELEX method is a known technique, and a specific method may be implemented in accordance with, for example, Pan et al. (Proc. Natl. Acad. Sci. U.S.A., 1995, 92: 11509-11513), incorporated herein by reference.
Target DNA
[00169] In some embodiments, the target nucleic acid is a binding partner for a binding moiety. In such instances, the target nucleic acid may contain a nucleotide sequence that is specifically bound by a binding moiety, such as a nucleic acid-binding protein, other nucleic acids, or small molecules. A binding moiety may include a transcription factor, a restriction enzyme, etc. The target nucleic acid may contain any convenient nucleotide sequence that is specifically bound by a binding moiety. Exemplary nucleotide sequences that bind a binding moiety include, but are not limited to, binding sites for endogenous transcription factors, such as E2F, Stat3, cAMP response element, Ets-1, Ap-1, NF-κΒ, GATA-3, STAT-1, STAT-6, TCF; and binding sites for transcriptional regulators used by infectious organisms, such as WhiB7 (Actinomycetes), FadR (E. coli), YycG/YycF (S. aureus, B. subtilis, S. pneumoniae, S. pyogenes, Listeria monocytogenes), Sigma 54 or SigB (P. aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae), Fur (S. aureus, E. coli, Helicobacter pylori, B. subtilis), TcdR {Clostridium difficile, C. botulinium (where the homologue is BotR), C. tetani (TetR) and C. perfringens), Vfr (P. aeruginosa and E. coll), NtrC {Klebsiella pneumonia), and ArsR {Helicobacter pylori, H. acinonychis and H. felis).
[00170] In some embodiments, the target nucleic acid is a target DNA that contains a nucleotide sequence
encoding an RNA, such as any of the regulatory or inhibitory RNAs described above. In some embodiments, the target DNA contains a nucleotide sequence encoding for a polypeptide gene product, such as a therapeutic polypeptide. Exemplary polypeptides that may be encoded in the target DNA include, but are not limited to, the peptidyl hormones activin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, calcitonin gene -related peptide, calcitonin N-terminal flanking peptide, ciliary neurotrophic factor (CNTF), corticotropin (adrenocorticotropin hormone, ACTH), corticotropin-releasing factor (CRF or CRH), epidermal growth factor (EGF), follicle-stimulating hormone (FSH), gastrin, gastrin inhibitory peptide (GIP), gastrin-releasing peptide, gonadotropin-releasing factor (GnRF or GNRH), growth hormone releasing factor (GRF, GRH), human chorionic gonadotropin (hCH), inhibin A, inhibin B, insulin, luteinizing hormone (LH), luteinizing hormone -releasing hormone (LHRH), a-melanocyte- stimulating hormone, β-melanocyte-stimulating hormone, γ-melanocyte- stimulating hormone, melatonin, motilin, oxytocin (pitocin), pancreatic polypeptide, parathyroid hormone (PTH), placental lactogen, prolactin (PRL), prolactin-release inhibiting factor (PIF), prolactin-releasing factor (PRF), secretin, somatotropin (growth hormone, GH), somatostatin (SIF, growth hormone -release inhibiting factor, GIF), thyrotropin (thyroid-stimulating hormone, TSH), thyrotropin-releasing factor (TRH or TRF), thyroxine, vasoactive intestinal peptide (VIP), and vasopressin. Other polypeptides suitable to be encoded in the target DNA include the cytokines, e.g., colony stimulating factor 4, heparin binding neurotrophic factor (HBNF), interferon-a, interferon a-2a, interferon a-2b, interferon a-n3, interferon-β, etc., interleukin- 1 , interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, etc., tumor necrosis factor, tumor necrosis factor-a, granuloycte colony-stimulating factor (G-CSF), granulocyte-macrophage colony- stimulating factor (GM- CSF), macrophage colony-stimulating factor, midkine (MD), and thymopoietin. Still other polypeptides suitable to be encoded in the target DNA include endorphins (e.g., dermorphin, dynorphin, a-endorphin, β- endorphin, γ-endorphin, sigma-endorphin, [Leu5]enkephalin,
[Met5]enkephalin, substance P), kinins (e.g., bradykinin, potentiator B, bradykinin potentiator C, kallidin), LHRH analogues (e.g., buserelin, deslorelin, fertirelin, goserelin, histrelin, leuprolide, lutrelin, nafarelin, tryptorelin), and the coagulation factors, such as ai-antitrypsin, a2-macroglobulin, antithrombin III, factor I (fibrinogen), factor II (prothrombin), factor III (tissue prothrombin), factor V (proaccelerin), factor VII (proconvertin), factor VIII (antihemophilic globulin or AHG), factor IX (Christmas factor, plasma thromboplastin component or PTC), factor X (Stuart-Power factor), factor XI (plasma thromboplastin antecedent or PTA), factor XII (Hageman factor), heparin cof actor II, kallikrein, plasmin, plasminogen, prekallikrein, protein C, protein S, and thrombomodulin, and combinations thereof.
[00171] In some embodiments, a target DNA contains a nucleotide sequence encoding a gene product that is used in laboratory research, such as fluorescent proteins (e.g., green fluorescent protein (GFP)); indicators (e.g., calcium indicators, voltage indicators); and other genetically engineered proteins (e.g., light-sensitive ion channels, voltage-gated ion channels, transcription factors, signaling proteins, cytoskeletal proteins, enzymes, etc.).
