EP4658280A2 - Cycle à six chaînons contenant des oligomères - Google Patents

Cycle à six chaînons contenant des oligomères

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Publication number
EP4658280A2
EP4658280A2 EP24751025.8A EP24751025A EP4658280A2 EP 4658280 A2 EP4658280 A2 EP 4658280A2 EP 24751025 A EP24751025 A EP 24751025A EP 4658280 A2 EP4658280 A2 EP 4658280A2
Authority
EP
European Patent Office
Prior art keywords
nucleotide
antisense strand
formula
oligonucleotide
counting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24751025.8A
Other languages
German (de)
English (en)
Inventor
Muthiah Manoharan
Masaaki AKABANE-NAKATA
Dhrubajyoti Datta
Jayanta KUNDU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alnylam Pharmaceuticals Inc
Original Assignee
Alnylam Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alnylam Pharmaceuticals Inc filed Critical Alnylam Pharmaceuticals Inc
Publication of EP4658280A2 publication Critical patent/EP4658280A2/fr
Pending legal-status Critical Current

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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/28—Oxygen atom
    • C07D473/30—Oxygen atom attached in position 6, e.g. hypoxanthine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
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    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • C—CHEMISTRY; METALLURGY
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    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3235—Chemical structure of the sugar modified ring structure having the O of the ribose replaced by another atom
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present disclosure relates generally to six-membered nucleosides and oligonucleotides and oligomers comprising the same.
  • BACKGROUND There is a need in the art for monomer for modulating oligonucleotide characteristics and/or functionality. The present disclosure addresses some of these needs.
  • SUMMARY [0005] Despite their recent success in the oligotherapeutic field, phosphorodiamidate morpholinos (PMOs) have several drawbacks. The synthesis of PMOs has difficult to make at scale.
  • oligonucleotide comprising at least one nucleoside of Formula (IV) (e.g., one):
  • B’ is an optionally modified nucleobase.
  • X M can be CH2, O, NR N or S, where R N is aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars.
  • X M is CH2, O or NH.
  • X M is CH2.
  • X M is O.
  • X M is S.
  • R 43 can be can be a bond to an intemucleotide linkage to a subsequent nucleotide hydrogen, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxycarboxy
  • R 43 is a bond to an intemucleotide linkage to a subsequent nucleotide. In some other embodiments of any one of the aspects described herein, R 43 is a soild support or a linker covelantly linked to a solid support. In yet some other embodiments of any one of the aspects described herein, R 43 is H. In still some other embodiments of any one of the aspects described herein, R 43 is a nitrogen protecting group, e.g., triphenylmethyl (trityl). In yet some other embodiments of any one of the aspects described herein, R 43 is hydroxyl or a protected hydroxyl. In yet some other embodiments of any one of the aspects described herein, R 43 is hydroxyl. In yet some other embodiments of any one of the aspects described herein, R 43 is a protected hydroxyl.
  • R 45 can be a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or 5’-O- C(O)CH2CH2C(O)-) to a solid support hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, optionally substituted 3-8 membered heterocyclyl (e.g., morpholin-l-yl, piperidin-l-yl, or pyrrolidin-l-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxy
  • R 45 is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, phosphate mimic, or a bond to an intemucleotide linkage to a preceding nucleotide, or or R 45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
  • VP vinylphosphonate
  • R 45 is hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate, or R 45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
  • R 45 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • R 45 is hydroxyl or protected hydroxyl.
  • R 43 and R 45 can be a solid support or a linker covalently bonded to a solid support.
  • the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide. It is noted that the nucleoside of Formula (IV) at 3 ’-end of the oligonucleotide can be linked to the preceding nucleoside by a phosphodiester intemucleotide linkage or a modified intemucleotide.
  • R 45 is a bond to a phosphodiester intemucleotide linkage.
  • R 45 is a bond to a phosphorothioate intemucleotide linkage.
  • an oligonucleotide described herein comprises two or more consective nucleosides of Formula (IV).
  • the oligonucleotide comprises: where: n D is an interger from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; or 10-50, or 10-40, or 10-30, or 20-50, or 20-45, or 20-40, or 20-35, or 20-30, or 25-50, or 25- 45, or 25-40, or 25-35, or 25-30);
  • X D is O or S; each R P2 is independently optionally substituted Ci-ealkyl (e.g., methyl); and
  • X M , B’, R 43 and R 45 are as defined for Formula (IV). Such may be prepared according to methods in the art, including, for example, Kundu et al., J. Org. Chem. 2022, 87, 9466-9478.
  • n D is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n D is 1, 2, 3, 4, 5 or 6.
  • n D is 1, 2, 3 or 4.
  • n D is 1 or 2.
  • X D is O.
  • all X M are same.
  • all X M are CH2.
  • all X M are O.
  • all X M are S.
  • at least one X M is not O.
  • the oliognucleotides is 4 nucleotides in length, n D is 4, and the oligonucleotide does no comprise the sequence 5’-UCAG- 3’.
  • At least one of X M is CH2 and at least one X M is O.
  • B’ is an optionally modified nucleobase.
  • X M is CH2, O, NR N or S, where R N is aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars.
  • X M can be CH2, O or NR N .
  • X M is CH2, O or NH.
  • X M is CH2.
  • R 33 can be hydrogen, hydroxyl, protected hydroxyl, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, alkoxy alkyl (e.g., methoxy ethyl), alkoxyalkylamine, al
  • R 33 is a nitrogen protecting group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support, a ligand, or a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids.
  • R 33 is a reactive phosphorous group, a solid support, a linker, a linker covalently bonded to a solid support, or a nitrogen protecting group.
  • R 33 is a reactive phosphorous group, e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)] -thiophosphorami dite.
  • R 33 is a soild support or a linker covelantly linked to a solid support.
  • R 33 is H.
  • R 33 is a nitrogen protecting group, e.g., colorhenylmethyl (trityl).
  • R 35 can be a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or 5’-O-C(O)CH2CH2C(O)- ) to a solid support, hydroxy, protected hydroxy, phosphate group, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl-ON(CH2R 8 )(CH2R 9 ), -0-C4-3oalkyl- ON(CH2R 8 )(CH2R
  • R 35 is hydroxy or protected hydroxy.
  • R 35 is a protected hydroxyl (e.g., 4,4'-dimethoxytrityl- protected).
  • R 35 is a phosphate group.
  • R 35 taken together with the carbon to which it is attached can form is a vinyl phosphonate group.
  • R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group, a C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate) group.
  • R 35 is a reactive phosphorous group.
  • the R 35 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is Cl and each R P2 is methyl.
  • R 33 and R 35 can be a reactive phosphorous group, a solid support, or a linker covalently bonded to a solid support.
  • R 33 is a solid support, or a linker covalently bonded to a solid support.
  • R 33 is a solid support, or a linker covalently bonded to a solid support, and R 35 is hydroxyl or protected hydroxyl group.
  • X M is CH2, O, NR N or S;
  • R 33 is a solid support, or a linker covalently bonded to a solid support; and
  • R 35 is hydroxyl or protected hydroxyl group (e.g., dimethoxy trityl).
  • R 33 is a reactive phosphorous group.
  • the R 33 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is Cl and each R P2 is methyl.
  • R 33 is a reactive phosphorous group and R 35 is hydroxyl or protected hydroxyl group.
  • R 33 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is Cl and each R P2 is methyl; and R 35 is hydroxyl or protected hydroxyl group (e.g., dimethoxytriryl).
  • the compounds (III) are useful in the synthesis oligonucleotides. Accordingly, in another aspect, provided herein is an oligonucleotide prepared using a compound of Formula (III). For example, an oligonucleotide comprising nucleoside of Formula (IV).
  • the oligonucleotide described herein comprises a nucleotide of Formula (IV) at one of positions 2-9, counting from the 5 ’end of the oligonucleotide.
  • the oligonucleotide described herein comprises a nucleotide of Formula (IV) at one of positions 2- 8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5 ’-end of the oligonucleotide.
  • the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 5, counting from the 5’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 6, counting from the 5 ’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 7, counting from the 5 ’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 8, counting from the 5 ’-end of the oligonucleotide.
  • the oligonucleotide described herein is double-stranded.
  • the oligonucleotide described herein is comprised in a double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein one of first or second oligonucleotide strand is an oligonucleotide described herein.
  • a double-stranded nucleic acid comprising a first strand and a second strand complementary to the first strand, and wherein at least one of the first and second strand is an oligonucleotide comprising a nucleotide of Formula (IV) described herein.
  • the double-stranded nucleic acid is a double-stranded RNA.
  • the double-stranded nucleic acid is an siRNA.
  • the double-stranded nucleic acid is an siRNA comprising a sense strand and an antisense strand substantially complementary to the sense strand, wherein the antisense strand comprises a nucleotide of Formula (IV).
  • the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at one of positions 2-9, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at one of positions 2-8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5’- end of the antisense strand).
  • the antisense strand comprises a nucleotide of Formula (IV) at one of positions 2-8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5’- end of the
  • the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 5, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 6, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 7, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 8, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA comprising a sense strand and an antisense strand substantially complementary to the sense strand, wherein the sense strand comprises a nucleotide of Formula (IV).
  • the double-stranded nucleic acid is an siRNA comprising a sense strand and an antisense strand substantially complementary to the sense strand, wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) one of positions 2-9 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) one of positions 2-9 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) one of positions 2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or one of positions 6-8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) one of positions 2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or one of positions 6-8 of the antis
  • the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 5 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 5 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 6 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 6 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 7 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 7 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).
  • a method for inhibiting or reducing the expression of a target gene in a subject comprises administering to the subject: (i) a double- stranded RNA described herein, wherein one of the strands of the dsRNA is complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.
  • R 43 and R 45 are a bond to a intemucleotide linkage.
  • R 43 is not a bond to an intemucleotide linkage to a subsequent nucleotide
  • R 45 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • R 43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
  • FIG. 1 depicts some exemplary compounds comprising C5 or C4 modified pyrimidines according to some embodiments of the disclosure.
  • FIG. 2 depicts some exemplary compounds comprising N2 modified purines according to some embodiments of the disclosure.
  • FIG. 3 depicts some exemplary compounds comprising N6 modified purines according to some embodiments of the disclosure.
  • FIG. 4 depicts some exemplary compounds comprising N7-deaza or C-8 modified purines according to some embodiments of the disclosure.
  • FIG. 5 depicts some exemplary modified car-morpholino, vinylphosphonate and CPG compounds comprising N2 modified purines according to some embodiments of the disclosure.
  • FIG. 6 depicts exemplary synthesis scheme for preparing C5-modified pyrimidine car- PMO compounds according to some embodiments of the disclosure.
  • FIG. 7 depicts some exemplary ligands.
  • FIGS. 8A-8C depict structures of chlorophosphoramidate carbocyclic morpholino monomers (FIG. 8A); PMO, carPMO, and PMO-carPMO chimers (FIG. 8B); and thio-PMO, and PiperazinoPMO chimers (FIG. 8C).
  • FIG. 9 depicts crystal structures of selected intermediates.
  • FIG. 10 shows overlaid X-ray crystal structures of compound 5, 7, and 8. Atoms are colored teal, pink, and yellow for carbons of 5, 7, and 8, respectively. Oxygen, nitrogen, and silicon are colored gray, blue, and red, respectively.
  • FIGS. 11A and 11B are general schemes for synstheis of exemplary car-morpholino and morpholino amidites.
  • FIG. 12 are general synthesis schemes for car-morpholino-VP, morpholino-VP- amidite and CPG.
  • FIG. 13 show exemplary control sequences with GNA and TNA at Position 7 for off- target mitigation evaluation. Sequences shown from top to bottom are SEQ ID NO: 230 (si-72 sense strand), SEQ ID NO: 231 (si-72 antisense strand), SEQ ID NO: 232 (si-75 sense strand) and SEQ ID NO: 233 (si-75 sense strand).
  • FIG. 14 shows structures of some monomer abbreviations used in the nucleic acid sequences described herein.
  • FIG. 15A shows exemplary oligonucleotides comprising six membered ring monomers I-IV shown in FIG. 14.
  • FIG. 15B shows exemplary oligonucleotides comprising six membered ring monomers Y271-Y274 shown in FIG. 14.
  • FIG. 16 shows in vitro results of some exemplary siRNAs targeting mTTR and comprising six membered ring monomers described herein. Sequences shown from top to bottom are SEQ ID NOs: 1 and 2 (control), SEQ ID NOs: 1 and 98 (si-2), SEQ ID NOs: 121 ans 2 (si-25) and SEQ ID NOs: 116 and 2 (si-20).
  • FIG. 17 shows in vitro results of some exemplary siRNAs comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strand. Sequences shown from top to bottom are SEQ ID NOs: 1 and 2.
  • FIG. 18 shows in vitro results of some exemplary siRNAs comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strandSequences shown from top to bottom are SEQ ID NOs: 1 and 2.
  • FIG. 17 shows in vitro results of some exemplary siRNAs comprising comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strandSequences shown from top to bottom are SEQ ID NOs: 1 and 2.
  • FIG. 18 shows in vitro results of some exemplary siRNAs comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strandSequences shown from top to bottom are SEQ ID NOs: 1 and 2.
  • FIG. 21 depicts a scheme showing 5’-5-morpholino-U monomer synthesis.
  • FIG. 22 depicts a scheme showing 5’-5-morpholino-C monomer synthesis.
  • FIG. 23 depicts a scheme showing 5’-5-morpholino-A monomer synthesis.
  • FIG. 24 depicts a scheme showing 5’-5-morpholino-G monomer synthesis.
  • FIG. 25 depicts a scheme showing 5’-5-morpholino (phosphorami dites) monomers synthesis and oligonucleotide ynthesis strategy.
  • FIG. 26 depicts a scheme showing 5’-5-morpholino (chlorophosphorami dates) monomers synthesis and oligonucleotide synthesis strategy.
  • FIG. 27 depicts a scheme showing A-acetyl piperazino (chlorophosphoramidates) monomers synthesis.
  • FIG. 28 depicts a scheme showing A-acetyl piperazino (phosphoramidites) monomers synthesis.
  • FIG. 29 depicts a scheme showing A-acetyl piperazino (chlorophosphoramidates) monomers synthesis and oligonucleotide synthesis scheme.
  • FIG. 30 depicts a scheme showing N- acetyl piperazino (phosphoramidites) monomers synthesis and oligonucleotide synthesis scheme.
  • FIG. 31 depicts some exemplary modified PMO sequences. Seqeunces shown are, from top to bottom, SEQ ID NOs: 241-244.
  • B’ is an optionally modified nucleobase.
  • the nucleobase can be a natural or non-natural nucleobase.
  • a “non-natural nucleobase” is meant a nucleobase other than adenine, guanine, cytosine, uracil, or thymine.
  • non-natural nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substi
  • purines and pyrimidines 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, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, content of all which is incorporated herein by reference.
  • the non-natural nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,
  • a non-natural nucleobase is a modified nucleobase, i.e., the nucleobase comprises a nucleobase modification described herein, e.g., the nucleobase is a substituted or modified analog of any of the natural nucleobases.
  • nucleobase modifications include, but not limited to: C-5 pyrimidine with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities, N 2 - and N 6 - with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities of purines, G-clamps, guanidinium G-clamps, and pseudouridine known in the art.
  • the non-natural nucleobase is a universal nucleobase.
  • a universal nucleobase is any modified or unmodified natural or non-natural nucleobase that can base pair with all of adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide comprising the universal nucleobase.
  • Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza- 7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3 -methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6- methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4, 5 -trimethylphenyl, 4- methylinolyl, 4,6-dimethylindolyl, phen
  • the non-matural nucleobase is a protected nucleobase.
  • a “protected nucleobase” referes to a nucleobase comprising a nitrogen protecting group, and/or an oxygen protecting group, and/or a sulfur protecting group.
  • the non-natural nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.
  • the nucleobase is a pyrimidine modified at the C4 position.
  • the nucleobase is a pyrimidine modified at the C5 position.
  • the nucleobase is a purine modified at the N2 position. In some embodiments of any one of the aspects described herein, the nucleobase is a purine modified at the N6 position.
  • the nucleobase is a purine modified at the C6 position.
  • the nucleobase is a N-7 deaza purine, optionally modified at the N7 position.
  • nucleobase is selected from the group consisting of
  • alkylester independently liphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars.
  • X M can be CH2, O,
  • X M is CH2.
  • X M is O.
  • X M is S.
  • X M is NR N .
  • X M is NH.
  • R 33 can be hydrogen, hydroxyl, protected hydroxyl, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialky
  • R 33 is a nitrogen protecting group.
  • R 33 ' can be triphenylmethylamine (Tr), [(4- methoxyphenyl)diphenylmethyl]amine (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr) or trifluoroacetamide.
  • R 33 is triphenylmethylamine or trifluoroacetamide.
  • R 33 is triphenylmethylamine.
  • R 33 is a reactive phosphorus group.
  • R 33 is -OP(OR P )(N(R P2 ) 2 ), -OP(SR P )(N(R P2 ) 2 ), - OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(NR P2 ) 2 , -OP(O)(OR P )H, - OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 .
  • R 33 is -OP(OR P )(N(R P2 ) 2 ), - OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), - OP(O)(OR P )H, -OP(S)(OR P )H, where R P is an optionally substituted C 1-6 alkyl, each R P2 is independently optionally substituted C1-6alkyl; and each R P3 is independently optionally substituted C1-6alkyl.
  • R 33 is -OP(OR P )(N(R P2 )2).
  • the R 33 is -OP(OR P )(N(R P2 )2), where R P is cyanoethyl (-CH2CH2CN) and each R P2 is isopropyl.
  • R 33 is a solid support or a linker covalently attached to a solid support.
  • R 33 is –OC(O)CH2CH2C(O)NH-Z, where Z is a solid support.
  • R 33 is - O(CH2CH2O)rCH2CH2OR 334 , where r can be 1-50; R 334 is independently for each occurrence H, C1-C30alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 335 ; and R 335 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 33 is - (CH 2 CH 2 NH) s CH 2 CH 2 -R 335 , where s can be 1-50 and R 335 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 33 is hydrogen.
  • R 33 is hydroxyl or protected hydroxyl. In some embodiments of any one of the aspects described herein, R 33 is hydroxyl.
  • R 33 is protected hydroxyl.
  • R 33 is C1-C30alkoxy optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy.
  • R 33 is –O(CH 2 ) t CH 3 , where t is 1-21.
  • t is 14, 15, 16, 17 or 18.
  • t is 16.
  • R 33 is –O(CH2)uR 337 , where u is 2-10; R 337 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 337 is -CH 3 or NH 2 .
  • R 33 is –O(CH 2 ) u - OMe or R 33 is –O(CH 2 ) u NH 2 .
  • u is 2, 3, 4, 5 or 6.
  • u is 2, 3 or 6.
  • u is 2.
  • u is 3 or 6.
  • R 33 is a C 1 - C 6 haloalkyl.
  • R 33 is a C 1 -C 4 haloalkyl.
  • R 33 is –CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.
  • R 33 is – OCH(CH 2 OR 338 )CH 2 OR 339 , where R 338 and R 339 independently are H, optionally substituted C 1 - C30alkyl, optionally substituted C2-C30alkenyl or optionally substituted C2-C30alkynyl.
  • R 338 and R 339 independently are optionally substituted C1-C30alkyl.
  • R 33 is – CH2C(O)NHR 3310 , where R 3310 is H, optionally substituted C1-C30alkyl, optionally substituted C2- C30alkenyl or optionally substituted C2-C30alkynyl.
  • R 3310 is H or optionally substituted C1-C30alkyl.
  • R 3310 is optionally substituted C1-C6alkyl.
  • R 33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support.
  • R 33 is a reactive phosphorous or a linker covalently attached to a solid support.
  • R 33 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(ß- thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).
  • a reactive phosphorous group e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,
  • R 35 can be hydroxy, protected hydroxy, phosphate group, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group.
  • VP vinylphosphonate
  • R 35 is R 551 , optionally substituted C 1-6 alkyl-R 551 , optionally substituted -C 2-6 alkenyl-R 551 , or optionally substituted -C 2- 6alkynyl-R 551 , where R 551 can be –OR 552 , -SR 553 , hydrogen, a phosphorous group, a solid support or a linker to a solid support.
  • R 551 is –OR 552
  • R 552 can be H or a hydroxyl protecting group.
  • R 551 is –SR 553
  • R 553 can be H or a sulfur protecting group.
  • R 35 is –OR 552 or - SR 553 .
  • R 552 is a hydroxyl protecting group.
  • Exemplary hydroxyl protecting groups for R 552 include, but are not limited to, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4′-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9- yl (MOX).
  • DMT 4,4′-dimethoxytrityl
  • Pixyl 9-phenylxanthine-9-yl
  • MOX 9-(p-methoxyphenyl)xanthine-9- yl
  • R 35 is –OR 552 and R 552 is 4,4′-dimethoxytrityl (DMT), e.g., R 35 is –O-DMT.
  • DMT 4,4′-dimethoxytrityl
  • R 35 is –O-DMT.
  • R 552 is a phosphate group, e.g., R 552 is dimethylaminochlorophosphate (-P(O)(NMe 2 )Cl).
  • the methylene connecting the R 35 to the rest of the compound of Formula (III) is absent and R 35 is connected directly to the rest of the compound of Formula (III).
  • R 35 is –CH(R 554 )- R 551 , where R 554 is hydrogen, halogen, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl, optionally substituted C 2 -C 30 alkynyl, or optionally substituted C 1 -C 30 alkoxy.
  • R 35 is –CH(R 554 )-R 551
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 554 is H. In some other non-limiting examples, R 554 is C 1 -C 30 alkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy.
  • R 551 is a reactive phosphorous group.
  • each R 555 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group; and each R 556 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or a sulfur-protecting group.
  • At least one R 555 in -P(O)(OR 555 )2, - P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 )2, -SP(S)(OR 555 )2, and -SP(S)(SR 556 )(OR 555 ) is hydrogen.
  • At least one at least one R 555 in P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ) is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group.
  • At least one R 555 is H and at least one R 555 is other than H in -P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ).
  • all R 555 are H in -P(O)(OR 555 )2, - P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), - OP(S)(SR 556 )2, -SP(O)(OR 555 )2, -SP(S)(OR 555 )2, -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 555 are other than H in in - P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 )2, -OP(S)(OR 555 )2, - OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 )2, -SP(O)(OR 555 )2, -SP(S)(OR 555 )2, -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 )2.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 )2, - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is H.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 )2, - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 )2 is other than H.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 )2, - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 )2 is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an sulfur-protecting group.
  • At least one R 556 is H and at least one R 556 is other than H in -P(S)(SR 556 ) 2 , -OP(S)(SR 556 ) 2 and -SP(S)(SR 556 ) 2 .
  • all R 556 are H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 )2, - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 556 are other than H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 )2, -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • R 35 is hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate or phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group.
  • VP vinylphosphonate
  • R 35 is hydroxyl, protected hydroxyl, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, or a phosphate mimic.
  • R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group.
  • R 35 is a monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma- thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic; or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • R 35 is a reactive phosphorus group.
  • R 33 is -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), - OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )H, - OP(S)(OR p )H, -OP(O)(SR p )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , -OP(S)(OR P )R P3 , -OP(O)(SR P )R P3 , -OP(O)(SR P )R P3 , -OP(O)(R P3 )(
  • R 35 is -OP(OR P )(N(R P2 )2), - OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), - OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(R P3 )(N(R P4 )2), or -OP(S)(R P4 )(N(R P2 )2), where each R P is independently an optionally substituted C 1-6 alkyl, each R P2 is independently optionally substituted C 1-6 alkyl; and each R P3 is independently optionally substituted C 1-6 alkyl.
  • R 35 is OP(O)(R P3 )(N(R P4 ) 2 ), or -OP(S)(R P4 )(N(R P2 ) 2 ).
  • R 35 is –OP(O)(R P4 )(N(R P2 ) 2 ).
  • the R 35 is –OP(O)(R P4 )(N(R P2 ) 2 ), where R P4 is Cl and each R P2 is methyl.
  • R 35 is a solid support or a linker covalently attached to a solid support.
  • R 35 is –OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • R 33 is H, hydroxyl, protected hydroxyl, nitrogen protecting group, a linker, a ligand or a ligand covalently attached to one or more ligands; and R 35 is reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support.
  • R 33 is H or a nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., –OP(O)(R P4 )(N(R P2 )2).
  • R 33 is a nitrogen protecting group (e.g., trityl); and R 35 is –OP(O)(R P4 )(N(R P2 )2), where R P4 is halogene (e.g., Cl) and each R P2 is independently C 1 -C 6 alkyl, e.g., each R P2 is independently methyl.
  • X M is O, S or CH2; R 33 is H, hydroxyl, protected hydroxyl, nitrogen protecting group, a linker, a ligand or a ligand covalently attached to one or more ligands; and R 35 is reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support.
  • X M is CH 2 ; R 33 is H or a nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., – OP(O)(R P4 )(N(R P2 )2).
  • X M is CH2; R 33 is a nitrogen protecting group (e.g., trityl); and R 35 is —OP(O)(R P4 )(N(R P2 )2), where R P4 is halogen (e.g., Cl) and each R P2 is independently C 1 -C 6 alkyl, e.g., methyl.
  • R P4 is halogen (e.g., Cl) and each R P2 is independently C 1 -C 6 alkyl, e.g., methyl.
  • X M is CH2; R 33 is hydroxyl or protected hydroxyl; and R 35 is a reactive phosphorous group (e.g., –OP(O)(R P4 )(N(R P2 )2).
  • X M is CH2; R 33 is protected hydroxyl; and R 35 is —OP(O)(R P4 )(N(R P2 ) 2 ), where R P4 is halogen (e.g., Cl) and each R P2 is independently C 1 -C 6 alkyl, e.g., methyl.
  • X M is O; R 33 is H or a nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., – OP(O)(R P4 )(N(R P2 ) 2 ).
  • X M is O; R 33 is a nitrogen protecting group (e.g., trityl); and R 35 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is halogen (e.g., Cl) and each R P2 is independently Ci- Cealkyl, e.g., methyl.
  • R P4 is halogen (e.g., Cl) and each R P2 is independently Ci- Cealkyl, e.g., methyl.
  • X M is O; R 33 is hydroxyl or protected hydroxyl and R 35 is a reactive phosphorous group (e.g., - OP(O)(R P4 )(N(R P2 )2).
  • R 33 is a nitrogen protecting group (e.g., trityl); and R 35 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is halogen (e.g., Cl) and each R P2 is independently Ci- Cealkyl, e.g., methyl.
  • X M is S; R 33 is H or a nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., - OP(O)(R P4 )(N(R P2 )2).
  • R 33 is a nitrogen protecting group (e.g., trityl); and R 35 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is halogen (e.g., Cl) and each R P2 is independently Ci- Cealkyl, e.g., methyl.
  • X M is S; R 33 is hydroxyl or protected hydroxyl; and R 35 is a reactive phosphorous group (e.g., - OP(O)(R P4 )(N(R P2 )2).
  • R 33 is a nitrogen protecting group (e.g., trityl); and R 35 is -OP(O)(R P4 )(N(R P2 )2), where R P4 is halogen (e.g., Cl) and each R P2 is independently Ci- Cealkyl, e.g., methyl.
  • R 33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R 35 is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • R 33 is a reactive phosphorous or a linker covalently attached to a solid support; and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma- thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • R 33 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)]-thiophosphoramidite); and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • a reactive phosphorous group e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diiso
  • R 33 is a solid support, a linker or a linker covalently attached to a solid support;
  • R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • X M is CEE;
  • R 33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and
  • R 35 is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • X M is CEE; R 33 is a reactive phosphorous or a linker covalently attached to a solid support; and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • X M is CEE
  • R 33 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrohdinyl)]-thiophosphoramidite); and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e (e.g., E or Z vinylphosphonate) group.
  • a phosphoramidite such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-
  • X M is CEE
  • R 33 is a solid support, a linker or a linker covalently attached to a solid support
  • R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • X M is O;
  • R 33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and
  • R 35 is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • X M is O;
  • R 33 is a reactive phosphorous or a linker covalently attached to a solid support; and
  • R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'- dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • X M is O;
  • R 33 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)]-thiophosphoramidite); and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • a reactive phosphorous group e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,
  • X M is O;
  • R 33 is a solid support, a linker or a linker covalently attached to a solid support;
  • R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • X M is S;
  • R 33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and
  • R 35 is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • X M is S; R 33 is a reactive phosphorous or a linker covalently attached to a solid support; and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'- dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • X M is S
  • R 33 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)]-thiophosphoramidite); and R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • a phosphoramidite such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphorami
  • X M is S;
  • R 33 is a solid support, a linker or a linker covalently attached to a solid support;
  • R 35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R 35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
  • R 43 can be a bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, -0-C4-3oalkyl- ON(CH2R 8
  • R 43 is bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, or a nitrogen protecting group.
  • R 43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
  • R 43 is a solid support, or a linker (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) covalently bonded to a solid support.
  • a linker e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-
  • R 43 is hydrogen or a nitrogen protecting group. [00142] In some embodiments of any one of the aspects described herein, R 43 is hydroxy or protected hydroxyl. [00143] In some embodiments of any one of the aspects described herein R 43 is – P(X D )(N(R P2 )2)-R 43’ , where X D is O or S; each R P2 is independently optionally substituted C1-6alkyl (e.g., methyl); and R 43’ is a bond to a subsequent nucleoside, e.g., a bond to 5’ oxygen of a subsequent nucleoside.
  • R 43 is –P(O)(N(CH3)2)-R 43’ .
  • R 45 can be a bond to an internucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded a solid support, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R 8 )(CH2R 9 ), -O-C4-30alkyl- ON(CH2R 8 )(
  • R 45 can be a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate (phosphorodithioate), phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, or alkylphosphonates; or R 45 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • VP vinylphosphonate
  • R 45 is a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 1-30 alkoxy; or R 45 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
  • R 45 is a bond to an internucleotide linkage to a preceding nucleotide.
  • R 45 is a hydroxyl or protected hydroxyl.
  • R 45 is optionally substituted C 2-30 alkenyl or optionally substituted C 1-30 alkoxy.
  • R 45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
  • VP vinylphosphonate
  • the methylene connecting the R 45 to the rest of the nucleoside of Formula (VI) is absent and R 45 is connected directly to the rest of the nucleoside of Formula (IV).
  • R 45 is –CH(R 451 )-X 5 - R 452 , where X 5 is absent, a bond or O; R 451 is hydrogen, optionally substituted C 1-30 alkyl, optionally substituted -C 2-30 alkenyl, or optionally substituted -C 2-30 alkynyl, and R 452 is a bond to an internucleoside linkage to the preceding nucleotide.
  • X 5 is O or a bond.
  • X 5 is O.
  • X 5 is absent, i.e., R 45 is–CH(R 451 )R 452 .
  • R 45 is –CH(R 451 )-X 5 - R 452 .
  • R 451 is H.
  • R 451 is Ci-Csoalkyl optionally substituted with a NH2, OH, C(0)NH2, COOH, halo, SH, or Ci-Cealkoxy.
  • R 451 is H.
  • R 451 is Ci-Csoalkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or Ci-Cealkoxy.
  • R 452 is a bond to an intemucleoside linkage to the preceding nucleotide.
  • R 45 is optionally substituted Ci-ealkyl-R 453 , optionally substituted -C2-6alkenyl-R 453 , or optionally substituted -C2-6alkynyl-R 453 .
  • R 453 can be -OR 454 , -SR 455 , -P(O)(OR 456 )2, -P(S)(OR 456 )2, -P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 )2, -OP(O)(OR 456 )2, -OP(S)(OR 456 )2, -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 )2, -SP(O)(OR 456 )2, -SP(S)(OR 456 )2, -SP(S)(SR 457 )(OR 456 ), or - SP(S)(SR 457 )2; where R 454 is hydrogen or oxygen protecting group; R 455 is hydrogen or sulfur protecting group; each R 456 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkeny
  • At least one at least one R 456 in P(O)(OR 456 ) 2 , -P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), -OP(O)(OR 456 ) 2 , - OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), SP(O)(OR 456 ) 2 , -SP(S)(OR 456 ) 2 , and -SP(S)(SR 457 )(OR 456 ) is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group.
  • At least one R 456 is H and at least one R 456 is other than H in -P(O)(OR 456 ) 2 , -P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), -OP(O)(OR 456 ) 2 , - OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), SP(O)(OR 456 ) 2 , -SP(S)(OR 456 ) 2 , and -SP(S)(SR 457 )(OR 456 ).