[00172] In certain embodiments, a target DNA containing a nucleotide sequence encoding a gene product is configured such that the gene product is expressed in a target cell. In such instances, the nucleotide sequence encoding a gene product may be operably linked to one or more control elements, e.g. a promoter sequence, enhancer sequence, introns, etc., that promote expression of the gene product from the target DNA in a target cell. Such control elements can include control sequences normally associated with the selected gene product (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter that is heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, can also be used. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif). In some cases, the target DNA includes an expression vector that contains a nucleotide sequence encoding a gene product and one or more control elements that promote expression of the gene product in a target cell.
[00173] In some embodiments, a cell type-specific or a tissue-specific promoter will be operably linked to the nucleotide sequence encoding a gene product, such that the gene product is produced selectively or preferentially in a particular cell type(s) or tissue(s). In some embodiments, an inducible promoter will be operably linked to the nucleotide sequence encoding a gene product.
Nucleic acid modifications
[00174] In some embodiments, a target nucleic acid (e.g., a target DNA, a target RNA, an antisense nucleic acid) comprises one or more modifications, e.g., a base modification, a backbone modification, etc. As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. Modified backbones and modified internucleoside linkages
[00175] Examples of suitable nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
[00176] Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
[00177] In some embodiments, a subject target nucleic acid (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) comprises one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular -CH2-NH-0-CH2-, -CH2-N(CH3)-0-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-0-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -0-N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as -0-P(=0)(OH)-0-CH2-). MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Suitable amide internucleoside linkages are disclosed in t U.S. Pat. No. 5,602,240.
[00178] Also suitable are target nucleic acids (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) having morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506. For example, in some embodiments, a subject nucleic acid (e.g., a subject target RNA; a subject antisense nucleic acid) comprises a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.
[00179] Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have
backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Mimetics
[00180] A subject target nucleic acid (e.g., e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) can be a nucleic acid mimetic. The term "mimetic" as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
[00181] One polynucleotide mimetic that has been reported to have excellent hybridization properties is a peptide nucleic acid (PNA). The backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262.
[00182] Another class of polynucleotide mimetic that has been studied is based on linked morpholino units
(morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups has been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey,
Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.
[00183] A further class of polynucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The
furanose ring normally present in an DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.
[00184] A further modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2-), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3'-exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).
[00185] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Modified sugar moieties
[00186] A subject target nucleic acid (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) can also include one or more substituted sugar moieties. Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O- alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub. l to do alkyl or C2 to do alkenyl and alkynyl. Particularly suitable are 0((CH2)nO) mCH3, 0(CH2)nOCH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)nONH2, and 0(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: d to do lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A suitable modification includes 2'-methoxyethoxy (2'-0-CH2 CH2OCH3, also known as 2'-0- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitable modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'- DMAEOE), i.e., 2'-0-CH2-0-CH2-N(CH3)2.
[00187] Other suitable sugar substituent groups include methoxy (-0-CH3), aminopropoxy (—0 CH2 CH2
CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-0- CH2— CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Base modifications and substitutions
[00188] A subject target nucleic acid (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
[00189] Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are useful for increasing the binding affinity of an oligomeric compound (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid). These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable base substitutions, e.g., when combined with 2'-0-methoxyethyl sugar modifications. Conjugates
[00190] Another possible modification of a subject target nucleic acid (e.g., a subject target DNA; a subject target RNA; a subject antisense nucleic acid) involves chemically linking to the polynucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject antisense nucleic acid or target protector nucleic acid.
[00191] Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.
METHODS
[00192] The present disclosure provides a method of delivering a gene product to an individual, the method
comprising administering to the individual an effective amount of a lipidoid composition of the present disclosure. The present disclosure provides a method of delivering a target nucleic acid to a cell, the method comprising contacting a cell with the lipidoid composition of the present disclosure to intracellularly deliver the target nucleic acid to the cell.
[00193] A lipidoid composition of the present disclosure can be provided together with a pharmaceutically
acceptable excipient. Pharmaceutically acceptable excipients are known to those skilled in the art, and have been amply described in a variety of publications, including, for example, A. Gennaro (1995) "Remington: The Science and Practice of Pharmacy", 19th edition, Lippincott, Williams, & Wilkins. In the discussion, below, of formulations, dosages, and routes of delivery, an "active agent" will refer to an agent discussed herein, e.g., a lipidoid composition of the present disclosure, unless otherwise specified.
[00194] A lipidoid composition of the present disclosure can be formulated in a variety of ways. The form (e.g., liquid, solid, pill, capsule) and composition of the formulation will vary according to the method of administration used. For example, where the formulation is administered orally, the nucleic acid can be formulated as a tablet, pill, capsule, solution (e.g., gel, syrup, slurry, or suspension), or other suitable form. In some cases, a lipidoid composition of the present disclosure is formulated to facilitate delivery to the surface of the intestinal cells.
[00195] The formulation can contain components in addition to a lipidoid composition of the present disclosure, where the additional components aid in the delivery of the lipidoid composition, e.g., delivery to an intestinal cell. The lipidoid composition of the present disclosure acid can be present in a pharmaceutical composition with additional components such as, but not limited to, stabilizing compounds and/or biocompatible pharmaceutical carriers, e.g., saline, buffered saline, dextrose, or water. The lipidoid composition of the present disclosure can also be administered alone or in combination with other agents, including other therapeutic agents. The formulation can also contain organic and inorganic compounds to, for example, facilitate nucleic acid delivery to and uptake by the target cell (e.g., detergents, salts, chelating agents, etc.).
[00196] A formulation comprising a lipidoid composition of the present disclosure can include: a) a lipidoid
composition of the present disclosure; and b) one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2- hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3- propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N- tris(hydroxymethyl)methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc.). Suitable salts include, e.g., NaCl, MgCl2, KC1, MgS04, etc.