  • all R 456 are H in -P(O)(OR 456 )2, - P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), -OP(O)(OR 456 ) 2 , -OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), - OP(S)(SR 457 )2, -SP(O)(OR 456 )2, -SP(S)(OR 456 )2, -SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 ) 2 .
  • all R 456 are other than H in in - P(O)(OR 456 ) 2 , -P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), -OP(O)(OR 456 ) 2 , -OP(S)(OR 456 ) 2 , - OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 )2, -SP(O)(OR 456 )2, -SP(S)(OR 456 )2, -SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 )2.
  • At least one R 457 in - P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 ) 2 , -OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 ) 2 , - SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 ) 2 is H.
  • At least one R 457 in - P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 ) 2 , -OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 ) 2 , - SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 )2 is other than H.
  • At least one R 457 in - P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 )2, -OP(S)(OR 456 )2, -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 )2, - SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 )2 is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an sulfur-protecting group.
  • At least one R 457 is H and at least one R 457 IS other than H in -P(S)(SR 457 ) 2 , -OP(S)(SR 457 ) 2 and -SP(S)(SR 457 ) 2 .
  • all R 457 are H in -P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 )2, - OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 ) 2 , -SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 ) 2 .
  • all R 457 are other than H in -P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 )2, -OP(S)(OR 456 )2, -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 )2, -SP(S)(SR 457 )(OR 456 ), and -SP(S)(SR 457 )2.
  • R 45 is optionally substituted -C2-6alkenyl-R 453 .
  • R 454 is hydrogen or an oxygen protecting group.
  • R 454 is hydrogen or 4,4′-dimethoxytrityl (DMT).
  • DMT 4,4′-dimethoxytrityl
  • R 454 is H.
  • R 45 is optionally substituted –C1-6alkenyl-R 453 .
  • R 45 can be -CH(R 458 )- R 453 , where R 453 is -OR 454 , -SR 455 , -P(O)(OR 456 ) 2 , -P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), - P(S)(SR 457 ) 2 , -OP(O)(OR 456 ) 2 , -OP(S)(OR 456 ) 2 , -OP(S)(SR 457 )(OR 456 ), -OP(S)(SR 457 ) 2 , - SP(O)(OR 456 ) 2 , -SP(S)(OR 456 ) 2 , -SP(S)(SR 457 )(OR 456 ), or -SP(S)(SR 457 ) 2 ; and R 458 is H, optionally
  • R 458 is H. In some other non-limiting examples, R 458 is Ci-Csoalkyl optionally substituted with a substituent selected from NH 2 , OH, C(O)NH 2 , COOH, halo, SH, and Ci-Cealkoxy.
  • R 45 is -CH(R 458 )-O- R 459 , where R 459 is H, -P(O)(OR 456 ) 2 , -P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 ) 2 , - OP(O)(OR 456 ) 2 .
  • R 45 is -CH(R 458 )-O-R 459 , where R 458 is H or optionally substituted Ci-C 3 oalkyl and R 459 is H or -P(O)(OR 456 ) 2 .
  • R 45 is -CH(R 458 )-S- R 60 , where R 60 is H, -P(O)(OR 456 ) 2 , -P(S)(OR 456 ) 2 , -P(S)(SR 457 )(OR 456 ), -P(S)(SR 457 ) 2 , - OP(O)(OR 456 ) 2 .
  • ligands modify one or more properties of the attached molecule (e.g., the oligonucleotide described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Ligands are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound.
  • a preferred list of ligands includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Preferred ligands amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thi
  • Ligands can include naturally occurring molecules, or recombinant or synthetic molecules.
  • exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxylpropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine
  • porphyrins e.g., TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g., EDTA
  • lipophilic molecules e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1- pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxyt
  • biotin transport/absorption facilitators
  • transport/absorption facilitators e.g., naproxen, aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactors, lipopolysaccharide, an activator of p38 MAP kinase, an activator of NF-KB, taxon, vincristine, vinblastine, cytochalasin, nocodazole,
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, 0, or y peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
  • a peptidomimetic also referred to herein as an oligopeptidomimetic is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the peptide or peptidomimetic ligand can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • amphipathic peptides include, but are not limited to, cecropins, ly cotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins.
  • endosomolytic ligand refers to molecules having endosomolytic properties.
  • Endosomolytic ligands promote the lysis of and/or transport of the composition of the invention, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell.
  • Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and brached poly amines, e.g.
  • spermine cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.
  • Exemplary endosomolytic/fusogenic peptides include, but are not limited to, AALEALAEALEALAEALEALAEAAAAGGC (GALA, SEQ ID NO: 196);
  • AALAEALAEALAEALAEALAEALAAAAGGC (EALA, SEQ ID NO: 197);
  • ALEALAEALEALAEA SEQ ID NO: 198
  • GLFEAIEGFIENGWEGMIWDYG IDF-7, SEQ ID NO: 199
  • GLFGAIAGFIENGWEGMIDGWYG Inf HA-2, SEQ ID NO: 14
  • GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7, SEQ ID NO: 200); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3, SEQ ID NO: 201); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF, SEQ ID NO: 202); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3, SEQ ID NO: 203); GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine, SEQ ID NO: 204); LFEALLELLESLWELLLEA (JTS-1, SEQ ID NO: 205);
  • GLFKALLKLLKSLWKLLLKA ppTGl, SEQ ID NO: 206
  • GLFRALLRLLRSLWRLLLRA ppTG20, SEQ ID NO: 207
  • WEAI ⁇ LAI ⁇ ALAI ⁇ ALAI ⁇ HLAI ⁇ ALAI ⁇ ALI ⁇ ACEA KALA, SEQ ID NO: 208
  • GLFFEAIAEFIEGGWEGLIEGC HA, SEQ ID NO: 209;
  • GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin, SEQ ID NO: 210); HsWYG (SEQ ID NO: 211); and CHKeHC (SEQ ID NO: 212).
  • fusogenic lipids fuse with and consequently destabilize a membrane.
  • Fusogenic lipids usually have small head groups and unsaturated acyl chains.
  • Exemplary fusogenic lipids include, but are not limited to, l,2-dileoyl-sn-3- phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-l,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z, 12Z)-octadeca
  • Exemplary cell permeation peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin, SEQ ID NO: 213); GRKKRRQRRRPPQC (Tat fragment 48-60, SEQ ID NO: 214); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based peptide, SEQ ID NO: 215); LLIILRRRIRKQAHAHSK (PVEC, SEQ ID NO: 216); GWTLNSAGYLLKINLKALAALAKKIL (transportan, SEQ ID NO: 217);
  • KLALKLALKALKAALKLA amphiphilic model peptide, SEQ ID NO: 218); RRRRRRRRR (Arg9, SEQ ID NO: 219); KFFKFFKFFK (Bacterial cell wall permeating peptide, SEQ ID NO: 220); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37, SEQ ID NO: 221); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin Pl, SEQ ID NO: 222);
  • ACYCRIPACIAGERRYGTCIYQGRLWAFCC (a-defensin, SEQ ID NO: 223);
  • DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK P-defensin, SEQ ID NO: 224
  • RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 PR-39, SEQ ID NO: 225
  • ILPWKWPWWPWRR-NH2 ILPWKWPWWPWRR-NH2
  • AAVALLPAVLLALLAP RFGF, SEQ ID NO: 227
  • AALLPVLLAAP RFGF analogue, SEQ ID NO: 228
  • RKCRIVVIRVCR bactenecin, SEQ ID NO: 229).
  • NEE alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid
  • NEI(CEECEENEI)nCEECEE-AMINE NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).
  • targeting ligand refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment.
  • Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.
  • Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins.
  • the term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.
  • PK modulating ligand and “PK modulator” refers to molecules which can modulate the pharmacokinetics of oligonucleotides described herein.
  • Some exemplary PK modulator include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2, 4, 6-triiodophenol and flufenamic acid).
  • lipophilic molecules bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, car
  • Oligomeric compounds that comprise a number of phosphorothioate intersugar linkages are also known to bind to serum protein, thus short oligomeric compounds, e.g. oligonucleotides of comprising from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).
  • ligands e.g. as PK modulating ligands
  • the PK modulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate linkages. In some embodiments, all intemucleoside linkages in PK modulating oligonucleotide are phosphorothioate and/or phosphorodithioates linkages.
  • aptamers that bind serum components e.g. serum proteins
  • Binding to serum components can be predicted from albumin binding assays, scuh as those described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27.
  • the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties.
  • a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties.
  • all the ligands have different properties.
  • the ligand has a structure shown in any of Formula (IV) - (VII):
  • repeating unit can be the same or different; p2A p2B p3A p3B p4A p4B p5A p5B p5C y2A y2B y3A y3P> y4A y4B> y5A y5P> y5C each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;
  • L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, tri saccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and
  • R a is H or amino acid side chain.
  • the ligand is of Formula (VII): wherein L 5A , L 5B and L 5C represent a monosaccharide, such as GalNAc derivative.
  • Exemplary ligands include, but are not limited to, the following:
  • the ligand is a ligand described in US Patent No. 5,994,517 or US Patent No. 6,906,182, content of each of which is incorporated herein by reference in its entirety.
  • the ligand can be a tri-antennary ligand described in Figure 3 of US Patent No. 6,906,182.
  • the ligand is selected from the following tri-antennary ligands:
  • ligands are same or different. Accordingly, in some embodiments of any one of the aspects described herein, all ligands are same. In some other embodiments of any one of the aspects described herein, ligands are different.
  • the ligand is selected from the group consistof ligands shown in FIG. 27.
  • linker means an organic moiety that connects two parts of a compound.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR 1 , C(O), C(O)O, C(O)NR 1 , SO, SO2, SO2NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl
  • alkynylheterocyclylalkynyl alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R 1 ) 2 , C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R 1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • the linker is a cleavable linker.
  • Cleavable linkers are those that rely on processes inside a target cell to liberate the two parts the linker is holding together, as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by specific enzymes (e.g. proteases) within the cell.
  • cleavable linkers allow the two parts to be released in their original form after internalization and processing inside a target cell.
  • Cleavable linkers include, but are not limited to, those whose bonds can be cleaved by enzymes (e.g., peptide linkers); reducing conditions (e.g., disulfide linkers); or acidic conditions (e.g., hydrazones and carbonates).
  • the cleavable linker comprises at least one cleavable linking group.
  • a cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.
  • the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood or serum of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
  • a first reference condition which can, e.g., be selected to mimic or represent intracellular conditions
  • a second reference condition which can, e.g., be selected to mimic or represent conditions found in the blood or serum.
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.
  • degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g
  • a cleavable linkage group such as a disulfide bond can be susceptible to pH.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1- 7.3.
  • Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0.
  • Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
  • a linker can include a cleavable linking group that is cleavable by a particular enzyme.
  • the type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
  • the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • a degradative agent or condition
  • the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals.
  • useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
  • cleavable linking groups are redox cleavable linking groups, which may be used according to the present invention that are cleaved upon reduction or oxidation.
  • An example of reductively cleavable linking group is a disulfide linking group (-S-S-).
  • a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein.
  • a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell.
  • the candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions.
  • candidate compounds are cleaved by at most 10% in the blood.
  • useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
  • Phosphate-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by agents that degrade or hydrolyze the phosphate group.
  • agents that degrade or hydrolyze the phosphate group are enzymes such as phosphatases in cells.
  • phosphate-based linking groups are -O-P(O)(ORk)-O-, -O- P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O- P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(
  • Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S- P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(O)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-, -O-P(S)(H)-S-.
  • a preferred embodiment is -O-P(O)(OH)-O-.
  • Acid cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are linking groups that are cleaved under acidic conditions.
  • acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
  • specific low pH organelles such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups.
  • acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids.
  • a preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.
  • Ester-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as esterases and amidases in cells.
  • ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups.
  • Ester cleavable linking groups have the general formula - C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.
  • Peptide-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.
  • Peptide-based cleavable groups do not include the amide group (-C(O)NH-).
  • the amide group can be formed between any alkylene, alkenylene or alkynylene.
  • a peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins.
  • the peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group.
  • Peptide-based cleavable linking groups have the general formula - NHCHR A C(O)NHCHR B C(O)-, where R A and R B are the R groups of the two adjacent amino acids.
  • the linker is - C(O)CH 2 CH 2 C(O)-, -OC(O)CH 2 CH 2 C(O)-, -OC(O)CH 2 CH 2 C(O)O-, -C(O)CH 2 CH 2 C(O)NH- or -OC(O)CH 2 CH 2 C(O)NH-.
  • the linker is -OC(O)CH 2 CH 2 C(O)NH-
  • Internucleoside linkages refers to a covalent linkage between adjacent nucleosides.
  • the two main classes of intemucleoside linkages are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino ( — CH2-N(CH3)-O — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ); siloxane ( — O — Si(H)2-0 — ); and N,N'- dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-).
  • Modified intemucleoside linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide compound.
  • linkages having a chiral atom can be prepared as racemic mixtures, as separate enantiomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing linkages are well known to those skilled in the art.
  • the phosphate group in the intemucleoside linkage can be modified by replacing one of the oxygens with a different substituent.
  • One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown.
  • modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • one of the non-bridging phosphate oxygen atoms in the phosphodiester intemucleoside linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an aryl group, etc...), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl).
  • the phosphorous atom in an unmodified phosphate group is achiral.
  • replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral.
  • a phosphorous atom in a phosphate group modified in this way is a stereogenic center.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
  • Phosphorodithioates have both non-bridging oxygens replaced by sulfur.
  • the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers.
  • modifications to both non-bridging oxygens, which eliminate the chiral center, e.g. phosphorodithioate formation can be desirable in that they cannot produce diastereomer mixtures.
  • the non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • a phosphodiester intemucleoside linkage can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the nucleosides), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • bridging oxygen i.e. oxygen that links the phosphate to the sugar of the nucleosides
  • nitrogen bridged phosphoroamidates
  • sulfur bridged phosphorothioates
  • carbon bridged methylenephosphonates
  • Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorous group are also referred to as “non-phosphodiester intersugar linkage” or “non-phosphodiester linker.”
  • the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers.
  • Dephospho linkers are also referred to as non- phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.
  • Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amides, carbamate and ethylene oxide linker.
  • a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.
  • Preferred non-phosphodiester intemucleoside linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer, phosphorodi thioates, phsophotriesters, aminoalky Iphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphorami dates (e.g., N-alkylphosphoramidate), and boranophosphonates.
  • the oligonucleotides described herein comprise one or more neutral intemucleoside linkages that are non-ionic.
  • the non-phosphodiester backbone linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate and phosphoramidate backbone linkages.
  • the intemucleoside linkage where R IL1 and R IL2 are each independently for each occurrence absent, O, S, CEE, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the end; and R IL3 and R IL4 are each independently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR3 (R is hydrogen, alkyl, aryl), BEE' , C (i.e.
  • R IL1 and R IL2 are replacing the oxygen linked to 5’ carbon of a first nucleoside sugar and the other of R IL1 and R IL2 is replacing the oxygen linked to 3’ (or 2’) carbon of a second nucleoside sugar.
  • R IL1 , R IL2 , R IL3 and R IL4 all are O.
  • R IL1 and R IL2 are O and at least one of R IL3 and R IL4 is other than
  • R IL3 and R IL4 are S and the other is O or both of R IL3 and R IL4 are S.
  • one of R 43 or R 45 is a bond to a modified intemucleoside linkage, e.g., an intemucleoside linkage of structure: where at least one of R IL1 , R IL2 , R IL3 and R IL4 is not O.
  • R IL3 and R IL4 is S.
  • both of R 43 and R 45 are a bond to a modified intemucleoside linkage.
  • R 43 is a bond to phosphodiester intemucleoside linkage.
  • R 45 is a bond to phosphodiester intemucleoside linkage.
  • R 43 is a bond to a modified intemucleoside linkage and R 45 is a bond to phosphodiester intemucleoside linkage.
  • R 45 is a bond to a modified intemucleoside linkage and R 43 is a bond to phosphodiester intemucleoside linkage.
  • the intemucleotide linkage is -P(X D )(N(R p )2)-, where X D is O or S; and each R P2 is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl.