[00197] A formulation comprising a lipidoid composition of the present disclosure can include a lipidoid composition of the present disclosure in an amount of from about 0.001 % to about 90% (w/w).
[00198] A lipidoid composition of the present disclosure can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
[00199] A lipidoid composition of the present disclosure, or a formulation comprising a lipidoid
composition of the present disclosure, can be administered to an individual in need thereof by any of a variety of routes of administration. Suitable routes of administration include enteral and parenteral routes. Administration can be via a local or a systemic route of administration. A lipidoid composition of the present disclosure, or a formulation comprising a lipidoid composition of the present disclosure, can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; and intracranial, e.g., intrathecal or intraventricular, administration. Intratumoral and peritumoral administration is also contemplated.
[00200] The formulation of compositions and their subsequent administration (dosing) is within the skill of those in the art. Dosing is dependent on several criteria, including severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual lipidoid compositions, and can generally be estimated based on EC50s or IC50s found to be effective in vitro and in vivo animal models.
[00201] For example, a suitable dose of a lipidoid composition of the present disclosure is from 0.01 μg to
100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein a lipidoid composition of the present disclosure is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight.
[00202] In some embodiments, multiple doses of a lipidoid composition of the present disclosure are administered. The frequency of administration of a lipidoid composition of the present disclosure can vary depending on any of a variety of factors, e.g., severity of the symptoms, etc. For example, in some embodiments, a lipidoid composition of the present disclosure is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).
[00203] The duration of administration of lipidoid composition of the present disclosure, e.g., the period of time over which an active agent is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, a lipidoid composition of the present disclosure can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more. METHODS FOR PRODUCING A LIPIDOID COMPOSITION
[00204] The present disclosure includes methods of making a lipidoid compound, e.g., as described herein. In some embodiments, the method includes: contacting a fatty acid ester with an amino linker to produce a fatty acid contacted mixture; and heating the mixture under an inert atmosphere for a time sufficient to produce the lipidoid compound, e.g., as described herein. The subject methods of making may provide for preparation of the lipidoid compounds from fatty acid ester and amino linker starting materials in a single step without the need for extensive purification.
[00205] Any convenient esters of the fatty acids described herein may be utilized in the subject methods. In some embodiments, the fatty acid ester is an alkyl ester or a substituted alkyl ester. In some instances, the fatty acid ester is a lower alkyl ester. In certain instances, the fatty acid ester is a methyl ester or an ethyl ester of a fatty acid of interest. In some instances, the fatty acid ester is an aryl, substituted aryl, heteroaryl or substituted heteroaryl ester. In some instances, the fatty acid ester is an activated ester that comprises a convenient leaving group.
[00206] Any convenient synthetic precursors of the amino linkers of the lipidoid compounds described herein may be utilized in the subject methods. In some embodiments of the method, the amino linker is described by formula (VI):
Figure imgf000037_0001
(VI)
wherein R1, R2, T1, T2, Z and Y are as described for formula (I), and in formula (II) p is 1. It is understood that any of the embodiments described herein for the lipidoid compounds of Formula (I) to (V) may also be applied to the synthetic precursors of Formula (VI).
[00207] In certain embodiments of Formula (VI), Z is N. In certain embodiments of formula (VI), Y is H. In certain embodiments of formula (VI), Y is an alkyl. In certain embodiments of formula (VI), Y is methyl. In certain embodiments of formula (VI), Y is a substituted alkyl. In certain embodiments of formula (VI), R1 and R2 are each hydrogen. In certain embodiments of formula (VI), R1 and R2 are each an alkyl. In certain embodiments of formula (VI), R1 and R2 are each methyl. In certain embodiments of formula (VI), R1 and R2 are each a substituted alkyl.
[00208] Any convenient methods may be utilized in contacting the fatty acid with the amino linker to produce a fatty acid contacted mixture. In some embodiments, the fatty acid contacted mixture further includes a solvent. Any convenient solvents in which one or more of the components of the fatty acid contacted mixture is soluble may be utilized. In certain instances, one of the starting material components of the mixture can act as a solvent.
[00209] Any convenient methods may be utilized in heating the mixture under an inert atmosphere for a time sufficient to produce the lipidoid compound, e.g., as described herein. Heating may be performed at any convenient temperature between room temperature and the boiling point of one of the components of the mixture and/or the solvent. In some embodiments, the heating achieves a temperature of 50°C or more, such as 80°C or more, 90°C or more, 100°C or more, such as 110 °C or more, 120 °C or more, 130 °C or more, or even 150 °C or more. In certain embodiments, the heating is maintained at a temperature in the range of 100 to 200 °C, such as in the range of 100 to 160 °C, such as in the range of 130-160 °C. By under an inert atmosphere is meant that the mixture is heated in the absence of oxygen. The mixture may be purged and/or heated under an inert gas, such as nitrogen or helium. The heating may be maintained for any convenient length of time depending on a variety of factors, such as the particular components, the solvent, the temperature, etc. In certain embodiments, the heating is maintained for 1 hour or more, such as 3 hours or more, 6 hours or more, overnight, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or even more.
EXAMPLES
[00210] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
Example 1 :
[00211] The synthesis of a series of essential fatty acid lipidoids through the systematic substitution of selected amines with omega-3 and omega-6 fatty acid esters, for the purpose of delivering polynucleotides (siRNA, miRNA and plasmid DNA) in vitro and in vivo is described. Three of the lipidoids synthesized, designated as NKS 11, NKS21 and NKS22, were chosen for in vivo studies, as they were effective in in vitro studies. Essential fatty acid lipidoids were biodegradable and biocompatible and may be used in a variety of drug delivery systems. Given the amino moiety of these essential fatty acid lipidoids compounds, they were particularly suited for the delivery of polynucleotides. The safety and efficiency of these lipidoids to deliver complexes (micelles, liposomes or particles) containing the lipidoids and polynucleotides, both in vitro and in vivo, were demonstrated. They were useful in delivering labile agents given their ability to buffer the pH of their surroundings. In the studies performed in delivering miR124 in mice, spleen was found to be the primary target organ for these delivery agents in vivo. Results from studies on these lipidoids to deliver oligonucleotides intranasally and transdermally are also presented. MATERIALS AND METHODS
Synthesis of lipidoids
[00212] Essential fatty acid esters and amines were used to synthesize a combinatorial library of lipidoids.
Synthesis occurred through the nucleophilic acyl substitution reactions. There were two sites in the amino monomer amenable to substitution reactions in each of the amines, resulting in two tails in each lipidoid, as shown in the schemes.
[00213] Conventional lipid syntheses typically require individually optimized, multi-step synthesis, use of
catalysts, solvent exchanges and purifications (40). Using these traditional methods to synthesize a combinatorial library of substantial size is laborious and limits throughput. Therefore, click chemistry, defined by Sharpless and colleagues, was used as an approach that uses only the most practical and reliable chemical transformations (42, 43). The synthetic scheme used to develop these lipidoids was based on the nucleophilic substitution reaction of essential fatty acid esters with primary or secondary amines (Scheme 1-3). This particular chemistry, unlike the traditional lipid synthesis chemistries, was completed in a single step and allowed for reactions in the absence of solvents. Typically, the amines chosen contain short carbon chains with two to three amine moieties and the fatty acid ester compounds include a tail of varying chain lengths and optionally feature various functional groups and varying degrees of saturation. The resulting novel essential fatty acid lipidoid compounds were particularly useful in delivering negatively charged agents given the secondary and tertiary amines available for protonation thus forming a cationic moiety. For example, the essential fatty acid lipidoid compounds may be used to delivery DNA, RNA, or other polynucleotides to a subject or to a cell. Also, two different fatty acid esters may be used in the reaction mixture to prepare an essential fatty acid lipidoid compound with two different tails.
Figure imgf000039_0001
[00214] Scheme 1. Synthesis of NKS 11: Reaction scheme for the synthesis of NKS 11 : Ethyl linoleate (0.103 g, 1 mmol) was added to diethylenetriamine (0.616 g, 2 mmol) dropwise and heated with stirring at 140°C for 72 hours under N2 to give NKS 11 as a white crystalline product.
Figure imgf000040_0001
Figure imgf000040_0002
[00215] Scheme 2. Synthesis of NKS 21: Reaction scheme for the synthesis of NKS21 : Oleic acid methyl ester (0.592 g, 2 mmol) was added to N'-methyl-2,2'-diamino diethylamine (0.117 g, 1 mmol) dropwise and heated with stirring at 140°C for 72 hours under N2 to give NKS21 as a white crystalline product.
Figure imgf000040_0003
[00216] Scheme 3. Synthesis of NKS 22:Reaction scheme for the synthesis of NKS 22. Ethyl linoleate (0.103 g, 1 mmol) was added to diethylenetriamine (0.616 g, 2 mmol) dropwise and heated with stirring at 160°C for 72 hours under N2 to give NKS22 as a white crystalline product.
Chemistry
[00217] In general, NKS lipidoids were synthesized by addition of fatty acid esters to respective amines drop wise in a ratio of either, 1 : 1 or 1 :2 or 1 :3 according to the number of amine sites open for substitution in 3-inl Teflon-lined glass screw-ίορ vial. No solvent or catalyst was used for these reactions. The reaction vial was heated at 140-160°C for 72h while stirring under N2. The product was purified by recrystallization using hexanes and acetone. For higher purity, the reaction mixture was purified using flash
chromatography (CH2Cl2:MeOH, 20:1) to afford (in most cases) a white solid (80-90% yield) as the final product. The structures of NKS lipidoids were analyzed and verified using Ή, l3C nuclear magnetic resonance (NMR) and high-resolution mass spectrometry.
[00218] In vitro siRNA transfection assay. 12h after seeding HeLa/green fluorescent protein (GFP) cells (50000 cells/well) into each well of a 24 well clear bottom plate in 500μ1 growth medium, cells were transfected with NKS/Silencer® GFP (eGFP) siRNA complexes in 200 μΐ of media in each well. A ratio of 10: 1 (wt/wt) (NKS: silencer GFP siRNA) was found to be ideal for GFP silencing for this study. The dilutions were made in a sodium acetate buffer (pH=5.5, 25mM). Lipofectamine 2000 was used as the control transfection agent. 2 ng of silencer® GFP siRNA (per well) (^g pRK-9- flag®-eGFP plasmid) was added to 100 μΐ of opti-MEM® (minimum essential media) and in a separate vial, while NKS (20 ng) diluted in 100 μΐ of opti-MEM® media. Both were combined, sonicated briefly and incubated for 15 min. Growth media was removed from the cells and the NKS/Silencer® GFP (eGFP) siRNA complexes were added to the cells. Transfections were performed in triplicates. [00219] Quantification of GFP silencing/expression. Flow cytometry was used to characterize the transfection efficiency (TE) of NKS lipidoids. In the case of pRK-GFP or eGFP-siRNA, Hela or Hela-GFP cells were analyzed 48 hours after transfection. Cells were detached from the plate by trypsin treatment (100 uIJwell of 0.25% IX (Invitrogen/Gibco®)) at 37°C for 10 minutes, followed by addition of media. Samples were transferred into centrifuge tubes and centrifuged at 300g, 4°C for 10 minutes (Eppendorf 5417R). The supernatant (media) was removed and cells were washed with PBS (Dulbecco's Phosphate-Buffered Saline (Invitrogen/Gibco®)). The cells were then fixed with 4% formaldehyde (37% formaldehyde (Fischer Scientific, F79-500) diluted into PBS) and refrigerated at 4°C for 1 hour. The 4% formaldehyde was removed, and the cells were re-suspended in PBS. The resulting samples were assayed by flow cytometer (Beckman-Coulter, EPICS® XL-MCL™) and Beckman-Coulter System II software. Data from each sample was collected for up to 104 cells over 5 minutes of time.