  • R 43 is linked to an intemucleotide linkage of formula -P(X D )(N(R p )2)-@, where X D is O or S; each R P2 is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl; and @ is a bond to 5 ’-position of a subsequent nucleoside.
  • R 43 is linked to an intemucleotide linkage of formula -P(X D )(N(R p )2)-@, where X D is O or S; each R P2 is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl; and @ is a bond to R 45 of a subsequent nucleoside of Formula (IV).
  • the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more modified intemucleoside linkages.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5 or 6 modified intemucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3 or 4 modified intemucleoside linkages.
  • the oligonucleotide comprises at least two modified intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the oligonucleotide and further comprises at least two modified intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the oligonucleotide.
  • the oligonucleotide comprises modified intemucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the oligonucleotide.
  • the modified intemucleoside linkage is a phosphorothioate.
  • the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3, 4, 5 or 6 phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3 or 4 phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the oligonucleotide and further comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the oligonucleotide.
  • the oligonucleotide comprises modified intemucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the oligonucleotide.
  • Oxygen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (TEIP), 3 -bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclo
  • oxygen protecting group is benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX).
  • DMT 4,4'-dimethoxytrityl
  • Pixyl 9-phenylxanthine-9-yl
  • MOX 9-(p- methoxyphenyl)xanthine-9-yl
  • the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4′-dimethoxytrityl.
  • protected hydroxyl and “protected hydroxyl” as used herein mean a group of the formula -OR Pro , wherein R Pro is an oxygen protecting group as defined herein.
  • Nitrogen protecting groups [00232] Some embodiments of the various aspects described herein include a nitrogen protecting group (also referred to as an amino protecting group herein).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • Ts
  • Additional exemplary nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N'- phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N- 2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-l,3,5-triazacyclohexan-2
  • Sulfur protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • nucleoside of Formula (IV) can be located anywhere in the oligonucleotide. In some embodiments, the nucleoside of Formula (IV) is present at the 5’- or 3’- terminus of the oligonucleotide. In some embodiments, the nucleoside of Formula (IV) is present at an internal position of the oliogunculeotide. In some embodiments, when the nucleoside of Formula (IV) is present at the 3 ’-terminus of the oligonucleotide, R 43 is a hydroxyl or protected hydroxyl group.
  • R 43 when the nucleoside of Formula (IV) is present at the 3’- terminus of the oligonucleotide, R 43 is a hydroxyl. In other embodiments, when the nucleoside of Formula (IV) is present at the 3 ’-terminus of the oligonucleotide, R 43 is a hydrogen or a nitrogen protecting group. In other embodiments, when the nucleoside of Formula (IV) is present at the 3’- terminus of the oligonucleotide, R 43 is a hydrogen.
  • the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (IV), a nucleoside with a modified sugar.
  • a “modified sugar” is meant a sugar or moiety other than 2’-deoxy (i.e, 2’-H) or 2’-OH ribose sugar.
  • nucleotides comprising a modified sugar are 2’-F ribose, 2’-0Me ribose, 2’-O,4’-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5- anhydrohexitol nucleic acid, HNA), cyclohexene (Cyclohexene nucleic acid, CeNA), 2’- methoxyethyl ribose, 2’-O-allyl ribose, 2’-C-allyl ribose, 2'-O-N-methylacetamido (2'-0-NMA) ribose, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2’-F arabinose (2'-ara-F
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro (2’-F) nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-F nucleotides. It is noted that the 2’-F nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), and 2’-F nucleosides.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-0Me nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-0Me nucleotides. It is noted that the 2’-0Me nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises solely comprises solely comprises nucleosides of Formula (IV), and 2’-0Me nucleosides. In some other embodiments, the oligonucleotide comprises, e.g., solely comprises solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides and 2’-F nucleosides.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-deoxy, e.g., 2’-H nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of 2’-deoxy, e.g., 2’-H nucleotides. It is noted that the 2’- deoxy, e.g., 2’-H nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide can comprise a 2’-deoxy, e.g., 2’-H nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the oligonucleotide.
  • the oligonucleotide comprises a 2’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises solely comprises nucleosides of Formula (IV), and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides, and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-F nucleosides and 2’-deoxy (2’- H) nucleotides.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides, 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (IV), a non-natural nucleobase.
  • the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising an independently selected non-natural nucleobase.
  • a nucleotide comprising a non-natural nucleobase can be present anywhere in the oligonucleotide.
  • the oligonucleotide further comprises a solid support linked thereto.
  • the oligonucleotides described herein can range from few nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) in length to hunderes of nucleotides in length.
  • the oligonucleotide can be from 5 nucleotides to 100 nucleotides in length.
  • the oligonucleotide is from 10 nucleotides to 50 nucleotides in length.
  • the oligonucleotide is between 15 and 35, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length.
  • oligonucleotide In some embodiments, longer oligonucleotides of between 25 and 30 nucleotides in length are preferred. In some embodiments, shorter oligonucleotides of between 10 and 15 nucleotides in length are preferred. In another embodiment, the oligonucleotide is at least 21 nucleotides in length.
  • the oligonucleotide described herein comprises a pattern of backbone chiral centers.
  • a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 9 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 19 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration, and no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration, and no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration, and no more than 4 intemucleotidic linkages which are not chiral.
  • the intemucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages which are not chiral are optionally contiguous or not contiguous.
  • the oligonucleotide described herein comprises a stereochemistry block.
  • a block is an Rp block in that each intemucleotidic linkage of the block is Rp.
  • a 5 ’-block is an Rp block.
  • a 3 ’-block is an Rp block.
  • a block is an Sp block in that each intemucleotidic linkage of the block is Sp.
  • a 5 ’-block is an Sp block.
  • a 3 ’-block is an Sp block.
  • provided oligonucleotides comprise both Rp and Sp blocks.
  • provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each intemucleotidic linkage in a natural phosphate linkage.
  • the oligonculeotide described herein comprises a 5’-block is an Sp block wherein each sugar moiety comprises a 2 ’-fluoro modification.
  • a 5 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’- block is an Sp block wherein each of intemucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’-block comprises 4 or more nucleoside units.
  • a 5 ’-block comprises 5 or more nucleoside units. In some embodiments, a 5 ’-block comprises 6 or more nucleoside units. In some embodiments, a 5 ’-block comprises 7 or more nucleoside units. In some embodiments, a 3 ’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3 ’-block comprises 4 or more nucleoside units.
  • a 3 ’-block comprises 5 or more nucleoside units.
  • a 3 ’-block comprises 6 or more nucleoside units.
  • a 3 ’-block comprises 7 or more nucleoside units.
  • oligonucleotide described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of intemucleotidic linkage, e.g., natural phosphate linkage, modified intemucleotidic linkage, Rp chiral intemucleotidic linkage, Sp chiral intemucleotidic linkage, etc.
  • A is followed by Sp.
  • A is followed by Rp.
  • A is followed by natural phosphate linkage (PO).
  • U is followed by Sp.
  • U is followed by Rp.
  • U is followed by natural phosphate linkage (PO).
  • C is followed by Sp.
  • C is followed by Rp.
  • C is followed by natural phosphate linkage (PO).
  • G is followed by Sp.
  • G is followed by Rp.
  • G is followed by natural phosphate linkage (PO).
  • C and U are followed by Sp.
  • C and U are followed by Rp.
  • C and U are followed by natural phosphate linkage (PO).
  • a and G are followed by Sp.
  • a and G are followed by Rp.
  • the oligonucleotides described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5 '-diphosphate ((HO)2(O)P- O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5 1 - (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P- 0-5
  • 5 '-alpha-thiotriphosphate, 5 '-gamma-thiotriphosphate, etc.), 5'- phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (e.g., RP(OH)(O)-O-5'-, R alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonates (i.e.
  • exemplary 5 ’-modifications include where Z is optionally substituted alkyl at least once, e g., ((HO) 2 (X)P-O[-(CH 2 )a-O-P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH 2 )a- P(X)(OH)-O]b- 5', ((HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics: HO
  • the oligonucleotide comprises a 5’-vinylphosphonate group (i.e., the 4’-C of the 5’-terminal nucleotide is bonded to a vinyl phosphonate).
  • the oligonucleotide comprises a 5’-E-vinyl phosphonate group.
  • the oligonucleotide comprises a 5’-Z-vinylphosphonate group.
  • the oligonucleotide dscribed herein comprises a 5 ’-morpholino, a 5 ’-dimethylamino, a 5 ’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5 ’-end.
  • the oligonucleotide dscribed herein can comprise a thermally destabilizing modification, for example, a nucleoside of formula (IV), within the seed region of the antisense strand.
  • the oligonucleotide can comprise at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5’-end of the oligonucleotide (e.g., one thermally destabilizing nucleotide).
  • the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5 ’-end of the antisense strand.
  • thermally destabilizing modification is located in positions 2-9, or preferably positions 4-8, counting from the 5 ’-end of the oligonucleotide.
  • the thermally destabilizing modification is located at position 5, 6, 7 or 8, counting from the 5’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 7, counting from the 5 ’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 6, counting from the 5 ’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 5, counting from the 5 ’-end of the oligonucleotide.
  • thermally destabilizing modification(s) includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s).
  • Tm overall melting temperature
  • the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5 ’-end of the antisense strand.
  • the thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).
  • UUA unlocked nucleic acids
  • GNA glycol nucleic acid
  • the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
  • the destabilizing modification mUNA is selected from the group consisting of - alkyl; O-alkylamino;
  • R' H, Me
  • B A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.
  • the destabilizing modification mUNA is selected from the group consisting of ; O- alkyl; O-alkylamino;
  • R' H, Me
  • B A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines;
  • the destabilizing modification mUNA is selected from the group consisting of
  • B A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurines; and Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.
  • the destabilizing modification mUNA is selected from the group consisting of
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-wPr; O- alkyl; O-alkylamino;
  • R' H, Me
  • B A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers
  • the destabilizing modification mUNA is selected from the group consisting of alkyl; O-alkylamino;
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers
  • the modification mUNA is selected from the group consisting of
  • B A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurines; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers
  • Exemplary abasic modifications include, but are not limited to the following:
  • X OMe, F wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.
  • the thermally destabilizing modification of the duplex is selected from the mUNA and GNA building blocks described in Examples 1-3 herein.
  • the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
  • the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GN A, 2’-0Me, 3’-0Me, 5 ’-Me, Hy p-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h’GNA (Mod A-Mod K).
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., Cl’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or Cl’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., Cl’, C2’, C3’,
  • C4’ or 04’ are independently or in combination absent from the nucleotide.
  • independently are H, halogen, OR3, or alkyl; andR3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • the term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue.
  • UNA also encompasses monomers with bonds between CT-C4' being removed (i.e. the covalent carbon- oxygen-carbon bond between the Cl' and C4' carbons).
  • the C2'-C3' bond i.e.
  • the acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings.
  • the acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.
  • glycol nucleic acid refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:
  • the thermally destabilizing modification of the duplex can be mismatches (i.e., noncompl ementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex.
  • exemplary mismatch base pairs include G:G, GA, GU, G:T, A: A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof.
  • Other mismatch base pairings known in the art are also amenable to the present invention.
  • a mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides.
  • the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as:
  • the thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.
  • the thermally destabilizing modification includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand.
  • nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety.
  • Exemplary nucleobase modifications are: inosine nebularine 2-aminopurine
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more a-nucleotide complementary to the base on the target mRNA, such as: wherein R is H, OH, OCH3, F, NH2, NHMe, NM02 or O-alkyl
  • the alkyl for the R group can be a Ci-Cealkyl.
  • Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.
  • the oligonucleotide can comprise one or more stabilizing modifications.
  • the oligonucleotide can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • the oligonucleotide comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the oligonucleotide can be present at any positions.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5 ’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 6, 14 and 16, counting from the 5 ’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 14 and 16, counting from the 5 ’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 7, 10 and 11, counting from the 5 ’-end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 7, 9, 10 and 11, counting from the 5 ’-end.
  • the oligonucleotide comprises at least one stabilizing modification adjacent to a destabilizing modification.
  • the stabilizing modification can be the nucleotide at the 5 ’-end or the 3 ’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification.
  • the oligonucleotide comprises a stabilizing modification at each of the 5 ’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the oligonucleotide comprises at least two stabilizing modifications at the 3 ’-end of a destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • thermally stabilizing modifications include, but are not limited to 2’-fluoro modifications.
  • Other thermally stabilizing modifications include, but are not limited to LNA.
  • RNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer double-stranded oligonucleotides can be effective as well.
  • a double-stranded RNA comprising a first strand (also referred to as an antisense strand or a guide strand) and a second strand (also referred to as a sense strand or passenger strand, wherein at least one of the first (i.e., the antisense strand) or the second strand (i.e., the sense strand) is an oligonucleotide described herein.
  • at least one of the first (i.e., the antisense strand) or the second strand (i.e., the sense strand) comprises at least one nucleotide of Formula (IV),
  • the sense strand is an oligonucleotide described herein.
  • the sense strand comprises at least one nucleotide of Formula (IV),
  • the antisense strand is an oligonucleotide described herein.
  • the antisense strand comprises at least one nucleotide of Formula (IV).
  • the antisense strand is substantially complementary to a target nucleic acid, e.g., a target gene or mRNA gene and the dsRNA is capable of inducing targeted cleavage of the target nucleic acid.
  • Each strand of the dsRNA molecule can range from 15-35 nucleotides in length.
  • each strand can be between, 17-35 nucleotides in length, 17-30 nucleotides in length, 17- 25 nucleotides in length, 18-30 nucleotides in length 18-25 nucleotides in length, 25-35 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17- 19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
  • the sense and antisense strands can be equal length or unequal length.
  • the sense strand and the antisense strand independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • the antisense strand is of length 15-35 nucleotides. In some embodiments, the antisense strand is 15-35, 17-35, 17-30, 17-25, 18-30, 18-25, 25-35, 27-30, 17- 23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length.
  • the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
  • the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length.
  • the antisense strand is 22, 23 or 24 nucleotides in length.
  • the antisense strand is 23 nucleotides in length.
  • the sense strand can be, in some embodiments, 15-35 nucleotides in length. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 17-25 nucleotides in length, 18-30 nucleotides in length 18-25 nucleotides in length, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length.
  • the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or
  • the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 19, 20, 21, 22 or 23 nucleotides in length.
  • the sense strand is 20, 21 or 22 nucleotides in length.
  • the sense strand is 21nucleotides in length.
  • the sense strand can be 15-35 nucleotides in length, and the antisense strand can be independent from the sense strand, 15-35 nucleotides in length.
  • the sense strand is 15-35, 17-35, 17-30, 17-25, 18-30, 18-25, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length
  • the antisense strand is independently 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19- 21, 21-25, 21-25, or 21-23 nucleotides in length.
  • the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 19, 20, 21, 22 or 23 nucleotides in length and the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 20, 21 or 22 nucleotides in length and the antisense strand is 22, 23 or 24 nucleotides in length.
  • the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
  • the sense strand and antisense strand typically form a double-stranded or duplex region.
  • the duplex region of a dsRNA agent described herein can be 12-35 nucleotide (or base) pairs in length.
  • the duplex region can be between 14-35 nucleotide pairs in length, 17-30 nucleotide pairs in length, 17-25 nucleotide pairs in length, 18-25 nucleotide pairs in length, 18-23 nucleotide pairs in length, 25-35 nucleotides in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.
  • the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length. In some embodiments, the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length. For example, the duplex region is 19, 20, 21, 22 or 23 nucleotide pairs in length. In some embodiments, the the duplex region is 20, 21 or 22 nucleotide pairs in length. For example, the dsRNA molecule has a duplex region of 21 base pairs.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of nucleotide of Formula (IV), Without limitations, the nucleotides of Formula (IV), all can be present in one strand.
  • the nucleotide of Formula (IV) may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides of Formula (IV) described herein.
  • the nucleotide of Formula (IV) described herein can be present at any position of the sense strand.
  • the nucleotide of Formula (IV) described herein can be present at a terminal region of the sense strand.
  • the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3 and
  • nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3 and 4, counting from the 3 ’-end of the sense strand. In some embodiments, the nucleotide of Formula (IV) can be present at one or more of positions 18, 19, 20 and 21, counting from 5 ’-end of the sense strand.