In vivo toxicity
[00220] Studies in animals. Four- to six-week-old BALB/c mice (Jackson Laboratory) were used to determine the toxicity and efficiency of delivering RNAi facilitated by omega-fatty acid lipidoids. In these experiments, mice were first anesthetized with isoflurane, and then the formulations developed using lipidoids NKS 11, NKS21 and NKS22 were then slowly injected in separate experiments into the tail veins of the animals by using a syringe with hypodermic needle of 27 -gauge size.
[00221] In vivo lipidoid-miR124 formulation. For the in vivo delivery of miR124 via a tail vein injection, a simple two-component formulation consisting of NKS 1 lor NKS21 or NKS22 and C16 mPEG
(polyethylene glycol) 2000 Ceramide was developed. To optimize the formulation, the concentration of the NKS lipidoids 11, 21 & 22 was varied (5: 1, 7.5: 1, 15: 1), while keeping the concentration of miR124 constant (lmg/kg body weight). A ratio of 5: 1 provided the best results. 15 g of miR124 in ΙΟΟμΙ PBS was added to a solution of 75 g NKS lipidoid and 1 % C16 mPEG 2000 Ceramide in ΙΟΟμΙ acetate buffer (25mM, pH=5.5). The mixture was sonicated at room temperature for 20 minutes before injecting.
[00222] Quantification of miR124 using qRT-PCR. Total RNA from liver, spleen, kidney, lung and salivary glands was isolated using Qiagen RNeasy® kit following the manufacturer's protocol. miR98 was used as an internal control for reverse transcription. Taqman® miRNA Reverse transcription kit (Applied Biosystems) was used for reverse transcription. Quantification of miRNA was performed using Taqman® miRNA assay kit (Applied Biosystems, per protocol).
[00223] Intranasal delivery of miR124. For the intranasal delivery of miR124, a formulation containing ^g of miR124 in 25μ1 added to 5μg NKS 11 and 1 % C16 mPEG 2000 Ceramide in 25μ1 acetate buffer (25mM, pH=5.5) was developed. The complex was sonicated at room temperature for 5 minutes. The mouse was anaesthetized and its head was immobilized and the NKS 1 l-miR124 complex was administered using a micro capillary tip into the nostril. The solution was delivered slowly over a 2-3 minute period, allowing the mouse to breathe the liquid in. The mouse was then observed in the cage for at least an hour to avoid depression of cardiac and/or respiratory functions. Five mice were used for experimental conditions and 3 mice were used as controls that received the miR124 via PBS. 24h after the administration, the mice were euthanized and the lungs were collected. To make sure that the miR124 was indeed taken up by the cells, the lungs were perfused using 1 % saline solution before collecting the RNA. Other organs like kidneys, liver and spleen were tested for any traces of miR124.
RESULTS
[00224] A library of lipidoid molecules (Figure 1) was generated using nucleophilic acyl substitution reactions.
These lipidoids were tested for their ability to complex with small interfering RNA (siRNA). (35) This led to the identification of three molecules that were efficiently complexing with nucleic acids such as siRNA, miRNA and plasmids. The results from these in vitro studies were either comparable or better than the current accepted industry standard, Lipofectamine® 2000. While Lipofectamine® 2000 is contraindicated for in vivo delivery, NKS 11, NKS21 and NKS22 proved to be safe and efficient for delivery of polynucleotides.
Essential fatty acid lipidoid-mediated delivery in vitro
[00225] Upon the successful synthesis and structural analysis of the lipidoids presented earlier, their ability to deliver siRNA to HeLa cell line (HeLa-GFP) that stably expresses green fluorescent protein (GFP) was tested. The efficiency of delivery was verified by treating HeLa-GFP cells with siRNA-lipidoid complexes containing GFP-targeting siRNA (siGFP), and then measuring the reduction in GFP expression by HeLa- GFP cells. The complexes were prepared by simple mixing and sonicating at room temperature for 5 minutes.
[00226] Flow cytometry was used to analyze the transfection efficiency (TE) of the lipidoids, 48h after
transfection. Lipofectamine® 2000, a commercially available transfection regent approved for in vitro studies was used as a positive control. The results of the in vitro transfection studies are presented in Figures 2-6.
NKS11
[00227] Figure 2 depicts GFP knockdown via siRNA delivered by NKS11.
[00228] Figure 2. In vitro delivery of siRNA. a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS 11 mediated siRNA delivery is overlaid with
Lipofectamine® 2000 mediated transfection and untreated HeLa cells expressing GFP, 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
[00229] As a further proof of TE of NKS 11 , the delivery of a 5.5kBp plasmid pRLK-GFP into HeLa cells was tested. As shown in Figure 3 below, NKS 11 successfully delivered pRLK-GFP at the levels of lipofectamine® 2000 (Invitrogen, San Diego, CA), 48h after transfection. As shown in figure 3, the lipidoids NKS 11 is efficiently transfected and expressing GFP protein in HeLa cells.
[00230] Figure 3. In vitro delivery of pRK-9-flag®-EGFP plasmid in HeLa cells, a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS 11 mediated siRNA delivery (black) is overlaid with Lipofectamine® 2000 mediated transfection (blue) and untreated HeLa cells expressing GFP (red), 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells. The pRK-9-flag®-EGFP plasmid is labeled as "GFP plasmid"; the pRK-9-flag®-EGFP plasmid and "GFP plasmid" are the same plasmids. NKS21
[00231] Figure 4 depicts in vitro delivery of siRNA using NKS21.
[00232] Figure 4. In vitro delivery of siRNA. a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS21 mediated siRNA delivery is overlaid with
Lipofectamine® 2000 mediated transfection and untreated HeLa cells expressing GFP, 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
NKS22
[00233] Figure 5 depicts GFP knockdown via siRNA delivered by NKS22.
[00234] Figure 5. In vitro delivery of siRNA. a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS22 mediated siRNA delivery is overlaid with
Lipofectamine® 2000 mediated transfection and untreated HeLa cells expressing GFP (red), 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells.
[00235] Figure 6 depicts GFP plasmid update using NKS22.
[00236] Figure 6. In vitro delivery of pRK-9-flag®-EGFP plasmid in HeLa cells, a. Flow cytometer data for the silencing of GFP in HeLa cells by delivery of Silencer® GFP (eGFP) siRNA. NKS22 mediated siRNA delivery (black) is overlaid with Lipofectamine® 2000 mediated transfection (blue) and untreated HeLa cells expressing GFP (red), 48h after transfection. Geometric mean of fluorescence intensities is reported with respect to the total population of cells. The pRK-9-flag®-EGFP plasmid is labeled as "GFP plasmid"; the pRK-9-flag®-EGFP plasmid and "GFP plasmid" are the same plasmids.
In vivo toxicity studies
[00237] Toxicity assays were carried out in BALB/c mice. (All of the experiments were carried out in triplicates).
Each mouse received 15mg/Kg, 30mg/Kg, 60mg/Kg, or lOOmg/Kg. The mice were observed for any loss of weight or any drastic observable health changes. No death or other observable changes in weight was observed. It was concluded that these delivery agents were safe for further experiments.
[00238] Even though no toxicity was observed physically, assays were conducted to determine whether there is any immunogenic toxicity observed when a neutral lipidoid like NKS 11, NKS21, NKS22 are administered systemically. intravenous administration of cationic lipidoids is known to provoke a dramatic proinflammatory response by inducing Thl cytokine expression, in particular, interferon-γ (!FN γ) and tumor necrosis factor (TNF a), 10-75-fold higher than treatment with control particles (28, 44). THe levels of these cytokines was determined at various time points after injecting NKS 11, NKS21 or NKS22.
[00239] Female BALB/c mice were injected intravenously (lateral tail vein) with 100 μg of NKS 11, NKS21,
NKS22 formulation prepared by adding 1 % PEG in acetate buffer, in separate experiments. As a positive control for cytokine induction, a group of mice were injected with 20μg of lipopolysaccharide (LPS) from Escherichia coli 0111 :B4. As a negative control, mice were injected with PBS to get a base level of these cytokines. Since there is usually a dramatic increase in cytokine levels within a few hours of
administration of the stimulant, cytokine levels were evaluated 2h and 6h after injecting the delivery agents NKS 11, NKS21, NKS22 and the controls. Mice were then anaesthetized with isoflurane and blood was collected via cardiac stick at set time points after injection (2h and 6h). Two mice were used for each time point. Serum was separated by centrifugation for cytokine analysis. Serum cytokine levels (TNF-a and IFN-γ) were determined using eBioscience mouse ELISA kits and Spectramax® M2 microplate reader. It was found that the immunostimulation due to NKS 11, NKS21 and NKS 22 were at the level of PBS or lower (Figures 7 and 8).
[00240] Figure 7: Cytokine levels in the serum in Balb/c mice at 2h and 6 h post intravenous injection of a single dose (4mg/Kg body weight of mice) of formulated NKS 11, NKS21, NKS 22 and PBS. 0.8 mg/Kg of Lipopolysaccharide (LPS) was used as a positive control for TNF-a induction. Averages and standard deviations of 2 mice per group are shown.
[00241] Figure 8: Cytokine levels in the serum in BALB/c mice at 2h and 6 h post intravenous injection of a single dose (4mg/Kg body weight of mice) of formulated NKS 11, NKS21, NKS 22 and PBS. 0.8 mg/Kg of lipopolysaccharide (LPS) was used as a positive control for IFN- γ induction. Averages and standard deviations of 2 mice per group are shown.
In vivo delivery
[00242] Micro RN As (miRNAs) are non-coding RNAs of around 19-25 nucleotide long transcripts that are known to negatively regulate gene expression, that affects processes like cell proliferation, differentiation, survival and motility (45). Aberrantly expressed miRNAs lead to human diseases and correcting these miRNA deficiencies by antagonizing or restoring miRNA function by delivering endogenous miRNAs may result in therapeutics for various diseases (46). For example, systemic delivery of miR-34a mimics has resulted in robust inhibition of non-small cell lung cancer (NSCLC) xenografts in mice using neutral lipid emulsion (47). Later studies reported the utility of delivering miRNA let-7 and miR-34 in inhibiting NSCLC in an autochthonous KRASG12D transgenic mouse model of lung cancer (48). Hence, the successful delivery of miRNA and similar other RNA interference agents holds the key to realizing of RNAi therapeutics.