  • the nucleotide of Formula (IV) described herein can also be located at a central region of sense strand. For example, the nucleotide of Formula (IV) described herein can be located at one or more of positions 6, 7, 8, 9, 10, 11, 12 and 13, counting from 5 ’-end of the sense strand. In some embodiments, the nucleotide of Formula (IV) is at the 5-terminus of the sense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of nucleotides of Formula (IV) described herein.
  • the nucleotide of Formula (IV) described herein can be present at any position of the antisense strand.
  • the nucleotide of Formula (IV) described herein can be present at a terminal region of the antisense strand.
  • the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3, 4,
  • nucleotide of Formula (IV) described herein nucleotide can be present at one or more of positions 2, 3, 4, 5, 6, 7, and 8, counting from the 3 ’-end of the antisense strand. In some embodiments, the nucleotide of Formula (IV) described herein nucleotide can be present at one or more of positions 6, 7, 8, and 9, counting from 5 ’-end of the antisense strand.
  • the sense strand comprises a nucleotide of Formula (IV) described herein at a position complemenatry to position 1, 2, 3, 4, 5, 6, 7, 8, or 9, counting from the 5 ’-end of the antisense strand.
  • the sense strand comprises a nucleotide of Formula (IV) described herein at a position complemenatry to position 2, 3, 4, 5, 6, 7, or 8, counting from the 5 ’-end of the antisense strand.
  • the sense strand comprises a nucleotide of Formula (IV) described herein at a position complemenatry to position 6, 7, 8, or 9, counting from the 5 ’-end of the antisense strand.
  • the dsRNA agent can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising a modified sugar. Accordingly, in some embodiments, the dsRNA agent can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides independently selected from the group consisting of 2’-F, 2-OMe, acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2 ’-methoxy ethyl, 2’-O-allyl, 2’-C-allyl, 2'-O-N- methylacetamido (2'-0-NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O- aminopropyl (2'-O-AP), and 2'-ara-F.
  • LNA locked nucleic acid
  • CeNA locked nucleic acid
  • CeNA HNA
  • a nucleotide comprising modified sugar can be present anywhere in the dsRNA molecule.
  • a nucleotide comprising a modified sugar can be present in the sense strand or a nucleotide comprising a modified sugar can be present in the antisense strand.
  • two or more nucleotides comprising a modified sugar are present in the dsRNA molecule, they can all be in the sense strand, antisense strand or both in the sense and antisense strands.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro (2’-F) nucleotides.
  • the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro nucleotides.
  • the 2’-fluoro nucleotides can be located anywhere in the sense strand.
  • the sense strand comprises a 2 ’-fluoro nucleotide at position 10, counting from 5 ’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at position 10, counting from 5’- end of the sense strand and the sense strand further comprises a 2’-fluoro nucleotide at position 8, 9, 11 or 12, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2 ’-fluoro nucleotide at positions 9 10, counting from 5 ’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at positions 10 and 11, counting from 5 ’-end of the sense strand.
  • the sense strand comprises a 2 ’-fluoro nucleotide at positions 9, 10 and 11, counting from 5 ’-end of the sense strand. In some other embodiments, the sense strand comprises a 2’-fluoro nucleotide at positions 8, 9 and 10, counting from 5’-end of the sense strand. In yet some other embodiments, the sense strand comprises a 2’-fluoro nucleotide at positions 10, 11 and 12, counting from 5 ’-end of the sense strand.
  • the antisense comprises 2 ’-fluoro nucleotides at positions 7, 10 and 11 from the 5’-end.
  • the sense strand comprises 2’-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5’-end of the sense strand (e.g., when the sense strand is 21-23 nucleotides in length).
  • the sense strand comprises 2’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand or the first paired nucleotide at the 5 ’end of the antisense strand.
  • the sense strand comprises 2’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 13 of the antisense strand, counting from the 5 ’-end of the antisense strand, or the first paired nucleotide at the 5 ’end of the antisense strand.
  • the sense strand can comprise 2’-fluoro nucleotides at positions 7, 8, and 9, counting from the 5 ’-end of the sense strand, when the sense strand is 19 nucleotides in length; or positions 8, 9, and 10, counting from the 5 ’-end of the sense strand, when the sense strand is 20 nucleotides in length; or positions 9, 10, and 11 counting from the 5 ’-end of the sense strand, when the sense strand is 21 nucleotides in length.
  • the sense strand comprises 2 ’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand or the first paired nucleotide at the 5 ’end of the antisense stran .
  • the sense strand can comprise 2’-fluoro nucleotides at positions 5, 7, 8, and 9, counting from the 5 ’-end of the sense strand, when the sense strand is 19 nucleotides in length; or positions 6, 8, 9, and 10, counting from the 5 ’-end of the sense strand, when the sense strand is 20 nucleotides in length; or positions 7, 9, 10, and 11 counting from the 5’-end of the sense strand, when the sense strand is 21 nucleotides in length.
  • the sense strand comprises a block of two, three or four 2’- fluoro nucleotides.
  • the sense strand can comprises a block of four 2’-fluoro nucleotides, such as at positions 9, 10, 11, and 12; or 8, 9, 10, and 11, when the sense strand is 21- 23 nucleotides in length (e.g., 21 nucleotides in length).
  • the sense strand does not comprise a 2’-fluoro nucleotide in position opposite or complimentary to a thermally destabilizing modification of the duplex in the antisense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro nucleotides.
  • the 2’-fluoro nucleotides can be located anywhere in the antisense strand.
  • the antisense strand can comprise a 2’-fluoro nucleotide at position 14, counting from 5 ’-end of the antisense strand.
  • the antisense comprises 2 ’-fluoro nucleotides at positions 2 and 14, counting from the 5 ’-end of the antisense strand.
  • the antisense comprises 2’-fluoro nucleotides at positions 2, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense comprises 2’-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5 ’-end. In some other embodiments, the antisense comprises 2’- fluoro nucleotides at positions 2, 4, and 14 counting from the 5 ’-end of the antisense strand. In some other embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 4, 14 and 16 counting counting from the 5 ’-end of the antisense strand. In still some embodiments, the antisense comprises 2’ -fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 counting from the 5’- end. of the antisense strand In still some embodiments, the antisense comprises 2 ’-fluoro nucleotides at positions 2, 4, 8, 9, 14 and 16 counting from the 5 ’-end of the antisense strand.
  • the antisense strand comprises at least one 2’-fluoro nucleotide adjacent to a destabilizing modification.
  • the 2’-fluoro nucleotide can be the nucleotide at the 5 ’ -end or the 3 ’ -end of a destabilizing modification, i. e. , at position - 1 or +1 from the position of the destabilizing modification.
  • the antisense strand comprises a 2 ’-fluoro nucleotide at each of the 5 ’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the antisense strand comprises at least two 2’-fluoro nucleotides at the 3 ’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • both the sense and the antisense strands comprise at least one 2 ’-fluoro nucleotide.
  • the 2 ’-fluoro modification can occur on any nucleotide of the sense strand or antisense strand.
  • the 2’-fluoro modification can occur on every nucleotide on the sense strand and/or antisense strand; each 2’-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2’- fluoro modifications in an alternating pattern.
  • the alternating pattern of the 2’-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2’-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2’-fluoro modifications on the antisense strand.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides.
  • the 2’-OMe nucleotides all can be present in one strand.
  • the 2’-OMe nucleotide may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’- OMe nucleotides.
  • the 2’-OMe nucleotides can be located anywhere in the sense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides.
  • the 2’-OMe nucleotides can be located anywhere in the antisense strand.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-deoxy, e.g., 2’-H ribose nucleotides.
  • the dsRNA can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-deoxy, e.g., 2’-H nucleotides.
  • the 2’-deoxy nucleotide may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the dsRNA can comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2’-deoxy modifications in a central region of the sense strand and/or the antisense strand.
  • At least one of the sense stand and the antisense can comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2’-deoxy modification in positions 5-17, e.g., positions 6-16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5 ’-end of the sense strand or the antisense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides.
  • antisense strand can comprise 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides.
  • the 2’-deoxy nucleotides can be located anywhere in the antisense strand.
  • the antisense strand comprises a 2 ’-deoxy nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5 ’-end of the antisense strand.
  • the antisense strand comprises a 2 ’-deoxy nucleotide at 1, 2, 3 or 4 of positions 2, 5, 7, and 12, counting from 5 ’-end of the antisense strand.
  • the antisense comprises a 2 ’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’- deoxy nucleotide at positions 5, 7 and 12, counting from 5 ’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5 and 7, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7 and 12, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7, 12 and 14, counting, from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’- deoxy nucleotide at positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the antisense strand [00316]
  • the antisense comprises a 2’-deoxy nucleotide at position 2 or 12, counting from 5’-end of the antisense strand.
  • the antisense comprises a 2’-deoxy nucleotide at position 12, counting from 5 ’-end of the antisense strand.
  • the dsRNA comprises at least three 2’-deoxy modifications, wherein the 2 ’-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5 ’-end of the antisense strand, and at position 11 of the sense strand, counting from 5 ’-end of the sense strand.
  • the dsRNA comprises at least five 2’-deoxy modifications, wherein the 2 ’-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5 ’-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5’- end of the sense strand.
  • the dsRNA comprises at least seven 2’-deoxy modifications, wherein the 2 ’-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5 ’-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5 ’-end of the sense strand.
  • the antisense strand comprises at least five 2’-deoxy modifications at positions 2, 5, 7, 12 and 14, counting from 5 ’-end of the antisense strand.
  • the sense strand does not comprise a 2 ’-deoxy nucleotide at position 11, counting from 5 ’-end of the sense strand.
  • the dsRNA can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising a non-natural nucleobase
  • a nucleotide comprising a non-natural nucleobase can be present anywhere in the dsRNA molecule.
  • a nucleotide comprising a non-natural nucleobase can be present in the sense strand or a nucleotide comprising a non-natural nucleobase can be present in the antisense strand.
  • two or more nucleotides comprising a non-natural nucleobase are present in the dsRNA molecule, they can all be in the sense strand, antisense strand or both in the sense and antisense strands.
  • the dsRNA molecule described herein can further comprise at least one phosphorothioate or methylphosphonate intemucleoside linkage.
  • the phosphorothioate or methylphosphonate intemucleoside linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the intemucleoside linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each intemucleoside linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both intemucleoside linkage modifications in an alternating pattern.
  • the alternating pattern of the intemucleoside linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the intemucleoside linkage modification on the sense strand may have a shift relative to the alternating pattern of the intemucleoside linkage modification on the antisense strand.
  • the dsRNA molecule comprises the phosphorothioate or methylphosphonate intemucleoside linkage modification in the overhang region.
  • the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate intemucleoside linkage between the two nucleotides.
  • Intemucleoside linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region.
  • the overhang nucleotides may be linked through phosphorothioate or methylphosphonate intemucleoside linkage, and optionally, there may be additional phosphorothioate or methylphosphonate intemucleoside linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide.
  • these terminal three nucleotides may be at the 3 ’-end of the antisense strand.
  • the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5 or 6 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3 or 4 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the dsRNA molecule described herein further comprises one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand.
  • one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand.
  • at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate or methylphosphonate intemucleoside linkage at one end or both ends of the sense and/or antisense strand.
  • the dsRNA molecule described herein comprises one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the internal region of the duplex of each of the sense and/or antisense strand.
  • at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate methylphosphonate intemucleoside linkage at position 8-16 of the duplex region counting from the 5 ’-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the termini position(s).
  • the dsRNA molecule described herein further comprises one to five phosphorothioate or methylphosphonate intemucleoside linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate intemucleoside linkage modification(s) within the last 3 positions of the sense strand (counting from the 5 ’-end), and one to five phosphorothioate or methylphosphonate intemucleoside linkage modification at positions 1 and 2 and one to five phosphorothioate or methylphosphonate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 and one phosphorothioate or methylphosphonate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate intemucleoside linkage modifications within the last six the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and two phosphorothioate intemucleoside linkage modifications within the last four positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and two phosphorothioate intemucleoside linkage modifications within the last four positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last four positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 and one within the last six positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modification at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 (counting from the 5’- end) of the sense strand, and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 (counting from the 5’- end) of the sense strand, and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one within the last six positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 20 and 21 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 20 and 21 the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 21 and 22 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one phosphorothioate intemucleoside linkage modification at position 21 of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 21 and 22 the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 22 and 23 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one phosphorothioate intemucleoside linkage modification at position 21 of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 22 and 23 the antisense strand (counting from the 5 ’-end).
  • the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand.
  • the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand.
  • the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand.
  • the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’ end of the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides n and n-1, and between nucleotides n-1 and n-2, where n is length of the antisense strand, i.e, number of nucleotides in the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.
  • the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisense strand and at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.
  • the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand and the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the antisense strand.
  • the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand.
  • the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand and the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the antisense strand.
  • the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.
  • dsRNA molecule described herein comprises a pattern of backbone chiral centers.
  • a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 9 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 19 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration, and no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration, and no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration, and no more than 4 intemucleotidic linkages which are not chiral.
  • the intemucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages which are not chiral are optionally contiguous or not contiguous.
  • dsRNA molecule described herein comprises a block is a stereochemistry block.
  • a block is an Rp block in that each intemucleotidic linkage of the block is Rp.
  • a 5 ’-block is an Rp block.
  • a 3 ’-block is an Rp block.
  • a block is an Sp block in that each intemucleotidic linkage of the block is Sp.
  • a 5 ’-block is an Sp block.
  • a 3 ’-block is an Sp block.
  • provided oligonucleotides comprise both Rp and Sp blocks.
  • provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each intemucleotidic linkage in a natural phosphate linkage.
  • dsRNA molecule described herein comprises a 5 ’-block is an Sp block wherein each sugar moiety comprises a 2 ’-fluoro modification.
  • a 5 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’- block is an Sp block wherein each of intemucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’-block comprises 4 or more nucleoside units.
  • a 5 ’-block comprises 5 or more nucleoside units. In some embodiments, a 5 ’-block comprises 6 or more nucleoside units. In some embodiments, a 5 ’-block comprises 7 or more nucleoside units. In some embodiments, a 3 ’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3 ’-block comprises 4 or more nucleoside units.
  • a 3 ’-block comprises 5 or more nucleoside units.
  • a 3 ’-block comprises 6 or more nucleoside units.
  • a 3 ’-block comprises 7 or more nucleoside units.
  • dsRNA molecule described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of intemucleotidic linkage, e.g., natural phosphate linkage, modified intemucleotidic linkage, Rp chiral intemucleotidic linkage, Sp chiral intemucleotidic linkage, etc.
  • A is followed by Sp.
  • A is followed by Rp.
  • A is followed by natural phosphate linkage (PO).
  • U is followed by Sp.
  • U is followed by Rp.
  • U is followed by natural phosphate linkage (PO).
  • C is followed by Sp.
  • C is followed by Rp.
  • C is followed by natural phosphate linkage (PO).
  • G is followed by Sp.
  • G is followed by Rp.
  • G is followed by natural phosphate linkage (PO).
  • C and U are followed by Sp.
  • C and U are followed by Rp.
  • C and U are followed by natural phosphate linkage (PO).
  • a and G are followed by Sp.
  • a and G are followed by Rp.
  • the dsRNA molecule described herein comprises one or more overhang regions and/or capping groups of dsRNA molecule at the 3 ’-end, or 5 ’-end or both ends of a strand.
  • the overhang can be 1-10 nucleotides in length.
  • the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length.
  • the overhang is 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length.
  • the overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.
  • the overhang can form a mismatch with the target sequence or it can be complementary to the gene sequences being targeted or it can be the other sequence.
  • the first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
  • the nucleotides in the overhang region of the dsRNA molecule described herein can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-Fluoro 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl- 5 -methyluridine, 2 ’-O-methoxy ethyladenosine, 2’-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h’GNA, and any combinations thereof.
  • dTdT can be an overhang sequence for either end on either strand.
  • the overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.
  • the 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule described herein may be phosphorylated.
  • the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different.
  • the overhang is present at the 3 ’-end of the sense strand, antisense strand or both strands. In some embodiments, this 3 ’-overhang is present in the antisense strand. In some embodiments, this 3 ’-overhang is present in the sense strand.
  • the dsRNA molecule described herein may comprise only a single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability.
  • the single-stranded overhang is located at the 3 '-terminal end of the sense strand or, alternatively, at the 3'-terminal end of the antisense strand.