[00243] To test the ability of the lipidoids (NKS 11 , NKS 21 and NKS 22) to deliver oligonucleotides like siRNA, miRNA and plasmids in vivo, microRNA 124 (miR-124) was selected. miR-124 was chosen for study because it is expressed only by the cells of the central nervous system and hence facilitates differentiation between mimics of miR-124 delivered using these lipidoids from those that are endogenously expressed (48). Mice were injected with 15μg of miR-124 using formulation developed using 75 μg NKS lipidoids and 0.1 % PEG, which is equivalent to about 5 mg/Kg of mouse body weight, assuming a mouse weighs around 25g. The mice were euthanized after 24h; and five organs- salivary gland, lungs, spleen, kidneys and liver, were collected, subjected to RNA isolation and quantitative reverse transcriptase PCR (qRT- PCR). Unlike the other delivery agents like cationic lipid particles, which end up mostly being delivered to the liver (35), which has been the primary target organ, NKS 11, NKS21 and NKS 22 are delivering substantial amounts of miR-124 to spleen (Figures 9, 10, and 11). As shown in Figure 9-11, a common theme of this particular series of lipidoids is delivery to spleen. [00244] Figure 9: Biodistribution of systemically delivered microRNAs (miRNA) mimics using NKS11. (a)
Delivery of miR124 to various organs- lungs, liver, spleen, kidneys and salivary glands. BALB/C mice (n=5) through IV tail vein injection. The control group mice (n=3) received miR124 delivered via PBS. Each mouse received a dose of lmg/kg of miR124. 24h after the administration, the mice were euthanized and total RNA was extracted from the organs. Organs were perfused with 1 % saline solution before subjected to RNA isolation. Mice that received naked miR124 acted as negative controls for endogenous miR-124 expression levels in these tissues under these conditions. miR-124 copy numbers were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph.
[00245] Figure 10: Biodistribution of systemically delivered microRNAs (miRNA) mimics using NKS21. (a) Delivery of miR124 to various organs- lungs, liver, spleen, kidneys and salivary glands. BALB/C mice (n=5) through IV tail vein injection. The control group mice (n=3) received miR124 delivered via PBS. Each mouse received a dose of lmg/kg of miR124. 24h after the administration, the mice were euthanized and total RNA was extracted from the organs. Organs were perfused with 1 % saline solution before subjected to RNA isolation. Mice that received naked miR124 acted as negative controls for endogenous miR-124 expression levels in these tissues under these conditions. miR-124 copy numbers were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph.
[00246] Figure 11: Biodistribution of systemically delivered microRNAs (miRNA) mimics using NKS22. (a) Delivery of miR124 to various organs- lungs, liver, spleen, kidneys and salivary glands. BALB/C mice (n=5) through IV tail vein injection. The control group mice (n=3) received miR124 delivered via PBS. Each mouse received a dose of lmg/kg of miR124. 24h after the administration, the mice were euthanized and total RNA was extracted from the organs. Organs were perfused with 1 % saline solution before subjected to RNA isolation. Mice that received naked miR124 acted as negative controls for endogenous miR-124 expression levels in these tissues under these conditions. miR-124 copy numbers were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph.
Intranasal delivery of miRNA to lungs
[00247] The ability of NKS 11 to facilitate intranasal delivery of miR124, for delivery to the lung, was tested.
Intranasal delivery of RNAi agents is a promising non-invasive strategy for delivering directly to the lungs by avoiding the challenges of systemic delivery. Theoretically, the dose of RNAi required for efficacy is substantially less as this route provides direct access to lung epithelial cells, making it a very attractive delivery route for several respiratory tract disorders like chronic obstructive pulmonary disease, cystic fibrosis, asthma, as well as lung cancers and viral infections in the lung (49).
[00248] Local delivery of miR-124 in lungs was tested 24h after intranasal administration in BALB/C mice using a procedure that was reported earlier (50). To confirm that the miR-124 mimic was indeed taken up by the cells and is not just in the blood found in the tissues, the lungs were perfused with 1 % saline solution before isolating RNA from the organs. The absence of miR-124 was verified in other organs like kidneys, spleen and liver, after intranasal administration (data not shown). The data shows that the amount of miR- 124 administered using NKS 11 was one log higher than when naked miR124 was administered (Figure 12).
[00249] Figure 12: Intranasal delivery of miR-124 into lungs 24h after administration in BALB/C mice using
NKS 11. A formulation containing ^g of miR124 in 25μ1 added to 5 g NKS 11 and 1 % C16 mPEG 2000 Ceramide in 25μ1 acetate buffer (25mM, pH=5.5). Averages and standard deviations of readings from 3 mice per group are shown.
Transdermal delivery of GFP expressing plasmid
[00250] Topical application of medications for various ailments ranging from skin diseases to rheumatoid arthritis has been known for centuries (51-54), making skin a very attractive route for drug delivery. For topically administered drugs to be effective, they should be able to penetrate stratum corneum, the outermost layer of skin that acts as a barrier (52). Several chemical compounds that act as penetration-enhancers and their mechanisms of action have been reported (44). Fatty acids of carboxylic acids, especially the unsaturated ones like oleic acid, linoleic acid, and lauric acid have been found to be enhancing penetration of stratum corneum, because of their potential to disturb the lipid packing order within the bilayer (51, 52).
[00251] The incorporation of linoleic acid in the structure of NKS 11 led us to explore its ability to deliver a
plasmid DNA across the skin of a mouse. The transfection studies on HeLa cells mentioned earlier already proved the ability of NKS 11 to deliver a eGFP expressing plasmid DNA, pRK-9- flag®-EGFP (5520Bp) into the cells.
[00252] In the experiment, a formulation containing 9 mg/kg NKS 11 and 1 % PEG 4000 was developed and was incubated with 1.3 mg/kg plasmid, for application on the skin of a BALB/C mouse. After 72h, the eGFP started expressing (Figure 13, panels A and B) on the skin of the mouse while the control, which received the plasmid DNA pRLK without the delivery formulation containing NKS 11 did not show any GFP expression.