  • the dsRNA can also have a blunt end, located at the 5 ’-end of the antisense strand (or the 3 ’-end of the sense strand) or vice versa.
  • the antisense strand of the dsRNA has a nucleotide overhang at the 3 ’-end, and the 5 ’-end is blunt. While not bound by theory, the asymmetric blunt end at the 5 ’-end of the antisense strand and 3 ’-end overhang of the antisense strand favor the guide strand loading into RISC process.
  • the single overhang is at least one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in length.
  • the dsRNA has a 2 nucleotide overhang on the 3 ’-end of the antisense strand and a blunt end at the 5 ’-end of the antisense strand.
  • the dsRNA described herein can comprise one or more modified nucleotides. For example, every nucleotide in the sense strand and antisense strand of the dsRNA molecule can be modified.
  • Each nucleotide can be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar; replacement of the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.
  • nucleic acids are polymers of subunits, many of the modifications occur at aposition which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not.
  • a modification may only occur at a 3’ or 5’ terminal position, may only occur in a central region, may only occur at a non-terminal region, or may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
  • a modification may occur in a double strand region, a single strand region, or in both.
  • a modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA.
  • a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini.
  • the 5’ end or ends can be phosphorylated.
  • Modifications can include, e.g., the use of modifications at the 2’ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2 ’-deoxy-2’ -fluoro (2’-F) or 2’-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.
  • the dsRNA molecule described herein comprises modifications of an alternating pattern, particular in the Bl, B2, B3, Bl’, B2’, B3’, B4’ regions.
  • alternating motif or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand.
  • the alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern.
  • A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “AB AB AB AB AB AB... ,” “AABB AABB AABB ... ,” “AAB AABAAB AAB ... ,” “AAAB AAABAAAB ... ,”
  • the type of modifications contained in the alternating motif may be the same or different.
  • the alternating pattern i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “AB AB AB...”, “AC AC AC...” “BDBDBD...” or “CDCDCD... ,” etc.
  • the dsRNA molecule described herein comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted.
  • the shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa.
  • the sense strand when paired with the antisense strand in the dsRNA duplex the alternating motif in the sense strand may start with “AB AB AB” from 5 ’ -3 ’ of the strand and the alternating motif in the antisense strand may start with “BAB AB A” from 3’-5’of the strand within the duplex region.
  • the alternating motif in the sense strand may start with “AABB AABB” from 5 ’-3’ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3’-5’of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.
  • the oligonucleotides described herein or at least one e.g., both strand of a dsRNA described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'- monophosphate ((HO)2(O)P-O-5'); 5 '-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5 '-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O- 5'); 5 '-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'
  • 5'- alpha-thiotriphosphate, 5 '-gamma-thiotriphosphate, etc.), 5 '-phosphorami dates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (e.g., RP(0H)(0)-0-5'-, R alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonates (i.e.
  • exemplary 5 ’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH 2 )a-P(X)(OH)-O]b- 5', ((HO)2(X)P-[-(CH 2 )a-O-P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics: H0
  • the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-vinylphosphonate group.
  • the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-E-vinyl or at least one (e.g., both) strand of a dsRNA described herein phosphonate group.
  • the oligonucleotide comprises a 5’-Z- vinylphosphonate group.
  • the 5 ’-modification can be placed in the antisense strand of a double- stranded nucleic acid, e.g., dsRNA molecule.
  • the antisense comprises a 5’-E- vinylphosphonate.
  • the antisense strand comprises a 5’-Z- vinylphosphonate group.
  • the 5 ’-terminal nucleotide of the antisense strand comprises a 5’- cyclopropylphosphonate group, that is, a group of the formula or a salt thereof, that is connected to the 4’-C of the 5 ’-end nucleotide.
  • the 5 ’-terminal nucleotide of the antisense strand comprises a group of the formula wherein each R pp is independently hydrogen or a Cl-6alkyl (e.g., methyl) or a salt thereof, connected to the 4’-C of the 5’-terminal nucleotide.
  • the group is of the formula or a salt thereof.
  • the sense strand comprises a 5 ’-morpholino, a 5’- dimethylamino, a 5 ’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5 ’-end.
  • the 5 ’-terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphodiester linkage to an abasic nucleotide.
  • the 5’- terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphorothioate linkage to an abasic nucleotide.
  • the sense strand comprises an inverted abasic acid modification at the 3 ’-end.
  • the 3 ’-terminal nucleotide of the sense strand comprises a 3’- >3’ phosphodiester linkage to an abasic nucleotide.
  • he 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide.
  • the 5 ’-terminal nucleotide of the sense strand comprises a 5’- >5’ phosphodiester linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphodiester linkage to an abasic nucleotide.
  • the 5 ’-terminal nucleotide of the sense strand comprises a 5’- >5’ phosphorothioate linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphodiester linkage to an abasic nucleotide.
  • the 5 ’-terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphodiester linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide.
  • the 5 ’-terminal nucleotide of the sense strand comprises a 5’- >5’ phosphorothioate linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide.
  • the abasic nucleotide may be optionally substituted, for example, by any of the modifications or ligand described herein.
  • the dsRNA agents of the invention can comprise thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2- 9 of the 5 ’-end of the antisense strand or positions 2-9 counting from the first paired nucleotide)of the duplex region at the 5 ’-end of the antisense strand) to reduce or inhibit off-target gene silencing.
  • dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5’ end, of the antisense strand have reduced off-target gene silencing activity.
  • the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5’ region of the antisense strand.
  • thermally destabilizing modification of the duplex is located in positions 2-9, or preferably positions 4-8, from the 5 ’-end of the antisense strand.
  • the thermally destabilizing modification of the duplex is located at position 5, 6, 7 or 8 from the 5’-end of the antisense strand. [00388] In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5 ’-end of the antisense strand.
  • the dsRNA can also comprise one or more stabilizing modifications.
  • the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • the stabilizing modifications all can be present in one strand.
  • both the sense and the antisense strands comprise at least two stabilizing modifications.
  • the stabilizing modification can occur on any nucleotide of the sense strand or antisense strand.
  • the stabilizing modification can occur on every nucleotide on the sense strand and/or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern.
  • the alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.
  • the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the antisense strand can be present at any positions.
  • the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5 ’-end.
  • the antisense comprises stabilizing modifications at positions 2, 6, 14 and 16 from the 5’-end.
  • the antisense comprises stabilizing modifications at positions 2, 14 and 16 from the 5 ’-end.
  • the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.
  • the stabilizing modification can be the nucleotide at the 5 ’-end or the 3 ’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification.
  • the antisense strand comprises a stabilizing modification at each of the 5’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the antisense strand comprises at least two stabilizing modifications at the 3 ’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the sense strand can be present at any positions.
  • the sense strand comprises stabilizing modifications at positions 7, 10 and 11 from the 5 ’-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10 and 11 from the 5 ’-end.
  • the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four stabilizing modifications.
  • the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.
  • a thermally stabilizing modification can replace a 2’-fluoro nucleotide in the sense and/or antisense strand.
  • a 2’-fluoro nucleotide at positions 8, 9, 10, 11 and/or 12, counting from 5 ’-end, of the sense strand can be replaced with a thermally stabilizing modification.
  • a 2’-fluoro nucleotide at position 14, counting from 5’-end, of the antisense strand can be replaced with a thermally stabilizing modification.
  • the antisense strand must have some metabolic stability. In other words, for the dsRNA molecules to be more effective in vivo, some amount of the antisense stand may need to be present in vivo after a period time after administration. Accordingly, in some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 5 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 6 after in vivo administration.
  • at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 7 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 8 after in vivo administration.
  • at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 9 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 10 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 11 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 12 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 13 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 14 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 15 after in vivo administration.
  • the oligonucleotide described herein or the antisense strand of the dsRNA molecule described herein comprises a nucleotide sequence substantially complementary to a target nucleic acid, e.g., a target gene or mRNA.
  • the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene.
  • the present invention further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in vitro.
  • the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for use in inhibiting expression of a target gene in a subject.
  • the subject may be any animal, such as a mammal, e.g., a mouse, a rat, a sheep, a cattle, a dog, a cat, or a human [00399]
  • the oligonucleotide and/or dsRNA molecule described herein is administered in buffer.
  • oligonucleotide and/or dsRNA molecule described herein described herein can be formulated for administration to a subject.
  • a formulated oligonucleotide and/or dsRNA composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water).
  • the siRNA is in an aqueous phase, e.g., in a solution that includes water.
  • the aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • a delivery vehicle e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein.
  • the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
  • a oligonucleotide and/or dsRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and/or dsRNA, e.g., a protein that complexes with oligonucleotide and/or dsRNA.
  • another agent e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and/or dsRNA, e.g., a protein that complexes with oligonucleotide and/or dsRNA.
  • Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg 2+ ), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
  • the oligonucleotide and/or dsRNA preparation includes another dsRNA compound, e.g., a second dsRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene.
  • another dsRNA compound e.g., a second dsRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene.
  • Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species.
  • Such dsRNAs can mediate RNAi with respect to a similar number of different genes.
  • the oligonucleotide and/or dsRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA).
  • a second therapeutic agent e.g., an agent other than a RNA or a DNA.
  • a oligonucleotide and/or dsRNA composition for the treatment of a viral disease e.g., HIV
  • a known antiviral agent e.g., a protease inhibitor or reverse transcriptase inhibitor
  • a dsRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.
  • oligonucleotide and/or dsRNA preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle.
  • liposome refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers.
  • Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior.
  • the aqueous portion contains the oligonucleotide and/or dsRNA composition.
  • the lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the oligonucleotide and/or dsRNA composition, although in some examples, it may.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes.
  • the internal aqueous contents that include the oligonucleotide and/or dsRNA are delivered into the cell where the dsRNA can specifically bind to a target RNA and can mediate RNAi.
  • the liposomes are also specifically targeted, e.g., to direct the oligonucleotide and/or dsRNA to particular cell types.
  • a liposome containing oligonucleotide and/or dsRNA can be prepared by a variety of methods.
  • the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component.
  • the lipid component can be an amphipathic cationic lipid or lipid conjugate.
  • the detergent can have a high critical micelle concentration and may be nonionic.
  • Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine.
  • the dsRNA preparation is then added to the micelles that include the lipid component.
  • the cationic groups on the lipid interact with the siRNA and condense around the dsRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of oligonucleotide and/or dsRNA.
  • a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition.
  • the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.
  • Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413- 7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, etal. Biochim. Biophys.
  • Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta T15A69, 1984, which is incorporated by reference in its entirety). These methods are readily adapted to packaging oligonucleotide and/or dsRNA preparations into liposomes.
  • Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated by reference in its entirety).
  • liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Examples of other methods to introduce liposomes into cells in vitro include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90: 11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.
  • cationic liposomes are used.
  • Cationic liposomes possess the advantage of being able to fuse to the cell membrane.
  • Non-cationic liposomes although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver siRNAs to macrophages.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated siRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • a positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of siRNA (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA, which are incorporated by reference in their entirety).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride
  • a DOTMA analogue, l,2-bis(oleoyloxy)-3-(trimethylammonia)propane can be used in combination with a phospholipid to form DNA-complexing vesicles.
  • LipofectinTM Bethesda Research Laboratories, Gaithersburg, Md. is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive.
  • DOTAP cationic lipid, l,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane
  • cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5 -carboxy spermylgly cine dioctaoleoylamide (“DOGS”) (TransfectamTM, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5 -carboxy spermyl-ami de (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
  • DOGS 5 -carboxy spermylgly cine dioctaoleoylamide
  • DPES dipalmitoylphosphatidylethanolamine 5 -carboxy spermyl-ami de
  • Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating poly lysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. etal., Biochim. Biophys. Acta 1065:8, 1991, which is incorporated by reference in its entirety).
  • these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions.
  • Other commercially available cationic lipid products include DMRIE and DMRIE- HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland).
  • DOSPA Lipofectamine
  • Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
  • Liposomes are particularly suited for topical administration. Liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer siRNA, into the skin.
  • liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal tissues, e.g., into skin. Lor example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/ cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin.
  • Such formulations with dsRNA descreibed herein are useful for treating a dermatological disorder.
  • Liposomes that include oligonucleotide and/or dsRNA described herein can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome.
  • transfersomes are a type of deformable liposomes. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include oligonucleotide and/or dsRNA described herein can be delivered, for example, subcutaneously by infection in order to deliver dsRNA to keratinocytes in the skin.
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self- repairing, and can frequently reach their targets without fragmenting, and often self-loading. [00422] Other formulations amenable to the present invention are described in United States provisional application serial nos.
  • the oligonucleotide and/or dsRNA compositions can include a surfactant.
  • the dsRNA is formulated as an emulsion that includes a surfactant.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric.
  • Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • the use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, NY, 1988, p. 285).
  • Micelles and other Membranous Formulations are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
  • a mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the oligonucleotide and/or dsRNA composition, an alkali metal Cs to C22 alkyl sulphate, and a micelle forming compounds.
  • Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyl oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.
  • the micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to
  • a first micellar composition which contains the oligonucleotide and/or dsRNA composition and at least the alkali metal alkyl sulphate.
  • the first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition.
  • the micellar composition is prepared by mixing the dsRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
  • Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth.
  • phenol and/or m-cresol may be added with the micelle forming ingredients.
  • An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
  • micellar formulation For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant.
  • the propellant which is under pressure, is in liquid form in the dispenser.
  • the ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve.
  • the dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
  • Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether.
  • HFA 134a (1,1, 1,2 tetrafluoroethane) may be used.
  • the specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
  • dsRNA preparations can be incorporated into a particle, e.g., a microparticle.
  • Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.
  • the oligonucleotide and/or dsRNA described herein can be formulated for pharmaceutical use.
  • the present invention further relates to a pharmaceutical composition comprising the oligonucleotide and/or dsRNA described herein.
  • Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the dsRNA molecules in any of the preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.
  • terapéuticaally-effective amount means that amount of a compound, material, or composition comprising a dsRNA molecule described herein which is
  • I l l effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate;
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety -nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention.
  • an aforementioned formulation renders orally bioavailable a compound of the present invention.
  • Methods of preparing these formulations or compositions include the step of bringing into association an oligonucleotide and/or dsRNA with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • oligonucleotide and/or dsRNA described herein may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
  • treatment is intended to encompass therapy and cure.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the oligonucleotide and/or dsRNA described herein or a pharmaceutical composition comprising an oligonucleotide and/or dsRNA described herein can be administered to a subject using different routes of delivery.
  • a composition that includes an oligonucleotide and/or dsRNA described herein described herein can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
  • the oligonucleotide and/or dsRNA described herein may 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, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. [00450] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice.
  • Lung cells might be targeted by administering the oligonucleotide and/or dsRNA described herein in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the oligonucleotide and/or dsRNA described herein and mechanically introducing the oligonucleotide and/or dsRNA described herein.
  • a method of administering an oligonucleotide and/or dsRNA described herein, to a subject e.g., a human subject.
  • the present invention relates to an oligonucleotide and/or dsRNA described herein for use in inhibiting expression of a target gene in a subject.
  • the method or the medical use includes administering a unit dose of the oligonucleotide and/or dsRNA described herein.
  • the unit dose is less than 10 mg per kg of body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of body weight, and less than 200 nmole of RNA agent (e.g., about 4.4 x 10 16 copies) per kg of body weight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of oligonucleotide and/or dsRNA described herein per kg of body weight.
  • RNA agent e.g., about 4.4 x 10 16 copies
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target gene.
  • the unit dose for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application.
  • dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight.
  • the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days.
  • the unit dose is not administered with a frequency (e.g., not a regular frequency).
  • the unit dose may be administered a single time.
  • the effective dose is administered with other traditional therapeutic modalities.
  • a subject is administered an initial dose and one or more maintenance doses.
  • the maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose.
  • a maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 pg to 15 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day.
  • the maintenance doses are, for example, administered no more than once every 2, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
  • the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days.
  • the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracistemal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracistemal or intracapsular), or reservoir may be advisable.
  • the composition includes a plurality of dsRNA molecule species.
  • the dsRNA molecule species has sequences that are non- overlapping and non-adjacent to another species with respect to a naturally occurring target sequence.
  • the plurality of dsRNA molecule species is specific for different naturally occurring target genes.
  • the dsRNA molecule is allele specific.
  • the administration of the oligonucleotide and/or dsRNA composition described herein is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • the invention provides methods, compositions, and kits, for rectal administration or delivery of oligonucleotide and/or dsRNA composition described herein.
  • aspects of the disclosure also relate to methods for inhibiting the expression of a target gene.
  • the method comprises administering to the subject in an amount sufficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the first strand is complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.
  • the present disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell.
  • the present disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell in vitro.
  • the invention relates to a method of modulating the expression of a target gene in a cell, comprising administering to said cell an oligonucleotide and/or dsRNA molecule described herein.
  • the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu
  • Embodiment 1 An oligonucleotide comprising at least one nucleoside (e.g., one) of
  • Formula (IV) Formula (IV) , w h erein: jy j s an optionally substituted nucleobase; X M is CEE,
  • R N is aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars);
  • R 43 is a bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alky
  • Embodiment 2 The oligonucleotide of Embodiment 1, wherein R 43 is bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, or a nitrogen protecting group.
  • Embodiment 3 The oligonucleotide of Embodiment 2, wherein R 43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
  • Embodiment 4 The oligonucleotide of Embodiment 2, wherein R 43 is a solid support, or a linker (e.g., -C(O)CH2CH2C(O)-) covalently bonded to a solid support.
  • R 43 is a solid support, or a linker (e.g., -C(O)CH2CH2C(O)-) covalently bonded to a solid support.
  • Embodiment 5 The oligonucleotide of Embodiment 2, wherein R 43 is either (i) hydrogen or a nitrogen protecting group; or (ii) hydroxyl or a protected hydroxyl.
  • Embodiment 6 The oligonucleotide of any one of Embodiments 1-5, wherein R 45 is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, phosphate mimic, or a bond to an intemucleotide linkage to a preceding nucleotide, or R 45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
  • VP vinylphosphonate
  • Embodiment 7 The oligonucleotide of Embodiment 6, wherein R 45 is a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded a solid support, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate, or R 45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
  • VP vinylphosphonate
  • Embodiment 8 The oligonucleotide of Embodiment 7, wherein R 45 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • Embodiment 9 The oligonucleotide of Embodiment 7, wherein R 45 is hydroxylprotected hydroxyl, or optionally substituted C1-30 alkoxy, or R 45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) (E-vinylphosphonate) group.
  • VP vinylphosphonate
  • Embodiment 10a The oligonucleotide of any one of Embodiments 1-9, wherein X M is CEE.
  • Embodiment 10b The oligonucleotide of any one of Embodiments 1-9, wherein X M is O.
  • Embodiment 10c The oligonucleotide of any one of Embodiments 1-9, wherein X M is S.
  • Embodiment 11 The oligonucleotide of any one of Embodiments l-10c, wherein B’ is unmodified nucleobase (e.g., adenine, cytosine, guanine, thymine or uracil), a pyrimidine modified at the C4 position, a pyrimidine modified at the C5 position, a purine modified at the N2 position, a purine modified at the N6 position, a purine modified at the C6 position or a N-7 deaza purine, optionally modified at the N7 position.
  • B’ is unmodified nucleobase (e.g., adenine, cytosine, guanine, thymine or uracil), a pyrimidine modified at the C4 position, a pyrimidine modified at the C5 position, a purine modified at the N2 position, a purine modified at the N6 position, a purine modified at the C6 position or a N-7 de
  • Embodiment 12 The oligonucleotide of any one of Embodiments 1-11, wherein the oligonucleotide comprises from 3 to 50 nucleotides.
  • Embodiment 13 The oligonucleotide of any one of Embodiments 1-12, wherein the oligonucleotide comprises at least one ribonucleotide.
  • Embodiment 14 The oligonucleotide of any one of Embodiments 1-13, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.
  • Embodiment 15 The oligonucleotide of any one of Embodiments 1-14, wherein the oligonucleotide comprises at least one nucleotide with a modified or non-natural nucleobase in addition to the nucleotide of Formula (IV).
  • Embodiment 16 The oligonucleotide of any one of Embodiments 1-15, wherein the oligonucleotide comprises at least one nucleotide with a modified ribose sugar in addition to the nucleotide of Formula (IV).
  • Embodiment 17 The oligonucleotide of any one of Embodiments 1-16, wherein the oligonucleotide comprises at least one nucleotide comprising a group other than H or OH at the 2’- position of the ribose sugar in addition to the nucleotide of Formula (IV).
  • Embodiment 18 The oligonucleotide of any one of Embodiments 1-17, wherein the oligonucleotide comprises at least one nucleotide with a 2’-F ribose in addition to the nucleotide of Formula (IV).
  • Embodiment 19 The oligonucleotide of any one of Embodiments 1-18, wherein the oligonucleotide comprises at least one nucleotide with a 2’-OMe ribose in addition to the nucleotide of Formula (IV).
  • Embodiment 20 The oligonucleotide of any one of Embodiments 1-19, wherein the oligonucleotide comprises at least one nucleotide comprising a moiety other than a ribose sugar in addition to the nucleotide of Formula (IV).
  • Embodiment 21 The oligonucleotide of any one of Embodiments 1-20, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.
  • Embodiment 22 The oligonucleotide of any one of Embodiments 1-21, wherein the oligonucleotide is attached to a solid support.
  • Embodiment 23 The oligonucleotide of any one of Embodiments 1-22, wherein oligonucleotide comprises at least one ligand.
  • Embodiment 24 The oligonucleotide of any one of Embodiments 1-23, wherein the oligonucleotide comprises at least one hydroxyl, phosphate or amino protecting group.
  • Embodiment 25 The oligonucleotide of any one of Embodiments 1-24, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide.
  • Embodiment 26 The oligonucleotide of any one of Embodiments 1-25, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, and wherein the nucleoside of Formule (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphodiester intemucleotide linkage.
  • Embodiment 27 The oligonucleotide of any one of Embodiments 1-25, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, and wherein the nucleoside of Formule (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphorothioate intemucleotide linkage.
  • Embodiment 28 A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein the first or second strand is an oligonucleotide of any one of Embodiments 1-27.
  • Embodiment 29 The double-stranded nucleic acid of Embodiment 28, wherein the first and second strand are independently 15 to 25 nucleotides in length.
  • Embodiment 30 The double-stranded nucleic acid any one of Embodiments 28-29, wherein double-stranded nucleic acid is capable of inducing RNA interference.
  • Embodiment 31 The double-stranded nucleic acid of any one of Embodiments 28-30, wherein one or both strands have a 1 - 5 nucleotide overhang on its respective 5 ’-end or 3 ’-end.
  • Embodiment 32 The double-stranded nucleic acid of any one of Embodiments 28-31, wherein only one strand has a 2 nucleotide overhang on its 5 ’-end or 3 ’-end.
  • Embodiment 33 The double-stranded nucleic acid of any one of Embodiments 28-32, wherein only one strand has a 2 nucleotide overhand on its 3 ’-end.
  • Embodiment 34 A method of reducing the expression of a target gene in a subject, comprising administering to the subject either: (i) a double-stranded RNA according to any one of Embodiments 28-33 or 63-76, wherein the first strand or the second strand is complementary to a target gene; or (ii) an oligonucleotide according to any one of Embodiments 1-27 or 57-62, wherein the oligonucleotide is complementary to a target gene.
  • Embodiment 35 A compound of Formula (III): Formula (III) , w h erein: jy j s an optionally substituted nucleobase;
  • X M is CEE, O, NR N or S (where R N is aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars);
  • R 33 is hydrogen, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O
  • Embodiment 36 The compound of Embodiment 35, wherein R 33 is H, a linker, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, or a nitrogen protecting group.
  • Embodiment 37 The compound of Embodiment 35, wherein R 33 is a H or nitrogen protecting group.
  • Embodiment 38 The compound of any one of Embodiments 35-37, wherein R 35 is a reactive phosphorous group, linker, solid support, a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or 5’-O- C(O)CH2CH2C(O)-) to a solid support, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate or phosphate mimic or R 35 taken together with the carbon to which it is attached form avinylphosphonate group.
  • R 35 is a reactive phosphorous group,
  • Embodiment 39 The compound of any one of Embodiments 35-28, wherein R 35 is a reactive phosphorous group, linker, solid support, a linker covalently bonded to a solid support, hydroxyl, or a protected hydroxyl.
  • Embodiment 40 The compound of any one of Embodiments 35-39, wherein R 35 is reactive phosphorous group, solid support, or a linker covalently bonded to a solid support.
  • Embodiment 41 The compound of any one of Embodiments 35-40, wherein R 35 is - P(X D )(N(R P2 )2)-R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); and R P4 is halogen (e.g., Cl)
  • Embodiment 42 The compound of any one of Embodiments 35-41, wherein X M is CEE.
  • Embodiment 43a The compound of any one of Embodiments 35-41, wherein X M is O.
  • Embodiment 43b The compound of any one of Embodiments 35-41, wherein X M is S.
  • Embodiment 44 The compound of Embodiment 35, wherein: X M is CEE; R 33 is H or nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., - P(X D )(N(R P2 )2)-R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); andR P4 is halogen (e.g., Cl)), solid support, a linker covalently bonded (e.g., -C(O)CEECEEC(O)-) to a solid support, hydroxyl, or a protected hydroxyl.
  • X M is CEE
  • R 33 is H or nitrogen protecting group (e.g., trityl)
  • R 35 is a reactive phosphorous group (e.g., - P(X D )(N(R P2 )2)-R
  • Embodiment 45 The compound of Embodiment 35, wherein:X M is O; R 33 is H or nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., - P(X D )(N(R P2 )2)-R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); and R P4 is halogen (e.g., Cl)), solid support a linker covalently bonded (e.g., -C(O)CEECEEC(O)-) to a solid support, hydroxyl, or a protected hydroxyl.
  • X M is O
  • R 33 is H or nitrogen protecting group (e.g., trityl)
  • R 35 is a reactive phosphorous group (e.g., - P(X D )(N(R P2 )2)-R P4 ,
  • Embodiment 46 The compound of Embodiment 35, wherein:X M is S; R 33 is H or nitrogen protecting group (e.g., trityl); and R 35 is a reactive phosphorous group (e.g., - P(X D )(N(R P2 )2)-R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); and R P4 is halogen (e.g., Cl)), solid support a linker covalently bonded (e.g., -C(O)CH2CH2C(O)-) to a solid support, hydroxyl, or a protected hydroxyl.
  • X M is S
  • R 33 is H or nitrogen protecting group (e.g., trityl)
  • R 35 is a reactive phosphorous group (e.g., - P(X D )(N(R P2 )2)-R P4
  • Embodiment 47 The compound of Embodiment 35, wherein: X M is CEE; R 35 is hydroxyl or a protected hydroxyl; and R 33 is a reactive phosphorous group (e.g., -P(X D )(N(R P2 )2)- R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and R P4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • X M is CEE
  • R 35 is hydroxyl or a protected hydroxyl
  • R 33 is a reactive phosphorous group (e.g., -P(X D )(N(R P2 )2)- R P4 , where
  • Embodiment 48 The compound of Embodiment 47, wherein X M is CEE; R 35 is hydroxyl or a protected hydroxyl; and R 33 is a solid support or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • X M is CEE
  • R 35 is hydroxyl or a protected hydroxyl
  • R 33 is a solid support or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • Embodiment 49 The compound of Embodiment 35, wherein: X M is O; R 35 is hydroxyl or protected hydroxyl; and R 33 is a reactive phosphorous group (e.g., -P(X D )(N(R P2 )2)-R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); andR P4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., -C(O)CEECEEC(O)- or - OC(O)CEECEEC(O)-) to a solid support.
  • X M is O
  • R 35 is hydroxyl or protected hydroxyl
  • R 33 is a reactive phosphorous group (e.g., -P(X D )(N(R P2 )2)-R P4 , where X D is O or S;
  • Embodiment 50 The compound of Embodiment 49, wherein: X M is O; R 35 is hydroxyl or protected hydroxyl; and R 33 is a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • X M is O
  • R 35 is hydroxyl or protected hydroxyl
  • R 33 is a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • Embodiment 51 The compound of Embodiment 35, wherein: X M is S; R 35 is hydroxyl or protected hydroxyl; and R 33 is a reactive phosphorous group (e.g., -P(X D )(N(R P2 )2)-R P4 , where X D is O or S; each R P2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); andR P4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., -C(O)CEECEEC(O)- or - OC(O)CEECEEC(O)-) to a solid support.
  • X M is S
  • R 35 is hydroxyl or protected hydroxyl
  • R 33 is a reactive phosphorous group (e.g., -P(X D )(N(R P2 )2)-R P4 , where X D is O or S;
  • Embodiment 52 The compound of Embodiment 51, wherein: X M is O; R 35 is hydroxyl or protected hydroxyl; and R 33 is a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • X M is O
  • R 35 is hydroxyl or protected hydroxyl
  • R 33 is a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)- or -OC(O)CH 2 CH 2 C(O)-) to a solid support.
  • Embodiment 53 The compound of any one of Embodiments 35-52, B’ is an unmodified nucleobase (e.g., adenine, cytosine, guanine, thymine or uracil), a pyrimidine modified at the C4 position, a pyrimidine modified at the C5 position, a purine modified at the N2 position, a purine modified at the N6 position, a purine modified at the C6 position or a N-7 deaza purine, optionally modified at the N7 position.
  • an unmodified nucleobase e.g., adenine, cytosine, guanine, thymine or uracil
  • a pyrimidine modified at the C4 position e.g., adenine, cytosine, guanine, thymine or uracil
  • a pyrimidine modified at the C4 position e.g., adenine, cytosine, gu
  • Embodiment 54 The compound of any one of Embodiments 35-53, wherein B and B’ are independently adenine, cytosine, guanine, thymine, uracil, selected from 1 to 10; and R 1 is independently liphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars.
  • Embodiment 55 A compound selected from the group consisting of:
  • Embodiment 56 An oligonucleotide prepared using a compound of any one of
  • Embodiment 57 The oligonucleotide of any one of Embodiments 1-27, wherein the nucleotide of Formula (IV) is at one of positions 2-9, counting from the 5 ’end of the oligonucleotide.
  • Embodiment 58 The oligonucleotide of Embodiment 57, wherein the nucleotide of Formula (IV) is at one of positions 2-8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5’end of the oligonucleotide.
  • Embodiment 59 The oligonucleotide of Embodiment 57, wherein the nucleotide of Formula (IV) is at one of position 5, counting from the 5’end of the oligonucleotide.
  • Embodiment 60 The oligonucleotide of Embodiment 57, wherein the nucleotide of Formula (IV) is at one of position 6, counting from the 5’end of the oligonucleotide.
  • Embodiment 61 The oligonucleotide of Embodiment 57, wherein the nucleotide of Formula (IV) is at one of position 7, counting from the 5’end of the oligonucleotide.
  • Embodiment 62 The oligonucleotide of Embodiment 57, wherein the nucleotide of Formula (IV) is at one of position 8, counting from the 5’end of the oligonucleotide.
  • Embodiment 63 The double-stranded nucleic acid of any one of Embodiments 28-32, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the sense strand.
  • Embodiment 64 The double-stranded nucleic acid of Embodiment 63, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions 2-9 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • Embodiment 65 The double-stranded nucleic acid of Embodiment 64, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions 2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or one of positions 6-8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions 2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or
  • Embodiment 66 The double-stranded nucleic acid of Embodiment 64, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 5 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • Embodiment 67 The double-stranded nucleic acid of Embodiment 64, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 6 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • Embodiment 68 The double-stranded nucleic acid of Embodiment 64, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 7 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • Embodiment 69 The double-stranded nucleic acid of Embodiment 64, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • Embodiment 70 The double-stranded nucleic acid of any one of Embodiments 28-32, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the antisense strand.
  • Embodiment 71 The double-stranded nucleic acid of Embodiment 70, wherein the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-9, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • Embodiment 72 The double-stranded nucleic acid of Embodiment 71, wherein the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-8, at one of positions 2- 7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).
  • the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-8, at one of positions 2- 7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5’end of the antisense strand (or counting from the first paired nucle
  • Embodiment 73 The double-stranded nucleic acid of Embodiment 71, wherein the nucleotide of Formula (IV) is in the antisense strand at position 5, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand.