[00253] Figure 13: Transdermal Delivery of eGFP expressing plasmid pRLK. A formulation containing 9 mg/kg NKS 11 and 1 % PEG 4000 complexed with 1.3mg/kg plasmid pRLK, was applied on the skin of a BALB/C mouse, a) 72h after a single dose of application, control, which received the plasmid DNA pRLK without the delivery formulation, did not show any GFP expression, b) The experimental mice that received the plasmid DNA pRLK with the delivery formulation containing NKS 11, started expressing GFP on the skin of the mouse.
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[00308] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:
1. A lipidoid compound described by the formula (I):
Figure imgf000051_0001
(I)
wherein:
m is 0 or an integer of 1 to 6;
Y is selected from the group consisting of hydrogen, alkyl, substituted alkyl or a fatty acid-containing group described by the formula (II):
Figure imgf000051_0002
(Π)
wherein:
p is 0 or an integer of 1-6;
L1, L2 and L3 are each independently derived from an essential fatty acid;
T1, T2 and T3 are each independently a linker;
Z is N, sulfonium (i.e., S+) or phosphonium (i.e., PR+); and
R1, R2 and R3 are each independently hydrogen, an alkyl or a substituted alkyl.
2. The compound of claim 1, wherein the essential fatty acid is an omega-3 fatty acid or an omega-6 fatty acid.
3. The compound of any one of the preceding claims, wherein the essential fatty acid is selected from the group consisting of linoleic acid, linolenic acid and oleic acid.
4. The compound of any one of the preceding claims, wherein the compound is described by the formula
(III):
Figure imgf000051_0003
(HI)
wherein L1, L2, R1, R2, T1, T2, Z and Y are as described for formula (I), and in formula (II) p is 1.
5. The compound of any one of the preceding claims, wherein Z is N.
6. The compound of any one of claims 1-4, wherein Z is sulfonium.
7. The compound of any one of claims 1-5, wherein Z is phosphonium.
8. The compound of any one of claims 1-5, wherein Y is H.
9. The compound of any one of claims 1-4, wherein Y is an alkyl or a substituted alkyl.
10. The compound of any one of claims 1-4, wherein Y is a fatty acid-containing group described by formula (II).
11. The compound of any one of claims 1-5 and 8-9, wherein the compound is described by the formula (IV):
Figure imgf000052_0001
(IV)
wherein:
L1, L2, R1, R2, T1 and T2 are as described for formula (I); and
R4 is H, an alkyl or a substituted alkyl.
12. The compound of any one of claims 1-5 and 10, wherein the compound is described by the formula (V):
Figure imgf000052_0002
(V)
wherein L1- L3, R1- R3 and T1 - T3 are as described for formulae (I) and (II).
13. The compound of any one of claims 11-12, wherein L1- L3 are each independently selected from the group consisting of linoleic acid, linolenic acid and oleic acid.
14. The compound of any one of claims 11-13, wherein T1- T3 are each independently a
Figure imgf000052_0003
The compound of any one of the preceding claims, wherein R1- R4 are each independently hydi
16. The compound of claim 1, wherein the compound has one of the following structures:
Figure imgf000053_0001
17. A lipidoid composition, comprising a lipidoid compound according to claim 1 non-covalently bound to a target nucleic acid.
18. The composition of claim 17, wherein the target nucleic acid is a target RNA.
19. The composition of claim 17, wherein the target nucleic acid is selected from the group consisting of: a DNA, an siRNA, an shRNA, a miRNA, and an antisense nucleic acid.
20. The composition of claim 17, wherein the DNA comprises a nucleotide sequence encoding a gene product.
21. The composition of claim 20, wherein the gene product is a polypeptide.
22. The composition of claim 21, wherein the polypeptide is a therapeutic polypeptide.
23. The composition of claim 20, wherein the gene product is an RNA.
24. The composition of claim 17, further comprising nanoparticles of the lipidoid compound.
25. A method of delivering a target nucleic acid to a cell, the method comprising:
contacting a cell with the lipidoid composition of any one of claims 17-24 to intracellularly deliver the target nucleic acid to the cell.
26. The method of claim 25, wherein the cell is in vitro.
27. The method of claim 25, wherein the cell is in vivo, and the contacting comprises administering the lipidoid complex to a subject.
28. A method of making a lipidoid compound, the method comprising:
contacting a fatty acid ester with an amino linker to produce a fatty acid contacted mixture; and heating the mixture under an inert atmosphere for a time sufficient to produce the lipidoid compound of claim 1.
29. The method of claim 28, wherein the fatty acid contacted mixture further comprises a solvent.
30. The method of any one of claims 28-29, wherein the heating achieves a temperature of 100°C or more.
31. The method of any one of claims 28-30, wherein the amino linker is described by formula (VI):
Y
T1-7-T2
R R2
(VI)
wherein R1, R2, T1, T2, Z and Y are as described for formula (I), and in formula (II) p is 1.
32. The method of claim 31 , wherein Z is N.
33. A method of delivering a gene product to an individual, the method comprising administering to the individual an effective amount of a lipidoid composition of any one of claims 17-24.
34. The method of claim 33, wherein the gene product is a nucleic acid.
35. The method of claim 33, wherein the gene product is a polypeptide.
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US12065396B2 (en) 2017-08-17 2024-08-20 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2024209060A1 (en) * 2023-04-07 2024-10-10 Université D'aix-Marseille Polyaminated fatty acids for use as potentiators of antibacterial activity

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US12065396B2 (en) 2017-08-17 2024-08-20 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
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