  • Embodiment 74 The double-stranded nucleic acid of Embodiment 71, wherein the nucleotide of Formula (IV) is in the antisense strand at position 6, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand.
  • Embodiment 75 The double-stranded nucleic acid of Embodiment 71, wherein the nucleotide of Formula (IV) is in the antisense strand at position 7, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand.
  • Embodiment 76 The double-stranded nucleic acid of Embodiment 71, wherein the nucleotide of Formula (IV) is in the antisense strand at position 8, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand.
  • the practice of the present invention can employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.
  • alkyl refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms.
  • alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl.
  • “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl.
  • Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.
  • a “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH2 group to an NH group or an O group).
  • the term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof.
  • the heteroatom(s) is placed at any interior position of the heteroalkyl group.
  • up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3
  • alkenyl refers to an alkyl group containing at least one carbon-carbon double bond.
  • the alkenyl group can be optionally substituted with one or more “alkyl group substituents.”
  • Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-l-yl and heptadec-8,l l-dien-l-yl.
  • alkynyl refers to an alkyl group containing a carbon-carbon triple bond.
  • the alkynyl group can be optionally substituted with one or more “alkyl group substituents.”
  • exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl.
  • Useful alkynyl groups include the lower alkynyl groups.
  • cycloalkyl refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and/or alkylene.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl.
  • Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • Heterocyclyl refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively).
  • Cxheterocyclyl and C x -C y heterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system.
  • 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent.
  • exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4- morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1 ,4-diazaperhydroepinyl, 1,3- dioxanyl, 1 ,4-dioxanyland the like.
  • Aryl refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms.
  • the aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and — NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl.
  • Heteroaryl refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • O, N, or S e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzisothi
  • halogen refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen radioisotope or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen-substituted moiety or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.
  • haloalkyl refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different.
  • fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.
  • Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g.
  • halosubstituted (Ci-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).
  • amino means -NHz.
  • alkylamino means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl).
  • dialkylamino means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -N(alkyl)(alkyl).
  • alkylamino includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.”
  • arylamino means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and — N(aryl)z.
  • heteroarylamino means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example — NHheteroaryl, and — N(heteroaryl)2.
  • two substituents together with the nitrogen can also form a ring.
  • the compounds described herein containing amino moieties can include protected derivatives thereof.
  • Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like.
  • Exemplary alkylamino includes, but is not limited to, NH(Ci- Cioalkyl), such as — NHCH3, — NHCH2CH3, — NHCH2CH2CH3, and — NHCH(CH 3 ) 2 .
  • Exemplary dialkylamino includes, but is not limited to, — N(Ci-Cioalkyl)2, such as N(CH 3 )2, — N(CH 2 CH3) 2 , — N(CH 2 CH 2 CH3)2, and — N(CH(CH 3 ) 2 ) 2 .
  • aminoalkyl means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms ( — N — ) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl.
  • an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.
  • hydroxyl and “hydroxyl” mean the radical — OH.
  • alkoxy!” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O- alkenyl, and -O-alkynyl.
  • Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
  • the alkoxy and aroxy groups can be substituted as described above for alkyl.
  • Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n- propyl, O-isopropyl, O-w-butyl, O-isobutyl, O-sec-butyl, O-/c/7-butyl, O-pentyl, O- hexyl, O- cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.
  • carbonyl means the radical — C(O) — . It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.
  • carboxy means the radical — C(O)O — . It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., carboxyl group.
  • cyano means the radical — CN.
  • nitro means the radical — NO2.
  • heteroatom refers to an atom that is not a carbon atom.
  • heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens.
  • alkylthio and thioalkoxy refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur.
  • the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • alkylthio also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups.
  • sulfinyl means the radical — SO — . It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfmic acids, sulfmamides, sulfinyl esters, sulfoxides, and the like.
  • sulfonyl means the radical — SO2 — . It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.
  • thiocarbonyl means the radical — C(S) — . It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.
  • acyl refers to an alkyl-CO — group, wherein alkyl is as previously described.
  • exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms.
  • Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
  • Aroyl means an aryl-CO — group, wherein aryl is as previously described.
  • Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.
  • Arylthio refers to an aryl-S — group, wherein the aryl group is as previously described.
  • exemplary arylthio groups include phenylthio and naphthylthio.
  • Aralkyl refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described.
  • Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
  • Aralkyloxy refers to an aralkyl-0 — group, wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxy group is benzyloxy.
  • Aralkylthio refers to an aralkyl-S — group, wherein the aralkyl group is as previously described.
  • An exemplary aralkylthio group is benzylthio.
  • Alkoxycarbonyl refers to an alkyl-0 — CO — group.
  • exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-0 — CO — group.
  • exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-0 — CO — group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an H2N — CO — group.
  • Alkylcarbamoyl refers to a R'RN — CO — group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.
  • Dialkylcarbamoyl refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.
  • “Acyloxy” refers to an acyl-0 — group, wherein acyl is as previously described.
  • “Acylamino” refers to an acyl-NH — group, wherein acyl is as previously described.
  • “Aroylamino” refers to an aroyl-NH — group, wherein aroyl is as previously described.
  • substituted means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified.
  • substituted refers to a group “substituted” on a substituted group at any atom of the substituted group.
  • Suitable substituents include, without limitation, halogen, hydroxyl, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxylalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.
  • two substituents, together with the carbons to which they are attached to can form a ring.
  • an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.
  • An “isocyanato” group refers to a NCO group.
  • a “thiocyanato” group refers to a CNS group.
  • An “isothiocyanate” group refers to a NCS group.
  • RNA e.g., mRNA
  • mRNA e.g., a transcript of a gene that encodes a protein
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein
  • target gene e.g., a target gene
  • RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNAs, and viral RNAs, can also be targeted.
  • RNAi refers to the ability to silence, in a sequence specific manner, a target gene, e.g., mRNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
  • nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary or 100% complementarity means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • nucleoside units of two strands can hydrogen bond with each other.
  • Substantial complementarity refers to polynucleotide strands exhibiting 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be noncomplementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
  • the non-target sequences typically differ by at least 5 nucleotides.
  • the term “off-target” and the phrase “off-target effects” refer to any instance in which an effector molecule against a given target causes an unintended affect by interacting either directly or indirectly with another target sequence, a DNA sequence or a cellular protein or other moiety.
  • an “off-target effect” may occur when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of an siRNA.
  • nucleoside means a glycosylamine comprising a nucleobase and a sugar. Nucleosides includes, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups.
  • nucleotide refers to a glycosomine comprising a nucleobase and a sugar having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.
  • locked nucleic acid or “LNA” or “locked nucleoside” or “locked nucleotide” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.
  • Locked nucleic acids are also referred to as bicyclic nucleic acids (BNA).
  • methyleneoxy LNA alone refers to P-D-methyleneoxy LNA.
  • MOE refers to a 2'-O-methoxyethyl substituent.
  • the term “gapmer” refers to a chimeric oligomeric compound comprising a central region (a “gap”) and a region on either side of the central region (the “wings”), wherein the gap comprises at least one modification that is different from that of each wing.
  • modifications include nucleobase, monomeric linkage, and sugar modifications as well as the absence of modification (unmodified).
  • the nucleotide linkages in each of the wings are different than the nucleotide linkages in the gap.
  • each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification.
  • nucleotides in the gap and the nucleotides in the wings all comprise high affinity modifications, but the high affinity modifications in the gap are different than the high affinity modifications in the wings.
  • the modifications in the wings are the same as one another. In certain embodiments, the modifications in the wings are different from each other.
  • nucleotides in the gap are unmodified and nucleotides in the wings are modified.
  • the modification(s) in each wing are the same.
  • the modification(s) in one wing are different from the modification(s) in the other wing.
  • oligomeric compounds are gapmers having 2'-deoxynucleotides in the gap and nucleotides with high-affinity modifications in the wing.
  • BNA refers to bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
  • BNA can contain a 5-, 6- membered, or even a 7-membered bridged structure with a “fixed” Cs’-endo sugar puckering.
  • the bridge is typically incorporated at the 2’-, 4 ’-position of the ribose to afford a 2’, 4’-BNA nucleotide (e.g., LNA, or ENA).
  • BNA nucleotides include the following nucleosides: oxyamino BNA vinyl-carbo BNA
  • LNA refers to locked nucleic acid, and is often referred as constrained or inaccessible RNA.
  • LNA is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge (e.g., a methylene bridge or an ethylene bridge) connecting the 2' hydroxyl to the 4' carbon of the same ribose sugar. Lor instance, the bridge can “lock” the ribose in the 3'-endo North) conformation:
  • ENA refers to ethylene-bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
  • the “cleavage site” herein means the backbone linkage in the target gene or the sense strand that is cleaved by the RISC mechanism by utilizing the iRNA agent.
  • the target cleavage site region comprises at least one or at least two nucleotides on both side of the cleavage site.
  • the cleavage site is the backbone linkage in the sense strand that would get cleaved if the sense strand itself was the target to be cleaved by the RNAi mechanism.
  • the cleavage site can be determined using methods known in the art, for example the 5 ’-RACE assay as detailed in Soutschek et al., Nature (2004) 432, 173-178, which is incorporated by reference in its entirety.
  • the cleavage site region for a conical double stranded RNAi agent comprising two 21 -nucleotides long strands (wherein the strands form a double stranded region of 19 consecutive base pairs having 2-nucleotide single stranded overhangs at the 3 ’-ends)
  • the cleavage site region corresponds to positions 9-12 from the 5 ’-end of the sense strand.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • a “terminal region” of a strand refers to positions 1-4, e.g., positions 1, 2, 3, and 4, counting from the nearest end of the strand.
  • a 5 ’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 5’-end of the strand.
  • a 3 ’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 3 ’-end of the strand.
  • a 5 ’-terminal region for the antisense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the antisense strand.
  • a preferred 5 ’-terminal region for the antisense strand is positions 1 , 2 and 3 counting from the 5 ’ -end of the antisense strand.
  • a 3 ’ -terminal region for the antisense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand.
  • a preferred 3 ’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 3’- end of the antisense strand.
  • a 5 ’-terminal region for the sense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the sense strand.
  • a preferred 5 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 5 ’-end of the sense strand.
  • a 3 ’-terminal region for the sense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand.
  • a preferred 3 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 3 ’-end of the sense strand.
  • a “central region” of a strand refers to positions 5-17, e.g., positions 6- 16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5’-end of the strand.
  • the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand.
  • a preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5 ’-end of the sense strand.
  • a more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5 ’-end of the sense strand.
  • a preferred central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 15 16 and 17, counting from 5 ’-end of the antisense strand.
  • a more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5’- end of the antisense strand.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g. animal or a plant).
  • ex vivo refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).
  • in vivo refers to events that occur within an organism (e.g. animal, plant, and/or microbe).
  • the term "subject" or "patient” refers to any organism to which a composition disclosed herein can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • a subject can be male or female.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders.
  • compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.
  • the subject is human.
  • the subject is an experimental animal or animal substitute as a disease model.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice.
  • the term subject is further intended to include transgenic species.
  • the subject can be of European ancestry.
  • the subject can be of African American ancestry.
  • the subject can be of Asian ancestry.
  • parenteral administration refers to administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • subcutaneous administration refers to administration just below the skin.
  • Intravenous administration means administration into a vein.
  • a dose refers to a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • a dosage unit refers to a form in which a pharmaceutical agent is provided.
  • a dosage unit is a vial comprising lyophilized antisense oligonucleotide.
  • a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
  • PMOs Phosphorodiamidate morpholino oligonucleotides
  • carbocyclic nucleosides were developed for use in oligonucleotide therapeutics.
  • PMO-based drugs eteplirsen, golodirsen, viltolarsen, and casimersen have been approved by the US FDA; all are used to treat patients with Duchenne muscular dystrophy.
  • 3 PMOs have also been shown to be effective against viral 4a and bacterial infections 411 and cancers 5 in cell-based and preclinical models.
  • PMOs must be chemically modified to improve cellular uptake and pharmacokinetics. 7,8 Among the reported modifications, incorporation of a guanidinium linkage or guanidinium-functionalized nucleobase into PMOs is notable. 9 Sinha’s group demonstrated the cell-penetrating and gene silencing properties of self-transfecting guanidinium morpholino-PMO chimeras in an vitro model and in zebrafish. 9d The Hayes group reported that triazole-linked morpholino-DNA chimeras were resistant to enzymatic degradation. 10 The Caruthers group developed thiophosphoramidate morpholino oligomers and their phosphorothioate DNA chimeras.
  • car-morpholino monomers were synthesized from commercially available, optically pure cyclopentenyl-amino-methanol 1.
  • car-U 2 was synthesized following the procedure previously reported for synthesis of car-RNA (Scheme l). 2c After silyl protection, the double bond of 3 was oxidized with OsCU to yield diol 4. The oxidative cleavage of 4 by NaIO-i and subsequent reductive cyclization with benzylamine gave N- benzyl car-U morpholino monomer 6 in 60% yield.
  • (NH ⁇ I ⁇ ChM ⁇ O as a nitrogen source following the original procedure, 12 7 was obtained. After A-tritylation, 5 was obtained.
  • Reagents and conditions (i) TBDPSC1, imidazole, DMF, room temperature; (ii) OsCU, N- methylmorpholine X-oxide, acetone, H2O, room temperature; (iii) (a) NalO-i, silica gel, MeOH, H2O, room temperature, (b) benzyl amine, NaCNBHr, AcOH, MS4A, MeOH, room temperature; (IV) 10% Pd on carbon, HCO2NH4, EtOH, reflux; (v) trityl chloride, EtsN, DMF, room temperature; (vi) TBAF, THF, room temperature.
  • Tr trityl
  • TBDPS /e/7-butyldiphenylsilyl.
  • Scheme 2c The triazole substituted U moiety of 5 was converted to 9 upon treatment with aqueous NFUOH solution at room temperature. Acetyl protection of the exocyclic amine followed by silyl deprotection of 10 by TBAF gave the car-C morpholino monomer 11 in 60% yield from 5.
  • TLC was performed on Merck silica gel 60 plates coated with F254. Compounds were visualized under UV light (254 nm) or after spraying with the p-anisaldehyde staining solution followed by heating. Flash column chromatography was performed using a Teledyne ISCOCombi Flash system with pre-packed RediSep Teledyne ISCO silica gel cartridges. All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. The microwave reactions were performed using a Discover® SP microwave system (CEM Corporation) in sealed glass tubes at 200 W with a 30-s premixing times with reaction temperature monitored using an internal infrared probe.
  • CEM Corporation Discover® SP microwave system
  • ESI-MS spectra were recorded on a Waters Q-TOF Premier instrument using the direct flow injection mode. 'H NMR spectra were recorded at 300, 400, 500, or 600 MHz. 13 C NMR spectra were recorded at 75, 101, 126, or 151 MHz. 31 P NMR were recorded at 121 MHz.
  • reaction mixture was extracted with CH2Cl2 and ethyl acetate. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0– 10% MeOH in CH 2 Cl 2 ) to obtain compound 24 as a brown solid (5.28 g, 99%).

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Abstract

La présente divulgation concerne de manière générale un cycle à six chaînons contenant des nucléosides, en particulier, des nucléosides pipéridino et des oligonucléotides, des oligomères dérivés des monomères cycliques à six chaînons comprenant ceux-ci.
EP24751025.8A 2023-02-01 2024-02-01 Cycle à six chaînons contenant des oligomères Pending EP4658280A2 (fr)

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PCT/US2024/014036 WO2024163762A2 (fr) 2023-02-01 2024-02-01 Cycle à six chaînons contenant des oligomères

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US9914745B2 (en) * 2009-08-14 2018-03-13 Indian Association For The Cultivation Of Science Morpholino-based antisense agent
AU2016302009B2 (en) * 2015-08-05 2021-09-30 Eisai R&D Management Co., Ltd. Chiral reagents for preparation of homogeneous oligomers
BR112018074346B1 (pt) * 2016-05-24 2023-01-03 Sarepta Therapeutics, Inc. Composto oligomérico e processo para preparar um composto oligomérico
US12502432B2 (en) * 2017-10-17 2025-12-23 Sarepta Therapeutics, Inc. Bicyclic peptide oligonucleotide conjugates
US10758629B2 (en) * 2018-05-29 2020-09-01 Sarepta Therapeutics, Inc. Exon skipping oligomer conjugates for muscular dystrophy

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